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Isolation and characterization of mouse Zac1, a novel co-regulator for transcriptional activation by nuclear receptors and p53
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Isolation and characterization of mouse Zac1, a novel co-regulator for transcriptional activation by nuclear receptors and p53
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ISOLATION AND CHARACTERIZATION OF MOUSE ZAC1, A NOVEL CO-REGULATOR FOR TRANSCRIPTIONAL ACTIVATION BY NUCLEAR RECEPTORS AND P53. by Shih-Ming Huang A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA in Partial Fulfillment o f the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOCHEMISTRY) December 2000 Copyright 2000 Shih-Ming Huang Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY O F SOUTHERN CALIFORNIA T h e G rad u ate Sch ool U n iv e r sity Park LOS ANGEI.ES, CALIFORNIA 90089^1695 This dissertation, written by S h ih -M in g Huang Under the direction o f Ais... Dissertation Committee, and approved b y ail its members, has been presented to and accepted by The Graduate School, in partial fulfillment: of requirements for the degree o f DOCTOR OF PHILOSOPHY Dean o f Graduate Studies Date December 18, -2000 D IS S E R T A HON COMMITTEE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I sincerely appreciate my mentor Michael R. Stallcup for his guidance and support through my whole Ph. D program. I also thank him for providing me opportunities to develop interesting research projects and discussing questions when I need to talk. His kind and open-minded mentor style will be a good model for me to develop my career in the future. I also would like to thank my committee members Dr. Michael M. Lai and Dr. Deborah Johnson for their advices and supports during my thesis work. I would like to express my special thanks to Dr. Axel H. Schonthal for his help and discussion in my p53 study. I also thank all o f my coworkers in the lab for their help and share, especially, Dr. Xiu Feng Ding, Dr. Dagang Chen, and Dr. Heng Hong for their help in my yeast two- hybrid screening work. Finally, I appreciate my scholarship sponsor, Defense Department, Taiwan, Republic o f China, and thank Biochemistry Department, National Defense Medical Center, for providing this training opportunity and keeping a position for me. I really thank my wife, Yu-Chiao Peng, RN, for her love and support in my life. I also like to present this thesis to her and my family in Taiwan. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENETS A cknow ledgm ents..................................................................................................... ii List o f Figures ............................................................................................................ iv List o f Tables ............................................................................................................. vi Abstract ....................................................................................................................... vii Chapter 1: Introduction........................................................................................... 1 Chapter 2: Molecular cloning o f a mouse splicing variant o f Zacl (m Z aclb)cD N A .................................................................................. 12 Chapter 3: Mouse Zacl, a transcriptional coactivator and repressor for nuclear receptors.................................................................................... 30 Chapter 4: Enhancement of p53-dependent gene activation in HeLa cells by Zacl through coactivator effects and disruption o f the p53 complex with human papilloma virus protein E 6.18.................... 66 Chapter 5: Concluding Remarks............................................................................ 101 Bibliography ............................................................................................................. 106 iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure Page 2-1 DNA and protein sequences o f m Z aclb..................................................... 18 2-2 Amino acid sequence alignment o f mZac 1, rLot 1 and hZac 1/hLot I... 23 2-3 Domains o f mouse Z a c l................................................................................. 25 3-1 Binding o f mZac lb to NRs and NR coactivators..................................... 35 3-2 Transcriptional activation domain o f Zac 1............................................... 38 3-3 Synergistic enhancement o f AR function by mZac lb and GRIP 1.... 41 3-4 Synergistic enhancement o f AR AF-1 and AJF-2 function by m Zaclb and GRIP 1....................................................................................................... 45 3-5 Relationship o f AR activity to DHT concentration in the presence of mZac 1 b and/or GRIP 1.................................................................................. 47 3-6 Repression by m Zaclb o f ER function with the MMTV(ERE) promoter........................................................................................................... 51 3-7 Coactivator effect o f m Zaclb on ER function with the thymidine kinase promoter............................................................................................... 53 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3-8 Variable coactivator or repressor roles of m Zaclb with AR and ER in three cell lines........................................................................................... 58 3-9 Coactivator effects o f m Zaclb with the GaI4 DBD fused to GRIP I or a C-terminal fragment o f CBP............................................................... 61 4-1 Z acl specifically enhances transcriptional activation by p53 in HeLa cells................................................................................................................ 72 4-2 Z acl coactivator function on p53-responsive promoters requires p53.................................................................................................................... 75 4-3 Physical and functional interactions between p53 and Z a c l.................. 78 4-4 Repression o f Zacl function by over-expression of p53 in HeLa cells................................................................................................................... 81 4-5 Stimulation or inhibition o f Zacl function by different levels of p53, p53 fragments, or p53 mutants................................................................... 85 4-6 Competitive binding of p53 and Zacl to HPV-18 E6 protein................ 88 4-7 Functional interactions among p53, E6.18, Zacl, and CBP.................. 91 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table Page 2-1 The analysis o f positive clones interacting with the c-terminal region o f GRIP I in the yeast two-hybrid screening system............................. 15 3-1 The effects o f mZaclb on the CMV promoter and on NR function are independent.................................................................................................. 56 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Transcriptional activation by nuclear receptors is mediated by p i 60 coactivators. These coactivators associate with DNA-bound nuclear receptors and transmit activating signals to the transcriptional machinery through two activation domains. In screening for mammalian proteins that bind the C-terminal activation domain o f the nuclear receptor coactivator GRIP1, I identified a new variant of mouse Zacl (m Zaclb). In yeast two- hybrid assays and in vitro, m Zaclb bound to GRIP1, to CBP/p300, and to nuclear receptors themselves in a hormone-independent manner. In transient-transfection assays, m Zaclb exhibited a transactivation activity when fused with the Gal4 DNA-binding domain, and it enhanced transcriptional activation by the Gal4 DNA-binding domain fused to GRJP1 or CBP fragments. More importantly, m Zaclb was a powerful coactivator for the hormone-dependent activity of nuclear receptors, including androgen, estrogen, glucocorticoid, and thyroid receptors. However, with some reporter genes and in some cell lines, m Zaclb acted as a repressor rather than a coactivator of nuclear receptor activity. Thus, m Zaclb can interact with nuclear receptors and their coactivators and play both positive and negative roles in regulating nuclear receptor function. Zacl was previously discovered as a putative transcriptional activator involved in regulation of apoptosis and the cell cycle. Since these are common activities o f Zacl and p53, I tested for a functional interaction between these two proteins. Zacl dramatically and specifically enhanced the activity o f p53-dependent promoters in HeLa cells through at least two pathways. First, Zacl bound to p53 and acted as a coactivator for p53. Second, Zacl bound to HPV-18 E6 protein and disrupted the E6-p53 complex, which leads to vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inactivation and degradation of p53. p53 was necessary for the action o f Zacl on the p53- dependent promoters, but the concentration range o f p53 that is permissive for enhancement by Zacl is very narrow. This study suggests that a finely balanced combination o f physical and functional interactions among the p53, Z acl, E6, and CBP proteins in HeLa cells regulates the function o f p53 as a transcriptional activator and regulator o f the cell cycle and apoptosis. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1: Introduction The subject o f my thesis is the isolation and characterization o f mouse Zacl (zinc finger protein which regulates apoptosis and £ell cycle arrest) and its role in transcriptional activation by the nuclear receptors and p53. In the beginning of this project, I determined that Zacl could interact with the C-terminal region o f GRJP1 and serve as a co-regulator for nuclear receptors. Subsequently, I studied the coactivator function o f Zacl in p53-dependent transcriptional activation because Zacl and p53 have similar functions in apoptosis and cell cycle arrest. Here, I will introduce these two important sequence-specific transcription factors, nuclear receptors and p53, in this chapter as my prelude. RNA polymerase II and its transcription factors The complexity o f the RNA polymerase II transcription machinery is reflected by the existence o f numerous factors binding to elements upstream o f the TATA box and increasing or decreasing the level of transcription. Many o f these factors are constitutively active. However, other factors are active only in the presence o f an inducing stimulus or in a specific tissue, thereby producing a specific pattern of inducible or tissue-specific gene expression. Transcription factors modulate gene transcription by binding to specific enhancer elements associated with the promoters o f their target genes. The subsequent transcriptional activation o f the gene involves changes in local 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conformations in chromatin structure and recruitment o f a transcription initiation complex containing RNA polymerase II to the promoter. Recent studies have suggested a strong link between histone acetylation/deacetylation, chromatin remodeling, and gene regulation (Struhl 1998; Mizzen and Allis 1998; Cheung et al. 2000; Strahl and Allis 2000). In particular, a number o f transcriptional regulatory proteins, including GCN5, PCAF, CREB-binding protein (CBP)/p300, TAFII250, and the nuclear receptor coactivators called steroid receptor coactivator 1 (SRC-1) and activator o f thyroid and retinoic acid receptors (ACTR), have been found to possess intrinsic histone acetyltransferase (HAT) activity (Brownell et al. 1996; Yang et al. 1996, Ogryzko et al. 1996; Mizzen et al. 1996; Spencer et al. 1997; Chen et al. 1997). In addition to histones, these enzymes might acetylate other components o f the basal transcription machinery and some transcription factors, like p53, GATA-1, and MyoD, and directly affect transcription (Imhof et al. 1997; Gu and Roeder 1997; Boyes et al. 1998; Sartorelli et al. 1999) 1.1 Nuclear receptor superfamily 1.1.1 The structure and function of nuclear receptors Nuclear receptors (NRs) comprise a family of transcription factors that regulate gene expression in a ligand-dependent manner (McKenna et al. 1999). Members include the receptors for steroid, thyroid, retinoid, and vitamin D hormones. Some un-identified regulatory ligands may bind to so-called orphan receptors o f the NR superfamily. Most 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NRs activate transcription in response to their ligands by binding to enhancer elements in the promoters o f target genes as homo- or hetero-dimers. However, some members of the NR family regulate transcription by binding to other classes o f DNA-bound transcription factors. In addition to gene activation, a subset o f NRs, including TR and RAR, can actively repress target genes in the presence or absence o f ligand binding, and many NRs have been demonstrated to inhibit transcription in a ligand-dependent manner by antagonizing the transcriptional activities o f other classes o f transcription factors. The mechanism o f transcriptional regulation by the DNA-bound NRs and their regulation domains appear to involve their ability to recruit a variety o f co-regulatory proteins, including coactivator and corepressor. Transcriptional coactivators, recruited by sequence specific transcription factors, enhance transcriptional activation o f target genes via interaction with chromatin remodelling complexes, and components of the basal transcription machinery. Generally, coactivators are not DNA-binding proteins but rather are recruited to the promoter through protein-protein contacts with the transcriptional activators. They may be thought of as adaptors or components in a signaling pathway that transmits transcriptional activation signals from DNA-bound activator proteins to the chromatin and transcription machinery. Transcriptional corepressors could involve competition for limiting factors, displacement of positive factors, or the generation of a chromatin structure that limits promoter accessibility by histone deacetylation (Glass and Rosenfeld 2000). Therefore, the latest working model o f NRs is an initial association with transcriptional corepressors and subsequent ability to recruit coactivators in response to ligands and other signals. 