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Coactivator synergy for nuclear receptors: Regulatory roles of arginine methyltransferase activity in transcription

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Coactivator synergy for nuclear receptors: Regulatory roles of arginine methyltransferase activity in transcription

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Content COACTIVATOR SYNERGY FOR NUCLEAR RECEPTORS: REGULATORY ROLES OF ARGININE METHYLTRANSFERASE ACTIVITY IN TRANSCRIPTION by Young-Ho Lee A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOCHEMISTRY) DECEMBER 2002 Copyright 2002 Young-Ho Lee Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3093781 UMI UMI Microform 3093781 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA The G raduate School University Park LOS ANGELES, CALIFORNIA 90089-1695 Thi s dissertation, w ritte n b y Y&un£~ Ho L- j &L.___________ U nder th e d ire c tio n o f b.hS.. D issertatio n C om m i ttee, an d approved b y a ll its m em bers, has been presented to an d accepted b y The G raduate School , in p a rtia l fu lfillm e n t o f requi rem ents fo r th e degree o f D O C TO R O F P H IL O S O P H Y o f G raduate S tudies December 18, 2002 DI SSER TA H O N CO M M ITTEE C hairperson Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements For I am convinced that neither death nor life, neither angels nor demons, neither the present nor the future, nor any powers, neither height nor depth, nor anything else in all creation, will be able to separate us from the love of God that is in Christ Jesus our Lord. (Romans 8: 38 -39). I am willing to acknowledge this small accomplishment is done not by me but through the help of Father God. He gives me wisdom and insight, strength all the time, especially whenever I stumbled and got lost. I had difficult times during my thesis years but I was not afraid because of His companion with me. I could overcome all kinds of troubles through His unfailing love. I want to accept this gift and blessings from Him with thankfulness. I especially appreciate my mentor Dr. Michael R. Stallcup for giving me the opportunity to study this exciting project. He guided me to the right direction for the project, and taught me how to be a good scientist. I also admit his patience and his support throughout my thesis years. I also want to thank Drs. Deborah L. Johnson and Robert Maxson for their supports, precious advices, encouraging words. Their time and patience really help to bear fruits of my works. ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I would like to express my gratitude to my lab mates, Dagang Chen, Han Ma, Shihming Huang, Stephen S. Koh, Catherine Teyssier, Hongwei Li, Jeonghoon Kim, David Y. Lee, Daniel Gerke and the others. I had great time with this nice group of people. Lastly, I want to thank to my parents, brother, sister, uncle, aunt and friends in my homeland and here for their love and friendship. I also appreciate Pastors Ken, Paul and my church fellows’ for their constant praying. Thank you all. July 12, 2002. Young-Ho Lee Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Acknowledgements ---------------— -------------------------------- List of Table and Figures — --------------------- ---------------------------- Abstract------------------- — -------------------------------------- ------- Chapter 1: Introduction --------------------- —---------------------------------- Chapter 2: Materials and Methods-------------------------------------------- Chapter 3: Synergy among multiple nuclear receptor coactivators - Chapter 4: Selective requirements of arginine methyltransferase and protein acetyltransferases for coactivator synergy------------------ Chapter 5: Coactivator p300/CBP methylation by CARM1 arginine methyltransferase-------------------------------------------------------- Chapter 6: Characterization of CARM1 interacting proteins and investigation of flightless -I coactivator activity ----------- — Chapter 7: Concluding Remarks ---------------------------------------------- References --------------------------------— --------------------------------- — page jj v — vii 1 8 — 15 — 47 — 84 — 116 - 144 - 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Table and Figures page Fig. 3-1. Requirement for three coactivators (GRIP1, CARM1, and p300) at low levels of N R --------------------------------------------------- ------------25 Fig. 3-2. CARM1 and p30Q/CBP synergy is dependent on NR, NRE, hormone, and GRIP1----------------------------------------------------------- 27 Fig. 3-3. AF1 and AF2 of NR independently support coactivator Fig. 3-4. NR box motif of GRIP1 is essential for coactivator synergy for AR and TR -------------------------------- — 35 Fig. 3-5. Ternary coactivator complex formation among GRIP1, CARM1 and p300/CBP----------------------------------------- — — 39 Fig. 3-6. Coactivator complex formation is required for coactivator Fig. 4-1. Synergy among various combinations of three or four coactivators at low NR levels-----------—------------------------- — ------------ 55 Fig. 4-2. Role of protein acetyltransferase activities of p/CAF and p300 in coactivator synergy---------------------------------------------- 59 Fig. 4-3. Construction of methyltransferase defective CARM1 mutant, E/Q Fig. 4-4. Role of protein methyltransferase activity of CARM1 in coactivator synergy----------------------------- 66 Fig. 4-5. Selective synergy of protein arginine methyltransferases with p300 and p/CAF ------------------------ — -------------------------- — 71 Fig. 4-6. Different mechanisms of transcriptional activation at low and high NR levels —-------------------- 83 Fig. 5-1. p300/CBP is a substrate for CARM1 methyltransferase---------89 Fig. 5-2. Localization of methylated p300 fragments by CARM1 -------- 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-3. Determination of methylated Arginine residues in GBD of p300 by CARM1 -------------------------------— --------------------- 97 Fig. 5-4. Arginine residues of pSOOGBD are important for protein- protein interaction---------------------------------------------------------------------- 100 Fig. 5-5. R2 and R3 of pSOOGBD are important for transcription activation function ---------------- — ------------------------------- — — — -104 Fig. 5-6. Treatment of methylation inhibitor influences protein interaction of pSOOGBD and GRIP1.AD1 or p53---------------------- — ---------- 107 Table 1. Summary of CARM1 interacting proteins from yeast two-hybrid screening----------------------------------------------------------------- 121 Fig. 6-1. Direct interaction of CARM1 and flightless-l in vitro------------- 124 Fig. 6-2. Flightless-1 human homolog has coactivator activity for NRs — Fig. 6-3. Coactivator activity of flightless-l is dependent on AD1 domain of GRIP1---------------------------------------------- 131 Fig. 6-4. Flightless-l cooperates specifically with CARM1 at low NR condition----------------------------------------------------------------------------------- 134 Fig. 6-5. Arginine methyltransferase activity of CARM1 is essential for cooperation with flightless-l------------------------------------------------------137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract Hormone-activated nuclear receptors (NR) bind to specific regulatory DNA elements associated with their target genes and recruit coactivator proteins to remodel chromatin structure, recruit RNA polymerase, and activate transcription. The p160 coactivators (e.g., SRC-1 /GRIP1 /ACTR) bind directly to activated NR and can recruit a variety of secondary coactivators. We have established a transient-transfection assay system under which the activity of various NR is highly or completely dependent on synergistic cooperation among three classes of coactivators: a p160 coactivator, the protein methyltransferase CARM1, and any of the three protein acetyltransferases, p300, CBP, or p/CAF. The three-coactivator functional synergy was only observed when low levels of NR were expressed and was highly or completely dependent on the methyltransferase activity of CARM1 and the acetyltransferase activity of p/CAF, but not the acetyltransferase activity of p300. Other members of the protein arginine methyltransferase family, which methylate different protein substrates, could not substitute for CARM1 to act synergistically with p300 or p/CAF. A ternary complex of GRIP1, CARM1, and p300/CBP was demonstrated in cultured mammalian cells, supporting a physiological role for the observed synergy. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In addition to histone H3, p3Q0/CBP was identified as an efficient methylation substrate for CARM1-specific methyltransferase. One of the major methylation sites is localized to Arginine 2142 of pSOOGBD (GRIP1 binding domain). Mutations of arginines in pSOOGBD have effects on protein interactions with the other factors (GRIP1 and p53) and on transcriptional enhancement by pSOOGBD and GRIP1. The interaction between pSOOGBD and GRIP1.AD1 or p53 was also influenced by treatment of cells with Adox, a methylation inhibitor. These suggest that CARM1-catalyzed p300/CBP methylation is an important switch which is potentially important in protein-protein interaction and transcriptional activation by NR. To further understand CARM1 action, CARM1 -interacting proteins were isolated by yeast two- hybrid screening. One of the CARM1 binding proteins was a developmentally important protein, flightless-l. Flightless-l interacts with CARM1 in vitro and acts as a coactivator for ER and TR in the presence of GRIP1. Furthermore, flightless-l cooperates specifically with CARM1 and its methyltransferase activity. Our understanding of CARM1 action may be further deepened by characterization of CARM1- interacting proteins. viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1. introduction General characteristics of Nuclear Receptors and transcription Transcription regulation plays a central role in biological phenomena. Various internal or external outside stimuli produce ‘biological signals’ which could modulate the rate of producing specific RNA messages. Transcription regulation in eukaryotic cells is tightly and delicately regulated by a variety of factors and enzymes for this reason. Generally multiple factors, like RNA polymerase and basal transcription factors, are required for transcription initiation. Additionally, transcription coactivators and corepressors continuously regulate basal transcription in positive or negative manners (Glass et al, 2000; McKenna et al, 1999; McKenna et al; 2002). Transcription coactivators act as adaptor molecules between promoter-bound basal transcription factors and enhancer-bound activators. As a result, many factors in close proximity modulate the RNA synthesis coordinately. Coactivator molecules are actively involved both in recruitment of RNA polymerase II and its associated basal transcription factors and in chromatin remodeling. In this aspect, coactivator molecules hold the important key for the regulation of eukaryotic genes. Many coactivator molecules are identified and the functions of coactivator molecules are investigated recently. Especially coactivators for the nuclear receptor (NR) superfamily are the subject of intense study (Glass et al, 2000; McKenna et al, 1999; McKenna et al; 2002). i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Nuclear receptors are an attractive model system for studying transcription regulation and chromatin remodeling in eukaryotic cells. Nuclear receptors are hormone-dependent transcription activators (Beato et al, 1995; Mangelsdorf et al, 1995; Tsai et al, 1994). Steroids and non steroids like thyroid hormone and retinoids are important ligands for NRs. Ligand bound NRs play important roles in development, differentiation, apoptosis, metabolism and the other processes by activating specific genes in vertebrates. Members of the NR superfamily directly activate specific genes by binding to hormone response elements (HRE) in promoter or enhancer regions. In addition, NR can inhibit the transcriptional activities of other classes of transcription factors like AP1 by protein-protein interactions (Glass et al, 2000). The NR family has about 50 different family members and is classified into three groups depending on the mechanism of their activation (Beato et al, 1995; Tsai et al, 1994, McKenna et al; 2002, Evans et al, 1988). Type I nuclear receptors include androgen (AR), estrogen (ER), progesterone (PR), glucocorticoid (GR) and mineralocorticoid receptor (MR) etc. This type of NR is usually present in cytoplasm but is translocated into the nucleus after ligand binding, binds to specific DNA elements and activates transcription. Receptors of the type II family such as receptors for thyroid hormone (TR), retinoids (RAR, RXR) and vitamin D (VDR), remain bound to DNA elements as inactive forms. 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hormone binding leads to conversion of NR conformation to its active form and activates transcription. In addition, the family also includes a group of orphan receptors that appear to belong to the NR superfamily on the basis of sequence identity but whose ligands have yet to be found. Recent studies have identified natural and synthetic ligands for several orphan receptors and shed light on some of the new hormone signaling pathways in the regulation of lipid, cholesterol, glucose and xenobiotic metabolism (Mangelsdorf et al, 1995). Although three NR subfamilies have many family members, the structure of NRs is very similar and conserved (Beato et al, 1995; Tsai et al, 1994, Evans et al, 1988). NR can be broadly divided into three functional domains based on the amino acid sequence analyses. The central region of the NR is the most highly conserved and contains the DNA binding domain (DBD) that targets the receptors to specific DNA response elements. The C-terminal part of the receptors is similar in size and moderately conserved in sequence. It is generally called ligand-binding domain (LBD) because it possesses the important property of hormone recognition, which ensures both selectivity and specificity of physiological responses. The N-terminal parts of NRs are extremely variable in size and have an AF-1 activation domain. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The DNA binding domain (DBD) of NR targets the receptor to specific DNA sequences known as hormone response elements (HRE) (Beato et al, 1995; Tsai et al, 1994, Khorasanizadeh et al, 2001, Evans et al, 1988). HREs are palindromic DNA sequences, and are recognized by ligand bound NR dimers. GR, MR, PR, and AR recognize the same DNA sequence (AGAACA as half site) and ER binds to a different site (AGGTCA). TR, RAR, RXR, VDR recognize direct repeat of GGTCA sequence (Khorasanizadeh et al, 2001; Tsai et al, 1994). The structure of the DNA binding domain was determined by NMR and X-ray crystallography analysis. The DNA binding domain of NR is a globular structure that is composed of two modules. Each module has a zinc finger and amphipathic alpha helix. The first module has a P box, three amino acids, which is responsible for DNA recognition. The site directed mutation of this sequence results in a shift of NR specificity in DNA recognition. The second module has a D box, which is important for dimerization of NRs. The hinge region between the DNA and hormone binding domains may allow the protein to bend or alter conformation, and often contains a nuclear localization domain and/or transactivation domain. The ligand-binding domain (LBD) is located carboxy- terminal to the hinge region. The LBD region is relatively large and is functionally complex (Beato et al, 1995; Tsai et al, 1994, Weatherman et al, 1999). 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It usually contains regions important for heat shock protein association, dimerization, nuclear localization, transactivation, and ligand binding etc. Most of the functions require small portions of the LBD, but ligand binding function needs the involvement of the majority of LBD (Tsai et al, 1994). The major dimerization domain is also localized in the C-terminal part of the LBD. This region has coiled coil interactions with the other NRs. Association of heat shock protein with NR LBD occurs for the type I class of NRs. In the absence of hormone, type I NR exists as a complex with heat shock proteins (hsp 90, hsp 70, and hsp 56). For most cases, unliganded type I NRs do not bind to DNA and thus have neither transcription nor silencing activity. Upon binding hormone, NR is dissociated from heat shock proteins and is able to dimerize, bind to DNA and transactivate target genes. On the other hand, Class II NRs, such as TR, VDR and RAR, is not associated with heat shock proteins. Class II NRs already bind to their DNA response elements in the absence of hormone but they actively repress the target gene transcription until hormone binding transforms the receptors into active transcription factors. Three-dimensional structures of LBD of several NRs were also documented (Weatherman et al, 1999, Renaud et al, 1995; Bourget et al, 2000). The LBD structure of NRs is strikingly similar among NR members and is composed of twelve highly conserved a-helical structures. 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Upon ligand binding, most of the LBD structure remains unchanged, but the helix 12 is shifted and provides a binding site for coactivator molecules like p160 coactivators. Only agonist but not antagonist binding leads to the conformational changes that transforms the NRs into active transcription factors and subsequently promote target gene transcription. NRs have two activation domains called AF1 (activation function 1) and AF2 (activation function 2) (Evans et al, 1988; Kumar et al, 1987, Lees et al, 1989). AF2 domain is located at the C-terminal region of ligand binding domain and contains a highly conserved hormone-dependent transcriptional activation activity. AF2 domain has a role in recruiting coactivators in the presence of ligand. Additionally, the N-terminal part of NR has an additional activation domain called AF1. Several groups recently have clarified the importance of AF1 domain (Ma et al, 1999, Bevan et al, 1999). AF1 domain is a divergent domain of NR family members, and also has independent transcription activation activity. Some of the coactivators interact with NR molecules through AF1 domain. AF1 activity is also regulated by posttranslational modifications like phosphorylation (Kato et al, 1998). The relative importance of AF1 and AF2 is variable depending on the specific NR (Lees et al, 1989), cell type, and target promoters (Tzukerman et al, 1994). AF1 and AF2 domains of NR could be associated with same coactivators. 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For example, p160 coactivator GRIP1 binds to AF1 through their C- terminal domain of GRIP1. In contrast, AF2 associates with GRIP1 through NR box motifs LxxLL. Recent reports suggest the possibility of cooperation between AF1 and AF2 in NR function (Benecke et al, 2000). For this cooperation, p160 coactivator could play important roles in linking two activation domains of NRs. These domains are responsible for conveying hormone dependent activation signal to transcriptional machinery of NRs. In this thesis, NR transcription regulation by various coactivators will be investigated. Especially, the functions and action mechanism of CARM1 and its protein methyltransferase activity will be a primary focus in regarding to NR transcription. In chapter 3, recent coactivator research on NRs will be reviewed and cooperation of multiple coactivators will be demonstrated in transient reporter assay system. In chapter 4, histone- modifying activities like acetyltransferase and methyltransferase of coactivators will be reviewed and their activity will be tested in coactivator synergy. In chapter 5, protein factor modifications by coactivator molecules will be described, and the methylation of p300/CBP by CARM1 will be discussed. In the final chapter, protein molecules interacting with CARM1 will be investigated. Furthermore, one of the CARM1 interacting molecules, flightless -I, will be assessed as a novel coactivator in NR transcription. 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2. Materials and methods Plasmids. The mammalian cell expression vector pSGS.HA (Chen et al, 1999a), which has simian virus 40 and T7 promoters, was used to express proteins with an N-terminal hemagglutinin (HA) tag; plasmids encoding the following proteins were previously constructed in pSGS.HA as indicated: GRIP1, CARM1, CARMi(VLD) (Chen et al, 1999a); GRIP1AAD1 and GRIP1AAD2, GRIP1 ANRbox (Ma et al, 1999); GRIP1AAD1 plus AD2 (Chen et al, 2000a); and rat protein arginine methyltransferase 1 (PRMT1) (Koh et al, 2001). New pSGS.HA expression vectors encoding the following proteins were constructed by inserting the appropriate cDNA coding region (reviewed in Chen et al, 1999a) into the indicated restriction enzyme sites of the vector: human PRMT2, EcoRl-BamHI; rat PRMT3, Mfe\-Xho\ fragment inserted into EcoRl and Xho\ sites; and yeast arginine methyltransferase 1 (RMT1), EcoRI-BamHI. The mutation E267Q in CARM1 was generated with the Quickchange site-directed mutagenesis kit (Stratagene), using pSG5.HA- CARM1 as the template. The following mammalian expression vectors with cytomegalovirus promoters were used to express p300, CBP, and p/CAF: pCX-p/CAF, pCX-p/CAFA579-608, pCX-p/CAFA609-624, pCMV- p300, pCMV-p300A1603-1653 (Puri et al, 1997), and pcDNA3-CBP, kindly provided by T.-P. Yao (Duke University). The p300 vectors and p/CAF vectors included an N-terminal Flag epitope tag. 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pGEX4T vectors expressing CBP.C (1594-2441), pSOO.C (1571-2414), pSOO.N (1-596) and p300CH3 (1709-1934) fragments were also provided by T.-P. Yao and pSOO.KIX (568-828) was a gift from Lee Kraus. The other fragments, pSOOGBD (2042-2157) and p300Q (2158-2414), are amplified by PCR using pfu and inserted into BamHI/Xhol site of pGEX4T or EcoRI/BamHI of pM vector. A series of point mutants, R1K, R2K and R3K are generated with the Quickchange site-directed mutagenesis kit (Stratagene) using pGEX4T- or pM- p300GBD as the template. For mammalian expression vectors of flightless-l, human cDNA was isolated with Sall/BamHI digestion and inserted into pcDNAS vector. Fli-LRR (1- 494) and Fli-Gelsolin (495-1268) was amplified by PCR and the amplified fragments were digested with Mfel/Sall and were inserted into EcoRI/Xhol site of pSGS.FIag. Flightless-l cDNA was from H. Campbell and pSGS.Flag vector was obtained from M. Parker’s pSG5.Flag-TEF2. Mammalian expression vectors encoding NRs included pHEO for human ER, pSVARO for human androgen receptor (AR), pCMX.hTRUI for human thyroid hormone receptor (TR) R > 1 (Chen et al, 1999a), pM-AR,AF1 and pM-AR.AF2 (Ma et al, 1999). The following luciferase-expressing reporter genes were described previously (Chen et al, 1999a; Huang et al, 2000): for AR, MMTV-LUC with the native mouse mammary tumor virus (MMTV) promoter; 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for TR, MMTV(TRE)-LUC with a single thyroid hormone response element substituted for the native glucocorticoid response elements; For ER, MMTV(ERE)-LUC with a single estrogen response element (ERE) substituted for the native glucocorticoid response elements, EREII- LUC(GL45) containing a basal herpes simplex virus thymidine kinase (TK) promoter and two EREs, and TK-LUC containing the basal TK promoter without EREs; and for Gal4 DNA binding domain (DBD) fusion proteins, GK1. The vector pGAL-CBP8 encoding Gal4 DBD fused to full-length CBP was described previously (Chrivia et al, 1993). The vector pVP16.CARM1, encoding the activation domain VP16 fused to CARM1, was constructed by inserting an EcoR\-Bgl\\ fragment encoding CARM1 into pVP16 (Clontech). Cell culture and transient transfections. CV-1 cells (Gluzman, 1981) were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. Approximately 20 h before transfection, 105 cells were seeded into each well of six-well dishes. The cells in each well were transfected with SuperFect Transfection Reagent (Qiagen) or Targefect (Targeting Systems) according to the manufacturer's protocol; total DNA was adjusted to 2.0 jig by addition of the empty vector pSGS.HA (Chen et al, 1999a). After transfection, the cells were grown in medium supplemented with 5% charcoal-stripped fetal bovine serum (Gemini Bioproducts) for 40 h before harvest; 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. where indicated, the medium was supplemented with 20 nM dihydrotestosterone (DHT) for AR, 20 nM estradiol (E2) for ER, or 20 nM 3,5,5'-triiodo-L-thyronine (T3) for TR during the last 30 h of growth. Luciferase assays were performed with the Promega Luciferase Assay kit, and luciferase activities are shown as the mean and deviation from the mean of two transfected sets. The results shown are representative of at least three independent experiments. Because some coactivators enhance the activities of so-called constitutive promoters two- to threefold, internal controls by cotransfection of constitutive B-galactosidase expression vectors were not used to normalize luciferase data. However, internal controls were used strategically to show that variation in transfection efficiency was not a factor in key results (data not shown). Coimmunoprecipitation and immunoblot analyses. Cos-7 cells (Gluzman, 1981) were grown in 100-mm-diameter dishes seeded with 106 cells and transfected with combinations of the coactivator expression plasmids pCMV-p300, pSG5.HA-CARM1, and pSG.HA-GRIP1 as indicated. At 40 h after transfections, cell extracts were prepared by lysing the cells in 1.0 ml of RIPA buffer (50 mM Tris CI [pH 8.0], 150 mM NaCI, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) and were clarified by centrifugation for 15 min at the maximum speed of a microcentrifuge. li Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A portion of the supernatant (40 pi) was removed for direct immunoblot analysis of CARM1, using rat monoclonal antibody 3F10 against the HA epitope (Boehringer Mannheim) at 100 ng/ml as the primary antibody and horseradish peroxidase-conjugated anti-rat immunoglobulin G (sc-2006; Santa Cruz Biotechnology) at 160 ng/ml (1:2,500 dilution) as the secondary antibody. The remaining supernatant (800 pi) was incubated with anti-Flag antibody (no. F3165; Sigma) (1 pg) for 16 h at 4°C; the immunoconjugates were precipitated by incubation with protein G agarose (no. P7700; Sigma) (50 pi of a 50% suspension), followed by centrifugation. Immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis, and immunoblotting was performed as described previously (Ma et al, 1999) with antibody against HA epitope. Immunoprecipitation of CARM1 and methyltransferase assays. Vectors encoding wild-type or mutant HA-tagged CARM1 (2.5 pg) were transfected into 106 Cos-7 cells. After 40 h, the cells were lysed with 1.0 ml of RIPA buffer. After centrifugation, 40 pi of supernatant was removed for direct immunoblot analysis of CARM1 (using antibody against HA tag), and 800 pi was used for immunoprecipitation of CARM1 as described above, using 1 pg of antibody against HA tag and 50 pi of protein G agarose suspension. 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The pellet from the immunoprecipitation was resuspended in 50 pi of methyltransferase reaction mixture (20 mM Tris-HCI [pH 8.0], 200 mM NaCI, 0.4 mM EDTA, 8 pg of unfractionated core histones [no. H9250; Sigma], 500 pM S-adenosylmethionine) and incubated at 30°C for 2 h. The reaction products were resolved by SDS-polyacrylamide gel electrophoresis (10% acrylamide), and methylated histone H3 was visualized by immunoblot analysis with antiserum raised against a peptide representing histone H3 amino acids 1 1 to 21 with asymmetric dimethylarginine at position 17 (Upstate Biotechnology) Yeast two hybrid screening Yeast two-hybrid screening was performed as previously described (Chen et al, 1999a; Huang et al, 2000). 17-day-old mouse embryo cDNA library was used according to manufacturer’s protocols. Screening was performed using pGBT9.CARM1 as a bait in Hf7C yeast cell strain. GST pulldown assay GST pulldown was performed as previously described (Chen et al, 1999a; Ma et al, 1999; Koh et al, 2001, Huang et al, 2000). GST, GST-GRIP1.C, GST-CARM1, GST-p300GBD(WT), GST-p300GBD(R1K), GST- p300GBD(R2K), GST-p300GBD(R3K) and the other proteins were expressed in BL21 E.coli cells. 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These proteins are purified with glutathione sepharose 4B beads from E.coli cell lysates after sonication. S3 5 -Iabeled proteins were prepared with TNT T7 -coupled reticulocyte lysate system (Promega). For immunoblot detection, mammalian expression vectors of GRIP1.AD1, p53, Fli-LRR and Fli-Gelsolin which has HA- or Flag tag were transfected into Cos-7 cells and the expression and interaction with GST proteins was detected with HA- or Flag- specific antibody. HAT assay by immunoprecipitation of p300 and p/CAF This assay was similarly performed with methyltransferase assay of CARM1. Vectors for wild-type or mutant p300 and p/CAF (2.5 jig each) were transfected into Cos-7 cells. After 40 h, the transfected cells were lysed with 1 ml of RIPA buffer and 0.8 ml of lysates were immunoprecipitated with anti-Flag antibody (Sigma) and protein G agarose. The pellet was resuspended with HAT assay buffer (100 mM Tris-HCI [pH 8.0], 1 % glycerol, 0.1 mM EDTA, 1 mM Dithiothreitol, 10 mM butyric acid, 8 jig of unfractionated core histones [no. H9250; Sigma], 500 jiM acetyl-coA) and incubated with histones and acetyl-CoA (Sigma) at 30°C for 2 hours. Acetylated histones were detected by using acetylated lysine specific antibody (Cell signaling system). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3. Synergy among multiple nuclear receptor coactivators 3-1. Introduction Coactivators for Nuclear receptors In an effort to elucidate the mechanism of NR mediated transcription, diverse nuclear receptor coactivators have been identified (Glass et al, 2000; McKenna et al 1999; McKenna et al, 2002). Coactivator molecules do not bind to DNA directly, but assist transcription factors like NRs by protein-protein interaction. Coactivators are known to bridge between the transcription activators and the basal transcription machinery and facilitate the formation of transcription initiation complexes. Furthermore, coactivator molecules have crucial roles in reorganizing chromatin structure to facilitate this process. A growing list of coactivator molecules has been identified by biochemical purification or expression cloning methods like yeast two hybrid screening. Some coactivators have enzymatic activities like acetyltransferase, arginine methyltransferase, kinase (Glass et al, 2000; McKenna et al 1999; McKenna et al, 2002). The other coactivators like TRAP and DRIP seem to be only involved in protein-protein interaction. 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Some coactivators activate diverse NRs, while other coactivators activate only certain classes of NRs. Most of the coactivators show ubiquitous tissue expression, but other coactivators like PGC1 show tissue-specific expression and function. Clearly, the kinds of coactivators outnumber the NRs. For this reason, many efforts are focused on whether these coactivators act on NR sequentially, cooperatively or in parallel. It becomes clear that many of the coactivators exist as multisubunit complexes to regulate transcription. The well-characterized coactivator complexes include p160 coactivator complex, acetyltransferase coactivator family members, ATP dependent chromatin remodeling complexes, and TRAP-DRIP-ARC complex. A typical example of a chromatin remodeling coactivator is the ATP dependent chromatin remodeling complex Swi/Snf. Cofactor molecules of this family require ATP hydrolysis for remodeling chromatin templates and appear to be involved in both positive or negative regulation of transcription by NR (Glass et al, 2000). The Mammalian homolog of Swi/Snf, BRG1/Brm1, is one of the components of this large protein complex. Some components of the mammalian Swi/Snf complex interact directly with NRs. Transfection of these components into mammalian cells affects the activity of NRs. Remodeling complexes containing ISWI (imitation SWI) are also involved in NR function. There are additional ATP dependent remodeling complexes like Mi2a/CHD3 and Mi2p/CHD4. 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Another class of coactivator, the TRAP-DRIP-ARC complex, does not have nucleosome remodeling activity or histone modifying activities (Glass et al, 2000). This complex was identified by biochemical purification of molecules associating with TR or VDR. TRAP-DRI P-ARC is a mediator type complex of 14 - 16 factors, which also can activates NR function and other signal dependent transcription factors in vitro. One of the common components is 220 kD PBP/TRAP220/DRIP205, and this is recruited to NRs through their LXXLL NR interaction motifs. Some components of TRAP-DRI P-ARC complexes are also found in other transcription activator complexes. Since none of the components in this complex has enzymatic activities, these factors probably play roles in recruitment of RNA polymerase II complexes by protein-protein interaction. There are groups of coactivators harboring enzymatic activities. The most well known example are coactivators having HAT (histone acetyltransferase) activities (Sterner et al, 2000). HAT activities of coactivators usually modify the N-terminus of histones at the promoters of transcriptionally activated genes. This topic will be covered in more detail in the next chapter. Coactivators which have HAT activities include mammalian GCN5 homolog p/CAF, CREB-binding protein (CBP), adenovirus E1A binding 300 kD protein (p300), one of the general transcription factors TAFII250. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Two of the HAT coactivators, p300 and CBP, show highly conserved sequence homology and similar biochemical characteristics including HAT activities although these are produced by distinct genes. However, some of the unique characteristics of p300 or CBP are also being reported recently (Vo and Goodman, 2001). Although the HAT mediated function of these coactivators is a crucial part for their coactivator role, HAT- independent functions should not be ignored. Many HAT family coactivators have influence on transcription machinery by protein-protein interaction in addition to histone modifying function. For example, p300/CBP protein sequence has several domains such as a bromodomain, a KIX domain, and three cysteine-histidine rich (CH) domains, which provide binding sites for a variety of factors (Vo and Goodman, 2001). The other HAT coactivator p/CAF (p300/CBP- associated factor) also exists in a large cellular complex which does not include p160 coactivators, p300, or CBP (Ogrzyko et al, 1998), although p/CAF can bind directly to p160 coactivators, CBP, and p300, as well as some NRs (Glass et al, 2000). Coactivator multiprotein complexes with HAT activity can stimulate transcription at several levels, including stimulating the formation of the preinitiation complex and by remodeling chromatin. One of the most well characterized coactivators for NR is the p160 coactivator. p160 family coactivators bind to nuclear receptor in ligand dependent manner and play important roles in assisting NR functions. 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The p160 coactivator family has three members, SRC1, GRIP1 (TIF2), and ACTR (RAC3, pCIP, AIB1, TRAM1) (Glass et al, 2000; McKenna et al 1999; McKenna et al, 2002). The physiological importance of p160 coactivators in hormone regulation was demonstrated by knockout mouse study of SRC-1 and ACTR/RAC3/pCIP/ AIB1/TRAM1 (Xu et al, 1998; Xu et al 2000). p160 coactivators including GRIP1 have three conserved NR binding motifs. These NR boxes (LxxLL motifs, L represents Leucine and X represents any amino acid) of GRIP1 are primarily involved in association with NR AF2 domain in a ligand dependent manner (Voegel et al, 1998; Heery et al, 1997; Torchia et al, 1997; Ma et al, 1999; Bevan et al, 1999). Structural study of NR boxes of p160 further emphasizes the significance of these motifs in association with NR binding. Each LxxLL motif of these three NR boxes forms a short a-helix, and is involved in direct contact with NR homodimers or heterodimers cooperatively. The hydrophobic face of the LxxLL helix is targeted to a hydrophobic pocket in the C-terminal part of NRs (Darimont et al, 1998; Glass et al, 2000). This contact is modulated by the ligand dependent conformational change of NRs. Many other coactivators have more than one NR box like p160 coactivators. From functional domain characterization, it is known that p160 coactivator GRIP1 has at least two activation domains called AD1 (Activation domain 1) and AD2 (Activation domain 2) (Ma et al, 1999). 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AD1 is located between amino acids 1040 and 1120 in GRIP1 (1462 amino acids). The other activation domain, AD2 is located in the C- terminal part of full length GRIP1. These two activation domains play independent roles in the activation of NRs. Other coactivators are targeted to these activation domains of GRIP1. p300/CBP is one of the examples of coactivators targeting to GRIP1 AD1 domain, and further enhances the transcription of NRs through this domain. (Chen et al, 1997; Voegel et al, 1998; Torchia et al, 1997). Although p300/CBP binds to NRs independent of GRIP1 in vitro, the activity of p30Q/CBP is dependent on the presence of the primary p160 coactivator. For this reason, p300/CBP could be classified as secondary coactivators. Other secondary coactivators whose function is dependent on GRIP1 were also recently identified. Yeast two-hybrid library screening with the GRIP1 C-terminal domain led to the discovery of coactivator called CARM1 (Coactivator Associated Arginine Methyltransferase 1) (Chen et al, 1999a). Different from p300/CBP, CARM1 interacts with GRIP1 through the C-terminal AD2 domain of GRIP1. CARM1 also further enhances transcription activation by NR in GRIP1 dependent manner. PRMT1, another member of the arginine methyltransferase family of CARM1, also enhances NR dependent transcription through interaction with C-terminal part of GRIP1 (Koh et al, 2000). The roles of arginine methyltransferase activity in coactivator function for NR will also be discussed in the next chapter. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In this chapter, the cooperative roles of multiple coactivators in NR dependent transcription will be discussed. In order to test the effect of multiple coactivators in NR transcription, an optimized NR assay system was developed. The formation of a ternary coactivator complex and its importance in synergy were demonstrated in a mammalian cell culture system as well. 3-2. Results Dependence of NR function on three different coactivators: GRIP1, CARM1, and p300. To test coactivator function, expression vectors for various NRs were transfected into CV-1 cells (which have little or none of most NRs) along with expression vectors for one or more coactivators and a luciferase reporter gene containing one or more enhancer elements specific for the NR being used. The transfected cells were treated or not treated with the appropriate hormone, and cell lysates were subsequently tested for luciferase activity as a measure of NR and coactivator function. When the amounts of various expression vectors transfected were carefully titrated, it was observed that the amount of NR expression vector strongly influenced the degree of cooperation observed among multiple coactivators. For six-well petri dishes (3.3-cm-diameter wells), transfection of 100 ng of ER expression vector enhanced reporter gene expression in the presence of estradiol (Fig. 3-1 A, bars 1 and 2) but not in the absence of the hormone (Fig. 3-2B). 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The luciferase reporter gene had an MMTV promoter with a single ERE substituted for the glucocorticoid response elements found in the native promoter. Coexpression of GRIP1 with ER enhanced reporter gene expression severalfold (Fig. 3-1 A, bar 3), and addition of CARM1 with GRIP1 caused a further enhancement (bar 4). In this experiment, coexpression of p300 with GRIP1 caused no additional stimulation (bar 5); in multiple experiments, we have found the effect of p300 in the presence of GRIP1 to be variable within the range of no effect to twofold enhancement. Addition of p30Q in the presence of GRIP1 and CARM1 caused a decrease in the activity observed with GRIP1 plus CARM1 (bars 4 and 6); in multiple experiments, coexpression of p300 in this situation caused either no effect or a decrease in activity of as much as 50%. In contrast, when lower levels of ER expression vector (<10 ng) were transfected, reporter gene activity was much more highly dependent on the coexpression of multiple coactivators. In the absence of any cotransfected coactivator vectors, 10 ng of ER vector produced no increase in luciferase activity above the background observed with reporter gene alone, even in the presence of estradiol (Fig. 3-1 A, bars 7 and 8). Addition of GRIP1, GRIP1 plus CARM1, or GRIP1 plus p300 caused no enhancement or a very modest enhancement of reporter gene activity compared with the activity observed with ER alone (bars 9 to 11). 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, when ER was supplemented with all three coactivators, a synergistic enhancement of reporter gene function was observed (bar 12). Thus, three-coactivator synergy and a high degree of dependence on three coactivators were observed at low ER concentrations but not at higher ER concentrations. The selectivity of the coactivator effect was demonstrated by the failure of GRIP1, CARM1, and p300 to stimulate the activity of the basal TK promoter (Fig. 3-2A, bars 1 to 6) and an RSV-fi- galactosidase reporter plasmid (Fig 3-2C). Similar synergistic enhancement of NR function by these three coactivators was observed when low levels of AR (Fig. 3-1B) and TR (Fig. 3-2C) expression vectors were used. Systematic variation of the level of AR expression vector revealed the relationship between synergy and NR levels (Fig. 3-1B). At low levels of AR expression vector (10 to 50 ng), very little reporter gene activity was observed unless GRIP1, CARM1, and p300 were all coexpressed with AR (Fig. 3-1B). However, at 70 ng of AR vector, GRIP1 alone, GRIP1 plus CARM1, or GRIP1 plus p300 produced substantial activity; a lower degree of three-coactivator synergy was still observed at 70 ng of AR vector, but the requirement for three coactivators was not as stringent. At higher AR levels, the three-coactivator synergy was not observed (data not shown). 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-1. Requirement for three coactivators (GRIP1, CARM1, and p300) at low levels of NR. CV-1 cells in each well of six-well culture dishes were transiently transfected with plasmids as indicated below. The transfected cells were grown with 20 nM E2 for ER or DHT for AR. Cell extracts were prepared and assayed for luciferase activity. (A) MMTV(ERE)-LUC reporter plasmid, 250 ng; 10 ng (low ER) or 100 ng (high ER) of ER expression vector; 250 ng of pSG5.HA-GRIP1, 500 ng of pSG5.HA- CARM1, and 500 ng of pCMV-p300. (B) MMTV-LUC, 250 ng; 10 to 70 ng of AR expression vector; coactivator vectors as for panel A. The graph on the right shows the activity observed with 10 ng of AR vector on an expanded scale. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A >> J j S ' O o So gx to _J ^oc 3 ' - ' 120 80 40 H igh ER Low ER 7 I 1 2 3 4 5 6 7 8 9 10 1 1 12 ULJ + + + + + + + + + + GRIP1 + + + + + + + + CARM1 + + + + p300 + + + + > o C O o ®X 5 3 3 w B 600 400 200 - A R ♦ AR GRIP! A AR GRIP1 CARM1 | AR GRIP1 p300 < § AR GRIP1 CARM1 p300 / ~ 4 h — 1 '.-4 1 0 30 50 AR vector (ng) 70 12 AR - GRIF1 CARM1 p300 + t + + + + + + + + + T + 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-2. CARM1 and p300/CBP synergy is dependent on NR, NRE, hormone, and GRIP1. (A) EREII-LUC (GL45) or TK-LUC reporter plasmid, 250 ng; 1 ng of ER expression vector; coactivator vectors as for panel A. (B) EREII-LUC(GL45), 250 ng; ER expression vector, 1 ng; 500 ng of each coactivator expression vector (GRIP1, CARM1, p300, and CBP). +, present. (C) RSV promoter directed beta-galactosidase activity is not affected by coactivator expression. MMTV(TRE)-Luc reporter plasmids, 250 ng, 250 ng of RSV-BGAL, 1 ng of TR, 250 ng of pSG5.HA-GRIP1, 500 ng of pSG5.HA-CARM1, and 500 ng of pCMV-p300. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 8) X - Z 5 1 000 ‘ ! — 1 i. 1 2 3 4 5 6 7 8 9 10 1! 12 13 14 15 16 17 f k - L u c E R E - tk - L u c E R 4 . -]~ 4 . _j_ + + + + + + + G R I P ! + + + + + + + + + + + + CAR Ml + + + + + + + p300 + + + + + + + B X 3 2000 1000 Estradiol + Estradiol : ....J O M a m a J • m i® . i i 1 2 3 4 5 6 7 8 9 to 1 1 12 13 14 15 16 ER 4- 4- + 4 . 4- 4 4~ 4- 4~ 4* 4- 4~ 4- 4- 4 GRiPl 4- 4- + + 4~ 4 - 4~ 4~ 4- 4~ 4- CAR M l 4- + + + 4 4- 4* 4 . p 3 0 0 4- 4~ 4~ 4~ 4- CBP 4- + 4- 4~ 4- 16 ■ Luc activity □ P-GAL activity 12 1 2 3 4 TR GRIP1 CARM1 P300 + + + + 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The observed coactivator synergy depended on the presence of the entire hormone response system in addition to the three coactivators. Coexpression of ER (1 ng), GRIP1, CARM1, and p300 in the presence of estradiol caused activation of a reporter gene controlled by a basal herpes simplex virus TK promoter and two EREs (Fig. 3-2A, bar 16). Omission of the hormone response elements (bars 1 to 6), ER (bars 7 to 11), or GRIP1 (bar 17) resulted in a complete loss of reporter gene stimulation. Omission of CARM1 or p300 or both resulted in a dramatic but not complete loss of reporter gene stimulation (bars 13 to 15). If CBP was substituted for p300, a similar level of synergy was also observed (Fig. 3- 2B, lanes 11 to 16). The synergistic effect of coactivators was completely dependent on the presence of estradiol (Fig. 3-2B, compare lanes 2 to 7 with lanes 9 to 14). Similar results were obtained when the various controls in Fig. 3-2B and C were performed with AR and TR instead of ER (data not shown). Thus, at low levels of NR, efficient hormone-dependent activation of an NR- dependent reporter gene was almost completely dependent on the coexpression of three coactivators, and the synergistic effect of the three coactivators depended entirely on the presence of a hormone-activated NR bound to its cognate enhancer element. Furthermore, the coactivator effects of CARM1 and p300 depended entirely on the presence of GRIP1. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Titration of the various coactivator expression vectors demonstrated that the level of reporter gene activity increased as the level of each coactivator was increased (data not shown). The amount of reporter gene activity observed from day to day when GRIP1 alone or GRIP1 plus one other coactivator was coexpressed with low levels of NR varied from none to significant, as seen in the various figures in this report (e.g., compare Fig. 3-1 A, right, with 3-2A; also see later figures); but the activity observed with three coactivators was always synergistic, i.e., much higher than the additive effects of individual or pairs of coactivators. Coactivator synergy is independently supported by AF1 and AF2 of NRs NR has two activation domains called AF1 and AF2 (Evans et al, 1988; Kumar et al, 1987). In order to determine which activation domains of NR are required for coactivator synergy, two NR fragment constructs were introduced into the coactivator assay. AF1 domain and AF2 domain of AR were constructed as a GAL4 DBD (DNA binding domain) fused form and expressed in CV1 cells with GK1 reporter system which has GAL4 responsive elements. For testing pM-ARAF2 activity, 20 nM of DHT was added to transfected cells after transfection. After careful titration of pM- ARAF1 and ARAF2 vector amounts to optimize synergy, 1 ng of pM- ARAF1 and 10 ng of pM-ARAF2 were used for coactivator assay. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-3. AF1 and AF2 of NR independently support coactivator synergy. (A) CV-1 cells were transiently transfected with GK1 reporter, 1 ng of pM- AR.AF1 or 10 ng pM-AR.AF2, 250 ng of GRIP1 vector, 500 ng of CARM1 vector, 500 ng of p300 vector, as indicated. For pM-AR.AF2 sets, 20 nM of DHT was applied after transfection. (B) NR box mutation of GRIP1 abrogates AF2 dependent coactivator synergy, not AF1 dependent synergy. 1 ng of pM-AR.AF1 or 10 ng pM-AR.AF2, 250 ng of GRIP1 vector or GRIP1 NR box mutant, 500 ng of CARM1 vector, 500 ng of p300 vector, as indicated. For pM-AR.AF2 sets, 20 nM of DHT was applied after transfection. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 2 3 4 5 6 7 8 9 10 11 PM-ARAF1 + + + + + PM-ARAF2 + + + + + GRIP1 + + + + + + + + CARM1 + + + + P300 + + + + mo fi PM-ARAF1 PM-ARAF2 GRIP1 GRIP.ANRbox CARM1 P300 1 2 3 4 5 6 7 8 9 10 + + + + + + + + + + + + + + + + 11 12 13 14 15 16 17 18 19 + + + + + ++ + + + + + + + + + + + + + + + + + + 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The requirement of different amounts of vectors in this assay probably results from different expression levels of ARAF1 and ARAF2 in CV1 cells. Fig. 3-3A shows that each of the activation domains of AR, ARAF1 or ARAF2, independently acts as a transcription activator in the GK1 reporter assay system. One coactivator (GRIP1) or two coactivators (GRIP1 plus CARM1 or GRIP plus p300) has little effect on transcription at low ARAF1 or ARAF2 conditions (bars 2 to 5 and bars 7 to 10). However, the presence of three coactivators, GRIP1, CARM1 and p300, synergistically activates ARAF1 and ARAF2 function (bar 6 and bar 11). This coactivator synergy in the pM-ARAF1 or AF2 system is consistent with our previous results using low levels of full length NR (Fig.3-1 and 3- 2). This suggests that two activation domains of NR associate with coactivator molecules independently. AF1 and AF2 domain of NR interacts with GRIP1 coactivator through two different domains of GRIP1 (Ma et al, 1999). For example, the AF1 domain interacts with the C-terminal part of GRIP1, and the AF2 domain interacts with the NR boxes (LxxLL motifs) of GRIP1. In order to test this hypothesis, a NR box mutant of GRIP1 is introduced in this coactivator assay. All three of the GRIP1 NR boxes are mutated by changing the motif LxxLL to LxxAA (Ma et al, 1999). The three-coactivator combination of GRIP1, CARM1 and p300 activates synergistically AF1 and AF2 domains of AR (bar 6 and 15). 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. When the NR box mutant of GRIP1 is introduced in this assay, AF2 dependent coactivator synergy is completely abrogated (bar 19) but AF1 dependent activation is only partially affected (bar 10). This data indicates that the AF2 domain is regulated through contact with the GRIP1 NR boxes. In contrast, the AF1 domain is mostly independent on the NR box, presumably because it makes contact with other domains of GRIP1 like the C-terminal region. The effects of the NR box mutation of GRIP1 were also assessed by using low levels of full-length NR. Coexpression of coactivators GRIP1, CARM1, and p300 in the presence of hormone caused activation of reporter genes at low AR or low TR conditions (bar 6 of Fig. 3-4 A, B). This coactivator synergy was almost completely abrogated when the GRIP1 NR box mutant was transfected instead of GRIP1 wild type (bar 10 of Fig. 3-4 A, B). Considering that the NR box of GRIP1 is important for AF2 function of NR, Fig. 3-4 suggests that the interaction between the AF2 domain of full length NR and the GRIP1 NR box is absolutely required for coactivator synergy. The other interaction between NR AF1 and the GRIP1 C-terminal domain is not sufficient for activating full length NR by coactivators especially at low NR conditions. This data was similarly reproduced with ER (data not shown). 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.3-4. The NR box motif of GRIP1 is essential for coactivator synergy for AR and TR. 1 ng of NR (AR and TR) expression vectors, 250 ng of reporter vectors, 250 ng of GRIP1 wild type or NR box mutant, 500 ng of CARM1 vector, 500 ng of p300 vector were transfected into CV1 cells in the presence of 20 nM of DHT (for AR) or T3 (for TR). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A > * . 2 : o' o O < 0 ° T l O T X 2 d 3 2 0 AR 16 8 4 0 B rtf Ji ® o o «; O O J T ” m X c s ~ v~ Z5 & - 1 p w 12 TR 8 4 0 1 2 3 4 5 6 7 8 NR GRIP1 GRIPlANRbox CARM1 P300 + 4 " 4 ~ + + - I" + 4- + 4* 4 “ * 4 " 4 " 4 ™ 4" 4 " 4 " 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These results suggest that two contact sites between NR and GRIP1 are essential for efficient activation of NR. Especially, NR AF2 seems to be a primary site for conveying coactivator activation signals. It also appears that NR AF1 dependent activation is somehow influenced by NR AF2 function. Ternary coactivator complex formation by GRIP1, CARM1, and p300. CARM1 binds to the AD2 domain of GRIP1, while p30Q and CBP bind to the GRIP1 AD1 domain (Chen et al, 2000a). Since CARM1 and p300 can bind to different domains of GRIP1 and can function synergistically as coactivators, we tested whether these three proteins can exist as a ternary complex in mammalian cells. CARM1 with an HA epitope tag, p300 with a Flag epitope tag, and GRIP1 were coexpressed in Cos-7 cells by transient transfection. HA-CARM1 was expressed well in all of the transfections (Fig. 3-5A, bottom). When an antibody against the Flag tag was used for immunoprecipitation, coprecipitating CARM1 was detected by immunoblotting with an antibody against the HA tag (Fig. 3-5A, top, lanes 3 and 7). However, the coprecipitated HA-CARM1 signal was substantially reduced if vectors for p300 or GRIP1 were omitted from the transfection (lanes 2, 5, and 6). The requirement for GRIP1 expression indicates that CARM1 and p300 do not interact directly but associate indirectly through their contacts with GRIP1; thus, the results indicate a ternary complex of the three coactivators. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-5. Ternary coactivator complex formation among GRIP1, CARM1 and p300/CBP. (A) Coimmunoprecipitation. Cos-7 cells in 100-mm- diameter dishes were transfected with coactivator expression vectors as indicated: 2.5 pg of pCMV-p300 (produces p300 with a Flag tag); 2.5 pg of pSG5.HA-CARM1; 2.5 pg of pSG5.HA-GRIP1 (wild type [WT]) or the equivalent vector encoding GRIP1AAD1 (AAD1), GRIP1AAD2 (AAD2), or GRIP1AAD1 plus AAD2 (AAD1/2). Complexes containing p300 were immunoprecipitated from transfected cell extracts with anti-Flag antibody, and coprecipitated CARM1 was detected by immunoblotting with antibodies against the HA tag (top). To check CARM1 expression before immunoprecipitation, 5% of the transfected cell extract was directly tested by immunoblotting with antibodies against HA tag (bottom). Lanes 1 to 3 and 4 to 10 represent two independent experiments. The diagram indicates the proposed interaction sites among the three coactivators. IP, antibody used for immunoprecipitation; WB, antibody used for Western immunoblotting; IgG-H, immunoglobulin G heavy chain from the immunoprecipitation, which is recognized by the secondary antibody used in the immunoblot. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (B) Modified mammalian two-hybrid system. CV-1 cells in six-well dishes were transfected with 250 ng of GK1 reporter plasmid controlled by an E1b basal promoter (ElbTATA) and Gal4 response elements (Gal4RE) and, as indicated, 250 ng of pGAL-CBP8, encoding Gal4 DBD fused to full-length CBP; 250 ng of pVP16.CARM1, encoding VP16 activation domain (VP16AD) fused to CARM1; and 500 ng of pSG5.HA-GRIP1 or the corresponding vector expressing the AAD1, AAD2, or AAD1 plus AAD2 mutant of GR1P1. The luciferase (Luc) activity of the cell extract is shown. (C) Expression of GRIP1 and GRIP1 mutants in mammalian cells. Cos-7 cells were transfected with 2.5 pg of pSG5.HA expression vectors for GRIP1 WT (wild type), GRIP1AAD1, GRIP1AAD2 or GRIP1AAD1AD2. Cell lysates were resolved by SDS-PAGE and the expression of GRIP1 proteins were checked by western blot using anti-HA antibody. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CARM1 + + ■ + ■ + 4 r + + H h 4 “ GRIP1 WT W T WTAAD14AD2 AAD1/2 WB : anti-HA B GR1P1 CARM VP16 GAL4 ElbTATA ■ M o o o CO O 0 ) » X < 0 — £ Z i £ o c 400 200 o GAL4-CBP pVP16-CARM1 GRIP1-W T GRSP1-AAD1 GRIP1-AAD2 GR1P1-AA01AD2 ^ ^ & JP C ? < 0 < 0 < 0 < 0 ^ O (y cF < 5 ^ N O W: anti-HA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. When GRIP1 mutants lacking the AD2 domain or the AD1 and AD2 domains were substituted for wild-type GRIP1, the coprecipitation of CARM1 was also substantially reduced (lanes 9 and 10). These data are consistent with previous reports that CARM1 associates with GRIP1 through contact with the AD2 domain. Surprisingly, when only the AD1 domain was deleted from GRIP1, there was no decrease in the coprecipitation of CARM1 (lane 8). Immunoblots demonstrated that wild- type and mutant GRIP1 proteins were expressed at similar levels (Fig. 3- 5C). Since p300 did not bind directly to CARM1 in this experiment (lanes 2 and 6), and since CBP did not bind directly to CARM1 in a modified mammalian two-hybrid system (Fig. 3-5B), the integrity of the ternary complex with the GRIP1 AD1 deletion mutant suggests that p300 and CBP bind directly or indirectly to other regions of GRIP1 in addition to the AD1 domain. A modified mammalian two-hybrid system was used to demonstrate that GRIP1 and CARM1 can also form a ternary complex with CBP. Coexpression of a Gal4 DBD-CBP hybrid protein with a VP16- CARM1 hybrid protein failed to activate a luciferase reporter gene controlled by Gal4 response elements (Fig. 3-5B, bar 6). Coexpression of GRIP1 with the CBP and CARM1 hybrid proteins strongly enhanced reporter gene expression, indicating that CARM1 and p300 interact indirectly through GRIP1 (bar 7). Without VP16-CARM1, GRIP1 enhanced Gal-CBP activity only two- to threefold (data not shown). 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. As in the coimmunoprecipitation experiment (Fig. 3-5A), the ternary complex did not form when a GRIP1 mutant lacking AD2 was substituted for wild-type GRIP1 (Fig. 3-5B, bars 9 and 10). However, deletion of GRIP1 AD1 failed to prevent the formation of the complex (bar 8), supporting the conclusion from the immunoprecipitation experiments that CBP and p300 can interact with GRIP1 through the AD1 domain and also through another yet-undefined domain. The importance of the GRIP1 AD2 domain for the formation of the ternary coactivator complex and for the functional synergy of these three coactivators was also demonstrated by employing the low-NR coactivator assay system. GRIP1, CARM1, and p300 synergistically enhanced the activation of a reporter gene by low levels of TR, and the activity was dependent on the presence of all three coactivators (Fig. 3-6A, bars 1 to 6). When a GRIP1 mutant lacking AD1 was substituted for wild-type GRIP1, the synergistic activity among the three coactivators was reduced but not eliminated (bars 7 to 10). However, deletion of AD2 or of AD1 and AD2 from GRIP1 eliminated reporter gene activity (bars 11 to 18). Similar results were obtained when ER was used instead of TR (Fig. 3-6B). Thus, the AD2 domain of GRIP1 was necessary for the formation and synergistic coactivator function of the ternary CARM1-GRIP1-p300 complex; the AD1 domain may contribute to the total coactivator activity but was not essential for complex formation or synergy. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-6. Coactivator complex formation is required for coactivator synergy (A) TR Coactivator assays. CV1 cells in six-well dishes were transfected with 250 ng of MMTV(TRE)-LUC reporter plasmid, 1 ng of TR expression vector, 500 ng of CARM1 vector, 500 ng of p300 vector, and 250 ng of the indicated wild-type or mutant GRIP1 vector. The cells were grown with 20 nM T3, and the luciferase activity of the cell extract was determined. (B) ER coactivator assay; 250 ng of MMTV(ERE)-LUC reporter plasmid, 1 ng of ER expression vector, 500 ng of CARM1 vector, 500 ng of p300 vector, and 250 ng of the indicated wild-type or mutant GRIP1 vector. The cells were grown with 20 nM Estradiol. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 3f :> o o g to o « X 2 D ^ O ' 301 20 10 0 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 T R GRIP1-WT GRIP1-AAD1 GRIP1-AAD2 GRIP1-AAD1AD2 CARM1 p300 + + 4 - + + + + + + + + + + + + + + + + + + + + + 4 " + 4 * + + + + + + + + + + + + + + + + + + 4- + C O 35 30 > 2 25 g o o 2 20 C O o < 0 n w X ® - 1 15 -J 10 5 O ER 1 2 + GRIP1-WT GRIP1-AAD1 GRIP1-AAD2 CARM1 p300 3 4 5 7 8 9 10 11 12 13 14 + + + + 4 - 4 - 4 - 4 - + 4 - 4 - + + + + + + + + + + + + 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3-3. Discussion Role of p160 coactivator complexes in transcriptional activation by NRs. Presumably, many of the steps in the complex process of transcriptional activation are not mediated by a single protein but by multiple proteins, possibly existing as preformed complexes, which work together to accomplish a single task or step in the process. Thus, studying the effect of an individual protein on gene activation is unlikely to produce a full understanding of that protein's function in vivo and may in fact provide misleading clues about the protein's true physiological role in the overall process. In the system reported here, the activity of low levels of NRs was almost completely dependent upon synergistic action of a p160 coactivator, a coactivator of the protein acetyltransferase family (p300, CBP, or p/CAF), and the protein methyltransferase CARM1. The absolute dependence of NR function and the functions of the other coactivators (CARM1, p300, and p/CAF) on the coexpression of GRIP1 further supports the model (Chen et al, 2000a; Chen et al, 1999) that p160 coactivators play a central and direct role in bringing the coactivator complex into association with the NRs, whereas CARM1, p300, and p/CAF associate with the NRs through their association with GRIP1. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The absolute requirement for the AD2 domain of GRIP1 (Fig. 3-6) is also consistent with this model whereby CARM1 is recruited to the promoter through its association with the AD2 domain of GRIP1 (Fig. 3-6). This model will be discussed further in next chapter. The fact that the deletion of AD1 did not cause complete loss of coactivator synergy or disruption of the ternary complex of coactivators (Fig. 3-5 and 3-6) was surprising given the clear evidence that p30Q and CBP associate with the AD1 domain (Chen et al, 1997; Voegel et al, 1998). Thus, our results indicate that p300 and CBP can bind to GRIP1 through the AD1 domain, through another as yet undefined domain of GRIP1, or through contact with another component of the complex. Most previous studies which documented the binding of the p160 AD1 domain to the C-terminal region of CBP and p300 used fragments of these two proteins in their binding studies and thus apparently missed the secondary interaction of CBP/p300 with another region of p16Q proteins (Chen et al, 1997; Kamei et al, 1996; Voegel et al,1998; Yao et al, 1996). However, Torchia et al. (Torchia et al, 1997) reported that the C-terminal region of CBP bound strongly to the AD1 region and more weakly to the N-terminal region of a p160 coactivator. This secondary site may be responsible for the AD1-independent interaction we have observed between GRIP1 and p300/CBP. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The AD1-independent coactivator function of GRIP1 occurred under low- NR conditions in the presence of three coactivators (Fig. 3-6) but not at higher-NR conditions when GRIP1 and p300 were used without CARM1 (Chen et al, 2000a). Our results suggest that the ternary coactivator complex CARM1-GRiP1-p300 may stabilize an AD1-independent p160- p300 interaction which is otherwise too weak to support coactivator function. This chapter has shown robust synergy among coactivators like GRIP1, CARM1 and p300 for NR transcription. Since CARM1 and p300 have histone modifying activities, the implications of protein acetylating and protein methylating coactivators in coactivator synergy will be investigated in the next chapter. Furthermore, the implications of the low- NR system in testing the mechanism of coactivator function will be discussed. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 4. Selective requirements of the protein acetyltransferase and protein methyltransferase activities 4-1. Introduction Histone modifications by acetyltransferase and methyltransferase activities of coactivators Eukaryotic chromosomes have a highly structured assembly between DNA and basic proteins called histones. The fundamental unit of chromatin is called the nucleosome which is composed of histone octamers and DNA. Four different kinds of histone molecules (H2A, H2B, H3 and H4) form an octamer which is wrapped with 147 base pairs of DNA to form the core nucleosome structure. The histones have a similar structure with a basic N- terminal tail, a globular domain organized by the histone fold, and a C- terminal tail. The histone folds of core histones mediate histone-histone and histone-DNA interactions. The N-terminal tails of the core histones are involved in condensation of chromatin rather than maintaining the structural integrity of the nucleosome. In addition, linker histone H1 found between nucleosome core particles is involved in formation of higher structures like solenoids (Cheung et al, 2000; Luger et al, 1998). 