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The hallmark o f all NR structures is a highly conserved DNA-binding domain (DBD) located in the central part o f the polypeptide chain. The hormone-binding domain (HBD), which is also conserved but somewhat less than the DBD, is a large C-terminal domain. In addition to binding hormone, the HBD also contains an important highly conserved activation function, AF-2, which is one o f two domains primarily responsible for activation o f transcription by the hormone-activated, DNA-bound NR. The other activation function, AF-1, found in the N-terminal domains of most NRs, is not conserved in length or sequence. The relative importance o f A F-1 and AF-2 varies among different NRs and also can be influenced by ligand, cell type, and target gene promoter. A growing number o f candidate nuclear receptor coactivator proteins have been identified, by virtue of their ability to bind HBDs (AF-2 domains) o f hormone-activated NRs, un- liganded HBD, or DBD. The best characterized among these is a family of three structurally related but genetically distinct 160-kDa proteins called the NR coactivators or pl60 coactivators. The three family members are SRC-1, glucocorticoid receptor interacting protein 1 (GRIP1; also called TIF2), and pCIP(also called RAC3, ACTR, AIB1, and TRAM1) (Onate et al. 1995; Hong et al. 1996; Voegel et al. 1996; Torchia et al 1997; Li et al. 1997; Chen et al. 1997; Anzick et al. 1997; Takeshita et al. 1997). 1.1.2 The structure and function of nuclear receptor coactivator An understanding of the precise repertoire of co-regulators and co-regulator complexes required for physiological NR function will help us to link all functional 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. signals among co-regulators, NRs, chromatin, and transcription machinery. The early studies of p i 60 coactivators suggested that there was only one primary type o f signal input domain and signal output domain. The conserved AF-2 domain o f any hormone- activated NR binds to the conserved NR-interacting domain (NID) found in the central part o f the polypeptide chain o f the three p i 60 coactivators (Heery et al. 1997; Ding et al. 1998; M clnemey et al. 1998). The NID contains three conserved sequences called NR boxes, LXXLL, where L is leucine and X is any amino acid. Binding o f the NID to the NR AF-2 domain is sufficient to recruit the coactivator to enhance NR function. Similar to p i 60 coactivators, the studies o f corepressors, N-CoR and SMRT, show that two interaction domains containing a conserved sequence (referred to as the CoRNR box or as a LXXI/HIXXXI/L helix, where L is leucine, X is any amino acid, I is isoleucine and H is histine) are required and sufficient to permit binding to unliganded TR and RARs. Therefore, NR AF-2 helix has evolved to discriminate between the LXXLL helix in coactivators and the extended helix in the N-CoR/SMRT corepressors, permitting the ligand-dependent switch o f NR activity. Furthermore, the crystal structures o f unliganded and agonist-bound HBDs for several NRs have confirmed the hypothesis that the AF-2 region undergoes a ligand-dependent conformational change and permitted evaluation of coactivator and corepressor binding (Chen and Evans 1995; Feng et al. 1998; Glass and Rosenfeld 2000). The enhancement o f NR activity by the pl60 coactivators depends on CBP/p300 family because CBP/p300 family is necessary for the function o f activation domain 1 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (A D I) o f the p i60 coactivators. Therefore, ADI receives the activating signal from the DNA-bound NRs and recruits CBP/p300, which could activate the transcription machinery because of its HAT activity to modify chromatin structure (Yao et al. 1996; Voegel et al. 1998). Another activation domain (AD2) o f the pi 60 coactivators is in their C-terminal region (Voegel et al. 1998; Ma et al. 1999). Recent studies have shown that the AF-1 domains o f the progesterone receptor (PR), ER, androgen receptor (AR), TR and mineralocorticoid receptor (MR) also could directly interact with the p i60 coactivators (mostly through the C-terminal region) and CBP/p300 (Onate et al. 1998; Webb et al. 1998; Tremblay et al. 1999; Kobayashi et al. 2000; Ma et al. 1999; Bevan et al. 1999; Oberste-Berghaus et al. 2000; Fuse et al. 2000). However, it is unclear whether these A F -is bind to specific motif(s) on the coactivators (like LXXLL m otif binding to AF-2), but the AD2 activity should play an important role for the AF-1 enhancement through the interaction of AF-1 with the C-terminal region of p i60 coactivators. Recent studies in our lab may be relevant to the mechanism o f AD2 function o f GRIP 1. The C- terminal GRIP I region could directly interact with AR AF-1 m otif and then enhance AR AF-1 activity in the absence o f ligand by its AD2 function (Ma et al. 1999). Additional studies showed one novel protein, coactivator associated arginine methyltransferase 1 (CARM1), mainly binds to the C-terminal region of the p i 60 coactivators and stimulates the AD2 transactivation function o f p l6 0 (Chen et al. 1999). When co-expressed with p i 60 coactivators, CARM1 also enhances the activity o f NRs above that observed with the p i 60 coactivators alone. We propose CARM1 serves as a secondary coactivator for NRs and represents a downstream target o f the AD2 domain o f p i 60 coactivators. 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CARM1 also possesses intrinsic arginine methyltransferase activity and can methylate arginine residue(s) of histone H3 in vitro. Other arginine methyltransferase family members can also serve as secondary coactivators for NRs and act synergistically with other family members (Koh et al. 2000). The binding o f CARM1 to AD2 o f p i 60 coactivators, like the binding o f CBP/p300 to ADI o f p i60 coactivators, might play an important role in nuclear receptor signaling. Therefore, p i 60 coactivators have at least two different functional pathways through ADI and AD2 which mediate NR AF-l and AF-2 functions. 1.2 p53 1.2.1 The functions and regulation of p53 The cellular tumor suppressor p53 exerts its tumor suppression functions largely by acting as a sequence-specific transcription factor (May and May 1999). In response to a variety of stimuli, such as DNA damage and expression o f cellular or viral oncoproteins, p53 accumulates in the nucleus of the cells and binds to and activates specific DNA sequences o f its target genes that can result in eithef cell cycle arrest at G1 or apoptosis. The genes activated by p53 include p21 (also called WAF1 or Cipl)(cyclin- dependent kinase inhibitor), cyclin G, GADD45, Mdm2, Baxl (apoptosis inducer), and IGF-BP3 (insulin-like growth factor binding protein 3). These gene products are implicated in the functions of p53 in regulation o f cell cycle progression, DNA replication, and apoptosis (Levine 1997). The inhibition o f DNA replication in cells 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. entering the S phase by p53 also could severely hinder the replication o f small DNA tumor viruses. Thus, it is not surprising that a number o f viral oncoproteins, such as simian virus 40 (SV40) large T antigen, the adenovirus (Ad) E1B 55-kDa protein, the human papillomavirus (HPV) E6, bind to and repress the biological functions o f p53 (Chang et al. 1979; Samow et al. 1982; Wemess et al. 1990). Furthermore, binding and inactivation o f p53 is required for the DNA tumor viruses to induce cellular transformation. SV40 large T antigen binds to the sequence-specific DNA-binding domain o f p53. This interaction interferes with sequence-specific DNA binding o f p53 and therefore inhibits p53-mediated transcriptional transactivation (Bargonetti et al. 1992). The E6 proteins o f highly oncogenic HPV type 16 and 18 associate with p53 and target it for ubiquitination and subsequent degradation, interfere with the DNA binding ability o f p53, and compete for coactivators o f p53 activation (Scheffner et al. 1990; Scheffner et al. 1993; Thomas et al. 1996; Patel et al. 1999; Zimmermann et al. 1999). There are several possible pathways for E1B 55kDa oncoprotein to inhibit the p53- mediated transcriptional transactivation functions. First, the E lB 55-kDa protein repression domain may be tethered to the transcription machinery through its interaction with DNA-bound p53 (Yew et al. 1994). Second, E lB 55-kDa protein might recruit histone deacetylases (HDACs) to the complex o f ElB and DNA-bound p53 in a manner similar to that o f a number o f known transcription repressors. Third, the E lB 55-kDa oncoprotein might inhibit post-translational modifications o f p53, as covalent modifications o f p53 such as phosphorylation and acetylation play important roles in activating p53 (Gu and Roeder 1997; Liu et al. 2000). In contrast to the p53-mediated 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transactivation, the p53-mediated transrepression does not involve an interaction between p53 and DNA, such as the c-fos, c-jun, P-actin, hsc-70 and IL-6 genes (May and May 1999). The possible repression by p53 may be the result o f an association between p53 and the cellular TATA-binding protein (TBP) or HDACs mediated by interaction with mSin3a (Farmer et al. 1996; Murphy et al. 1999). 1.2.2 The new family members o f p53 Since p53 has the power to either target a cell for death or allow it to survive, it must be under rigorous and complex control. p53 contains an N-terminal transactivation domain, a core DNA-binding domain in the central region, and a C-terminal multifunctional regulatory domain. Mutations o f p53 that affect DNA binding and functional inhibition o f the p53 activation domain both appear to play important role(s) in human tumorigenesis. For instance, the product o f the mdm2 oncogene, which binds and is believed to mask the activation domain o f p53, is overexpressed in certain tumors, resulting in the abrogation o f p53 function, accompanied by the proteolytic degradation o f p53, mediated by the ubiquitin pathway (Piette et al. 1997). The functions of p53 also are regulated by post-translational modification via phosphorylation, dephosphorylation or acetylation in highly conserved residues in its N- and C-terminal domains (Meek 1999). Two new families o f proteins (p73 and p63), very similar to p53 and displaying striking homology concerning the transactivation, DNA binding, and oligomerization domains, add a further level o f complexity. The finding o f p53 homologues may explain 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the fact that p53-null mice develop mostly normally. Interestingly, p73 and p63 have long extensions at the C-terminus and form homo-oligomers, not hetero-oligomers with p53. More data on differences between these proteins and p53 are emerging. For example, p73 is not induced by DNA damage and is not targeted by viral oncoproteins such as SV40 T antigen, adenovirus E 1B-55 kDa and HPV E6. The tumor suppressor function o f p73 and p63, to date, are unclear because neither p73 nor p63 appears to be frequently mutated in human cancer (Steegenga et al. 1999; Marin and Kaelin 2000). 1.3 The cross-talk between nuclear receptors and p53 NRs and p53 are two different types o f sequence-specific transcription factors which both respond to their specific extracellular stimuli. When NRs bind to their specific ligands (agonist or antagonist), the complex associates with components (coactivator or corepressor) responsible for turning on or turning off their target genes under the specific stimulus. The ability o f p53 to induce arrest within the G1 phase o f the cell cycle in response to DNA damage, such as ionizing radiation, UV and chemicals, is brought about by p53 stimulating transcription o f the gene for the cyclin-dependent kinase inhibitory protein p21. Another way in which p53 activation can result in the removal o f damaged cells is through the triggering o f apoptosis via transcriptional induction o f genes that encode pro-apoptotic factors, such as Bax. It is an interesting issue to dissect the relationship between NRs and p53. NRs have cross-talk with many signaling pathways, like NF-kB and AP-1 (McKay and Cidlowski 1999; Webb et al. 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1999). p53 also has cross-talk with the above two pathways and also has been found to interact with and repress the functions o f NRs (Webster and Perkins 1999; Bhat et al. 1997; Yu et al. 1997; Liu et al. 1999). p l6 0 coactivators, which mediate the function o f nuclear receptors, also could serve as coactivators for NF-tcB, AP-1, and p53 (Lee et al. 1999; Ko et al. 2000; Lee et al. 2000). One novel protein Zacl was identified along with p53 in a functional screening system by virtue o f their common ability to induce expression of the type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor gene (Spengler et al. 1997). In this thesis, I report the isolation o f Zacl in connection with the nuclear receptor system by using the GRIP1 C-terminus as the bait for yeast two-hybrid screening. Further, the functional role (as a transcriptional coactivator or repressor) o f Zac 1 in nuclear receptor activity depends on the type o f NR, promoter context, and cellular context (Huang and Stallcup 2000). However, the fact that p53 also is a NR repressor and that Zacl also has functions in cell-cycle arrest and apoptosis suggest that both p53 and Zacl might contain similar regulatory functions. Furthermore, I observed the enhancement o f p53-dependent gene activation in HeLa cells by Zacl through coactivator effects and the disruption of the p53 complex with human papilloma virus protein E 6.18 (Huang et al. Submitted). The linkage between NRs and p53 by Zacl in my work may provide a new direction for studying the functional relationship between these two sequence-specific transcription factors. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2: Molecular Cloning of a Mouse Splicing Variant of Zacl (mZaclb) cDNA. Introduction Transcriptional coactivators are components in a signaling pathway that emanates from DNA-bound transcriptional activator proteins and results in local modification of chromatin structure and recruitment o f a transcription initiation complex to the promoter o f a specific gene (Struhl 1998; Mizzen and Allis 1998; Cheung et al. 2000; Strahl and Allis 2000). NRs bind pl60 coactivators through two different interaction domains. The NR AF-2 domain undergoes conformational changes after binding to its agonist and recruits the p i 60 coactivator by binding the p i 60 coactivator’s NID (Feng et al. 1998). The AF-1 activation functions o f some NRs bind the C-terminal region of p i 60 coactivators (Onate et al. 1998; Webb et al. 1998; Tremblay et al. 1999; Kobayashi et al. 2000; Ma et al. 1999; Bevan et al. 1999; Oberste-Berghaus et al. 2000; Fuse et al. 2000). The p i 60 coactivators receive the activating signal through direct contact with DNA- bound NRs and transmit the signal onward through activation domains ADI and AD2. ADI binds CBP or p300, which serve as secondary coactivators, at least in part by acetylating histones or other proteins, or both, in the transcription initiation complex (Yao et al. 1996; Voegel et al. 1998). The AD2 domain o f p i60 coactivators propagates its activating signal at least partly by binding CA RM 1. The secondary coactivator function 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o f CARM1 correlated with the abilities of CARMl to bind the C-terminal region of GRIP I and methylate histones (Chen et al. 1999). However, the mechanism o f downstream signaling by AD2 was unclear three years ago. Therefore, our laboratory tried to identify potential proteins that physically and functionally interact with this domain. In this chapter, I will discuss the identification of proteins that can interact with the C-terminal region of GRIP1 (amino acids 1121-1462) by using the yeast two-hybrid system to screen a mouse 17-day embryo library. And I will further discuss the isolation o f a splicing variant o f Zacl (m Zaclb) and the sequence homology between m Zaclb and other related proteins. Materials and Methods Construction of plasmids: Yeast expression vector for the Gal4 DBD fused to the GPIP1 C terminus (amino acids 1121 to 1462), pGBT9.GRIPlc, was made by inserting a PCR amplified cDNA fragment encoding mouse GRIP 11121-1462 (kindly provided by Dr. XF Ding) into Smal/Sall sites in pGBT9. Isolation of m Zaclb cDNA clones by yeast two-hybrid system: The Matchmaker Two-Hybrid System Kit (Clontech) was used to screen a mouse 17-day embryo cDNA Matchmaker library (Clontech), by following the manufacture’s protocol. 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Construction of full-length of m Zaclb by PCR method: The full-length coding region o f mZaclb was synthesized by PCR, using the same mouse embryo library as a template, a 5’ sense primer (5’-TTGAATTC ATGGCTCCATTCCGCTGTC-3 ’ [underlined translation start codon]) representing the 5’ end o f the mZacla-coding sequence (GenBank accession number X95503 and X95504), and a 3’ antisense primer (5’-TTCTCGAGTTATCTAAATGCGTGATGG-3’ [underlined translation stop codon]) representing the 3’ end o f the coding region from the partial m Zaclb clones isolated from my two-hybrid screening work. Results 2.1 Isolation o f proteins which can interact with the C-terminus o f GRIP1 by yeast two- hybrid system. In our laboratory Dr. H Hong and Dr. D Chen used pGBT9.GRIPln2i.i462 (encoding Gal4DBD-GRIPl 1121- 1462) as bait to screen a mouse 17-day embryo library by yeast two-hybrid system. In this work, Dr. Chen isolated one novel protein, CARM1, which functions as a secondary coactivator through its association with C-terminal region o f p i 60 coactivators (Chen et al. 1999). Then, I used their residual positive clones to identify other candidates. 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In my isolation work, I extracted five different plasmid DNAs (Table 2-1) from a total o f 33 positive clones and then identified their sizes and sequences by restriction enzyme digestion and sequence analysis. Three non-identical positive clones with partially overlapping sequences (clone #24, 27 and 28), representing a total o f 3,343 unique basepairs (bps), were found to be almost identical to the sequence reported in GenBank under accession number X95503 and X95504, coding for m Zacl. Clone #2 had around 1,800 bps and shared 85% homology with reported mouse a-actinin 2 (GenBank under accession number AF248643). The total length o f Clone #26 was around 2,600 bps and shared 80% homology with human KIAA0099 gene (GenBank under accession number D43951). Table 2-1 The Analysts o f Positive Clones Interacting with the C-terminal region of GRIP1 in the Yeast Two-Hybrid Screening System. Colony Number Size (b p )a Candidate Gene Homology (%) to published sequence 2 1800 a-actinin 2 85 24 3000 Zacl .£ > o o 26 2600 KIAA0099 80 27 1800 Zacl 100 b 28 3000 Zacl o o cr a value is approximate, not exact. b an extra 33 bps in these three clones, compared with the previously published sequence in the GenBank (X95503 and X95504). 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2 Identification o f one 11 amino-acid insert in the m Zacl a. Three non-identical positive clones with partially overlapping sequences had two exceptions to the reported mZacl cDNA sequence. These three overlapping sequences extended the 3’ untranslated region o f the previously published sequence (GenBank under accession number X95504) by 50 nucleotides and also contained a 33-bp insert, encoding for amino acids PQMQLQPLQLQ, located after codon 567 o f the previously reported m Zacl sequences (GenBank under accession number X95503 and X90054). The insert was found in all three of the nonidentical, overlapping clones that I isolated. The 33-nucleotide insert (ccccagatgcagc/gcagccacfgcagctgcag) also could be re-checked by PstI restriction enzyme digestion because this insertion created three unique PstI recognition sequences (ctgcag) in the Zac 1 cDNA sequence. 2.3 Construction o f the full-length m Zaclb cDNA by the PCR method. Since the combined 3,343-nucleotide sequence o f my three clones lacked a complete 5’ coding region, I employed PCR to isolate the missing coding sequences from the same mouse 17-day embryo cDNA library. First, I got an additional PCR product representing the 5 ’ untranslated and 5’ coding region o f mZacl by using an upstream primer representing the published mZacl sequence (GenBank under accession number X95503) beginning at nucleotide -248 (relative to the translation start codon) and a downstream primer beginning at nucleotide +300. The sequence of the proximal 248 bps 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o f the 5’ untranslated region from mouse 17-day embryo cDNA library was identical to that of m Zacl, suggesting that the two transcripts come from the same promoter. Based on the same promoter and start coding region, then, I designed the upstream primer from the 5’ end of the previously reported mZacl start coding region and the downstream primer representing the 3’ end o f the coding region of my newly isolated clones. The PCR product contained a 704-codon open reading frame that was identical to that o f the previously reported mZacl sequence, except for the 11-codon insertion in my clones. In this screening work, I finally identified one 3,975-bp mZacl cDNA which contained 248- bp 5’ untranslated region, 2,115-bp translated region, and 1,612 bps 3’ untranslated region (Fig. 2-1). I named this new mZacl variant as m Zaclb, and I referred to the original iso form as mZacl a. The sequence for m Zaclb has been deposited in GenBank under accession number AJF147785. In Summary, m Zaclb differs from m Zacla by one 33-bp insert in the open reading frame and one extra 50-bp sequence in the 3’ untranslated region. 2.4 The sequence comparison among mZaclb, rLotl and hZacl. Before the study o f possible functional roles of m Zaclb in NR transcriptional activations, I used the National institutes of Health BLAST program (Website: www.ncbi.nlm.nih.gov/BLAST/) to analyze the homologous m Zaclb proteins from GenBank database. The analysis showed rat Loti (Lost on transformation, rLotl; GenBank under accession number U72620) and human Zacl/Lotl (GenBank under 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-1 DNA and protein sequences o f m Z aclb The 3,975 nucleotides o f m Zaclb cDNA and the predicted 704 amino-acid protein sequence are shown: 248 bps of 5 ’- untranslated region (italic small letter); 2,115 bps o f translated region (Capital letter); and a coding region for 704 amino-acid protein, including the 11 amino-acid (33-bp) insert shown in bold capital letters. The 1,612 bps o f 3 ’-untranslated region was in small letters and 50 bps extended was shown in bold small letter. 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (248 bp) tgtctctt -241 ctcacaggtt tgagtcttca gacttctaca gaactccata aCatctgcct cacagctggc -181 tttcctgctc tcacagaaga tacccagcta ttgtgctctg gatctctcct ggctgctagg -121 ctgtagcgct gcctttctgg agtcaggctg Cagtgactcc ccaccttctt tctgtctggg -61 cttaaatggc acagcagttc cCcagcacat ctgaagaaga aagtgtgaga accaaaggcc -1 MSGCTCCATTCCGCTGTCAAAAATGTGGCAAGTCCTTCGTCACCCTGGAGAAGTTCACC 6 0 M A P F R C Q K C G K S F V T L E K F T 2 0 ATTCACAATTATTCCCACTCCAGGGAGCGCCCATTCAAGTGCTCGAAGGCTGAGTGTGGC 1 2 0 I H N Y S H S R E R P F K C S K A E C G 4 0 AAAGCCTTCGTCTCCAAGTATAAGCTGATGAGACACATGGCCACACACTCGCCACAGAAG 1 8 0 K A F V S K Y K L M R H M A T H S P Q K 6 0 ATTCACCAGTGTACTCACTGTGAGAAGACATTCAACCGGAAGGACCACCTGAAGAACCAC 2 4 0 I H Q C T H C E K T F N R K D H L K N H 8 0 CTCCAGACCCACGATCCCAACAAGATCTCCTACGCGTGTGACGATTGCGGCAAGAAGTAC 3 0 0 L Q T H D P N K I S Y A C D D C G K K Y 1 0 0 CACACCATGCTGGGCTACAAGAGGCACCTGGCCCTGCACTCGGCGAGCAATGGCGATCTC 3 6 0 H T M L G Y K R H L A L H S A S N G D L 1 2 0 ACCTGTGGGGTGTGCACCCTGGAGCTGGGGAGCACCGAGGTCCTGCTGGACCACCTCAAG 4 2 0 TCGVCTLEL GST EV LLD HL K 1 4 0 TCTCACGCGGAAGAAAAGGCCAACCAGGCACCCAGGGAGAAGAAATACCAGTGCGACCAC 4 8 0 S H A E E K A N Q A P R E K K Y Q C D H 1 6 0 TGTGATAGATGCTTCTACACCCGGAAAGATGTGCGTCGCCACCTGGTGGTCCACACAGGA 5 4 0 C D R C F Y T R K D V R R H L V V H T G 1 8 0 TGCAAGGACTTCCTGTGTCAGTTCTGTGCCCAGAGATTTGGGCGCAAAGACCACCTCACT 6 0 0 C K D F L C Q F C A Q R F G R K D H L T 2 0 0 CGTCACACCAAGAAGACCCACTCCCAGGAGCTGATGCAAGAGAATATGCAGGCAGGAGAT 6 6 0 R H T K K T H S Q E L M Q E N M Q A G D 2 2 0 TACCAGAGCAATTTCCAACTCATTGCGCCTTCAACTTCGTTCCAGATAAAGGTTGATCCC 7 2 0 Y Q S N F Q L I A P S T S F Q I K V D P 2 4 0 ATGCCTCCTTTCCAGCTAGGAGCGGCTCCCGAGAACGGGCTTGATGGTGGCTTGCCACCC 7 8 0 MP P FQ L G A A PE N G L D G G L P P 2 6 0 GAGGTTCATGGTCTAGTGCTTGCTGCCCCAGAAGAAGCTCCCCAACCCATGCCGCCCTTG 84 0 E V H GL V L A A PE E A PQ PM PP L 2 8 0 GAGCCTTTGGAGCCTTTGGAGCCTTTGGAGCCTTTGGAGCCGATGCAGTCTTTGGAGCCT 9 0 0 E P L E P L E P L E P L E P M Q S L E P 3 0 0 TTGCAGCCTTTGGAGCCGATGCAGCCTTTGGAGCCAATGCAGCCTTTGGAGCCGATGCAG 9 6 0 L Q PL E PM Q P L E P M Q P L E P M Q 3 2 0 CCTTTAGAGCCTTTGGAGCCTCTGGAGCCGATGCAGCCTTTGGAGCCGATGCAGCCTTTG 1 0 2 0 P L EP L EP LE P MQ P LE P M QP L 340 (To be continued) 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Continued) GAGCCTATGCAGCCAATGCTGCCAATGCAGCCAATGCAGCCAATGCAGCCAATGCAGCCA 1 0 8 0 E P M Q P M L P M Q P M Q P M Q P M Q P 3 6 0 ATGCTGCCAATGCAGCCAATGCTGCCAATGCAGCCAATGCAGCCAATGCAGCCAATGCTG 1 1 4 0 M L P M Q P M L P M Q P M Q P M Q P M L 3 8 0 CCAATGCCAGAGCCGTCTTTCACTCTGCACCCTGGCGTAGTTCCCACCTCTCCTCCCCCA 1 2 00 P M P E P S F T L H P G V V P T S P P P 4 0 0 ATTATTCTTGAGGAGCATAAGTATAATCCTGTTCCTACCTCATATGCCCCATTTGTAGGC 1 2 6 0 I I L Q E H K Y N P V P T S Y A P F V G 4 2 0 ATGCCCGTCAAAGCAGATGGCAAGGCCTTTTGCAACGTGGGTTTCTTTGAGGAATTTCCT 1 3 2 0 M P V K A D G K A F C N V G F F E E F P 4 4 0 CTGCAAGAGCCTCAGGCGCCTCTCAAGTTCAACCCATGTTTTGAGATGCCTATGGAGGGG 1 3 8 0 L Q E P Q A P L K F N P C F E M P M E G 4 6 0 TTTGGGAAAGTCACCCTGTCCAAAGAGCTGCTGGTAGATGCTGTGAATATAGCCATTCCT 1 4 4 0 F G K V T L S K E L L V D A V N I A I P 4 8 0 GCCTCTCTGGAGATTTCCTCCCTATTGGGGTTTTGGCAGCTCCCCCCTCCTACTCCCCAG 1 5 0 0 AS LEI SS LLGFWQLPP PTPQ 5 0 0 AATGGCTTTGTGAATAGCACCATCCCTGTGGGGCCTGGGGAGCCACTGCCCCATAGGATA 1 5 6 0 NGFV NSTIPV GPG EPLPHR I 5 2 0 ACCTGTCTGGCGCAGCAGCAGCCACCGCCACTGCCGCCGCCACCACCGCTGCCACTGCCA 1 6 2 0 TC LAQ QQ PPP LPP PPP LP LP 5 4 0 CAGCCACTGCCAGTGCCACAGCCACTACCACAGCCACAGATGCAGCCACAGTTTCAGTTG 1 6 8 0 Q P L P V P Q P L P Q P Q M Q P Q F Q L 5 6 0 CAGATCCAGCCCCAGATGCAGCCCCAGATGCAGCTGCAGCCACTGCAGCTGCAGCTACCA 1 7 4 0 Q I Q P Q M Q P Q M Q L Q P L Q L Q L P 5 8 0 CAGCTGCTGCCGCAACTGCAACCTCAGCAGCAGCCTGATCCTGAGCCAGAGCCAGAGCCA 1 8 0 0 Q L L P Q L Q P Q Q Q P D P E P E P E P 6 0 0 GAGCCAGAGCCAGAGCCAGAGCCAGAGCCGGAACCGGAACCGGAGCCAGAGCCAGAGCCA 1 8 6 0 E P E P E P E P E P E P E P E P E P E P 6 2 0 GAACCAGAGCCAGAGGAAGAACAGGAAGAGGCAGAAGAAGAGGCAGAGGAAGGAGCAGAG 1 9 2 0 E P E P E E E Q E E A E E E A E E G A E 6 4 0 GAAGGAGCAGAACCAGAGGCACAGGCAGAAGAAGAGGAAGAGGAAGAGGAAGCGGAAGAG 1 9 8 0 E G A E P E A Q A E E E E E E E E A E E 6 6 0 CCACAGCCAGAAGAAGCCCAAATAGCAGTGAGTGCTGTGAATCTGGGCCAGCCCCCCCTA 2 0 4 0 P Q P E E A Q I A V S A V N L G Q P P L 6 8 0 CCCCCAACTCCCCATATTTTCACAGCTGGCTCCAACACTGCTATCCTGCCCCATTTCCAT 2 1 0 0 P P T P H I F T A G S N T A I L P H F H 700 (To be continued) 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Continued) CACGCATTTAGATAAa11gg tttttaagag ggtgcttctc ttgtgggaga tgttttaaac 2160 H A F R - 704 atcagttaca gtttgaggag aagcattgga aaacaggaat ggggttttag cttatttgtc 2220 ataagtagct tgagaaaaag aattctctaa ctgcatgcgt tgtgccaata tataccctta 2280 gtattcatgc ttcctaccaa atttagtgag cgtgtgtgca ttctgtaatc aaactgcaaa 2340 tattatcata ttatcctatt attacccttg tattattacc ctcatattat taccctcata 2400 ttatcctcat tatcttataa tcacgtgatt acgtgataag atccaaaaca tgagctgcta 2460 ttttgtaaat atcgtgttga gtgtaagctg ttgtagtgat gttagctatg taactgtgtg 2520 tagcctagga aggggatgat ggtaaagttt ggaattctcc aacttggaag gtgtttttaa 2580 gagaagggga taatctttgt atggcgttta taactaggct gtgtgtttct tttcagggac 2640 tcgtctataa gaaatggaca gtttagttcc tcttcttgtt agcttactct gtagtttctt 2700 cttcttgttg cccattgtgt agctttatag agtgtgacgc tattgatgtc tccatttttt 2760 aaagtgaatt taaatgtact gttcaatatt tttcatgtga tgttgttcca atgtgagtta 2820 cgacttcatt tatcttaaag acaaaactgg ttgtcagtca tatctgacag aagaaagaaa 2880 tcactgtgta accaagtcaa gtggccaact aattgaagaa gaatcaatca aagtgtttgt 2940 ggactgtgat actcattatg tttttaacag gaatttaaga aaatgtactg gaatttaaaa 3000 aaagcataag tatattagat aagaattttc tttgcctagc ttaacctact acttaagctg 3060 cttaagttct gaagtattgt ttgtaatcac caatagaaaa gtgtatctgt agatgatcaa 3120 tttaagtcat tgttagtttg tatcccaaga ggattgtgtt ttgcaatgta acctacttgt 3180 aatctccctt gataccttgt taatcgattt tgaagtgtaa acctaacctt tgaagactct 3240 gtatttcctt cttgagactg tatcccccag atatatctcc taacctttga agactctgta 3300 tttcattttt gagactgtat tccccaggat ttatctccta acctttgtag actctgtatt 3360 tcgtttttga gactgtcttt cccagcatat atctcctgac ctttgacaac tctgtatttc 3420 gtttttgaga ctgtattccc cagcatatat ctcctgacct ttgaagaccc tgcattttgt 3480 ttttgagatg gaattcaaca gcatatatct cctaatcttt gatgactctg tattttgttt 3540 ttgagattgt attccccagc atatatctcc taacctttga agactctgta tttcattttt 3600 gagactgtat tccccaacgt gtatctccta acctttgaat aatctccact ttgtttttga 3660 gactgtattc cccagcatat atctcctaac ctttgactct gtactttgtt tttgagagtg 3720 tattccc (3,727 bp) 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. accession number U72621) were highly homologous to m Zaclb with 83% and 69% identical amino acid sequences, respectively. Then, I further analyzed m Zaclb with rLotl and hZacl by the CLUSTAL- Multiple Alignment program (Website: expasy.cbr.nrc.ca/tools/#align). The alignment result showed that rLotl had one major missing region, amino acids 281-383 o f m Zaclb; hZacl had two major missing regions, amino acids 281-383 and 527-667 o f m Zaclb (Fig. 2-2). Thus, these three proteins in the Zacl family all contained seven copies o f C2H2 type zinc-fingers in the N-terminus, one proline-rich region located at amino acids 383-527 o f m Zaclb and one at the far C-terminus (amino acids 668-704) (Fig. 2-3). 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-2 Amino acid sequence alignm ent of m Z acl, rL o tl and h Z acl/h L o tl. The CLUSTAL-Multiple Alignment program (Website: expasy.cbr.nrc.ca/tooIs/#align) aligned the complete amino acid sequences o f m Zaclb, rLotl and hZacl/hLotl. Sequence alignment is shown with amino acid identity (*), highly conservative substitution (:) and conservative substitution (.) indicated in the bottom of three sequences. Gaps introduced between amino acids for optimal alignment are indicated (-) within the sequences. 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z aclb r L o t l h Z a c l m Z a clb r L o t l h Z a c l m Z aclb r L o t l h Z a c l MAPFRCQKCGKSFVTLEKFTIHNYSHSRERPFKCSKAECGKAFVSKYKLMRHMATHSPQK 60 MAPFRCQKCGKSFLTLEKFTIHNYSHTRERPFKCSKTECGKAFVSKYKLMRHMATHSPQK 60 MATFPCQLCGKTFLTLEKFTIHNYSHSRERPYKCVQPDCGKAFVSRYKLMRHMATHSPQK 60 IHQCTHCEKTFNRKDHLKNHLQTHDPNKISYACDDCGKKYHTMLGYKRHLALHSASNGDL 120 THQCTHCEKTFNRKDHLKNHLQTHDPNKMIYACEDCGKKYHTMLGYKRHMALHSASSGDL 120 SHQCAHCEKTFNRKDHLKNHFQTHDPNKMAFGCEECGKKYNTMLGYKRHLALHAASSGDL 120 TC'GVCTLELGSTEVLLDHLKSHAEEKANQAPREKKYQCDHCDRCFYTRKDVRRHLWHTG 180 TCGVCTLELGSTEVLLDHLKSHAEEKAHHAPREKKHQCDHCERCFYTRKDVRRHLWHTG 1 8 0 TCGVCALELGSTEVLLDHLKAHAEEKPPSGTKEKKHQCDHCERCFYTRKDVRRHLWHTG 1 8 0 CKDFLCQFCAQRFGRKDHLTRHTKKTHSQELMQENMQAGDYQSNFQLIAPSTSFQIKVDP 24 0 CKDFLCQFCAQRFGRKDHLTRHTKKTHSQELMQESLQAGEYQGGYQ PIAP - - P FQIKAD P 23 8 CKD FLCQFCAQRFGRKDHLTRHTKKTHSQELMKESLQTGDLLSTFHTIS P - - S FQLKAAA 23 8 MPPFQLGAAPENGLDGGLPPEVHGLVLAAPEEAPQPMPPLEPLEPLEPLEPLEPMQSLEP 3 0 0 MPPFQLEMPPESGLDGGLPPEIHGLVLASPEEVPQ--------------------------------- 2 7 3 LPPFPLGASAQNGLASSLPAEVHSLTLSPPEQAAQ- - ......................... 2 7 3 LQPLEPMQPLEPMQPLEPMQPLEPLEPLEPMQPLEPMQPLEPMQPMLPMQPMQPMQPMQP 3 60 ........................................... PMLSMPPMQ................... 2 8 2 ..................................................... - ................... PMQ-------------- 2 7 6 MLPMQPMLPMQPMQPMQPMLPMPEPSFTLHPGWPTSPPPIILQEHKYNPVPTSYAPFVG 4 2 0 .......................................... PMPEQPFTLHPGWPSSPPPIILQEHKYS PVPTSFAPFVS 3 2 2 .......................... ...........................PLPESLASLHPSVSPGSPPPP-LPNHKYNTTSTSYSPLAS 3 1 5 *'* * :***.* * * * * * * MPVKADGKAFCNVGFFEEFPLQEPQAPLKFNPCFEMPMEGFGKVTLSKELLVDAVNIAIP 4 80 MPMKADLKGFCNMGLFEEFPLQECQSPVKFSQCFEMAKEGFGKVTLPKELLVDAVNIAIP 3 8 2 LPLKADTKGFCNISLFEDLPLQEPQSPQKLNPGFDLAKGNAGKVNLPKELPADAVNLTIP 3 7 5 ASLEISSLLGFWQLPPPTPQNGFVNSTIPVGPGEPLPHRXTCLAQQQPPPLPPPPPLPLP 54 0 GSLEISSLLGFWQLPPPPPQNGFMNGTIPVGAGEPLPHRITCLAQQQPPPLLPPPP 4 3 8 ASLDLSPLLGFWQLPPPATQNTFGNSTLALGPGESLPHRLSCLGQQQQ...................... 4 2 3 QPLPVPQPLPQPQMQPQFQLQIQPQMQPQMQLQPLQLQLPQLLPQLQPQQQPDPEPEPEP 60 0 - PLPLPEPLPQPQLPPQFQLQLQPQPQ ----------- -MQPQMQLQPLQLQLPQLLPQL 4 8 5 EPEPEPEPEPEPEPEPEPEPEPEPEEEQEEAEEEAEEGAEEGAEPEAQAEEEEEEEEAEE 6 6 0 QPEPEPEPEPEEEEEEEEEIE EEEEIEEEEEAEPEAEE--EEEAEDEEEAEEEE-EE 53 9 ..................... EPP........................ - ................. 4 2 6 PQPEEAQIAVSAVNLGQPPLPPTPHIFTAGSNTAILPHFHHAFR 704 PQPEEAQIAMSAVNMGQPPLPPTPHVFTAGTNTAILPHFHHAFR 583 - - ............. LAMGTVSLGQLPLPPIPHVFSAGTGSAILPHFHHAFR 4 6 3 . ;*********** Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 2-3 Domains of mouse Z acl. Sequence motifs in mouse Zacl are indicated. The 11 amino acids at the top are found in m Zaclb but not mZacl a. The brackets at the bottom indicate regions missing in the homologous human and rat proteins, hZacl and rLotl. Numbers are amino acids numbers o f mZacla. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mZacl a Insert found in mZaclb: PQMQLQPLQLQ \ / . . . \ / . . . . 208 280 383 527 \/5S 4 656 693 7 C2H2 zinc fingers PLE repeats PMQ repeats P-rich (P) (Q)(L) PE&E repeats Missing in Missing in hZacl rLotl and hZacl Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion In my isolation work, I identified m Zaclb with the same bait and cDNA library used to identify CARM1 and in the same yeast two-hybrid screen (Chen et al. 1999). It is attractive to speculate whether the functional role of m Zaclb is the same as CARM1, like a secondary coactivator, in the NR functions. Intriguingly, the first group o f researchers isolated Zacl by using an expression-cloning method to screen simultaneously for different receptors positively coupled to adenylate cyclase (Spengler et al. 1997). Their work had proven that Z acl, like p53, was involved in apoptosis and cell cycle arrest. Therefore, it is also an interesting topic to dissect the functional relationship between p53 and Zacl in NR-dependent, p53-dependent, or other transcriptional activations. Zacl has seven-(C 2H2)-type zinc fingers at the N-terminus, and its central region (amino acids 275- 380) has 34 PLE, PMQ, or PML repeats. In its C-terminus, the region is rich in P, Q, and E. Previous work with hZacl/hLotl proved that hZacl still has the functions in apoptosis and cell cycle arrest (Varrault et al. 1998). A comparison o f the sequences between mouse and human species indicated that there are two missing regions in the hZac 1: 34 PLE, PMQ, or PML repeats in the central region and most of the P, Q, and E rich region in the C-terminus o f m Z acl. The studies of hZacl suggested that the two missing regions were not important domains for Z a cl’s functions in apoptosis and cell cycle arrest. Further, the study o f hZacl showed that it had transactivation activity and DNA-binding ability (Varrault et al. 1998; Kas et al. 1998). Thus, Zacl might serve as a sequence- 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. specific transcription factor. Unlike m Zacl, which is highly expressed only in the pituitary gland, hZac I was widely expressed. rLotl first was isolated in the rat ovarian surface epithelial cell lines by the differential display technique; decrease or loss o f rLotl expression was observed in five o f eight independently transformed cell lines (Abdollahi et al. 1997). Compared to m Zacl, rLotl has one missing region in the m Zacl central region (amino acids 275-380). The gene showed a limited distribution o f expression in normal rat tissues, including ovary, pancreas, testes, and uterus. Later, the group isolated the hLotl (also called hZacl), which had ubiquitous expression, and its chromosome localization was mapped at 6q25 (Abdollahi et al. 1997; Varrault et al. 1998). This chromosomal region has also been implicated in the genesis of breast, kidney, and pleural mesothelial cancers. Interestingly, hZacl /hLotl also was identified and was called PLAGLl (pleomorphic adenomas o f the salivary gland like I ) because it was highly homologous to PLA G1 in their amino-terminal zinc finger domain. PLAG1, a novel developmentally regulated C2H2 zinc finger gene at chromosomal location 8ql2 is a main target in pleomorphic adenomas o f the salivary gland (Kas et al. 1998). All studies o f Z acl, Loti, and PLAGLl support their functions to be transcription factors (Varrault et al. 1998; Kas et al. 1998). Recently, it was shown that type I PACAP receptor expression could be induced by Zac I and p53 (Hoffmann et al. 1998). The work provided the model for transactivation o f the cAMP-responsive receptor plasmid by Zacl and p53 through induction o f the type I PACAP receptor gene. Zacl may regulate type I PACAP gene expression by its DNA- 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. binding ability, located in the N-terminal seven zinc finger motifs. Thus, Zac 1/Lot 1 family functions in apoptosis and cell cycle arrest, the induction o f type I PACAP receptor expression, and their loss in some tumor samples suggests that they may be tumor suppressor proteins. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3: Mouse Zacl, a Transcriptional Coactivator and Repressor for Nuclear Receptors. Introduction NR coactivator proteins, recruited by ligand-bound NRs or their activation functions, modify local chromatin structure by catalyzing covalent histone modifications and direct assembly and or stabilization of the transcription pre-initiation complex (Glass and Rosenfeld 2000). A growing list o f putative NR coactivators has been identified by their abilities to bind and/or enhance the activity o f NRs (Freedman 1999; McKenna et al. 1999; Glass and Rosenfeld 2000). Three major protein families may function in a coactivator complex associated with the DNA-bound NRs: the p i60 coactivators; CBP and p300; and p/CAF (Onate et al. 1995; Hong et al. 1996; Voegel et al. 1996; Torchia et al 1997; Li et al. 1997; Chen et al. 1997; Anzick et al. 1997; Takeshita et al. 1997; Chakravarti et al. 1996; Blanco et al. 1998). The p i60 coactivators include SRC-1, GRIP 1, and p/CIP. They bind directly to the HBD of NRs by their LXXLL motifs and enhance NR AF-2 activity (Heery et al. 1997; Ding et al. 1998; Mclnemey et al. 1998). The p i 60 coactivators also bind some NR (PR, ER, TR, MR and AR) AF-1 regions and thereby enhance AF-1 function (Onate et al. 1998; Webb et al. 1998; Tremblay et al. 1999; Kobayashi et al. 2000; Ma et al. 1999; Bevan et al. 1999; Oberste-Berghaus et al. 2000; Fuse et al. 2000). CBP, p300, and p/CAF have been shown to bind both directly to the NR and to the p i60 coactivators (Chen et al. 1997). The HAT activity o f these 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. coactivators can acetylate histones, transcriptional activators, and components of the transcription initiation complex, leading to the proposal that CBP and p300 serve as platforms to integrate the effects o f multiple signaling pathways on many different transcriptional activator proteins (Kwok et al. 1994; Kamei et al. 1996; Swope et al. 1996). The p i60 coactivators have two activation domains, ADI and AD2, which transmit the activating signal from the DNA-bound NR to the chromatin and/or transcription machinery. The function o f ADI is due to its ability to bind CBP or p300 (Yao et al. 1996; Voegel et al. 1998). AD2 functions by an unknown mechanism that is independent of CBP and p300 (Voegel et al. 1998; Ma et al. 1999). Recently, CARM1 was found to play an important role in the AD2 function by direct interaction with AD2 and its intrinsic arginine methyltransferase activity which could methylate histones (Chen et al. 1999). In work described in the previous chapter, I screened a mouse cDNA library to identify proteins that bind to the C-terminal region of GRIP I and further identified a new GRIP 1-binding protein which is a variant o f a previously identified zinc finger transcription factor, Z acl. I named the new isoform as m Zaclb. In this chapter, I will further show that m Zaclb could serve as a primary coactivator or repressor of NR activity, and its functions depend on the type o f nuclear receptor, promoter context, and cell context. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods Construction of plasmids: The complete mZac lb-coding region (amino acids 1- 704) was cloned into the EcoRI and Xhol sites o f vector pSG5.HA (Chen et al. 1999), which has promoters for expression in vitro and in mammalian cells and provides an N- terminal hemagglutinin tag for the expressed protein. The pSG5.HA vector, coding for full-length G R IP1, was described previously (Chen et al. 1999). Vectors encoding the Gal4 DBD fused to various fragments of m Zaclb were constructed by inserting EcoRI- Xhol fragments o f the appropriate PCR-amplified m Zaclb cDNA into the EcoRI and Sail sites o f the pM vector (Clontech). A Gal4 DBD-GRIP1 expression vector was constructed by inserting an EcoRI-Sall fragment encoding GRIP 15-1 4 6 2 into pM. Bacterial expression vectors for glutathione S-transferase (GST) fused to G R IP1 and CBP fragments were constructed by inserting the appropriate PCR fragments into pGEX-4T l (Pharmacia) as follows: GRIP I5-479 into the EcoRI-BamHI sites; GRIP 15.765, CBP2041-2240, and CBP 1 5 9 1 -2 4 4 1 into the EcoRI-XhoI sites; and GRIP 11 3 0 5 -1 4 6 2 into the Smal-Sall sites. A vector encoding GST fused with full-length m Z aclb was constructed by inserting a mZac lb-encoding PCR fragment into the BamHI and Xhol sites o f pGEX-2TK. Cell culture, transient-transfection assays, and immuoblotting: For functional assays HeLa cells were grown in Dulbecco modified Eagle medium-F-12 supplemented with 10% charcoal-dextran-treated fetal bovine serum. CV-1 cells and 1471.1 cells were grown in Dulbecco modified Eagle medium with the same serum. Transient transfections 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and luciferase assays were performed as described previously (Ma et al. 1999) in six-well culture dishes. Total DNA was adjusted to 2 pg by adding the necessary amount o f vector pSG5.HA. Luciferase activity o f the transfected cell extracts is presented as relative light units (RLU), and values are the means and standard deviations for three transfected cultures. Since coactivators influence the expression o f many control vectors that are used to monitor transfection efficiency, internal controls were not used. Instead, reproducibility o f observed effects was determined in multiple independent transfection experiments. Immunoblotting o f transiently transfected COS7 cells was performed as previously described (Ma et al. 1999), using 10% o f the extract from a well o f a six-well culture dish and monoclonal antibodies 3F10 (Roche) against the hemagglutinin epitope and RK5C1 (Santa Cruz Biotechnology) against the GaI4 DBD. Protein-protein interaction assay: Radioactively labeled proteins were translated in vitro, incubated with immobilized GST fusion protein, eluted, and analyzed by gel electrophoresis as previously described (Ma et al. 1999). Radioactive protein in gels were detected in a Phosphorlmager 445SI and quantified by ImageQuaNT software (Molecular Dynamics). Quantitative yeast two-hybrid assays were performed as described previously (Ding et al. 1998). 