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The presence of the nucleosomal barrier causes limited access of transcription factors and basal transcription machinery to the DNA template. This is one of the major hurdles in transcription activation in vivo. However, chromatin structure is a dynamic structure, which is subject to global or local structural change according to transcription, repair or other needs. Chromatin is the integrating center of diverse molecular signals. The N- terminal parts of histones are subject to a variety of modifications like acetylation, phosphorylation, methylation, ubiqutination etc (Strahl and Allis, 2000). These modifications may influence the local or global structure of chromatin. As mentioned previously, many of the coactivator molecules have chromatin remodeling activities and characteristic enzymatic activities for modifying histone N-terminal tails. Histone modifications at N-terminal sequences cooperatively or exclusively regulate chromatin structure and the access of various factors to chromatin. Multiple modifications of histones become unique signals for regulating chromatin structure and regulating the rate of transcription. Specific combinations of histone modifications make unique “histone code” and regulate transcription accordingly (Strahl and Allis, 2000) 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Histone acetylation and histone methylation by coactivator molecules will be described among histone modifications in this chapter. Coactivators which have HAT activities include p300/CBP, and p/CAF etc. These coactivators acetylate the N-terminus of histones and facilitate local chromatin remodeling during transcription initiation process (Ogrzyko et al, 1998; Korzus et al, 1998; Cheung et al, 2000; Rice et al, 2001; Kraus et al, 1999). There are some reports which show that the C-terminal part of p160 coactivators has a weak HAT activity (Chen et al, 1997; Spencer et al, 1997). However, the substrates and characteristics for p160 HAT activity are not clearly elucidated. The link between histone acetylation and transcription activation is pretty well established (Pazin and Kadonaga, 1997). Hormone treated cells show increased NR recruitment to target promoter in vivo and the accompanying recruitment of p160 coactivator, HAT coactivators like p300, CBP, p/CAF and the others. Chromatin immunoprecipitation assays suggest that histone acetylation and HAT coactivators are associated with hormone- dependent NR recruitment and transcription initiation complex formation (Shang et al, 2000). However, the exact mechanism of how histone acetylation leads to enhancement of transcription activation remains unclear. It is simply proposed that acetylation of histone molecules neutralizes the positive charge of lysine and loosens the contact between histone molecules and DNA (Luger et al, 1998). 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Recent reports suggest the possibility that acetylated lysine could act as a binding site for bromodomain containing proteins like TAFII250. This suggests that histone acetylation could simply attract the other factors involved in chromatin remodeling (Jacobson et al, 2000; Jenuwein et al, 2001; Rice et al, 2001). HAT activity of p300/CBP and p/CAF shows interesting overlapping but distinct specificity (Sterner et al, 2000). For example, p/CAF acetylates primarily histone H3 but p300/CBP acetylates more diverse histone molecules like histone H2A, H2B, H3, and H4 etc. The presence of multiple HAT enzymes may play roles in different transcription initiation complexes or in different timing of NR transcription activation. Acetyl moieties of histones are removed by histone deacetylases (HDAC). NR transcription is positively or negatively regulated by recruitment of HAT coactivators or HDAC corepressors. Methylation of histone molecules was documented more than thirty years ago (Rice and Allis, 2001). Lysine and Arginine residues of histone N- terminal domains are targets of protein methylation. However, enzymes responsible for histone methylation were not identified until recently. One of the recently identified histone methylating enzymes, CARM1, was isolated by yeast two hybrid screening with the AD2 domain of p160 coactivator (Chen et al, 1999a). 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CARM1 has a highly conserved central arginine methyltransferase domain and belongs to the PRMT (protein arginine methyltransferase) family. There are several members in the arginine methyltransferase family, but the substrate specificity is very different among members (Stallcup et al, 2001). CARM1 and PRMT1 could uniquely methylate histone molecules among PRMT family members. Interestingly, CARM1 primarily methylates histone H3 (histone residues Arginine 2, 17 and 26) and PRMT1 methylates histone H4 (histone residues Arginine 3) (Strahl et al, 2001; Schurter et al, 2001; Stallcup et al, 2001). These two different arginine methyltransferases could synergize in NR transcription activation (Koh et al, 2001). Ligand induced arginine methylation of histone H3 was recently demonstrated by chromatin immunoprecipitation (CHIP) assay (Ma et al 2002). These agonist-induced, arginine methylations of histones are probably catalyzed by CARM1 and PRMT 1 in cells. However, the in vivo level of arginine methylation in histones is extremely low, and the physiological roles of arginine methylation and arginine methyltransferases are still to be explored. Histones are also targets for lysine methylating enzymes (Rice and Allis, 2001). Lysine methylation of histone molecules is also being widely documented. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Many SET domain containing proteins are histone lysine methyltransferase (Rea et al, 2000; O’Carroll et al, 2000; Rice and Allis, 2001). Su(var)39 is a histone methylating enzyme. Histone H3 (Lys4) is methylated by Set9 and histone H3 (Lys9) is methylated by Suv39H1. These methylations are antagonistic to each other. In addition, Set9 methylation has a stimulating effect on p300 mediated acetylation but Suv39H1 dependent methylation has an inhibitory effect. However, these effects of lysine methylations have not yet been demonstrated in NR transcription systems and will not be discussed here in this thesis. Methylation of histones could affect chromatin structure directly or provide binding sites for other factors. For example, lysine 9 methylation of histone H3 recruits silencing protein HP1 and may play a role in heterochromatin formation (Lachner et al, 2001, Bannister et al, 2001; Rice and Allis, 2001). Arginine methylation of histones appears to be involved in transcription activation by NR (Ma et al, 2001) but the role of arginine methylation of histones in chromatin structure is not well defined (Stallcup et al, 2001). In this chapter, the importance of histone acetylation and methylation activity of coactivators in synergistic enhancement of NR activity will be assessed using the low-NR assay system. The other members of the arginine methyltransferase family will also be checked for possible cooperation with p300 or p/CAF. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-2.Results Requirement for the acetyltransferase activity of p/CAF, but not of p300, for coactivator synergy with CARM1 and GRIP1. p/CAF, p300, and CBP are protein (including histone) acetyltransferases and have been shown to act as coactivators for NRs (Glass et al, 2000). We therefore tested whether p/CAF could substitute for p300 or CBP to act synergistically with GRIP1 and CARM1 as coactivators for low levels of NRs. The coactivator combination of GRIP1, CARM1, and p300 produced the highest reporter gene activity among the various combinations of three coactivators tested with ER (Fig. 4- 1A, bar 7). p/CAF also acted synergistically with GRIP1 and CARM1 (bar 8), but the activity was generally lower than that obtained with p300 (although no effort was made to ensure equal expression of p300 and p/CAF). As shown for the coactivator trio GRIP1, CARM1, and p300 (Fig. 3-6), the AD2 domain of GRIP1, but not the AD1 domain, was essential for coactivator synergy among GRIP1, CARM1, and p/CAF (data not shown). The combination of GRIP1 plus the two acetyltransferases (Fig. 4-1, bar 9) was modestly synergistic but less effective than the combinations of GRIP1 with CARM1 and one acetyltransferase (bars 7 to 8). The combination of CARM1, p300, and p/CAF was ineffective in the absence of GRIP1 (bar 10), presumably because GRIP1 is necessary for formation of the coactivator complex. 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-1. Synergy among various combinations of three or four coactivators at low NR levels. CV-1 cells were transiently transfected as for Fig. 3-1 and grown with 20 nM E2 for ER or 20 nM T3 for TR. The plasmids used were 250 ng of MMTV(ERE)-LUC, 1 ng of ER vector, 250 ng of GRIP1 vector, 500 ng of CARM1 vector, 500 ng of p300 vector, and 500 ng of p/CAF vector (A) and 250 ng of MMTV(TRE)-LUC, 5 ng of TR vector, and coactivator vectors as for panel A (B). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A & = o ^ £ 03 O w X 03 _ ir: - J 0 | 'o 0^ 3 1000 800 600 400 200 l l o ... . . « . - a ... .■ 1 .i :. Ji J ] 2 3 4 5 6 7 8 9 10 ER + + + + + + + + + GRIP1 + + + + + + + CARM1 + + + + p300 + + + + p/CAF + + + + ■ i = o ' m X C O — , i~ Z j .P O f s o s o -4 -0 - 2:0- I o. .—. . « ■. ..m. ■ ll .. .« ■ .. ..mi. 1 2 3 4 5 6 7 8 9 TR + + + + + + + GRIP1 + + + + + + + CARM1 + + + + p300 + + + p/CAF + + 4 - 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A similar pattern of relative activities for these coactivator combinations was also observed for AR and TR (data not shown). In this low-NR system, the coactivator function of p/CAF and its synergy with the other coactivators were absolutely dependent on the presence of GRIP1, suggesting that p/CAF may be recruited to the promoter through its association with GRIP1 or that p/CAF coactivator activity may depend indirectly on activities of p160 coactivators and/or CARM1. The fact that a strong dependence on three coactivators can be demonstrated suggests that a dependence on a larger number of coactivators may be established by careful titration of the levels of NR and coactivator expression vectors. In experiments using TR and a suitable reporter gene, we observed that the combination of GRIP1, CARM1, p300, and p/CAF produced a higher level of activity than any other combinations of three of these coactivators (Fig. 4-1B) To test whether the acetyltransferase activities of p300 and p/CAF are required for their synergistic coactivator function with GRIP1 and CARM1, we tested previously defined acetyltransferase-negative deletion mutants of p300 and p/CAF (Puri et al, 1997) in our coactivator synergy assay. The p300 mutant del/ lacks amino acids 1603 to 1653 in the acetyltransferase domain; the p/CAF mutants MT1 and MT2 lack amino acids 579 to 608 and 609 to 624, respectively. 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Histone acetylation assays, performed with wild-type and mutant p300 immunoprecipitated (via the Flag tag) from transiently transfected cells, confirmed the previously defined lack of acetyltransferase activity in the mutant p300 protein (lane 2 of Fig. 4-2A, upper panel). Histone band acetylated by p300 is rather broad indicating that p300 acetylated various histone molecules H2A, H2B, H3 and H4 (lane 1 of Fig. 4-2A, upper panel). In contrast, p/CAF is known to acetylate preferentially histone H3 and shows a distinct acetylated histone band (lane 1 of Fig. 4-2A, lower panel). HAT deficient p/CAF mutants, MT1 and MT2, showed significantly reduced levels of histone acetylation, but still has basal levels of activity (lanes 2, 3 of Fig. 4- 2A, lower panel). As shown above, the combination of GRIP1, CARM1, and p/CAF acted synergistically to enhance the ability of low levels of AR or TR to activate their respective reporter genes in the presence of the appropriate hormone (Fig. 4-2B, bars 7). Substitution of either p/CAF mutant for the wild- type p/CAF caused a substantial reduction in the ability of p/CAF to cooperate with GRIP1 and CARM1 as coactivators for TR and completely eliminated the ability to cooperate with GRIP1 and CARM1 as coactivators for AR (compare bars 3 and 7 to 9). Immunoblot studies indicated that the mutant and wild-type p/CAF proteins were expressed at similar levels in transfected cells (data not shown). 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-2. Role of protein acetyltransferase activities of p/CAF and p30Q in coactivator synergy. (A) HAT activity of p300 and p/CAF wild types and mutants. HAT activity of p300 and p/CAF are tested by transfecting flag- tagged p300 and pCAF plasmids into Cos- 7 cells. Transfected cells were lysed with RIPA buffer and cell extracts were immunoprecipitated (IP) with antibody against Flag tag (anti-Flag), and immunoprecipitates were incubated with mixed histones and acetyl-coA to allow acetylation. Acetylated proteins were detected by Western immunoblotting (WB), using antibodies specific for proteins containing acetylated lysine. CV-1 cells in six-well dishes were transfected with 1 ng of AR or TR expression vector, 250 ng of MMTV- LUC or MMTV(TRE)-LUC, 250 ng of GRIP1 vector, 500 ng of CARM1 vector, and 500 ng of a vector encoding the indicated wild-type or mutant form of p/CAF (B) or p300 (C). The cells were grown with 20 nM DHT or T3, and cell extracts were assayed for luciferase activity. WT, wild-type p/CAF or p300; MT1, p/CAF579-608; MT2, p/CAF609-624; MT, p3001603-1653. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Histones Histones e A ■ Q r < r / r r r f y < ? ° I P : a n ti- F la g W B : a n ti- a c e ty l- ly s i n e • B S' S8 m X 2 D 'o ^ 3 w 3 0 20 10 3 0 20 10 ‘ A R T R l l i ■ 1 1 1 6 0 1 20 8 0 4 0 8 0 6 0 20 AR TR KJ --- I 2 3 4 5 6 2 8 9 2 3 4 5 6 7 NR + + + + + + + + + NR -f -F + + + + GRIPl + J- + + + + f + GRIP! + + + + + CARMl + r + + C A R M l T * + + p/CAF WT M il MT2 wr MTi MT2 p300 WT W T MT Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. When other NRs were tested in this system, the p/CAF mutations caused severe loss of synergistic coactivator function for glucocorticoid receptor (like AR) but caused only a partial loss of function for ER (like TR) (data not shown). The reason for the different effects of these mutations on different NRs is not clear at this time but may reflect different coactivator requirements to mediate effectively the activities of different NRs. In contrast to p/CAF, elimination of the acetyltransferase activity of p300 caused little or no loss of the ability of p300 to cooperate with GRIP1 and CARM1 as coactivators for AR and TR (Fig. 4-2C). When the amounts of wild-type and mutant p300 expression vectors were varied, their activities were indistinguishable over a broad range of vector amounts (data not shown). Thus, the acetyltransferase activity played an important role in the synergistic coactivator function of p/CAF, but the ability of p300 to cooperate with GRIP1 and CARM1 was independent of the acetyltransferase activity. These results indicate that p300 and p/CAF contributed by different mechanisms to the synergistic enhancement of NR function. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Requirement for the methyltransferase activity of CARM1 for coactivator synergy with GRIP1 and p300 We previously demonstrated that substitution of alanine for three highly conserved residues (valine-leucine-aspartate, or VLD; amino acids 189 to 191) in CARM1 caused loss of methyltransferase activity and loss of the ability of CARM1 to cooperate with GRIP1 as a coactivator for NRs (Chen et al, 1999a). The VLD sequence is conserved among members of the protein arginine methyltransferase family, indicating that it plays an important role in the structure or function of the methyltransferase domain. A recent three- dimensional crystal structure of the related protein arginine methyltransferase PRMT3 (Zhang et al, 2000) indicates that the VLD sequence is important for tertiary structure of the core methyltransferase domain and thus may affect the overall structure as well as the methyltransferase activity of the protein. Based upon the same crystal structure, we designed a mutation which is predicted to eliminate the methyltransferase activity without affecting the overall structure of the protein. The crystal structure allowed the prediction of a binding site for the arginine residue to be methylated and suggested that two glutamate residues, which are highly conserved among the protein arginine methyltransferase family, form salt bridges with the two terminal guanidino group nitrogen atoms of the substrate arginine residue. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In CARM1, these conserved glutamate residues are located at amino acid positions 258 and 267. In a new CARM1 mutant, we converted glutamate 267 to glutamine (E267Q). Wild-type CARM1 and both CARM1 mutants (VLD-to-AAA and E267Q) were expressed at equivalent levels in transfected cells by immunoblot analysis with an antibody against the HA tag on the CARM1 proteins (Fig. 4-3A, top). The antibody against the HA tag was used to immunoprecipitate the CARM1 proteins from the transfected cell extracts in order to test their histone methyltransferase activities. In this assay, wild- type CARM1 methylated histone H3, but neither mutant CARM1 protein had any detectable histone methyltransferase activity (Fig. 4-3A, bottom). Methylated histone H3 was detected by immunoblot analysis with antibodies which recognize CARM1 -methylated histone H3 but not unmethylated H3. Both mutants retained the ability to bind GRIP1 (Fig. 4-3B and Chen et al, 1999a). The CARM1 wild-type and mutant proteins were tested for the ability to cooperate with GRIP1 and p300 as coactivators for NRs. When various amounts of CARM1 expression vectors were cotransfected with a low level (1 ng) of TR vector (conducive for three-coactivator synergy) and vectors encoding GRIP1 and p300, wild-type CARM1 strongly enhanced TR- mediated reporter gene expression in cooperation with GRIP1 and p300. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-3. Construction of methyltransferase defective CARM1 mutant, E/Q. (A) Methyltransferase activities of wild-type and mutant CARM1. Cos-7 cells (100-mm-diameter dishes) were transfected with 2.5 ng of the indicated CARM1 expression vector: Con, control with pSGS.HA; WT, pSGS.HA- CARM1 wild type; E/Q, pSG5.HA-CARM1(E267Q) mutant; VLD, pSG5.HA- CARMH(VLD) mutant. Cell extracts were immunoprecipitated (IP) with antibody against HA tag (anti-HA), and immunoprecipitates were incubated with mixed histones and S-adenosylmethionine to allow methylation. Incubated reactions were analyzed by Western immunoblotting (WB), using antibodies specific for the CARM1 -methylated form of histone H3 (anti- MeH3; bottom). Expression of CARM1 was assessed before immunoprecipitation by immunoblotting with antibodies against HA tag (top). (B) E267Q mutation of CARM1 does not affect the interaction between CARM1 and GRIP1. CARM1 wild type and CARM1 (E267Q) mutant proteins are produced in reticulocyte lysate in the presence of S3 5 methionine. The production of CARM1 proteins are checked by directly loading to SDS - PAGE (10% input). In vitro translated CARM1 and CARM1 (E/Q) protein are incubated with GST-GRIP.C protein and its interaction is analyzed by SDS- PAGE and autoradiograph. 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A WB: anti-HA i IP: anti-H A W B : a n ti-M e H 3 Con WT E/Q VLD O S' b . / < r ■ S X ° r # & KT O 0 CARM1 CARM1(E/Q) 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-4. Role of protein methyltransferase activity of CARM1 in coactivator synergy. (A) Coactivator synergy with different amounts of wild-type and mutant CARMl. CV-1 cells in six-well dishes were transfected with 1 ng of TR vector, 250 ng of MMTV(TRE)-LUC reporter plasmid, 250 ng of GRIP1 vector, 500 ng of p300 vector (line plots), or no p300 vector (bars) and the indicated amount of CARM1 wild-type or mutant vector. The cells were grown with 20 nM T3, and the luciferase activities of the cell extracts were determined. (B) Coactivator activities of mutant and wild-type CARM1 at low versus high NR levels. CV-1 cells in six-well dishes were transfected with 1 (Low NR) or 100 (High NR) ng of ER vector, 250 ng of MMTV(ERE)-LUC reporter plasmid, 250 ng of GRIP1 vector, 500 ng of p300 vector, and 500 ng of wild-type or mutant CARM1 vector. The cells were grown with 20 nM E2, and the luciferase activities of the cell extracts were determined. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CO o wX 2 d 3 8 GRIPl CARM1 GRIP I CARM1 p 3 0 0 I GRIPl E /Q p 3 0 0 j GRIPl VLD p 3 0 0 ! 0- B 0 200 400 CAR M l vector (ng) 600 ■— o t3 o C O o 2 d 3 ' ' 1 2 8 L o w N R i t ■ ■ . . GRIP! CARM1 E/Q p300 60 40 20 H ig h N R 1 2 3 4 5 6 7 + + + + + + + + + + + + + + + + I 10 11 12 13 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The E267Q mutant of CARM1 had dramatically reduced but detectable activity as a coactivator, while the VLD mutant of CARM1 had no detectable coactivator activity (Fig. 4-4A). Similar results were obtained when ER was substituted for TR (Fig. 4-4B, left). We also investigated the ability of the mutant and wild-type CARM1 proteins to act as coactivators in the presence of low versus high levels of ER. With 1 ng of ER vector, wild-type CARM1 cooperated effectively with GRIP1 and p300 to enhance ER function (Fig. 4- 4B, bar 7), but the CARM1 E267Q mutant was essentially inactive (bar 8). Similar results were observed when p/CAF was substituted for p300 (data not shown). In contrast, when 100 ng of ER vector was used (with GRIP1 vector but without p300 vector), the coactivator function of the E267Q mutant of CARM1 was almost as good as that of wild-type CARM1 (bars 12 to 13). Thus, the methyltransferase activity of CARM1 was required for synergistic coactivator action with GRIP1 and p300 or p/CAF. However, at higher NR levels, the methyltransferase activity of CARM1 was not required for CARM1 to cooperate with GRIP1 in the absence of p300, suggesting that the coactivator function of CARM1 may involve two different activities of CARM1, one methyltransferase dependent and one methyltransferase independent. In addition, the results shown in Fig. 4-4B and 3-1A indicate that the requirements for coactivator function are different at high and low levels of NR. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. C A R M 1 , compared with other protein arginine methyltransferases, has a unique ability to cooperate with p300 and p/CAF as coactivators for NRs. In addition to CARM1, the current list of mammalian arginine-specific protein methyltransferases includes PRMT1, PRMT2, PRMT3, and JBP1 (Zhang et al, 2000). All of the proteins except PRMT2 have been shown to methylate specific proteins in vitro, but each enzyme methylates different proteins (Stallcup et al, 2001). CARM1, as indicated above, can methylate histone H3 (Chen et al, 1999a). PRMT1 can methylate a variety of RNA binding proteins, as well as histone H4 and STAT1 (Mowen et al, 2001; Stallcup et al, 2001, Shen et al, 1998). A yeast enzyme, RMT1/Pmt1/ODP1, has a protein substrate specificity similar to that of mammalian PRMT1. With the exception of PRMT1 and RMT1, which have been shown to methylate various RNA binding proteins (Stallcup et al, 2001), the in vivo targets for these methyltransferases have not been established. We have shown previously that, in the presence of GRIP1, PRMT1 can act as a coactivator for NRs and can function synergistically with CARM1 (Koh et al, 2001). We therefore tested whether PRMT1 and other members of the protein arginine methyltransferase family could substitute for CARM1 to function in synergy with GRIP1 and p300 as coactivators for low levels of TR. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the presence of GRIP1 and absence of p300, each methyltransferase enhanced the reporter gene activity above the level observed with TR plus GRIP1 (Fig. 4-5A, bars 3 and 5 to 9), with the degree of enhancement ranging from two- to ninefold in this experiment. Similar results were observed at higher NR levels in the absence of p300 (data not shown). Thus, in the absence of p300, all of the methyltransferases exhibited similar, although not quantitatively identical, coactivator functions with GRIP1. Addition of p300 as a third coactivator caused a synergistic increase in activity when CARM1 was present but not when any of the other methyltransferases was present (bars 10 to 14). Essentially all of the activity observed was eliminated when GRIP1 (bars 15 to 19) or TR (bars 20 to 24) was omitted. Like p300, p/CAF cooperated synergistically with CARM1 but not with any of the other methyltransferases (Fig. 4-5B). Thus, p300 and p/CAF have a specific and synergistic functional relationship with CARM1 but not with the other protein arginine methyltransferases. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-5. Selective synergy of protein arginine methyltransferases with p300 and p/CAF. CV-1 cells in six-well dishes were transfected with 1 ng of TR vector, 250 ng of MMTV(TRE)-LUC reporter plasmid, and coactivator vectors as indicated: 250 ng of GRIP1 vector, 500 ng of p300 (A) or p/CAF (B) vector, and 500 ng of the indicated methyltransferase vector encoding CARM1, PRMT1, PRMT2, PRMT3, or RMT1. The cells were grown with 20 nM T3, and the luciferase activities of the cell extracts were determined. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 1 2 0 >* S' o o c c o w X ttj & o£ 8 0 4 0 1 2 3 4 5 6 7 i . I I 10 11 12 13 14 15 16 17 IS 19 20 21 22 23 24 T R + + H ~ + + + + + + + + + + + + + + + GRIP! + + + + + + + + + + + + + + + + + p300 + + + + + + + + + + + + + + + + CARM1 + + + + P R MU + + + + PRMT2 + + + + PRMT3 + + + + R M T1 + + + + B 20 K s to o < D m X Ed 3 1 o O 1 2 3 4 ■ s 6 7 8 9 .m . o n 1 2 I 1 3 I 1 4 TR + + + i + + + + + ; + + + + + GRIPl + + : + + + + + t - + + + + p/CAF "F i + + + ■4* + CARM1 + + PRMT1 + + PRMT2 + + PRMT3 + + RMT1 + 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-3. Discussion The role of protein acetyltransferase activity in p300 and p/CAF coactivator function. Histone acetylation by p300, CBP, and p/CAF plays an important role in transcriptional activation of native genes (Chen et al, 1999a; Cheung et al, 2000; Rice et al, 2001; Shang et al, 2000). In addition, the ability of these coactivators to acetylate and make contact with other components of the transcription machinery may also help to transmit the activating signal from the transcriptional activator-coactivator complex to the transcription machinery (Chen et al, 1997; Chen et al, 1999b; Sartorelli et al, 1999). Thus, in different circumstances these multifunctional coactivators may use different portions of their signaling repertoire. In fact, various recent studies have reached different conclusions as to whether the protein acetyltransferase activities of p 3 Q 0 and CBP are required for their coactivator function (Chen et al, 1999b; Korzus et al, 1998; Kraus et al, 1999; Li et al, 2000). In our system, NR function required the acetyltransferase activity of p/CAF but not that of p300 (Fig. 4-2). Some of the other investigators (Korzus et al, 1998) reached similar conclusions using a microinjection assay. The diverse results in various studies as to whether the acetyltransferase activity of p300 and CBP is required may not be contradictory but may rather reflect the use of different transcriptional activators, reporter genes. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (e.g., different promoters or transiently transfected versus chromosomally integrated reporter genes), combinations of coactivators, cell types, or assay methods. The different requirements for the acetyltransferase functions of p3Q0 versus p/CAF (Fig. 4-2), as well as our demonstration that GRIP1, CARM1, p300, and p/CAF can all cooperate synergistically to enhance NR function (Fig. 4-1), suggest that p300 and p/CAF contribute to transcriptional activation by different mechanisms: p/CAF through its acetyltransferase function and p300 through protein-protein interactions. Role of protein methyltransferase activity of CARM1 in coactivator function and synergy. The results with the two CARM1 mutants suggest several conclusions relevant to the mechanism of CARM1 coactivator function. First, as predicted from the crystal structure of the CARM1 homologue PRMT3 (Zhang et al, 2000), the VLD mutation apparently affected more than simply the methyltransferase activity, since both mutants lacked methyltransferase activity but the VLD mutant had a more severe loss of coactivator function (Fig. 4-4). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Second, the fact that the E267Q mutant was expressed at normal levels, binds GRIP1, and had wild-type coactivator activity when tested under high- NR conditions (Fig. 4-3 and 4-4B) indicates that the overall protein structure is probably not adversely affected by the E267Q mutation and thus suggests that the mutation caused a selective loss of methyltransferase function. Thus, the severe loss of coactivator function by this mutant when tested under low- NR conditions reinforces the importance of the methyltransferase activity in the coactivator function of CARM1. Third, the fact that the methyltransferase-negative mutant E267Q retains substantial coactivator function when tested under high-NR conditions indicates that another domain of CARM1 (which is functionally separable from the methyltransferase activity) can contribute to downstream signaling by CARM1, at least under high-NR conditions. CARM1 has an autonomous transcriptional activation activity (Koh et al, 2001), which is separable from its methyltransferase activity (our unpublished results) and is an obvious candidate for the additional downstream signaling domain. Another indication of the importance of the CARM1 methyltransferase domain is the fact that CARM1, alone among the various arginine-specific methyltransferases tested, was capable of participating in three-coactivator synergy with p300 and p/CAF (Fig. 4-5). 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Each of the protein arginine methyltransferase family members methylates different protein substrates in vitro (Stallcup et al, 2001). The protein(s) methylated by CARM1 in connection with its coactivator function remains to be determined. Since CARM1 can methylate histone H3 in vitro (Chen et al, 1999a; Schurter et al, 2001) and cooperates synergistically as a coactivator with a histone acetyltransferase (Fig. 4-2 and 4-4), an attractive hypothesis is that methylation of arginine residues of histone H3 by CARM1 cooperates with acetylation of histone H3 (or another histone) to promote chromatin remodeling (Fig. 4-6). In fact, recent in vitro and in vivo evidence has provided strong support for the hypothesis that histone acetylation, phosphorylation, and methylation (on lysine) can regulate each other and/or cooperate in promoting chromatin remodeling (Cheung et al, 2000; Jenuwein et al, 2001; Rice et al, 2001). These histone modifications may alter chromatin structure by neutralizing the positive charge of the N-terminal histone tails (Luger et al, 1998) or by creating new binding sites to recruit proteins which contribute to chromatin remodeling (Jacobson et al, 2000; Jenuwein et al, 2001; Rice et al, 2001). Our results suggest that arginine- specific methylation of histone H3 or another protein by CARM1 is an important part of the transcription activation process, at least for NRs. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In fact, we have recently shown by chromatin immunoprecipitation that CARM1 is recruited to steroid hormone-responsive promoters in a steroid hormone-dependent manner. Furthermore, using antibodies that specifically recognize the CARM1 -methylated form of histone H3, we also observed that methylated histone H3 is preferentially associated with glucocorticoid- activated versus transcriptionally inactive MMTV promoters which are stably integrated into chromatin (Ma et al, 2001). These findings further support the role of CARM1 and its histone methyltransferase activity in transcriptional regulation. We cannot rule out the possibility that other proteins, in addition to histones, are methylated by CARM1 in connection with its coactivator function. Low NR levels provide stringent conditions for studying coactivator synergy; different coactivator requirements indicate differences in the mechanism of transcriptional activation at low and high NR levels. It is important to note that the levels of NR vector required for three- coactivator synergy in our system are well below those normally used for transient-transfection studies with NRs. The requirements for and effects of various combinations of the three coactivators (GRIP1, CARM1, and p300 or GRIP1, CARM1, and p/CAF) were quite different when low versus high levels of NR vectors were employed in the transient-transfection assays. 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. At low NR levels, (i) NR alone failed to activate the reporter gene, even when hormone was present; (ii) use of one or two of the coactivators with NR caused modest to no activation of reporter gene; (iii) expression of all three coactivators resulted in a strong synergistic enhancement of NR function and in fact was required for efficient NR function; (iv) the methyltransferase activity of CARM1 was required for the coactivator synergy and thus for reporter gene activation; and (v) substitution of another member of the protein arginine methyltransferase family for CARM1 eliminated the synergistic coactivator activity and most or all of the observed reporter gene activity. In contrast, when high NR levels were used, (i) NR alone activated the reporter gene in a hormone-dependent manner; (ii) GRIP1 alone enhanced NR function, and addition of one of the secondary coactivators (CARM1, p300, or p/CAF) produced a further enhancement in most cases; (iii) the activity achieved by adding all three coactivators was no more effective, and was often less effective, than the activity observed with two coactivators; (iv) the methyltransferase activity of CARM1 was not required for its ability to cooperate with GRIP1 to enhance NR activity; and (v) all of the protein arginine methyltransferases, when coexpressed with GRIP1, were approximately equivalent in their abilities to enhance NR function. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thus, at high versus low NR levels, the degree to which coactivators were required, the number of coactivators and types of coactivators which could effectively contribute to NR function, and even the specific functional domains of individual coactivators which were required to enhance reporter gene activation by NRs were dramatically different. In other words, the mechanisms of transcriptional activation at high versus low NR levels are different. While the exact mechanistic differences remain to be determined, we speculate that overexpression of NRs, which almost certainly occurs in transient-transfection experiments, may allow NRs to accomplish directly tasks which normally require assistance from coactivators (Fig. 4-6). Recent fluorescence-quenching studies of fluorescently labeled steroid receptors bound to large tandem promoter arrays suggest that these receptors rapidly and repeatedly associate with and dissociate from their cognate enhancer elements (McNally et al, 2000). NR overexpression may lead to a higher level of enhancer element occupancy by the NR. NRs and many other transcriptional activators can bind to various basal transcription factors, such as TFIIB and TBP (Baniahmad et al, 1993, Ing et al, 1992; Sadovsky et al, 1995; Schulman et al, 1995). Perhaps at concentrations high enough to force almost constant NR occupancy of enhancer elements, the NRs can recruit and activate the transcription initiation complex without additional assistance from some coactivators (Fig. 4-6). More work is required to test such ideas. 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. W e have previously shown that coactivator synergy between CARM1 and a related protein methyltransferase, PRMT1, also requires low levels of NR (Koh et al, 2001). Other investigators have also reported that high levels of NRs reduce the abilities of coactivators to enhance reporter gene activity further (Chen et al, 2000b). Which set of experimental conditions (i.e., high versus low NR levels) may produce results which more accurately reflect the physiological roles of the coactivators? Since transient transfections almost certainly cause substantial overexpression of the proteins encoded by the transfected plasmids, it seems reasonable to conclude that the lower NR concentrations are more likely to approach physiological conditions. While there are some differences in the mechanism of transcriptional activation for stably integrated versus transiently transfected reporter genes (Smith et al, 1997), it seems unlikely that the stringent and specific coactivator requirements observed in our transient-transfection system are entirely an artifact of the experimental system or are irrelevant to the physiological function of these coactivators. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thus, it seems likely that the extremely high degree of synergy observed among the three coactivators and the stringent requirement for the methyltransferase activity of CARM1 (which cannot be replaced by any other methyltransferase protein) provide valid insights about the function of these coactivators in vivo. p16Q coactivators, p300 and CBP (Chen et al, 1999b, Shang et al, 2000), and more recently CARM1 (Ma et al 2001) have all been shown to be recruited in response to steroid hormones to the promoters of natural target genes in their native chromosomal locations; thus, these proteins play important roles in the transcriptional activation process on native genes in vivo. This provides a strong validation for the synergy we observed for these three coactivators in our transient-transfection assays. Thus, the low-NR conditions represent a powerful and convenient new system for studying the mechanism of coactivator function; such quantitative mechanistic studies are an essential complement for techniques such as chromatin immunoprecipitation, which examine physical recruitment of proteins to stably integrated reporter genes. Our demonstration that GRIP1, CARM1, and p300 form a ternary complex in vivo and that the methyltransferase activity of CARM1 is required for transcriptional activation provide important insights into the mechanism of coactivator function. 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapters 3 and 4 have dealt with the roles of histone modifying activities of various coactivators in the cooperative enhancement of NR function. CARM1 specific arginine methyltransferase activity and p/CAF HAT activity were required for NR activation. To further understand the role of CARM1 methylatransferase in its synergy with p300/CBP, coactivators p300/CBP will be tested as substrates for methylation by CARM1 in chapter 5. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-6. Different mechanisms of transcriptional activation at low and high NR levels. (Top) At low NR levels, NRs shuttle on and off of the hormone response element (HRE), and occupancy of the HRE is relatively infrequent. Transcriptional activation by the bound NRs requires the assistance of multiple coactivators to remodel chromatin structure and recruit and activate RNA polymerase II (Pol II complex). (Bottom) High NR levels may force almost constant occupancy of the HREs by NRs. Perhaps such high occupancy allows NRs to recruit RNA polymerase through direct contact with TATA binding protein (TBP), TFIIB, or other components of the RNA polymerase II complex, independent of the action of some or many coactivators. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cooperation of coactivators at low NR HRE SAM Acetyl J F I 1 B) Pol II tbp 'i complex Methyl Acetyl TATA Nucleosome Activation at high NR HRE "frail) Pol II s'T B P ') complex TATA Nucleosome Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5. Coactivator p300/CBP methylation by CARM1 arginine methyltransferase 5-1. Introduction Many of the transcription factors and signal transduction molecules are posttranslationally modified, and their activity is enhanced or repressed by their modifications. Posttranslational modifications involved in factor modulations include phosphorylation, acetylation, ubiquitination, and methylation. In spite of the importance of histone modifications by coactivators, histones are not the only target molecules for these kinds of protein modification. For example, HAT activities of these coactivators also acetylate other substrates including many transcription factors and modify their biological functions (Sterner et al, 2000). Both p/CAF and p300/CBP can acetylate non-histone factors, a n d modulate their activities. For example, one of the p160 coactivator ACTR is acetylated by p300 and this results in dissociation of the receptor-coactivator complex and may lead to decrease in NR dependent transcription activation or contribute to the process of activation in some way (Chen et al, 1999b). ER (estrogen receptor) is a target of p300 mediated acetylation as well (Wang et al, 2001). p300/CBP or p/CAF mediated factor acetylation provides the possibility that NR dependent transcription can be positively or negatively regulated by coactivator or NR acetylation. 8 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. p300/CBP and p/CAF are also involved in acetylation of the other transcription factors like tumor suppressor p53 or muscle specific factor MyoD (Gu et al, 1997; Puri et al, 1997). Factor methylation by arginine methyltransferases (PRMT) is one of the unexplored fields. Arginine methyltransferase family members are found in various species from yeast to mammals (Zhang et al, 2000; Weiss et, 2000, McBride et al, 2001). These members have a characteristic S- adenosylmethionine binding motif and less conserved flanking motifs. Knockout of PRMT1 results in embryonic lethality, suggesting the importance of arginine methyltransferase in cellular and developmental processes (Pawlak et al, 2000). Several substrates for arginine methyltransferases have been identified, but in vivo implications need further investigation. One of the most well known examples of arginine methylated factors is RNA binding proteins like hnRNP (heterogeneous ribonucleoproteins) (Shen et al, 1998; Stallcup et al, 2001). The effects of arginine methylation of these RNA binding proteins are not well known, but some of the cases suggest that arginine methylation of RNA binding proteins could have an effect on RNA transport and stability etc. The helicase domain of hepatitis C virus (HCV) viral factor NS3 is methylated by PRMT 1 in vitro and in vivo (Rho et al, 2001). 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This arginine methylation of factors could also influence RNA- protein interaction. Furthermore, STAT1 (Signal Transducer and Activator of Transcription) factor in the Jak/STAT signal transduction pathway could be modulated by protein arginine methylation. Arginine methylation of STAT1 is mediated by PRMT1 in vitro and in vivo and involved in the activation of STAT1 factors (Mowen et al, 2001). One of the STAT inhibitor molecules, PIAS1, is discharged from STAT1 transcription complex and STAT1 dependent transcription is elevated as a result of arginine methylation. Their data suggest that arginine methylation of factors could be involved in protein-protein interaction and transcription regulation (Stallcup et al, 2001; McBride et al, 2001). In addition, arginine methylation of factors seems to play a role in protein-RNA interaction, RNA transport and the other biological functions. However, the physiological roles of protein arginine methylations still need a lot of investigation. The previous chapter has shown that arginine methyltransferase activity of CARM1 is essential for the coactivator role of CARM1 in NR transcription. It is possible to speculate that non-histone substrates of CARM1 could be involved in NR transcription regulation. The possibility of coactivator methylation and the implications in NR transcription function will be explored in this chapter. Especially, CARM1 catalyzed p30Q/CBP methylation will be primarily examined, and the role of p300/CBP methylation in coactivator function, and complex formation will be discussed as well. 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5-2. Results Coactivator p300/CBP is a substrate for CARM1 arginine methyltransferase activity To test the possible methylation of p300/CBP by CARM1, purified and full length p300/CBP proteins were used for in vitro methylation reaction. Substrate p300 protein was produced in a baculovirus expression system and purified on His-tag specific column. Since purified p300 was already demonstrated to have coactivator and HAT activity by in vitro ER- dependent transcription assay (Kraus et al, 1999), this p300 protein used in this assay is likely to have a functional structure. CARM1 and PRMT1 methyltransferases are expressed as GST-fused forms in E. coli BL21 and purified by Glutathione sepharose bead. Mixed histones are included as a control substrate for CARM1 and PRMT1 reactions. 0.2 pg of p300 protein is co-incubated with GST-CARM1 or GST-PRMT1 enzyme in the presence of radiolabeled SAM (S-Adenosylmethionine), the methyl group donor. Fig.5-1A shows that full length p300 is strongly methylated by CARM1 but not by PRMT1. This indicates that purified p300 protein is a specific substrate for CARM1. Both CARM1 and PRMT1 methylate mixed histones, but methylate different sizes of histones (lane 1 and 2 of Fig.5- 1A). The different sizes of methylated histones confirms previous reports that CARM1 primarily methylates histone H3 and PRMT1 methylates histone H4 (Chen et al, 1999a; Stallcup et al, 2000; Ma et al, 2001; Strahl et al, 2001). 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-1. p300/CBP is a substrate for CARM1 methyltransferase. (A) Purified p300 protein is methylated specifically by CARM1, not by PRMT1. Full length p300 protein was expressed in baculovirus expression system and purified by Ni column. 200 pg of purified p300 or 500 pg of mixed histones was incubated with GST-CARM1 or GST- PRMT1 in the presence of 3 5 S-labeled methionine. Methylations of proteins were detected by SDS-PAGE and autoradiography. (B) CBP/p300 C-terminal part is methylated by CARM1. p300N (N-terminal fragment, a.a 1-596), p300C (C-terminal fragment, 1571-2414) and CBPC (C-terminal fragment, 1594-2414) were used for in vitro methylation assay. Each protein was expressed and purified as a GST- fusion protein. Purified proteins were incubated with GST-CARM1 and 3 5 S-labeled methionine for methylation reaction. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Substrates: Histone p300 CARM1 + + PRMT1 + + B o < ^ 0 . < ? ° CARM1: + + + + Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This also shows that both CARM1 and PRMT1 proteins used in this assay are enzymatically active. Although different amounts of p300 (200 ng) and histone (500 ng) proteins are used for this in vitro methylation, p300 and histones are methylated at similar intensity. Considering the different molecular weights of p300 and histones, smaller numbers of p300 protein molecules are included in this assay. In a sense, p300/CBP is a better substrate for CARM1 at least in the methylation reaction in vitro. CARM1 was also tested as a possible acetylation substrate by p300 protein. Purified p300 has strong acetylating activity for histones but does not acetylate CARM1 (data not shown). In addition, the HAT activity of p3Q0 is not influenced by CARM1-catalyzed p300 methylation (data not shown). Next, we tested whether CBP (CREB binding protein), a homolog molecule of p300, is also a substrate for CARM1 methylation. In order to test the possibility of CBP as a CARM1 substrate, the C-terminal part (a.a 1594-2441) of CBP was used for in vitro methylation assay. The C- terminal part (a.a 1571-2414) of p300 and the N-terminal part (1-596) of p300 protein were also expressed and purified in GST-fused form and tested for methylation substrates by CARM1. Fig. 5-1B shows that approximately 100 kD size of methylated proteins are clearly detected in lane 2 and 3. This data suggests that C- terminal parts of p300 and CBP are strongly methylated by CARM1. However, the N-terminal part of p300 in lane 1 is not or very weakly methylated by CARM1. 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-1B also indicates that the major methylation site of p300 and CBP is located in the C-terminal part of p300/CBP. Furthermore this suggests that methylation site(s) of p300 and CBP may be a conserved arginine residue(s) between p300 and CBP. GBD (GRIP1 binding domain) of p300 is one of the major sites methylated by CARM1 In order to narrow down the region(s) methylated by CARM1, three smaller fragments of p300 C-terminal molecules were generated in GST- fused form. The three fragments of p300 protein are p300CH3 (cysteine - histidine rich 3 sequence), p300GBD (GRIP1 binding domain), and p300Q (glutamine rich sequence); these fragments cover most of the p300 C- terminal part (1571-2414) which was shown to be methylated in Fig. 5-1B. All of three constructs are well expressed in E.coli BL21 cells, and we obtained enough amounts of substrate proteins for CARM1 methyltransferase assays. In vitro methylation assays in Fig. 5-2A show that p300GBD (GRIP1 binding domain, p160 coactivator binding sequence) is strongly methylated by CARM1. The CHS domain is not methylated by CARM1 at all, and the Q-rich sequence at the C-terminal end of p300 is methylated very weakly by CARM1 protein (Fig. 5-2A). This data indicates that the major methylation site of p300 by CARM1 is located in the GBD sequence of p300. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-2. Localization of methylated p300 fragments by CARM1. (A) pSOOGBD (GRIP1 binding domain) is one of the major methylation sites of CARM1. Smaller p300 protein fragments, p300CH3 (cysteine- histidine rich 3 sequence), pSOOGBD (GRIP1 binding domain), and p300Q (glutamine-rich sequence), were generated. Purified proteins were utilized as substrates for CARM1 catalyzed in vitro methylation reaction. Methylation of proteins was detected by protein labeling with [3 H] SAM. (B) Summary of p300 protein fragments used in CARM1 catalyzed- methylation assay. Domains and lengths of p300 fragments are indicated in diagram, and methylated p300 fragments are marked with asterisks (*). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CARM1 + KIX 1-596 HAT CH 3 IG B C S Q p300 (1-2414) ,744-1571 1571-2414 1709-1934 2158-2414 * __ 2042-2157 *** 568-828 P300CH3 p300Q pSO O G BD pSO O KIX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In a separate experiment, CBP fragments harboring the GBD were also methylated by CARM1 (data not shown). This raises the possibility that CARM1 catalyzed methylation could be involved in the modulation of interaction between p300/CBP, GRIP1, and other factors, and therefore the regulation of NR transcription. Fig. 5-2B shows the summary of all of the p300 fragments used in the methylation assay with CARM1. The CARM1 methylated fragments of p300 are designated by asterisks (*). As shown in Fig. 5-2B, the major methylation sites of p300 are localized to two domains of p300. One is the GRIP1 binding domain (GBD) of p300, and the other is the KIX domain (a.a 568-828) of p300 (Data not shown). Both GBD and KIX domains of p300 are involved in protein-protein interaction and the function of p300 (Vo and Goodman, 2001). However, the KIX domain is mainly important in CREB interaction and the cAMP signaling pathway (Parker et al, 1996; Vo and Goodman, 2001). The methylation of the KIX domain has been shown not to be involved in NR transcription and coactivator synergy (Xu et al, 2001). For this reason, the methylation of p300 GBD is further analyzed in this thesis. Determination of arginine residue(s) methylated by CARM1 pSOOGBD has four arginine residues and CBP-GBD has three arginine residues. Three arginine residues among these are conserved between pSOOGBD and CBP-GBD. 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The non-conserved arginine of p300 is converted to the other basic amino acid lysine in CBP. In order to check which of the arginine(s) of p300/CBP-GBD is (are) methylated by CARM1, each individual arginine (R) residues of pSOOGBD was substituted with lysine (K) by site directed mutagenesis. The point mutants of pSOOGBD are named as R1K (Arg 2056 Lys), R2K (Arg 2088 Lys) and R3K (Arg 2142 Lys) (Fig. 5-3A). Fig. 5-3B bottom part shows that wild type and mutant versions of pSOOGBD are expressed in similar amounts in E.coli. The upper part of Fig. 5-3B is the result of an in vitro methylation assay using pSOOGBD wild and mutant types as substrates for CARM1. The data shows that pS O O WT (wild type), R1K, and R2K proteins are strongly methylated by CARM1. But R3K mutant is not methylated by CARM1 and probably lacks the methylation site. This indicates that the major methylation site of pSOOGBD is R3 (Arginine 2142) of pSO O . The p300 C-terminal region harboring GBD is an important domain which associates with p160 coactivators including GRIP1, tumor suppressor p53, Smad, IRF1, IRF3, PCNA (proliferating cell nuclear antigen), TBP (TATA binding protein), YY1 and Ets2 among other proteins (Torchia et al, 1998; Livengood et al, 2002; Vo and Goodman, 2001; Lin et al, 2001; Hasan et al, 2001). 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-3. Determination of methylated Arginine residues in GBD of p300 by CARM1. (A) A series of R to K mutants of pSOOGBD. Three arginines of pSOOGBD were changed to lysine residues one by one. The mutant series of p300 proteins were expressed in GST fusion form. (R1K, Arg2056Lys of p300; R2K, Arg2088Lys; R3K, Arg2182Lys). (B) in vitro methylation assay. GST-p300GBD and GST-p300GBD (R to K) mutant proteins were expressed and purified. The expressions of these proteins were confirmed by coomassie blue staining of proteins (lower panel). Each protein was incubated with methyltransferase CARM1 and 3H- radiolabeled S-Adenosylmethionine and analyzed by autoradiography (upper panel). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. R1K R2K R3K P300GBD GST GST GST B ,< P , < ? / V ' TSj"1 £ / / < / O o3 a 5 c? CARM1 + + + + 200 % input 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pSOOGBD is a small domain which interacts with diverse proteins by induced-folding of flexible polyglutamine loops (Lin et al, 2001). Since pSOOGBD is one of the major contact sites for GRIP1 and p53, we became interested in whether arginine residues of pSOOGBD are involved in the interaction between pSOOGBD and GRIP1 or p53. For this purpose, the interactions between GRIP1.AD1 or p53 and pSOOGBD mutants were tested by GST-pulldown assay. Mammalian expression vectors encoding GRIP.AD1 or p53 were transfected into Cos7 cells and the cell extracts were prepared after a forty-hour incubation. Each cell extract was separated into five different tubes and mixed with bacterially expressed pSOOGBD wild type or mutant proteins. The GST or GST-pSOOGBD associated fractions were analyzed by Western blot using HA-specific antibody. Fig. 5-4 shows that both GRIP1.AD1 and p53 interact with purified p300GBD WT (wild type) as reported. Especially GRIP1.AD1 shows strong association with pSOOGBD (the third lane of Fig. 5-4 upper panel). p53 and p300 interaction is clear but the interaction strength is weaker (the third lane of Fig. 5-4 lower panel). Similar binding assays were performed when R to K mutant versions of pSOOGBD were introduced in this system, (lane 4 to 6 of Fig. 5-4). R1K mutant of pSOOGBD binds to both GRIP.AD1 and p53 similar to pSOOGBD wild type (lane 4). R2K mutant of pSOOGBD lost ability to interact with GRIP1.AD1 and p53 (lane 5). And R3K mutant is impaired in interaction with p53 but retains interaction with GRIRAD1 (lane 6). 9 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-4. Arginine residues of pSOOGBD are important for protein-protein interaction. HA tagged GRIP1.AD1, GRIP.N, and p53 vectors were transfected into Cos-7 cells. Transfected cells were lysed by RIPA lysis buffer and co-incubated with Glutathione sepharose bound GST- pSOOGBD, -pSOOGBD (R1K), -pSOOGBD (R2K), and -pSOOGBD (R3K) as indicated. Protein-protein interactions were checked by SDS-PAGE and western blot with HA-specific antibody. (A) GRIP1.AD1 (B) p53. For detection, immunoblots were exposed to X-ray film for 1 minute (A) or 10 minutes (B). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GRIP.AD1 WB : anti-HA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These data suggest that arginine residues R2 and R3 are essential for pSOOGBD protein interaction with specific partner proteins. Especially R2 (Arg 2088) of pSO O is important for association of GRIP1 .AD1 and p53. This is well correlated with recently published NMR structure data of p300-p160 coactivator complex (Demarest et al, 2002). They have shown that the positive charge of R2 (Arg 2088) of p300 is involved in forming a salt bridge with one of the Aspartic acids of the p160 coactivator. Their data supports that R2 is a critical residue for certain protein-protein interactions in Fig. 5-4. However, it is intriguing that R2K has lost its binding ability although it retains the positive charge by lysine instead of Arginine 2088. Fig. 5-4 also shows that R3 (Arg 2142) is important for protein-protein interactions with specific p300 protein partners such as p53. Since R3 is the residue methylated by CARM1, pSOOGBD and p53 interaction could be regulated by CARM1 mediated pSOOGBD methylation. Next, the effects of R to K mutations were investigated in a one-hybrid protein-protein assay in mammalian cells (Fig. 5-5). pSOOGBD and R to K mutants were constructed in the Gal4 DBD (DNA binding domain) fused form. When these constructs were introduced into CV1 cells with the GK1 luciferase reporter which has Gal4 binding elements, similar levels of transcription activation were observed for wild type and mutant proteins (Fig. 5-5A, bars 2-5). 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. When GRIP1.AD1 expression vector was transfected along with GAL4 DBD fused pSOOGBD, transcription activity was enhanced by approximately twofold for wild type and the R1K mutant of pSOOGBD (bars 6-7). In contrast, the activity of the R2K mutant was defective in further transcription activation by GRIP.AD1 (bar 8). Thus, lack of interaction between pSOOGBD R2K and GRIP1.AD1 (Fig. 5-4) results in loss of function of coactivator complex GRIP1-p300 in reporter activation (Fig. 5- 5). Unexpectedly, R3K mutant of pSOOGBD also showed lowered transcription enhancing activity when co-transfected with GRIP1.AD1 (Fig. 5-5, bar 9). This mutant behavior is also similar in the presence of CARM1 (Fig. 5-5A, bar 10 to 13). The effect of R2K and R3K on transcription activation was confirmed by using different does of GRIP1.AD1 expression plasmids (Fig. 5-5B). Wild type GAL4DBD-p300GBD enhanced the reporter activity with increasing amount of GRIP.AD1 vector. However, R2K mutant only showed basal level activity as the expression GRIP1.AD1 was increased. Interestingly, R3K mutant of pSOOGBD had partial transcription enhancing activity when 0.4 pg or 0.6 jug of GRIP1.AD1 vectors was introduced. However, the activity between pSOOGBD WT and pSOOGBD R3K is not distinguishable in the absence of GRIP.AD1 or in the presence of small amount (0.2 jig) of GRIP1.AD1 vector. This data suggests that R3 residue of pSOOGBD is potentially involved in the interaction between pSOOGBD and GRIP.AD1. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-5. R2 and R3 of pSOOGBD are important for transcription activation function. (A) pSOOGBD and pSOOGBD mutants were constructed in pM vector (GAL4 DNA binding domain fusion form). One hybrid assay: 250 ng of GK1 reporter which has GAL4 DNA binding sites, 500 ng of pM- pSOOGBD or pSOOGBD mutants (R1K, R2K, R3K), 250 ng of GRIP1.AD1, 250 ng of CARM1 expression vectors were transfected into CV-1 cells. The luciferase activity was checked forty hours after transfection. (C) Dose experiment; 250 ng of GK1 reporter, 500 ng of pM-p300GBD or pSOOGBD mutants (R1K, R2K, R3K), and 200, 400, 600 ng of GRIP1.AD1 expression vectors were transfected and luciferase activity was analyzed. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 2 3 4 5 6 7 8 9 10 11 12 13 pM-p300GBD + + + pM-p300R1 K + + + pM -p300R2K + + + pM -p300R3K + + + GRIP1.AD1 + + + + + + + + CARM1 + + + + pM-p300WT I pM-p300R2Ki it- pPv1-p300R3K GRIP.AD1 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This interaction could be weak and may not have influence on the physical interaction between the two factors but could be involved in their functional interaction. The loss of activity might be an indirect effect resulting from destabilizing the interaction of pSOOGBD with other basal transcription factors. Effects of Adox methylation inhibitor on p300 protein interaction in mammalian two hybrid system The effect of methylation of pSOOGBD arginine residue was investigated by using Adox (Adenosine dialdehyde) methylation inhibitor. Adox is known to be an inhibitor of S-adenosylhomocysteine hydrolase. Inhibition of this enzyme causes the elevation of SAH (S-Aenosylhomocysteine) which inhibits SAM (S-Adenosylmethionine)-dependent methylation. (Najbauer et al, 1993; Chen et al, 2002). In order to test Adox for protein- protein interaction in a quantitative manner, a mammalian two hybrid assay system was established. GAL4 DNA binding domain-fused pSOOGBD and the counterpart VP16 activation domain-fused GRIP.AD1 and p53 were tested for interaction in the mammalian two hybrid assay. When the wild type of GAL4DBD-p300GBD is introduced with VP 16- GRIP1.AD1, pSOOGBD and GRIP1.AD1 show strong interaction in this assay (bar 5 of Fig. 5-6A). However, GRIP1.AD1 does not interact with a different part of the pSO O protein, the glutamine rich domain of pSO O (p300Q) (bar 10 of Fig. 5-6A). 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5-6. Treatment with a methylation inhibitor influences protein interaction between pSOOGBD and GRIP1.AD1 or p53. (A). Effects of methyltransferase Adox on interaction of pSOOGBD and GRIP1.AD1. Mammalian two hybrid assay: CV-1 cells in six-well dishes were transfected with 250 ng of GK1 reporter, 250 ng of pM-p300GBD encoding GAL4DBD-fused pSOOGBD wild type and mutants, 250 ng of pVP16-GRIP1.AD1 encoding VP16AD fused to GRIP1.AD1 domain. Methylation inhibitor Adox was added to transfected CV1 cells at the concentration indicated. After forty hours, the luciferase (Luc) activity of the cell extract was measured. (B) Effect of methylation inhibitor (Adox) on the interaction of pSOOGBD and tumor suppressor p53. 250 ng of GK1 reporter, 250 ng of pM-p300GBD, 250 ng of pVP16-p53. Methylation inhibitor Adox was included with concentration from 20 pM to 80 pM. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1000 ! § 600 < D sS f f l _ J IS . 400 pM-p300GBD PM-p300Q VP-GRIP1.AD1 Adox (pM) 2 + 3 4 5 6 7 8 9 10 ■ f + + + 4- 4- 4 - 4 - 4- 4- 20 40 60 80 B > O o O (0 O T « _ m X § ^ .2 — i o & ■ 3 2 1 0 pM-p300GBD VP-GRIP1.AD1 Adox (pM) 2 + 4 + 5 + + 20 6 + + 40 7 + + 60 8 + + 80 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The association between VP16-p53 and pSOOGBD was also detected, but the association was much weaker (bar 4 of Fig 5-6B). These interaction data in the mammalian two hybrid system are very similar to the data in GST pulldown experiments in Fig. 5-4. Next, methylation inhibitor Adox was added to the transfected cells in order to block the arginine methylation reaction by endogenous CARM1. When 20 jjiM Adox was applied to transfected CV1 cells, the interaction between pSOOGBD and GRIP1 AD1 in the mammalian two- hybrid system was increased by twofold (bar 6 of Fig. 5-6A). Higher levels of Adox (40- 80 jiM) further stabilized this interaction (bars 7-9). Similarly, the introduction of Adox also influenced the interaction between pSOOGBD and tumor suppressor p53. As the concentration of Adox was increased (20-80 pM), the interaction between pSOOGBD and tumor suppressor p53 was enhanced (bars 5-8 of Fig. 5-6B). These data show that arginine methylation of pSO O may influence the interaction of p300GBD with GRIP1.AD1, p53 and other factors. According to Adox data in Fig. 5-6, arginine methylation of pSOOGBD is likely to have an inhibitory effect on these protein-protein interactions. As shown in this chapter, an arginine residue of pSOOGBD is methylated by CARM1. This arginine is important in some protein-protein interactions and the function of the coactivator complex of pSO O and GRIP1. 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These data indicate that the CARM1 catalyzed methylation of pSOOGBD could be one of the key regulatory steps in coactivator complex formation and synergistic transcription activation. 5-3. Discussion Substrate specificity of CARM1 methyltransferase In an effort to elucidate the mechanism of coactivator synergy between CARM1 and pSO O , p300 was identified as a good substrate for CARM1 methyltransferase activity. However, pSO O was not methylated by the other arginine methyltransferase PRMT1. Since many of the PRMTs have distinct substrate specificity, the biological roles of various members of the arginine methyltransferase family might be diverse in cells. Identification of substrates for PRMTs is one of the key concerns for understanding the biological functions of these enzymes. The common methylated motifs of substrates could be determined by comparing substrate molecules of PRMTs. CARM1 shows a unique substrate specificity among arginine methyltransferase family members. CARM1 is known to methylate ‘KAXRK’ (lys-ala-any amino acid-arg-lys) sequence of proteins (Shurter et al, 2001). This conserved sequence of arginine methylations by CARM1 was suggested based on methylation sites of histone H3. However, another protein, PABP1 (poly(A) binding protein 1) is also methylated by CARM1 but at a somewhat different motif. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The CARM1 mediated methylation site of PABP1 was defined as “RPAAPR” (arg-pro-ala-ala-pro-arg) (Lee and Bedford, 2002). The pSO O methylation sites identified in this chapter and by the others do not match well with the sequences of histone H3 or PABP1. The sequence of strongly methylated arginines of p300 is “VQRAG” (val-gln-arg-ala-gly) at Arg 2142 in GBD and “DLRNH" (asp-leu-arg-asn-his) at Arg 580 in KIX (Fig. 5-3 and Xu et al, 2001). Furthermore, Arg 580 of the GST-KIX fragment (568-828) of p300 is strongly methylated but Arg 580 of GST- p300N fragment (1-596) is not or very weakly methylated (Fig. 5-1B and Fig. 5-2B). Probably, the flanking sequence of the target region and the structure of substrates could be key determining factors for CARM1 substrates. More examples of CARM1 substrates are necessary for drawing conclusions about the conserved sequence motifs of CARM1 substrate. The other PRMTs seem to have less strict substrate specificity. For example, PRMT1 methylates a wide variety of protein substrates, which have arginine and glycine rich sequences, but CARM1 methylation is restricted to smaller numbers of protein molecules (Stallcup, 2001; Lee and Bedford, 2002). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Implications of coactivator methylation by CARM1 pSOOGBD was identified as one of the major methylation domains by CARM1 methyltransferase activity in this chapter. pSOOGBD was originally defined as the binding site for p160 coactivators, and this domain is required for coactivator activity of pSOO with NR (Torchia et al, 1998; Li et al, 2001). This prompted us to test the importance of arginine residues in pSOO physical and functional interactions with GRIP1. Intriguingly, mutation of the methylation site, arginine R3 (Arg 2142) does not affect the interaction with GRIP.AD1 in a protein binding assay in vitro (Fig. 5-4) but the treatment with Adox methylation inhibitor influences this interaction between the two molecules (Fig. 5-6A) in cells. Moreover, mutation of R3 in pSOOGBD partially blocks the activity of p300-GRIP1 coactivator complex (Fig. 5-5), which could be due to reduced binding affinity. It is possible that the failure to observed reduced binding of the p300GBD (R3K) mutant in Fig. 5-4 is due to use of high concentrations of he two binding proteins. These observations suggest that arginine methylation of pSOOGBD could influence the interaction of p300 and GRIP1. It is also possible R3 methylation of pSOOGBD modulates the interaction between other factors and p300 and affects the interaction of pSOO and GRIP1 indirectly. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since the C-temninal part of p 3 0 C S interacts with other important factors S ike PCNA (proliferating cell nuclear antigen), TBP (TATA binding protein), Smad, YY1, IRF1, IRF3, and Ets2, it is very interesting to test the importance of arginines of pSOOGBD in association with these factors (Torchia et al, 1998; Livengood et al, 2002; Vo and Goodman, 2001; Lin et al, 2001; Hasan et al, 2001). The effect of pSOOGBD methylation on the association with tumor suppressor p53 was also tested in this chapter. Compared with the GRIP.AD1 case, pSOOGBD binds to p53 weakly. Furthermore, two arginine residues, R2 and R3, of pSOOGBD are involved in the association with p53 (Fig. 5-3). This suggests that p53 function and its interaction with pSO O could be directly affected by arginine methylation of pSOOGBD. Tumor suppressor protein p53 has a transcription factor function, which is regulated by association with coactivators like pSOO/CBP and also by pSOO/CBP mediated acetylation. (Gu et al, 1997). The interaction domains of p53 in pSO O are located in several independent sites, KIX domain, CHS domain and GBD of pSO O (Livengood et al, 2002). Since KIX and GBD domains are methylated by CARM1, protein methylation of pSOOGBD and KIX by CARM1 might play important roles in regulation of transcription activity and tumor suppressor functions of p53. This potentially suggests that CARM1 mediated coactivator methylation may play a key regulatory role in coactivator complex formation and in transcriptional regulation by NR, p53, and other proteins. 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Previously there were reports that coactivator acetylation regulates protein-protein interaction of the p160 coactivator complex with NR (Chen et al, 1999b). As we observed (Fig. 5-2B), CARM1 mediated methylation of the pSOOKlX domain was reported by other investigators (Xu et al, 2001). The methylation of the KIX domain by CARM1 reduced p300 binding to CREB; this might be an important negative regulatory mechanism in the cAMP signaling pathway. From these observations, protein acetylation and methylation modulate protein-protein interactions and transcription. These data support the concept that arginine methylation of pSOOGBD may regulate protein-protein interaction and transcription. CARM1 catalyzed protein methylation is a potential code for regulating histone and coactivator molecules. In spite of its potential importance, investigation of protein arginine methylation in vivo has technical limitations. Metabolic labeling of ceils and subsequent immunoprecipitation is one of the most sensitive ways of determining protein methylation in vivo. However, this method could not distinguish whether the primary methylation site is arginine or lysine. The better approach is to utilize methylated arginine specific antibodies like Ab412 (AbCAM) for western blot or immunoprecipitation (Mowen et al, 2001). 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Although arginine methylations of some proteins are efficiently detected by this antibody, the methylation of the other substrates including histone H3 are poorly detected by this antibody (unpublished results). One of the best ways of overcoming this dilemma is to develop methylated arginine specific pSOOGBD antibody by synthesizing a methylated peptide centered on R3 peptide. This antibody will make possible the investigation of the behavior of methylated p300 after hormone treatment by CHIP (Chromatin immunoprecipitation) assay. This approach also could be applied for investigation of the p300-regulated p53 function in vivo. CARM1 methylates two important domains of p300, and this methylation potentially regulates transcriptional activation by NR and p53 as well as the cAMP signaling pathway. This indicates that CARM1 mediated protein methylation could have regulatory roles in diverse signaling pathways. Although methylation of arginine does not affect the overall charge of proteins, CARM1 mediated protein methylation could enhance or block binding by changing hydrogen bonds or the local topography. CARM1 catalyzed protein methylation thus contributes both to the “histone code” and the “coactivator code” and acts as an important switch in transcription regulation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This chapter has dealt with CARM1 mediated protein methylation. Especially p3Q0 was tested as a CARM1 substrate, and the possible implication of methylation in the function of p300 was discussed. In the final chapter, the focus will be shifted to yeast two hybrid screening using CARM1 as a bait. To further understand the action mechanism of CARM1, CARM1 associating molecules were identified and characterized. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 6. Characterization of CARM1 interacting proteins and investigation of flightless-1 coactivator activity. 6-1. Introduction Previous chapters have shown that CARM1 has unique arginine methyltransferase activity, methylating histone H3 and p300. This activity could be involved in NR function and synergy with other coactivators. However, CARM1-specific protein interactions should not be ignored as a contributing factor in CARM1 function. This chapter will be focused on CARM1 specific protein interactions to further elucidate CARM1 functional mechanisms. The arginine methyltransferase family has many family members identified in diverse species (Stallcup et al, 2001; Zhang et al, 2000). Recent X-ray crystallography studies of two different members, PRMT3 and RMT1, have revealed the core structure of arginine methyltransferases (Zhang et al, 2000; Weiss et al, 2000). The core domains of these arginine methyltransferases are strikingly similar; for example, two conserved glutamic acid residues of PRMTs are important in forming salt bridges with the arginine residue of the protein substrates. However, this conserved core structure of the arginine methyltransferase does not explain the distinct substrate specificities and diverse roles of each arginine methyltransferase family member. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Interestingly, arginine methyltransferase family members have N-terminal and C-terminal extensions in addition to the conserved, central arginine methyltransferase domain. These structural differences could explain the substrate specificity and different biological functions of arginine methyltransferase family members. CARM1 also has unique N-terminal and C-terminal domains flanking the conserved methyltransferase core domain. The roles of the N-terminal and C-terminal parts of CARM1 are not characterized; these domains could account for the unique methyltransferase activity and coactivator activity of CARM1. In order to define CARM1- specific interacting proteins and substrates, yeast two hybrid screening was employed in this chapter. A partial list of clones isolated is shown in Table 6-1. The developmentally important, flightless-l protein was identified as one of the CARM1 interacting proteins among the clones obtained. Flightless-l protein was originally reported as an essential factor in Drosophila melanogaster development (Campbell et al, 1993). Mutation of this flightless-l gene leads to “loss of flying” phenotype of Drosophila. In severe cases, the mutation of this gene leads to defects in cellularization of the embryo and impaired gastrulation. Flightless-l is a well-conserved protein among diverse species like human, mouse and zebra fish (Campbell et al, 1993; Campbell et al, 2000). The genetic locus of human flightless-l is mapped to a region, which relates with Smith-Magnis syndrome (Chen et al, 1995). 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Smith-Magnis syndrome is a genetic disease showing developmental and behavioral abnormalities in humans. However, it is not yet clear whether the mutation of human flightless-l in this locus causes the Smith-Magnis syndrome. In addition, recent knockout mouse experiments for flightless-l gene shows impairment in early embryo development of mouse (Campbell et al, 2002). These data indicate that the flightless-l gene is necessary for development of Drosophila and probably for other species. However, the molecular action mechanism of flightless-l protein remains obscure. Protein sequence analysis of flightless-l shows LRR (leucine rich repeat) motifs in the N-terminal region and gesolin like motifs in the middle and C- terminal regions (Campbell et al, 1993). The 16 tandem 23-amino acid leucine rich motifs (LRR) are found in flightless-l protein and this motif is also found in other kinds of protein molecules. These LRR family molecules also show diversity in their locations and their functions. The LRR motif is regarded as a module involved in protein-protein interaction. Some groups tried to find the molecules interacting with LRR motifs of flightless-l (Liu et al, 1998; Wilson et al, 1998). Interestingly, they found molecules like FLAP (FLI LRR associated protein) and LRRFIP, which have coiled-coil structures. Gelsolin motifs are also found in many different kinds of protein molecules. 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Gelsolin motifs of flightless-l are known to interact with actin molecules and may play roles in cytoskeletal organization of cells and cytoskeleton associated signal transduction pathways (Davy et al, 2000, Davy et al, 2001). In this chapter, CARM1 associating proteins, especially flightless-l will be investigated for possible roles in transcriptional regulation by NRs. The possibility of a developmentally important protein, flightless-l as a coactivator and its synergy with CARM1 and other coactivators was assessed. 6-2. Results Yeast two hybrid screening using CARM1 as a bait In order to further analyze the role of CARM1, CARM1 associated protein clones were isolated by yeast two hybrid screening, using full-length CARM1 as bait. pGBT9-CARM1 bait plasmid was transformed into HF7c yeast strain. A 17-day mouse embryo cDNA library was subsequently transformed into this CARM1 yeast transformant. 126 positive clones were isolated by selecting for yeast colonies that grow on histidine- negative plates. Finally, filter lift assay showed that 25 clones were (3 - galactosidase positive among the histidine positive colonies (Table 6-1). Table 6-1 indicates that some clones were isolated several times from screening. For example, one of the novel clones, KIAA 0675, was obtained five times by screening. 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Interestingly, CARM1 itself is obtained two times from screening and this suggests that CARM1 exists as a homodimer in yeast cells. This confirms the previous structural and functional studies that PRMTs are present as homodimers (Weiss et al, 2000). This also indicates that the yeast two hybrid screening in Table 6-1 was performed at optimal screening conditions and the clones isolated are potentially the partner molecules of CARM1 in mammalian cells. Some of the clones like KIAA 0675 are non characterized, functionally unknown clones. Others are known proteins like PIAS1 (Protein inhibitor of Activated STAT 1), ubiquitin conjugating enzyme, and CHD3 (chromodomain- helicase-DNA binding 3)/Mi2a, and protein phosphatase 4 regulatory factor. Contrary to our initial expectation, many of the clones interacting with CARM1 in yeast are not transcription- related molecules. This suggests that the roles of CARM1 are not restricted to the nucleus or to transcriptional regulation roles. For example, CARM1 associated protein phosphatase 4 regulatory factor seems to be involved in certain signal transduction pathways. CARM1 association with CHD3/Mi2a suggests the possibility of CARM1 as one of the members of chromatin remodeling complexes. Some CARM1 associated factors like PIAS1 seem to have more diverse roles. PIAS1 was originally known as an inhibitor for the Jak/STAT signaling pathway but is also listed as a coactivator for NRs and sumoylating enzyme for p53 and other factors (Kotaja et al, 2000; Gross et al, 2001; Kahyo et al, 2001). 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 6-1. Partial list of CARM1 interacting clones in yeast two hybrid screening Name of clones Number of isolations CARM1 2 Flightless-l 2 CHD3/Mi2a 1 PIAS1 1 GC-box binding Zinc finger protein 1 Protein phosphatase 4 regulatory subunit 1 Ubiquitin conjugating enzyme E2 1 Profilin 1 1 KIAA 0675 5 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Another class of molecules includes flightless-l, which is known to be developmental^ important both in Drosophila and mouse (Campbell et al, 1993; Campbell et al, 2002). However, the biochemical roles of this protein are not well defined. This mammalian homologue of flightless-l protein was obtained twice as a CARM1 interacting protein in the yeast two hybrid screening. Since flightless-l interacts with CARM1 in the yeast two hybrid system and CARM1 is a classified as a coactivator, flightless-l protein was tested as a coactivator for NR. Flightless-l as a coactivator for NR Full-length cDNA for human flightless -I was provided by Dr. H. Campbell at Australian National University, and this cDNA was subcloned into mammalian expression vector pcDNAS. First, the interaction between CARM1 and flightless-l protein was confirmed by GST-pulldown experiments. Purified GST-CARM1 protein and in vitro translated flightless -I were prepared for this assay. Lane 1 of Fig. 6-1A shows that the flightless -I protein was synthesized in vitro. Because of its large size (-145 kD) and some unknown biochemical nature of this protein, the efficiency of protein synthesis in vitro was not very high and many degradation products of flightless-l were also produced. This unstable in vitro translation of flightless-l was reported previously by another group (Davy et al, 2001). Their immunoprecipitation data have shown that 145 kD size flightless-l protein is the full length flightless-l protein. 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 6-1. Direct interaction of CARM1 and flightless-l in vitro. (A) The Human homolog of flightless-l was synthesized in reticulocyte lysate with 3 5 S labeled methionine. In vitro synthesized flightless-l protein was incubated with bacterially expressed GST protein or GST-CARM1 protein. Bound fractions were analyzed by SDS-PAGE and autoradiography. (B) Two fragments of flightless-l were constructed in mammalian expression vector as flag-tag fused form, Fli-LRR and Fli-Gelsolin. Two flightless-l constructs were transfected into Cos-7 cells and incubated for forty hours. Cell lysates of transfectants were mixed with purified GST-CARM1 or GST protein. Bound fractions were analyzed with SDS-PAGE and western blot with anti-flag antibody. (Fli-LRR, 1-494 fragment of flightless-l; Fli- Gelsolin, 495-1268). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Flightless-l.FL ......... •*— 145kD L B c N ° <§> £ £ K Fli-LRR Fli-Gelsolin W: anti-Flag Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lane 2 of Fig. 6-1A shows that flightless-l protein was not associated with GST protein beads. Pulldown experiment with GST-fused CARM1 showed a weak association with flightless-l proteins (lane 3 of Fig. 6-1). Especially full-length, non-degraded flightless-l binds stronger to CARM1 protein than the other, smaller-sized fragments. From this data, the direct interaction of CARM1 and flightless-l in vitro was demonstrated. Flightless-l has LRR motifs in its N-terminal regions and gelsolin motifs in the middle and C-terminal regions. To determine the CARM1 interacting domain of flightless-l protein, two mammalian expression plasmids expressing fragments of flightless-l were designed. Fli-LRR covers the N- terminal (amino acids 1-494) part of flightless-l and Fli-Gelsolin includes middle and C-terminal (amino acids 495-1268) part. Mammalian cell expression of the LRR domain and Gelsolin domain of flightless -I was detected by western blot using anti-flag antibody (lane 1 of Fig. 6-1B upper and lower parts). LRR or Gelsolin-expressing cell extracts were incubated with GST or GST-CARM1 protein. The upper panel of Fig. 6-1B shows that the LRR fragment of flightless-l interacts specifically with GST- CARM1 but not with GST protein. In contrast, the interaction between the Gelsolin fragment and GST-CARM1 was very weak and showed similar intensity of binding to the GST protein. Fig. 6-1 indicates that flightless-l directly interacts with CARM1, probably through its N-terminal LRR motifs. In addition, flightless-l associates with the p160 coactivator GRIP1 and with ER in vitro (data not shown). 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These results suggests that flightless-l is probably a constituent of the p160 coactivator complex and enhance NR-dependent transcription in cooperation with other factors. Next, flightless-l protein was tested for activity as a NR coactivator, using a NR -dependent transient luciferase reporter assay. To test coactivator function of flightless-l, 100 ng of ER or TR expression vector (high NR conditions) was transfected into CV-1 cells along with an expression vector for one or more coactivators and an appropriate luciferase reporter gene containing ER or TR specific enhancer elements. Transfected cells were treated with the appropriate hormone, and cell lysates were subsequently tested for luciferase activity as a measure of NR and coactivator activity. Fig. 6-2A shows that expression of flightless-l causes further enhancement of NR dependent transcription (bar 5 of Fig.6-2A, upper and lower panel). This coactivator activity of flightless-l is dependent on GRIP1 and NR. In the absence of GRIP1 or NRs, flightless- I failed to enhance NR transcription (bar 7 and 9 of Fig. 6-2A). This absolute dependence of flightless -I on GRIP1 in coactivator function is similar to CARM1 (bar 6, 8 of Fig.6-2A) and p300/CBP. For this reason, flightless-l could be classified as a secondary coactivator which needs a primary p160 coactivator for its function. 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 6-2. Flightless-l human homolog has coactivator activity for NRs. (A) Flightless-l coactivator activity is dependent in GRIP1 and NR. 250 ng of MMTV(ERE)-luc or MMTV(TRE)-luc were used as reporters. 100 ng of ER or TR expression vectors, 250 ng of GRIP1 expression vector, 500 ng of CARM1 expression vector, and 500 ng of flightless-l expression vector were transfected into CV-1 cells. (B) Coactivator activity of flightless-l is enhanced by increased amounts of expression vector. 1X 105 cells of CV- 1 were transfected with 250 ng of MMTV(ERE)-luc reporter, 100 ng of ER expression vector, 250 ng of GRIP1 expression vector, 200 ng of CARM1 expression vector, and 200-600 ng of flightless-l expression vectors. Transfected cells were incubated with 20 nM estradiol or T3 for 40 hours and luciferase activity was measured. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TR 20 10 0 8 9 2 3 4 5 6 7 1 NR GRIP1 CARM1 Fli-I + + + + + + + B > . : > o t 5 o to O 05 X * K -3 0 5 I 3 w 40 30 20 10 0 IER • ER GRIP1 CARM1 ■ ER GRIP1 Fli-I Fli-I(ng) 0 200 400 600 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. As the amount of flightless-! is increased from 200 ng to 600 ng, the coactivator activity of flightless-l is accordingly enhanced. In Fig. 6-2B, Flightless-l is a poor coactivator at low concentration of expression vector, but becomes more efficient at higher concentration. Probably this results from low level expression and unstable nature of flightless-l protein. Alternatively, another factor present in limiting amounts may be required for efficient action of flightless-l. Fig. 6-2B further confirms the coactivator activity of flightless-l for ER transcription activation. Since flightless-l shows GRIP1 dependence for its coactivator function, we decided to test which domains of GRIP1 are required for flightless-l coactivator activity. GRIP1 mutants lacking activation domains (GRIP1 AAD1, GRIP1 AAD2 and GRIP1 AAD1AD2) were introduced along with flightless-l in the transient reporter assay system. Flightless-l again showed the two to three-fold TR transcription enhancing activity in the presence of wild type GRIP1 (bar 4 of Fig. 6-3). When the AD1 deletion mutant or the AD1AD2 deletion mutant of GRIP1 was introduced with flightless-l, the TR enhancing activity was reduced to basal level (bar 6, 10). In contrast, GRIP1 AAD2 mutation did not abrogate flightless-l coactivator activity for TR transcriptional activation (bar 8). These results suggest that the coactivator function of flightless-l is dependent on activation domains of GRIP1, especially GRIP1 AD1 domain. 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.6-3. Coactivator activity of flightless-! is dependent on AD1 domain of GRIP1. CV-1 cells in six-well dishes were transfected with 250 ng of MMTV (TRE)-Iuc, 100 ng of TR expression vector, 500 ng of flightless-l and 250 ng of GRIP1, GRIP1AAD1, GRIP1AAD2, GRIP1AAD1AD2 as indicated. After transfection, cells were maintained with 20 nM of T3 until luciferase activity was measured. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■ £ o o o c a o MX £=> <D _J □ w TR GRIP1 GRIP.AAD1 GRIP.AAD2 GRIP.AAD1 AD2 Fli-I + + 4 + + 6 7 8 9 + + + + + + 10 + + + 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This is very similar to the requirement of GRIP1 AD1 domain for p300/CBP coactivator activity. In contrast, the coactivator activity of CARM1 requires the AD2 domain of GRIP1 (Chen et al, 1999a; Chen et al, 2000a). Fig. 6-3 shows that flightless-l coactivator interacts functionally with the AD1 domain of GRIP1 and raises the possibility of collaboration with the AD2 targeting- coactivator CARM1. Synergy between CARM1 and flightless-l for NR transcription Flightless-l and CARM1 interact in vitro and cooperate with different activation domains of GRIP1 in coactivator assays for TR. To test their possible collaboration in NR coactivation, a low NR assay system was introduced again (Chapter 3). A small amount of NR expression vector (1 ng) was introduced with two or three combinations of coactivators in a transient reporter assay. As shown in Fig. 3-1, transfection of one or two coactivators has little effect on luciferase activity at low NR conditions (bars 3-7 and 1 1 of Fig. 6-4). Coexpression of flightless-l and CARM1 synergistically enhances TR activity in the presence of GRIP1 (bar 8). GRIP1 is also required for coactivator synergy between CARM1 and flightless-l at low NR conditions (bar 11). However, flightless-l protein does not cooperate with the other arginine methyltransferase PRMT1 (bar 9) or the other coactivator p300 (bar 10) in this assay. Fig 6-4 shows that flightless-l has specific cooperation with CARM1 in the transient reporter assay. 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 6-4. Flightless-l cooperates specifically with CARM1 at low NR condition. CV-1 cells were transfected with 250 ng of MMTV(TRE)-luc, 1 ng of TR expression vector, and combinations of coactivator expression vectors as indicated. 250 ng of GRIP1 vector, 500 ng of CARM1 vector, 500 ng of flightless-l vector, 500 ng of p300 vector, 500 ng of PRMT1 vector. After transfection, cells were maintained in the presence of 20 nM T3 until luciferase activity was measured. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TR + + + + + + + + GRIP1 + + + + + + + CARM1 + + PRMT1 + + P300 + Fli-I + + + Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This probably results from physical and functional interaction between two molecules, CARM1 and flightless-l. Next, the role of arginine methyltransferase activity of CARM1 in this CARM1 and flightless-l synergy was investigated. E/Q mutant of CARM1, which is selectively defective for methyltransferase activity (see chapter 4), was introduced into the low NR synergy assay. Flightless-l and CARM1 specifically cooperate for NR-dependent transcription in the presence of GRIP1 (Fig. 6-5, bar 8). As a positive control for this synergy assay, the combination of CARM1 and p300 were included in this assay (bar 7). Coactivator synergy between CARM1 and flightless-l is dramatic but less effective than the CARM1 and p300 combination. Introduction of the E/Q mutant instead of wild type CARM1 leads to almost complete loss of activity of flightless-l and CARM1 (bar 12). Similarly, arginine methyltransferase activity is also required for p300 and CARM1 synergy (bar 1 1 and Fig. 4-4). This suggests that the arginine methyltransferase activity of CARM1 is essential in cooperation with flightless-l as coactivator for NR- dependent transcription. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.6-5. Arginine methyltransferase activity of CARM1 is essential for cooperation with flightless-l. CV-1 cells were transfected with 250 ng of MMTV(TRE)-luc, 1 ng of TR expression vector, 250 ng of GRIP1 expression vector, 500 ng of flightless-l vector, 500 ng of p300 vector, 500 ng of CARM1 vector or 500 ng of CARM1 (E/Q) expression vector in the presence of 20 nM T3. Transfected cells were lysed and checked for luciferase activity after forty hours. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 2 3 4 5 6 7 8 9 10 11 12 TR + + + + + + + + + + + GRIP1 + + + + + + + + + GARM1 + + H b E/Q + + + Fli-I + + + + P300 + " 1 " H h + 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6-3. Discussion CARM1 interacts with diverse kinds of protein factors potentially suggesting diverse function of CARM1 Identification of CARM1 associated proteins could reveal hidden roles of CARM1 and help to elucidate its mechanism of action. As discussed earlier, CARM1 seems to have both arginine methyltransferase dependent and independent functions (Chapter 4). Isolation of CARM1 interacting proteins could provide us insights about both methyltransferase- dependent and -independent roles of CARM1. Some CARM1 interacting proteins may be substrates for CARM1 methyltransferase activity. Others may mediate the function (e.g. coactivator function) of CARM1 through protein-protein interactions with CARM1. The other proteins may act as a regulator for CARM1 activity through interaction with CARM1. Table 6-1 shows that CARM1 associates with diverse kinds of molecules in yeast cells. This possibly implies diverse functional roles of CARM1 in vivo. The subcellular location of CARM1 is not restricted to the nucleus, since CARM1 is also present in cytoplasm (our unpublished data). This location of CARM1 in cells further supports the idea of diverse roles for CARM1 in cells. Some of the molecules identified through screening could act as a coactivator for NRs. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Many coactivators share some structural, functiona! and sequence motifs like LxxLL for binding to NRs (Voegel et al, 1998; Darimont et al, 1998; Heery et al, 1997; Torchia et al, 1997; Ma et al, 1999; Bevan et al, 1999) or enzymatic activities. However, other coactivators reported do not have any common structural, functional motifs and do not belong to any previously known classes of coactivators. In addition, many of the coactivators are present in a large complex and function cooperatively. For this reason, clones interacting with CARM1 could have coactivator activity. It is very possible CARM1 and the associating proteins have collaborative or antagonistic relationships. Characterization of these clones could also lead to elucidation of upstream regulators or downstream targets for CARM1 functions. Flightless-l is a novel class of coactivator for NR One of the developmentally essential genes in mouse and Drosophila, flightless-l, was demonstrated to have coactivator activity for NR (Fig. 6-2) and its activity is cooperative with CARM1 (Fig. 6-4). Flightless-l cooperation with GRIP1 primarily requires the AD1 domain of GRIP1 (Fig. 6-3). The AD2-dependent activity of CARM1 might be amplified by the AD1-dependent activity of flightless-l. However, it is not yet clear whether flightless-l binds directly with GRIP1 AD1 or interacts indirectly through the other AD1 coactivators like p300. However, flightless-l did not cooperate synergistically with p300 (Fig. 6-5). 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Flightless-l has two structurally interesting motifs; LRR sequences at the N terminus and two gelsolin motifs at the central region and C-terminus. The LRR sequence is probably involved in interaction with the other protein factors. CARM1 also interacts with flightless-l through this LRR domain. This hydrophobic module could be involved in recruitment of various coactivators in addition to CARM1. The protein sequence of flightless-l also includes two putative LxxLL NR binding sequences. One is located inside the LRR domain, and the other inside the Gelsolin motifs. The importance of LxxLL for flightless-l function is not determined yet. The gelsolin motifs of flightless-l have an ability to interact with actin molecules (Davy et al, 2000; Davy et al, 2001). Flightless-l protein interacts both with G-actin and with F-actin. This result might suggest a role for the gelsolin motif of flightless-l in association with cytoskeletal structures and might influence cytoskeleton-associated signal transduction pathways (Davy et al; 2001). It is plausible the flightless-l coactivator activity is related to its cytoskeleton signaling function. Significant amounts of actin and actin binding proteins are found in the nucleus (Ankenbauer et al, 1989). Several groups proposed roles for actin in the transcription process. Actin could be required for transcription by RNA polymerase II (Egly et al, 1984). Microinjection of actin antibodies into cells blocks the transcription processes and suggests the involvement of nuclear actin in transcription processes (Scheer et al, 1984). 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Recently, another actin binding protein, supervillin, was reported as a coactivator for the NRs like AR and GR (Ting et al, 2002). Supervillin has actin binding properties like flightless-l and furthermore this actin binding region is highly homologous (approximately 50%) to gelsolin motifs (Ting et al, 2002). Since gelsolin motifs of flightless-l have similar actin binding characteristics with supervillin, flightless-l and supervillin may act as coactivators by similar mechanisms. One of the other clones identified as a CARM1 binding protein is profilin 1 (Table 6-1). Profilin 1 is also known to interact with actin molecules and could have similar activity with flightless-l and supervillin. Considering these observations, the important roles of actin and actin binding proteins in NR transcription are strongly suggested. Actin is not simply a structural protein involved in cytoskeleton structure but could have functional roles in transcription. This chapter and papers from other investigators suggest that actin and actin binding proteins like flightless-l have a regulatory activity for NR transcription (Ting et al, 2002). We speculate that flightless-l is an important component involved both in cytoskeletal structure formation and in NR- dependent transcription. The characteristic of flightless-l and supervillin is very similar to another developmentally important protein (3-catenin. (3 - catenin also has a dual role both in cytoskeletal structure formation by interacting with adhesion molecules and in transcriptional regulation. p- catenin was also reported to interact with and act as a coactivator for AR (Truica et al, 2000). 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Furthermore, p-catenin associates with CARM1 in co-immunoprecipitation assays and cooperates with CARM1 in coactivator assays (Koh et al, 2002). In this respect, flightless-l, p-catenin and supervillin could comprise a new family of coactivators for NR. The action mechanism of flightless-l and the other actin binding molecules in transcription is not clear at this stage. One attractive hypothesis is the recruitment of ATP dependent chromatin remodeling complex through interaction of flightless-l and actin because actin was identified as an essential component in Swi/Snf complexes like BAF (Brg or Brm associated factor) (Zhou et al, 1998; Rando et al, 2002). Is CARM1 a regulator for the function of flightless-l? One of the interesting cellular features of flightless-l is the serum- regulated subcellular localization of flightless-l (Davy et al, 2001). Especially when the serum concentration is low, flightless-l is localized inside the nucleus. In contrast, when the serum level is increased, flightless-l is associated with the cytosol and cytoskeleton structure. This implies that the coactivator activity of flightless-l could be regulated by serum concentration and by the resulting differential subcellular localizations. CARM1 methyltransferase activity is required for synergistic action of flightless-l and CARM1 (Fig. 5-5). This data may have various implications for flightless-l function. Protein methyltransferase activity of CARM1 could affect the regulatory localization of flightless-l by serum. 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This activity could also influence the association between flightless-l and cytoskeletal components and the subsequent signaling events. In the broader view, the arginine methyltransferase activity of CARM1 may influence the role of flightless-l in developmental processes. CARM1 is already reported to have coactivation activity for muscle specific factors and is essential for muscle differentiation (Chen et al,, 2002). Knockout of flightless-l in Drosophila also causes defective development of flight muscle (Campbell et al, 1993). Developing a knockout mouse of CARM1 or knock-in E/Q mutation in the CARM1 methyltransferase domain will be helpful for elucidation of the possible roles of CARM1 in the physiological setting. These important roles in development, and interesting biochemical and cellular features of flightless-l leads the investigation of CARM1 and its methyltransferase activity to new directions. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 7. Concluding Remarks Transcriptional regulation by NR is modulated by a variety of coregulators. CARM1, coactivator associated arginine methyltransferase, becomes one of the central players in NR-dependent transcription regulation. The aim of my thesis was to further elucidate the function and the action mechanism of CARM1. For this purpose, a NR transient assay system was developed to test the activity of CARM1 in a multiple coactivator-context in chapter 3. Dramatic coactivator synergy between CARM1 and the other HAT coactivators like p300, CBP and p/CAF was observed in this low NR assay system. A ternary complex among these coactivators was demonstrated, and the essential role of GRIP1 in complex formation was observed as well. Based on this observation, the contribution of histone acetyltransferase activity and histone methyltransferase activity of coactivators was investigated in chapter 4. Arginine methyltransferase activity of CARM1 is required for coactivator synergy especially at low NR condition. It was proposed that CARM1 has arginine methyltransferase- dependent and also -independent functions by comparing two CARM1 mutants, E/Q and VLD. The CARM1 methyltransferase activity required for cooperation with p300 is not substituted by the other arginine methyltransferases. In addition, the HAT activity of p/CAF, but not that of p300/CBP, is important for coactivator synergy. In Chapter 5, a new methylation substrate of CARM1 was investigated because CARM1 specific methyltransferase activity is required for CARM1 action. 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Especially coactivator p30Q methylation by CARM1 was investigated, and the implications of arginine methylation were tested in protein-protein interaction and transcriptional activation assays. Chapter 6 focused on the proteins associating with CARM1. Diverse clones were identified by yeast two hybrid screening, which suggests multiple roles for CARM1. One of the clones, flightless -I, was tested for coactivator activity for NRs. Flightless -I was synergistic with CARM1 and its methyltransferase activity for enhancing NR -dependent transcription. This thesis is not the final answer for this exciting project. Instead, this raises lots of new questions and possibilities. Initially CARM1 and p300 were tested to investigate the possible collaboration of different histone modifications in the NR assay system. Likewise, histone methylation and the other modifications like phosphorylation, ubiquitination, sumoylation and the others could be tested in similar way. For example, the collaboration between methylation and ubiquitination could be tested by coexpressing CARM1 and histone ubiquitination factors. Interestingly, one member of the ubiquitin conjugating enzymes was identified as a CARM1 interacting protein in the yeast two hybrid assay. This ubiquitin conjugating enzyme could be tested for its histone modifying activities. Histone methylation by CARM1 and histone ubiquitination could be cooperative or exclusive in their function. 145 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This hypothesis could be tested by in vitro or in vivo ubiquitination assays of histones by using the ubiquitin conjugating enzyme and with CARM1. Furthermore, upstream regulators and downstream substrates of CARM1 remain obscure. CARM1 interacting proteins could modulate the arginine methyltransferase activity of CARM1 and could also be substrates for CARM1. For example, the ubiquitin conjugating enzyme could modify the activity of CARM1 by ubiquitination. It is also plausible that CARM1 methylates and modulates the function of the ubiquitin conjugating enzyme. The relationship between these two factors could be required for NR-dependent transcription and the other biological processes. Another important issue in the elucidation of CARM1 function is to characterize the methyltransferase-independent roles of CARM1. This issue may be approached by the analysis of proteins interacting with CARM1 through the N-terminal or C-terminal, that is the non-methyltransferase domain. It was already demonstrated that the C-terminal part of CARM1 has a transcription activation activity when this domain was tethered to GAL4 DNA binding domain (our unpublished data). Some of the proteins isolated by yeast two hybrid screening may bind to the N-terminal or C- terminal domain of CARM1 and could contribute to the methyltransferase -independent coactivator function of CARM1. For example, the ubiquitin conjugating enzyme could associate with the C-terminal part of CARM1 and be involved in the enhancement of CARM1 activation function. 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This hypothesis could be tested by protein -protein interaction assays and one-hybrid transcription assays by using the C-terminal fragment of CARM1. Both the methyltransferase-dependent and -independent activities of CARM1 are probably regulated by its associating factors. The effects of CARM1 interacting factors on NR-dependent transcription could be investigated by using low NR assay system (Chapter 3). The low NR assay system will be quite a useful tool for testing the synergy of CARM1 and the other histone modifying factors, CARM1 regulators and CARM1 substrates etc. The usefulness of the low NR system was again demonstrated by testing flightless-l coactivator activity in chapter 6. Two different kinds of factors like CARM1 and the ubiquitin conjugating enzyme could be cooperative in enhancing the NR-dependent transcription. As shown in this thesis, CARM1 and its arginine methyltransferase is clearly an important regulator of transcription in vitro and in cell line systems. However, the physiological significance of CARM1 and its methyltransferase activity is still much to be explored. 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Creator Lee, Young-Ho (author)

Core Title Coactivator synergy for nuclear receptors: Regulatory roles of arginine methyltransferase activity in transcription

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,OAI-PMH Harvest

Language English

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Advisor Stallcup, Michael R. (committee chair), Johnson, Deborah L. (committee member), Maxson, Robert E. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-252425

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