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results 3.1 Binding o f mZaclb to NR and their coactivators in vitro. In the GST pull-down assay, in vitro synthesized m Zaclb could bind to the C- terminal region of GRIP1 (amino acids 1122-1462, G RIPlc) fused to GST and immobilized on agarose beads (Fig. 3-1 A), which further confirmed the results from the two-hybrid screen (data not shown). Unlike CARM1, m Zaclb bound somewhat more weakly to an N-terminal fragment of GRIP1 containing the basic helix-loop-helix and PAS sequences (GRIP 15-479)- However, m Zaclb failed to bind to GST-fusion proteins representing central regions o f the GRIP1 polypeptide (GRIPI563-1121) which contain the CBP/p300 binding site and the NIDs that efficiently bind NR HBDs. These binding patterns were also similar to my quantitative yeast two-hybrid assays (data not shown). I also tested whether m Zaclb could directly interact with NRs by its one LXXLL m otif (amino acids 579-583) because all p i 60 coactivators interact with hormone- activated NR HBD through the LXXLL motif. A GST-mZaclb fusion protein could bind strongly in vitro to full-length AR, ER, and TR (Fig. 3 -IB). The binding was largely hormone-independent, but was reproducibly enhanced by hormone. In contrast, binding o f the same NRs to a GST-GRIP1 protein containing the NR HBD (AF-2)-binding domain was highly hormone-dependent. GST-m Zaclb also bound to the HBDs o f AR and MR in a hormone-independent manner (Fig. 3-1C), but bound very weakly or not at 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-1. Binding of m Z aclb to NRs and NR coactivators. The proteins indicated at the left o f each panel were translated in vitro and incubated with bead-bound GST fusion proteins (indicated at the top of each panel along with amino acid numbers for protein fragments fused to GST); bound proteins were eluted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. The percentage o f labeled protein bound, as determined by phosphorimager analysis, is shown below each lane. For comparison, the leftmost lane of each panel shows the indicated percentage o f input protein used in the binding reaction. In panel B and C, the following hormones were included where indicated: for AR, lpM DHT; for ER, lpM estradiol; for TR, lfaM T3; and for MR, IpM corticosterone. 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GST-GRIP1 C N cn cn cn so N O TT ^ « ■ « - « I - ~ Js o 5-479 5-765 563-11 o n r*- C N cn o — m m Z a c I b — > m — — — — 0.1 1.2 0.9 0.2 0.4 4.8 2.8 B r-t n n r — . .n .o w ~ , 'C W l C O N E o C O N E C L a l 2 oc a o ' O 3 n . c I S O r- C /3 a C /3 a h- C /3 a h- C /3 o H o n n o n e - - - + - -1 - AR— • ------- — — 0.9 10.5 19.2 1.4 3.2 E R — ► — — — ------ ------- 1.1 15.0 19.8 2.9 12.2 T R — ► — — ■ — 1.7 18.8 39.6 0.6 38.2 kDa -2 0 0 -97.4 - 46 3 C. JC GST i E — C/3 a i H C/3 o E — on O Hormone - - - + + AR AF2 — ► — — 4.7 27.5 32.5 8.3 MR AF2— ►----- ------ ----- ---- 0.6 11.6 13.1 4.8 n D r-'. N O X 5 W N 5? C J C 3 al vO N a £ w E O 3 h“ E “ H a. C/3 C/3 C/3 c a a a AR A F l1 r - v d H C/3 a CL CO U o o m Q. CL c n u mZac lb- 0.2 2.2 10.0 0.7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. all to the AR AF-1 region (Fig. 3 -ID). All o f the previous binding results could be observed in similar patterns with a C-truncated GST-mZaclb (amino acids 1-520) lacking o f the LXXLL motif; therefore, LXXLL was not the major interaction domain for m Zaclb to bind NRs (data not shown). Since LXXLL motifs usually bind in a hormone- dependent manner to NRs, this finding was consistent with the binding o f Zac 1 in the absence o f hormones. GST-mZaclb also could bind to C-terminal fragments o f CBP and p300 (Fig. 3 -IE); this region o f CBP and p300 also binds p i 60 coactivators. Thus, m Zaclb, similar to the binding partners o f p/CAF, could interact with NRs and two different classes o f NR coactivators, pl60 coactivator and CBP/p300 (Chen et al. 1997; Blanco et al. 1998). 3.2 Activation domain o f mZaclb. Since m Zaclb could directly bind NRs and their coactivators, I wanted to test whether m Zaclb had another property expected o f a coactivator, i.e. a putative activation domain. I further constructed many m Zaclb fragments fused with GaI4 DBD to identify which m Zaclb region is responsible for the ability to activate a Gal4-responsive reporter gene in transiently transfected HeLa cells. mZaclbio 3-52o had the maximum activity, and a slightly lower level of activity was observed with a sub-fragment, amino acids 103-380 (Fig. 3-2A). The N-terminal (amino acids 1-102) and C-terminal (amino acids 521-704) regions o f m Zaclb had a negative effect on this activity, since their presence, even in full-length m Zaclb, reduced the activity (e.g. compare fragments 1 and 2, and compare 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-2. T ranscriptional activation domain of Z a c l. (A) Expression vectors (1 pig) for the indicated fragments o f m Zaclb fused to the Gal4 DBD were transiently transfected into HeLa cells along with the GK1 reporter gene (l|ig ), which encodes luciferase and is controlled by Gal4 response elements. Luciferase activities o f the transfected cell extracts were determined. Numbers beside the bars indicate fold activation compared with that of the Gal4 DBD alone. RLU, relative light units. (B) The vectors encoding the Gal4 DBD- Zacl fusion proteins (2pg) listed in panel A were transiently transfected into COS7 cells and the cell extracts were subjected to immunoblot analysis using antibodies against the Gal4 DBD. Lane numbers correspond to the line numbers in panel A. 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A G al4D B D 1 G al4D B D -m Z aclb 1-704 2 G al4D B D -m Z ac 1 b 1-520 3 G al4D B D -m Z aclb 1-380 4 G aI4D B D -m Z aclb 1-220 5 G al4D B D -m Z aclb 103-704 6 G al4D B D -m Z ac 1 b 103-520 7 G al4D B D -m Z aclb 103-380 8 G aI4D B D -m Z aclb 221-520 9 G al4D B D -m Z aclb 521-704 Luciferase Activity (104 RLU) 0 1 2 3 4 - - i ■ . i < 1 lx i "■ 3.1x B 1 2 3 4 5 67 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fragments 3 and 7). This suggests a possible negative regulatory role for the N-terminal (seven zinc-fmger motifs) and C-terminal (negative charge rich region) domains that could be modulated by interaction o f m Zaclb with other proteins or provide sites for post-translational modification. By the immunoblot analyses conducted on extracts from transiently transfected COS7 cells, fusion proteins 4 and 9 were expressed at elevated levels compared with the others, suggesting that the lack o f activity in some fragments was not due to lack o f expression (Fig. 3-2B). Thus, my result was consistent with the notion that Zac 1 may be a transcription factor, as suggested by previous studies (Varrault et al. 1998; Kas et al. 1998) and, further, suggested an activation domain in the central region o f its polypeptide chain. 3.3 Enhancement o f AR function by mZaclb. Based on the findings that m Zaclb contained a putative activation domain and bound NRs and their coactivators, m Zaclb might have coactivator functions for NRs. I used transient transfections in HeLa cells to test the ability o f m Zaclb and GRIP1 to act separately and together as coactivators for AR with a reporter gene controlled by a mouse mammary tumor virus (MMTV) promoter. In this assay, GRIP1 enhanced hormone- activated AR function by 4.5-fold (Fig. 3-3A, sample c), while m Zaclb caused a 48-fold enhancement (sample d). Together GRIP1 and m Zaclb caused a 137-fold enhancement (sample e), and this activity was completely hormone-dependent (sample f). Thus, the presence o f hormone-activated AR was necessary for GRIP1 and m Zaclb to activate the 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-3. Synergistic enhancement o f AR function by mZaclb and GRIP1. (A) HeLa cells were transiently transfected with MMTV-LUC reporter gene (0.5p.g), pSVARo (0.5fig) encoding AR, and either 0.5pg o f pSG5.HA-GRIPl, 0.5ng o f pSG5.HA- m Zaclb, or both. Where indicated, transfected cultures were grown in lOOnM DHT. Luciferase activities o f the transfected cell extracts were determined. Numbers above the bars indicate activity relative to that o f hormone-activated A R with no added coactivators. A synergy ratio (SYN) was calculated as described in the text. The diagram at the top indicates the proposed mechanism o f reporter gene activation: recruitment of m Zaclb to the transcription complex could occur by m Zaclb binding either to AR or to GRIP1. ARE, androgen-responsive elements in the MMTV promoter, TATA, TATA box o f the MMTV promoter; LUC, luciferase-coding region; HA, hemagglutinin; rightward- pointing arrow, transcription start site. (B) HeLa cells were transfected as described above with the indicated amounts o f pSVARo and 0.4|ug of the MMTV-LUC reporter gene. Four micrograms of pSG5.HA-mZaclb and/or 0.4mg o f pSG5.HA-GRIPl was included or not as follows; open circles, no GRIP1 or mZaclb; closed squares, GRIP1; closed triangles, m Zaclb; closed circles, GRIP1 and m Zaclb. Transfected cells were grown with lOOnM DHT. The inset shows the lower two curves on an expanded scale. Synergy ratios calculated as for panel A are shown in parentheses. (C) HeLa cell transfections were performed with the indicated amounts of pSG5.HA-GRIPl and 0.3|ug each o f MMTV-LUC, pSVARo, and pSG5.HA-mZaclb; transfected cells were grown with lOOnM DHT. (D) HeLa cell transfections were performed with the indicated amounts of pSG5.HA-mZaclb and 0.3p.g each o f MMTV-LUC, pSVARo, and pSG5.HA- GRIP1; transfected cells were grown with DHT. RLU, relative light units. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. L uciferase A ctivity (104 R L U ) G> t o 4 ^ ON OO O to C O £ 2° (III C T Q cm o o Luciferase A ctivity a (10 RLU) o o o ♦ K o to o = > " § • ? 5 c > 3 7 3 H Luciferase Activity (10' RLU) > o o X & % % C /) X 1 L r. X s 1 I Luciferase A ctivity (10 RLU) N J 4^ T 3 c/o O y i o O £ *3 R e ra to * reporter gene; that finding is consistent with the role o f a coactivator. GRIP1 and m Zaclb acted synergistically; the effect of the two together was 2.7-fold greater than the sum of their individual effects. This synergy ratio (SYN) was calculated by dividing the extra activity observed when GRIP1 and m Zaclb were added together (e - b, in Fig. 3-3A) by the sum of the extra activities due to GRIP1 alone plus the extra activity due to m Zaclb alone (c + d - 2b, in Fig. 3-3A). Synergy was also observed when m Zaclb was tested with another p i 60 coactivator, SRC-la, and when a C-truncated m Zaclb (amino acids 1- 520) was tested with GRIP1 (data not shown). The result from the C-truncated m Zaclb confirmed again that the LXXLL of m Zaclb was not responsible for NR interaction. The stronger coactivator effect of m Zaclb was not due to a higher level o f expression because m Zaclb and GRIP1 were expressed at similar levels by immunoblot analysis in COS-7 cells (Fig. 3-3A, inset). The higher relative coactivator effect of m Zaclb compared with GRIP1 was maintained when the amount o f AR expression vector varied over an 8-fold range, as was the synergistic effect of the two coactivators (Fig. 3-3B). However, the synergy (Fig. 3-3 B, numbers in parentheses) was more pronounced at lower AR levels; the activity was approximately 7-fold more than additive at 0.1 gg o f AR vector and 2- fold more than additive at 0.8 gg of AR vector. The enhanced reporter gene activity observed with m Zaclb and/or GRIP1 was completely dependent on the presence o f AR. In the presence of m Zaclb, the reporter gene activity was directly proportional to the amount of GRIP1 vector over a 10-fold range of GRIP1 vector amounts (Fig. 3-3C), indicating that the amount of GRIP1 vector used in the synergy studies (0.4 - 0.5 gg, Fig. 3-3A & B) was non-saturating. However, in the presence of GRIP1, 0.2 pg o f m Zaclb 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vector produced an optimum response, and increasing the amount o f m Z aclb vector above this level reduced but did not eliminate the positive coactivator effect due to m Zaclb (Fig. 3-3D). To determine which AR activation function, AF-1 or AF-2, was regulated by m Zaclb, the N-terminal domain o f AR, containing AF-1, and the C-terminal AR domain, containing AF-2, were each fused to Gal4 DBD and expressed in HeLa cells with m Zaclb and/or GR1P1. m Zaclb enhanced AF-1 activity 6-fold (Fig. 3-4A) and AF-2 activity more than 100-fold (Fig. 3-4B). GRIP1 had little, if any, effect on A F-l activity but enhanced AF-2 activity 18-fold. The two coactivators had synergistic effects on AF-l and AF-2, but the synergy was more pronounced with AF-1 (SYN = 4.7) than on AF-2 (SYN = 1.6). Neither GRIP1 nor m Zaclb had any effect on reporter gene activity in the absence o f the AR-Gal4 DBD fusion proteins (data not shown). Thus, although m Zaclb bound AR AF-2 but not AR AF-1 (Figs. 3-1C & D), it enhanced the function o f both AR activation domains. I used varying concentrations o f DHT with full length AR in the transient transfection assays to examine whether m Zaclb and GRIP1 had effects on hormone potency. The synergistic effects o f GRIP 1 and mZac 1 b were observed at sub-saturating as well as saturating concentrations o f DHT (Fig. 3-5A, SYN). However, the coactivators affected the concentration o f DHT required to elicit half-maximal activity (ECso)- In the presence o f GRIP1, the E C 5 0 was 4-10 times lower than in the presence o f m Z aclb alone 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-4. Synergistic enhancem ent o f AR AF-1 and AF-2 function by m Z aclb and G R IP1. HeLa cells were transiently transfected with 0.5pg o f pM -ARAFl (A) or pM- ARAF2 (B) and 0.5pg o f the GK1 reporter gene. Where indicated, 0.5pg o f pSG5.HA- m Zaclb and/or 0.5pg o f pSG5.HA-GRJPl was also included, and cells transfected with pM-ARAF2 were grown with 1 OOnM DHT. SYN, synergy ratio calculated as for Fig. 3- 3A. Numbers above the bars indicate activity relative to that o f AR AF-1 or AR AF-2 in the absence o f added coactivators. RLU, relative light units. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4—» • > • 1 - M 4 -* o < < L > C /3 cd 5 - H ^4-1 ’o H-l pSG5.HA pSG5.HA-GRIPl pSG5.HA-mZaclb + SY N =4.7 27.5x SY N =1.6 196x + + + + Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-5. Relationship of AR activity to DHT concentration in the presence of m Zaclb and/or GRIP1. (A) HeLa cells were transiently transfected with 0.5pg o f the MMTV-LUC reporter gene, 0.5pg of pSVARo, and, where indicated, 0.5pg of pSG5.HA- m Zaclb and/or 0.5ptg of pSG5.HA-GRIPl. Transfected cells were grown with the indicated concentrations of DHT. Synergy ratios (SYN), calculated as for Fig. 3-3A, are shown at the top. CoA, coactivator. (B) The two lower curves from panel A are shown on an expanded scale. (C) The activity from panel A in the absence of DHT is shown. Numbers above the bars indicate activity relative to that observed in the absence of m Zaclb and GRIP1. RLU, relative light units. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A S Y N 0.2 2.2 2.3 2.0 1.8 1.7 • * — » o < < u C/3 C O J — V O £ 2 3 J D J B < 3 e « O S W in « ! 2 O ' ' 3 c 15 12 9 6 3 0 + GRIPl + mZaclb + mZac 1 b + GRIPl No CoA 4 T DHT + GRIPl 3 2 1 No CoA _ 0 No -Log [DHT] (M ) ’> o c~ < 3 % O f iS 2 ^ ,« 5 1 0 pSG5.HA pSG5.HA-GRIPl pSG5.HA-m Zaclb lx 1 + 12.4x 2.2x 3.3x ■ + + 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. or m Zaclb plus GRIPl (Fig. 3-5A & B). GRIPl and m Zaclb also had different effects on the hormone-independent activity of AR. GRIPl enhanced AR function even in the absence o f hormone, whereas m Zaclb had little if any effect by itself and even suppressed the hormone-independent activity caused by GRIPl (Fig. 3-5C). In the absence o f AR, neither GRIPl nor m Zaclb had any effect on the expression o f the reporter gene (data not shown). Thus, in the presence o f G RIPl, m Zaclb altered the effects o f DHT in two ways: m Zaclb suppressed the hormone independent activity o f AR caused by G RIPl; and it increased the EC50 value for DHT and thus had a more dramatic coactivator effect at higher DHT concentrations than at lower DHT concentrations. For example, when the activity o f GRIPl was compared with the activity o f GRIPl plus m Zaclb, m Zaclb caused a 4-fold enhancement at 10'1 1 M DHT, an 8-fold enhancement at 10'1 ° M DHT, and a 30-fold enhancement at 10'9 M and higher concentrations of DHT (calculated from Fig. 3-5A & B). 3.4 Promoter-selective coactivator or repressor effects o f mZaclb fo r ER. Similar coactivator functions of m Zaclb for GR or TR pi were observed in HeLa cells (data not shown). However, the enhancement of GR and TR function by m Zaclb (5 to 12-fold) was less dramatic than the enhancement o f AR function (up to 100-fold). In contrast, when estradiol-activated ERa was tested with a modified MMTV promoter having the endogenous GREs replaced by a single estrogen responsive element (ERE), m Zaclb had no coactivator effect or even slightly repressed the activity by itself (Fig. 3- 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6A). GRIPl alone enhanced ER function by as much as 60-fold (Fig. 3-6A & B), but m Zaclb repressed the GRIPl-enhanced ER activity by up to 10-fold (Fig. 3-6 A-C). The repression occurred at all amounts of GRIPl and m Zaclb expression vectors tested (Fig. 3-6B & C). The amounts of ER expression vector (0.04 - 0.1 jag) used in these experiments were just saturating or below saturating, because 0.1-0.2 pg o f plasmid was determined to be just saturating in the presence o f GRIPl (data not shown). In the absence o f ER, m Zaclb had no effect on the expression o f the reporter gene (data not shown). GRIPl and m Zaclb had a similar pattern of effects on tamoxifen-bound ER a, although the reporter gene activity with tamoxifen was less than 5% that observed with estradiol (data not shown). When a different reporter gene (EREII-LUC[GL45]) with a different promoter (herpes simplex virus thymidine kinase promoter) and two EREs was tested with ER in the same cells, m Zaclb enhanced the activity by about 27-fold (Fig. 3-7A). GRIPl also enhanced activity by 10-fold, but the enhancement caused by m Zaclb and G R IPl together was synergistic. In the absence o f ER, G R IPl and m Zaclb each caused less than 3-fold enhancement of the low basal activity o f this reporter gene (Fig. 3-7B). When similar reporter genes with the same thymidine kinase promoter and either one or two copies o f an ERE from the Xenopus vitellogenin gene were tested with ER, m Zaclb caused a similar enhancement of activity; m Zaclb and GRIPl together had additive or less than additive enhancing effects, i.e. no synergy was observed (data not shown). Thus, in the same cell line m Zaclb acted as a coactivator for ER with some reporter genes and 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-6. Repression by m Zaclb of ER function with the MMTV(ERE) promoter. (A) HeLa cells were transiently transfected with 0.5pg o f MMTV(ERE)-LUC reporter plasmid, 0.04pg o f pHEO (encoding hERa), and, where indicated, 0.5pg o f pSG5.HA- m Zaclb and/or 0.5pg o f pSG5.HA-GRIPl. Transfected cells were grown with lOOnM estradiol, and luciferase activities of the transfected cell extracts were determined. Numbers above the bars indicate activity relative to that o f ER in the absence o f GRIPl and m Zaclb vectors. (B) HeLa cells were transfected with 0.4pg o f MMTV(ERE)-LUC, 0.04mg o f pHEO, the indicated amounts o f pSG5.HA-GRIPl, and, where indicated, 0.4pg o f pSG5.HA-m Zaclb. Cells were grown with lOOnM estradiol. (C) HeLa cells were transfected with 0.4pg o f MMTV(ERE)-LUC, 0.1 pg o f pHEO, 0.4pg o f pSG5.HA- GRIP1, and the indicated amounts of pSG5.HA-mZaclb. Transfected cells were grown with lOOnM estradiol. RLU, relative light units. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Luciferase Activity 0 4 (10 RLU) Luciferase Activity ( j j (10 RLU) O' X 0 Q X I •a X 3 (A w C / 5 0 0 0 l/i t/i t/i X K > > > 3 G N p s o ■ x I T + + Luciferase Activity £)> (105 RLU) + - U ) o V O l/t X l/i o\ X 00 . . . ) — K ) — t- - O v — i U l N > Fig. 3-7. Coactivator effect o f m Zaclb on ER function with the thymidine kinase promoter. HeLa cells were transiently transfected with 0.5 jig o f the EREII-LUC(GL45) reporter gene in the presence (A) or absence (B) or 0.04 jig o f pHEO; where indicated, 0.5 mg o f pSG5.HA-mZaclb and/or 0.5 jig o f pSG5.HA-GRIPl was included. Transfected cells were grown with 100 nM estradiol. The number above each bar indicates activity relative to that in the absence o f coactivators. RLU, relative light units. 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o < 1 > •M o < D (fi c d s - .0 SH • w * O 3 J 1) > p S 13 P I % l P . 80 1 2 3 MMTV-LUC B ER MMTV(ERE)-LUC ER EREII(GL45)-LUC 1 2 3 1 2 3 l + G R I P l | |+m Zaclb [^j+ G R IP l+ m Z aclb Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.6 Enhancement o f GRIPl and CBP activity by mZaclb. The fact that mZaclb bound to G RIPl and p300/CBP as well as directly to NRs suggested the possibility that coactivator and repressor effects o f m Zaclb on NRs could result from any o f these physical interactions. To test the ability o f m Zaclb to enhance the function o f CBP, the C-terminal region o f CBP (amino acids 2041-2240) which binds to the ADI region of GRIPl and to m Zaclb (Fig. 3 -IE) was fused to Gal4 DBD and tested in transiently transfected HeLa cells in the presence and absence o f co-expressed m Zaclb and/or GRIPl. By itself, the GaI4 DBD-CBP fusion protein activated a reporter gene with Gal4 binding sites. Co-expressed GRIPl enhanced reporter gene activity 30- fold, m Zaclb caused a 38-fold enhancement, and the effects o f G RIPl and mZaclb together were approximately additive (Fig. 3-9A). In a similar experiment, mZaclb enhanced the activity of full length GRIP 1 fused to Gal4 DBD by about 2.5-fold (Fig. 3- 9B); the same degree of enhancement was obtained when the C-terminal region of GRIPl was fused with Gal4 DBD (data not shown). Thus, mZaclb dramatically enhanced the activity o f a fragment o f CBP that it binds to, but only modestly enhanced the activity of the GRIP 1 fragment that it binds to. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3-9. Coactivator effects of m Zaclb with the Gal4 DBD fused to G R IPl or a C- terminal fragment o f CBP. (A) HeLa cells were transiently transfected with 0.5pg of GK1 reporter plasmid, 0.5pg o f pM.CBP204.2240, and, where indicated, 0.5pg of pSG5.HA-mZaclb and/or 0.5pg o f pSG5.HA-GRIPl. Numbers above the bars indicate activity relative to that o f Gal4DBD-CBP204i-2240 in the absence o f coactivators. The diagram at the top indicates the proposed mechanism of reporter gene activation; recruitment o f m Zaclb to the transcription complex could occur by m Zaclb binding either to CBP or to G RIPl. Gal4RE, Gal4-responsive elements in the GK1 promoter; TATA box in the GK1 promoter; LUC, luciferase-coding region; rightward-pointing arrow, transcription start site. (B) HeLa cells were transfected with 0.5pg of GK1 reporter plasmid, 0.5pg o f pM .G RIPl, and, where indicated, 0.5pg o f pSG5.HA-mZaclb. RLU, relative light units. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GaI4DBD- 2041-2240y GaI4 RE TATA LUC pSG 5.H A pSG 5.H A -G R IPl pS G 5.H A -m Z aclb 64.5x 38.lx 30.5x B > > 4— * 16: ’ > 12| o < U * < o CO 2 B f > u r < u CO C 3 3 C l > • 3 2. o cL 20 pG13-LUC Zacl p53 10 0 20 pGI3-LUC o pCMV-Pgal 0.3 0.6 0.9 1.2 1 . Zacl orp53 (pg o f expression plasmid) D s s «c S ^ 2 2 5 pMG15-LUC - None Zacl p53 30] 1 ^ 0 20 CO C 3 u> £ G 3 p53 (jig o f expression plasmid) I p » i [HE- H P » r- 2 P21-LUC j p21-sm-LUC j p21<lm-LUC ; ^ ^ 4 I < 2 " 3 s d 2 ' o 3 30 4.5 . u d ■ none □ Zacl □ p53 4 0.5 1-6 x 0.1 0.1 0.1 p21 -LUC p21 -sm-LUC p21 -dm-LUC 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. same p53 expression vector caused an increase o f up to 30-fold on this promoter, while Zacl enhanced this promoter only by 4.5-fold (Fig. 4 -IF). Thus, the dramatically specific enhancement of pG13-LUC expression by Zacl could not be achieved by simply increasing p53 concentration, although different promoters had different levels of dependence on p53 concentrations. Zacl could possibly enhance gene expression by two different mechanisms: it directly binds to specific DNA sequences on its target promoter or just serves as a coactivator for another transcription factor. Therefore, I further tested whether the effects of Zacl on the pG13 and p21 promoters were direct (i.e. independent o f p53) or through p53. When the p53 responsive elements were deleted from the p21 promoter, the enhancement by Zacl was eliminated, suggesting the idea again that the enhancement by Zacl was specifically dependent on p53 responsive element(s) (Fig. 4 - IF). Zacl alone had no effect on pG13-LUC in the human colon carcinoma 116 (HCT116) p53 cell line, but caused an eight-fold enhancement in the HCT116 p53 + /+ cell line (Fig 4-2A). In another p53-null cell line, mouse embryonic fibroblast (MEF) p53 v\ Z acl alone also had no effect on the p21-LUC reporter plasmid, but could enhance reporter expression in the presence o f p53 by 6-fold (Fig 4-2B). Thus, the induction of p53-dependent promoters by Zacl depended on the presence o f p53 and p53-responsive element(s); these findings also ruled out the possibility that Zacl could directly bind on p53-responsive elements through its potential DNA binding domain in the N-terminal seven-zinc-finger-domain. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-2. Z acl coactivator function on p53-responsive promoters requires p53. (A) The indicated HCT116 cell line, expressing p53 or p53-null, was transiently transfected with 0.5jag o f pG13-LUC reporter plasmid and, where indicated, 0.5jag o f pSG5.HA- m Zaclb. (B) MEF p53'" cells were transiently transfected with 0.5pg o f reporter plasmid p21-LUC and, where indicated, 0.6pg o f pSG5.HA-mZaclb and/or pSG5.HA-p53. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. T3 G O O C /1 X > TJ C O 0 C/i X > 1 N T3 p on o U) h- + + + + -J O N xg'OI D d Luciferase Activity (RLU 105) > Luciferase Activity (RLU 106) o h o 4^ oo X On •£ - X o X C D N> - £ < * O n 0 0 O —i------ 1 j _ j____ i N p o □ ■ X ffi n o H H xs xs L h U l U ) u > + -L ^ 1 -1 u > 4.2 Z acl has direct physical and functional interactions with p53 p53 is a multiple functional domain protein: activation domain in the N-terminus, DNA-binding-domain in the core region, and regulatory and tetramerization domains in the C-terminus (May and May 1999). Since the Zacl-induced p53 transcriptional activity is dependent on the presence o f p53 and p53-responsive element, I further tested whether Zacl could directly interact with p53. Full-length p53 strongly bound to GST-Zacl fusion protein in the GST pull-down assay (Fig 4-3A). Deletion o f either the N-terminus o f p53 (amino acids 1-95) or the C-terminus (amino acids 301-390) did not cause loss o f binding, but deletion o f both the N-terminal and C-terminal regions eliminated binding. The N-terminal region o f p53 alone bound moderately to Z a c l, but the C-terminal region alone bound weakly. Thus, Zacl bound to the p53 N-terminal region alone or to the combined central and C-terminal regions. One possible mechanism o f Zacl action on p53 is to regulate the transactivation activity o f p53 through the interaction between Zacl and N-terminal region o f p53. Therefore, I tested whether Zacl could directly enhance the p53 transactivation activity. When full-length p53 or the p53 activation domain (amino acids 1-127, p53 1 -1 2 7 ) was fused to the yeast Gal4 DNA binding domain (DBD), each one was capable of activating transcription of a reporter gene containing Gal4 response elements. The Gal4.DBD.p53i. 127 had a 12-fold higher transactivation activity than Gal4.DBD.full-length p53, consistent with the presence o f an inhibitory domain located in the C-terminal region of p53 (Hupp 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-3. Physical and functional interactions between p53 and Z acl. (A) The diagram shows functional domains o f p53 and their approximate locations are indicated by the amino acid numbers; AD, activation domain; DBD, DNA binding domain; RD, regulatory domain; TD, tetramerization domain. Full length p53 or p53 fragments were translated in vitro and incubated with bead-bound GST or GST-Zacl; bound proteins were eluted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. The percentage o f labeled protein bound, as determined by phosphorimager analysis, is shown below each lane. For comparison, the leftmost lane o f each panel shows 20% of the input protein used in the binding reaction. (B) HeLa cells were transfected with 0.4pg GK.1 reporter plasmid (encoding luciferase and controlled by five Gal4 response elements), 0.8pg o f pM-53 or pM -p53 1.127 (encoding Gal4 DBD fused to p53 or p531.127), and where indicated (white histograms) 0.8pig pSG5.HA-mZaclb. 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 1 94 300 355 390 1 AD 1 DBD RDIlTD RD2 325 — 1 In p u t (20% ) C O O 2 o o r v i « 7 5 o 3 1 — In p u l (20% ) 55 0 2 C o G ST -Z ocl p53( 1-390) — ► = — p53(95-300) -► mm 1 29 0.5 2.7 p53(95-390) -► — — p53(l-94) 0.8 17.1 1.8 12 p53( 1-300) — ► ■ ■ • p53(301 -390) - 0.4 9.2 0.8 2.5 B D C /D 20 2 3 io < U c6 o 3 0 ■ none □ Zacl ( 12.6) 2045lx (3.9) lx l.lx 1 2 S x 4 9 jx 1623x pM pM -p53 pM -p53 M27 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al. 1995). Furthermore, Zacl not only enhanced the transactivation activity o f full- length p53 4-fold, but also enhanced the p53 1-127 13-fold in HeLa cells (Fig. 4-3B). Thus, these enhancements on full-length p53 and p53 1.127 transactivation activities through the interaction between Z acl and p53 support the hypothesis o f the coactivator role o f Zacl for p53 activation. 4.3 Selective repression o f Z acl function by p53 C-terminal fragments or over expression offull-length p53 The previous data suggested that Z acl could serve as a coactivator o f p53 through direct interaction with p53 and further enhance the transactivation activity o f p53. Curiously, Zacl alone had specific and dramatic enhancement on pG13-LUC activity in the HeLa cells, where there are extremely low levels o f p53, and this effect was dependent on the presence o f p53. Although the pG13-LUC activity was not affected by the exogenous p53 (Fig 4-1A and B), I further tested whether the amount o f p53 in the HeLa cells was an important factor for the coactivator function o f Z acl. When I introduced exogenous p53 expression into HeLa cells, the enhancement o f pG13-LUC activity by Zac 1 was completely blocked by co-transfection of relatively high levels (0.5 pg) of either mouse wildtype p53 or a human mutant that lacked DNA binding activity (R175H) (Fig 4-4A) (See below about effects o f different levels o f p53 on Z acl action). Thus, the repression by this human mutant p53 also suggested that the transactivation 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-4. Repression of Zacl function by over-expression of p53 in HeLa cells. Cells were transfected with reporter genes and expression plasmids as indicated below, with (unfilled histograms) or without (filled histograms) Zacl expression vector. (A) pG13- LUC, 0.5pg; pSG5.HA-p53 or pSG5.HA-p53(R175H), 0.5pg; pSG5.HA-mZaclb, 0.5pg. (B-D) pG13-LUC, 0.5pg; CMV.(3gal, 0.5 pg; AR, 0.2 pg; MMTV-LUC, 0.4 pg; pSG5.HA-m Zaclb, 0.3pg; 0.8pg o f a pSG5.HA vector expressing p53 or the p53 fragment indicated by amino acid numbers. (E) pG13-LUC, 0.5pg; pSG5.HA-mZaclb, 0.3pg; 0.6pg o f a pSG5.HA vector expressing the p53 fragment indicated by amino acid numbers. 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. r_ > < L > C /3 £2 c_> 9 pG13-LUC 245x ■ none □ Z acl 6 3 4x 7x o p53 p53(R 175H) none c3 s o c : •< s & ^ £2 —> 12 9 6 3 O 150 % | 3 - I O O *C 3 C3 o & >; f > ^-O S 23 = 5 S ,q-> o 4 50 O 2 B pG13-LUC 735x S50x 444x 43r8x * l x 8x 16x -sv 5x 0.4x0.Sx I Q x 0.3x 2.7x C pCMV.pgal 13.8x 8.6x 5.3x n lxl 1.8x1 0 ^ 8 x 5.9x 5^8 x 1.6x 6.9x 2.2x 2 -Z x o D AR with MMTV-LUC I X 43x lx 40x 0.03x0. lx 2 J x 2.3: 45x 1.3x 37x H=-| 39x n 1 2.4x 2.3x None 1-390 1-94 95-300 301-390 1-300 95-390 a> C /3 £ 2 8 6 < C = > 4 £ 2 O E pG13-LUC 793X 598 x l.4x 299x None 1-300 1-325 1-335 1-355 1-390 301-390 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and DNA-binding abilities o f p53 were not involved in the repression by p53 o f Zacl coactivator function. I tested whether physical interaction between p53 and Zacl was necessary for repression of Zacl coactivator function by p53 in HeLa cells. I used different p53 truncation constructs to determine that the C-terminus o f p53 (amino acids 301-390, p53C) was enough to repress most of the Z acl coactivator function for p53 transcriptional activity (also seen in the HCT116 p 53^ cell, data not shown) (Fig 4-4B). In contrast, other constructs lacking the p53 C-terminal region caused little or no repression o f the Z acl enhancement at pG13-LUC activity. However, none o f the p53 truncations could block Z a c l’s other coactivator functions with the CMV.p-gal reporter gene or the androgen receptor, except of wildtype full-length p53 (Fig 4-4C and D). Thus, p53C was necessary and sufficient for the inhibition o f Zacl coactivator function on the pG13 promoter, but not on CMV or MMTV promoter. However, the p53C and wildtype full-length p53 apparently inhibited Zacl function by different mechanisms, since they did not have the same effects on Zacl coactivator function with all o f the reporter genes tested. To further examine the p53 region required for repression, I used more p53 constructs lacking the extreme C-terminus (amino acids 356-390, RD2), tetramerization domain (amino acids 326-355, TD) and RD1 (amino acids 300-325, NLS) to define the minimal repression region. A p531.335 mutant still completely inhibited Zacl coactivator function on the pG13 promoter (Fig. 4-4E). A p53 1.325 mutant inhibited Zacl activity by 50%, while p53 1.3 0 0 had no inhibitory activity. Thus, both of the RDI and N- 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. terminal TD regions in p53 C-terminal region were required to inhibit Zacl coactivator function (Fig 4-4E). The previous data showed that the Zacl coactivator effect on pG13 promoter was dependent on p53 and p53-responsive elements (Fig. 4-1 and 4-2) and high levels o f p53 inhibited its coactivator function (Fig 4-4). These data suggested lower levels o f p53 expression vectors might still cooperate with Zacl to enhance pG13 expression. Indeed, levels of p53 expression vector below 20 ng could further enhance the coactivator effect o f Zacl in the pG13 promoter, but 40 ng or higher levels o f p53 expression vector inhibited Zacl (Fig. 4-5 A). This enhancement o f Zacl coactivator function by low doses o f p53 was dependent on wildtype full-length p53, and was not observed with other p53 mutants (p53C, N-terminal region o f p53, or R175H) (Fig 4-5A). Thus, a narrow range of functional p53 levels was required for the coactivator effect of Zacl on the pG13 promoter, but a broad range o f p53 levels facilitated the coactivator effect o f Zac 1 in p2 1 promoter. However, the C-terminal region of p53 was enough to inhibit the coactivator function of Zacl on these two promoters. I further determined that the repression o f Zacl coactivator function by p53C was partially removed by various doses o f wildtype full-length p53 in HeLa cells (Fig. 4-5B). Similarly, various doses o f p53C also could alleviate the repression effect by high levels o f wildtype full-length p53 on Zacl function (data not shown). Thus, the exact level of 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-5. Stimulation or inhibition of Z acl function by different levels of p53, p53 fragments, or p53 mutants. (A) HeLa cells were transfected with 0.3pg pG13-LUC reporter plasmid, 0.5pg pSG5.HA-mZaclb, and the indicated amount of a pSG5.HA expression vector for p53 or the indicated p53 fragment or mutant. (B) HeLa cells were transfected with 0.3p.g pG13-LUC reporter plasmid, 0.5pg pSG5.HA-mZaclb, 0.5pg pSG5.HA-p53 C and the indicated amount o f a pSG5.HA expression vector for p53. 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A R175H B 0.02 0.04 0.06 0.08 pg of expression plasmid > < 2 < D cd S - H .U < + - H o None £— Zacl+p53 C 0 0.05 0.1 0.15 0.2 0.25 0.3 p53 (pg o f expression plasmid) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. functional p53 is an important regulatory factor for the coactivator function o f Zacl in HeLa cells. 4.4 Binding and functional interactions among Z acl, p53, and HPV-18 E6 protein Zacl had a much more dramatic effect on the pG13 promoter in HeLa cells than in MEF or HCT cell lines (Fig. 4-1 and 4-2). One major difference among these three cell lines is the presence o f HPV-18 proteins (especially, E6 protein) in HeLa cells. High-risk HPV E6 proteins from HPV-16 and 18 not only reduce p53 amount by the E6-ubiquitin degradation system, but also can displace p53 from a number o f p53 recognition elements or compete for some p53 coactivators, like CBP and p300. HeLa cells are known to contain very low levels o f wildtype p53. In this study, elevation o f cellular p53 levels in HeLa cells could not account for the coactivator effect of Zacl on the pG13 promoter, since exogenous expression of p53 failed to produce the same effect on pG13 promoter by Zacl (Fig. 4-1 A, 4-4 and 4-5). Therefore, I tried to test whether Z acl had physical and functional interactions with E6 protein to reverse other inhibitory effects from HPV-18 E6 (E6.18) protein on p53 functions. First, I determined that Zacl and p53 or N-truncated p53 (amino acids 95-390) could, individually or altogether, bind to GST-E6.18 fusion protein in the GST pull-down assays (Fig 4-6A). However, when Zacl and p53 were incubated together with GST-E6.18, the binding o f Zacl and p53 protein were both reduced slightly, suggesting that Zacl and p53 might compete for binding to E6.18 protein. Furthermore, I used unlabelled recombinant His6-Zacl fusion protein to compete 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-6. C om petitive binding of p53 and Z acl to HPV-18 E6 protein. GST pull down assays were conducted as in Fig. 4-3A. Percent binding (determined by phosphorimager analysis) is given below each lane o f the autoradiograms. (A) Zacl and/or p53 were translated in vitro and incubated with bead-bound GST-E6.18 fusion protein. (B) The indicated p53 protein or CBP fragment was synthesized in vitro and incubated with bead bound GST-E6.18 protein. The input lanes represent 10% of the input for the p53 proteins and 12.5% for the CBP fragment. Other lanes contain increasing amounts of His6-Zacl as competitor; the top panel shows Coomassie Blue-stained His6-Zacl. 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A B Input (25%) GST-E6.18 P 5 3 1 . 3 9 0 + + + Zacl + + + Zacl p53. 1-390 5.8 4.3 4.1 2.3 3 ja l, His-Zacl competitor £ 0 15 30 45 P ^ 1-390 0,6 0.8 C B P ] 594-2441 12.5 7 6 5 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for the bindings o f p53 or N-truncated p53 with GST-E6.18 protein (Fig. 4-6B). The binding abilities between E6.18 and full-length p53 and its mutant could be reduced by the highest amount o f recombinant His-tag Zacl to 18% and 7%, respectively. The inhibition of binding by the recombinant Zac 1 protein was relatively specific, since the same amount o f Zac 1 protein only reduced binding o f CBP to E 6.18 to 40% of control values. Thus, the specific disruption o f p53-E6.18 complex by Z acl also provided another possible mechanism for the enhancement o f p53 activation. Detection of binding in HeLa cells was not possible because p53 is expressed at undetectable levels in HeLa cells and no Zacl antibody was available to use. Subsequently, I tested whether the interaction between Zacl and E6.18 had functional consequences and whether the p53 activation in HeLa cells involved reversal of the inhibitory effect o f E6.18 protein by Z acl. I reintroduced these proteins into a HPV-free cell lines, like HCT116 cells. Zacl enhanced the activity o f pG13 reporter in the HCT116 p53+ /+ cells 7-fold (Fig. 4-7A, histograms 1 and 4), and E6.18 protein caused a 5-fold inhibition o f reporter gene activity. Co-expression of Zacl with E6.18 reversed the inhibition by E6.18, although the activity did not reach that achieved by Zacl alone. E6 proteins are also known to bind and interfere with the activity of coactivators CBP and p300. However, p300 expression had no effect on pG13-LUC expression (Fig. 4-7A, histogram 2) and did not reverse the inhibitory effect o f E6.18 (histograms 10-11). CBP modestly enhanced the activity of pG!3-LUC (histogram 3), and it also partially reversed the inhibitory effect o f E6.18 (histogram 8-9), but it was not as effective as Z acl. In the presence o f E 6 .18 and Zacl proteins, expression o f CBP or p300 failed to cause any further reversal o f the inhibitory 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4-7. Functional interactions am ong p53, E6.18, Z a c l, and CBP. (A) HCT116 p5 3 ‘ r/+ cells were transfected with 0.3pg pG13-LUC; where indicated, the following expression vectors were also included: 0.5pg o f E6.18 vector and 0.4 or 0.8pg o f p300, CBP, or Zacl vectors (for these vectors, + indicates 0.8pg; the ramp indicates 0.4pg at the low end and 0.8pg at the high end). (B) HCT116 p 5 3 + /+ cells were transfected with 0.4pg o f GK1 reporter plasmid (controlled by Gal4 response elements); where indicated, expression vectors for Gal4 DBD (0.8pg), Gal4 DBD fused to E6.18 (0.8pg), and Zacl (0.2, 0.5 or 0.8pg; +indicates 0.8pg o f Zacl vector) were included. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A pG13-LUC D 10 i lx 0.9x 0.3x ° l x 0.2x 0.2x 10 11 E6.18 + + + 4- 4 - + + B y o /: < 2 6 a> C/3 03 J — fS o D n J Gal4 Gal4-E6.18 Zacl 19x Gal4-E6.18 lx Q .6 x 1 2 + 4 - 4 - 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. effect o f E6.18 (data not shown). Thus, the results (Fig. 4-6 and 4-7A) were consistent with the hypothesis that part o f the stimulatory effect o f Zacl on pG!3-LUC activity in HeLa cells is due to Z a c l’s ability to disrupt the p53-E6.18 complex, restoring the activity of p53. Furthermore, the results indicated that, while inhibition o f CBP or p300 function by E6.18 may also play a role in the down-regulation of p53 function, such a mechanism cannot account for the majority o f the inhibition caused by E 6.18 in HeLa cells. The idea w'as supported by the two following results. Exogenous expression of Zac 1 was much more effective than exogenous expression o f CBP and p300 in reversing the inhibition o f pG13-LUC activity by E6.18 protein in HCT116 p53+ /+ cells; and exogenous CBP and p300 in HeLa cells just enhanced (by 4 to 6 -fold) on the pG13 promoter activity (data not shown). Finally, to test more directly for a functional interaction between Zacl and E6.18 proteins, I examined the effect of Zacl on the autologous transcriptional activation activity of a Gal4DBD-E6.18 fusion protein. Gal4DBD-E6.18 had a weak autologous transcriptional activation activity in HCT116 p53w + cells (Fig. 4-7B), and Zacl strongly inhibited this activity (histograms 3-6). However, Zacl had no effect on the Gal4DBD (histograms 1-2). The ability of Zacl to reverse partially the inhibition o f p53 activity by E6.18 and to alter the transactivation activity o f E6.18 indicated a functional interaction between Zacl and E 6.18 proteins. 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion Over 50% o f all human tumors have defective cell cycle arrest or apoptotic activities caused by mutations of p53 (Hollstein et al. 1994). Like other tumor suppressor genes that exhibit mutations that lead to loss of gene function, the vast majority of mutations in p53 are mis-sense mutations that encode full-length proteins with amino acid substitutions within its DNA-binding domain. However, within cervical tumors, unlike most other cancers, p53 is wildtype, perhaps indicating that the effects of high-risk HPV E6 are analogous to an inactivating mutation (Scheffner et al. 1991). This possibility was supported by studies showing that pathways leading to p53 activation are functional in cells derived from cervical cells (Butz et al. 1995). Recently, studies have shown that inhibition o f E6 -induced p53 degradation might not be sufficient to overcome all the deleterious effects o f E6 upon p53 (Mantovani and Banks 1999). Indeed, in cervical cell lines, although blocking E6 -induced degradation o f p53 frequently results in increased levels o f p53 protein, the subsequent correct nuclear localization o f p53 appeared to be perturbed (Thomas et al. 1999). More recent studies suggest that E6 protein suppresses p53 activity through multiple mechanisms, including induction of p53 degradation, inhibition o f DNA binding by p53, and inhibition o f the activity of the coactivators CBP and p300 (Rapp and Chen 1998; Thomas et al. 1996; Patel et al. 1999; Zimmermann et al. 1999). 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In this study, I introduced mouse Zacl into HeLa cells derived from an HPV 18- positive cervical carcinoma to test whether Zacl could affect the subtle balanced relationship between p53 and its interacting factors, like E6 and CBP/p300, in HeLa cells. Surprisingly, Zacl caused a specific enhancement of one p53-responsive reporter plasmid (pG13-LUC) by several hundred-fold (Fig 4-1 A, 4-4ABE and 4-5AB). These data will open a new way to study how to improve low amounts o f functional wildtype p53 by adjusting the physical and/or functional balance with other p53-interacting factors in the cells. However, the enhancement effect by Zac 1 was dependent on wild-type p53 in the cells because Zacl alone had no effect on pG13-LUC activities in p53-null cell lines (H CT116 p53'/_ and MEF p53'/_ cells) (Fig 4-2AB). Furthermore, in MEF p53'/_ cells, I identified that the function of Zacl was a coactivator o f p53 (Fig 4-2B). These results indicate that Zac 1 was not acting as a sequence-specific transcription factor by protein- DNA interaction; these results also are consistent with a model whereby Zacl binds to p53 molecules bound to the enhancer elements of the p53-regulated genes and serves as a coactivator for p53. Zacl bound p53 efficiently in vitro and stimulated the transactivation activity of p53 (Fig. 4-3); these results further support the coactivator model. The coactivator function of Zac 1 also has been determined with nuclear receptors (Huang and Stallcup 2000). The fact that the coactivator effect of Zacl for p53 in HeLa cells was more dramatic than in two other cell lines, HCT116 and MEF cells, suggested other factors (e.g. expression o f HPV-E6.18 protein in HeLa cells but not in the other two cell lines) could influence the coactivator activity of Zacl. 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. One related study showed that high-risk HPV E2 could interact with p53 and activate the transcriptional activity of p53 in HeLa cells through at least two pathways (Desaintes et al. 1997). The first one involved the binding of E2 to its recognition elements located in the integrated viral P 105 promoter, which controls expression o f E6 and E7. E2 binding consequently repressed transcription o f the endogenous H PV -18 E6 oncogene and then elevated endogenous p53 in the HeLa cell. The second pathway did not require specific E2 DNA binding ability. However, over-expression of a p53 trans dominant-negative mutant (p53C) abolished both E2-induced p53 transcriptional activation and E2-mediated G1 growth arrest, but showed no effect on E2-triggered apoptosis. Further, the direct interaction between HPV-16 E2 transcriptional activator and p53 has been reported (Massimi et al. 1999). Considering the HPV E2 functions in the regulation o f p53 activity in HeLa cells, there were many reasons to support the idea that the Zacl coactivator function did not involve blocking the p53 degradation pathway. First, there were no detectable p53 or E6 changes by western blotting assay or RT-PCR assay (data not shown) in HeLa ceils transfected with Z a cl. Secondly, the dose curve o f p53 on the pG13-LUC reporter gene activity also showed that the dramatic effect by Zacl could not be achieved by only increasing p53 in HeLa cells (Fig 4-1 BE). Interestingly, I found that higher doses o f wildtype full-length p53, like the p53 trans-dominant-negative mutant, could abolish the Z acl coactivator function in p53 transcriptional activation (Fig 4-4A). Furthermore, the repression from one human p53 mutant (R175H) which loses transactivation and DNA-binding abilities suggested that the abolition by R175H was not by the transactivation and DNA-binding abilities of p53, but it could be through the 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. physical interaction between p53 and Zac 1. Titration o f p53 levels in HeLa cells revealed that different levels o f p53 had dramatically different effects on the coactivator activity o f Z acl. Low levels of p53 were required for Zacl activity on pG13 and p21 promoters, so transfection o f low levels o f p53 expression vector in HeLa cells increased the effectiveness o f Zacl on both promoters (Fig. 4-5 A and data not shown). However, high levels o f p53 expression vector completely inhibited the Zacl coactivator effect on pG13- LUC (Fig. 4-5A), although similar high concentration o f p53 expression vector enhanced the effect of Zac 1 on the p 2 1 promoter (data not shown). The N-terminal region and DNA-binding functions o f p53 were not required for its inhibition of Zacl coactivator function on the pG13 promoter (Fig. 4-4AB). Furthermore, I determined that the C-terminal fragment o f p53 was enough to repress the Zacl coactivator function and it was even more efficient to inhibit Zacl function than wildtype full-length p53 (Fig 4-4BE). Taken together, these findings suggested that p53C abolished the Zacl coactivation function for the p53 transcriptional activation by a mechanism that did not only involve competing for the p53-responsive elements with endogenous p53. p53C possibly could form hetero-oligomers with the endogenous p53 and, further, could titrate Zacl in the cells. However, more detailed experiments indicated that p53 and p53C inhibit Zacl function by different mechanisms. Low levels o f full- length p53 enhanced the coactivator effect o f Zacl on pG!3-LUC, but p53C efficiently inhibited Zacl coactivator function on pG13 at high and low concentrations (Fig. 4-5A). Full-length p53 inhibited the coactivator effect o f Zacl on CMV and AR, but p53C did 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. not (Fig. 4-4CD). Finally, p53 and p53C could neutralize each other’s inhibitory effects on the coactivator function o f Zacl in the pG13 promoter (Fig 4-5B). Interestingly, only wildtype full-length p53, not p53C, could repress all other tested coactivator functions of Zacl (Fig 4-4 BCD). This also supported the idea that the repression o f Zacl coactivator function in p53 transcription activation was dependent on the interaction between Zacl and p53. Therefore, the inhibition o f coactivator function o f Zacl in CMV and AR activations by wildtype full-length p53 is through a separate pathway from the inhibition o f coactivator function of Zacl in p53 activation. The coactivator function of Z acl for the p53 activation was much higher in HeLa cells than in other cells (e.g. HCT116) (Fig. 4-1A and 2A), indicating that the full effect o f Zacl involved more than just a simple p53-Zacl interaction; rather, other cellular components can influence the p53-Zac I functional interaction. HPV-E6.18, expressed in HeLa cells but not in the other two cell lines, and other common binding proteins (CBP/p300) of p53 and Zacl both are good candidates for such proteins. E6 proteins are known to suppress p53 activity by several mechanisms: E6 induces ubiquitin-mediated degradation o f p53 and thereby keep p53 levels extremely low in HeLa cells; E6 also binds to p53 and prevents it from binding to genes with p53 response elements. E6 proteins might inhibit p53 function indirectly by binding to and interfering with the activity o f CBP or p300. The fact that Zacl bound to E6.18 protein and specifically disrupted E6.18-p53 binding in vitro (Fig. 4-6) provides an attractive mechanism for Zacl to prevent p53 degradation and also to prevent E6.18 from inhibiting DNA binding 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and transcriptional activation by p53. This hypothesis was further supported by the demonstration that Zacl interacted functionally with E 6.18 protein. Zacl partially reversed the inhibition of p53-dependent gene activity by E 6.18 and altered the transcriptional activation activity o f E6.18 fused to Gal4DBD in the H CT116 cells (Fig. 4-7). These results also provided evidence that high-risk E6 proteins have other functions in addition to p53 degradation to regulate p53 activity, because the suppression of E6 functions by Zacl was not linked to an increase of p53 amount in HeLa cells. In my previous data, Zacl bound to and activated CBP/p300. However, CBP and p300 were not as effective as Zacl in reversing the inhibitory effect of E6.18 protein on p53 (Fig. 4-7A), and Zacl did not efficiently disrupt the binding of CBP to E 6.18 (Fig. 4-6B). CBP or p300 alone also did not play the same role as Zacl in the p53 transcriptional activation in HeLa cells (data not shown), although, they like Zacl also bound p53 and E6.18. However, the enhancement of p53 activity by Zacl was apparently not due to rescue of CBP and p300 activity. Thus, it is likely that Z acl, E6.18. CBP and/or p300, and other as yet undefined cellular components all play roles in the complex regulatory axis surrounding p53. In the previous chapter, Zacl was shown to serve as a co-regulator of nuclear receptor function. The fact that the effect o f Zacl on nuclear receptor function can vary dramatically, depending on the cell type, reporter gene, and type o f nuclear receptor used, is reminiscent o f my findings here on the effects of Zacl on p53 activation. The work also reinforced the notion that Zac 1 is part o f a complex regulatory axis in which many 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cellular proteins play important roles, and which can be influenced strongly by viral proteins like HPV E6, adenovirus E l A, and SV40 T antigen proteins. The participation o f Z acl, p53, and the viral proteins in such a complex and finely balanced regulatory system may help to explain how they can affect so many different cellular processes. 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5: Concluding Remarks 5.1 The co-regulator functions of Z acl in nuclear receptor activation The enzymatic activities o f CBP/p300 and CARM1 help them to fulfill their secondary coactivator functions through the modification o f histone proteins, transcription factors, or basal transcriptional initiators (McKenna et al. 1999; Chen et al. 1999). These studies support the hypothesis that the signaling pathway travels from NR through p i 60 coactivators and CBP/p300 to accomplish chromatin remodeling and transcription machinery (Freedman 1999). Thus, the functional domains, ADI and AD2, o f p i 60 coactivators provide a good tool to study the signaling pathway of coactivator action in the NR functions. In this thesis, I reported the isolation and characterization of m Zaclb, a transcriptional co-regulator for NR. m Zaclb served as a primary coactivator for some NR functions. However, CARM1 was a secondary coactivator for NR functions, even through both were isolated by the same yeast two-hybrid screening work. The primary coactivator functions by m Zaclb are supported by these findings: m Zaclb directly interacted with NRs, p i 60 coactivators, and CBP/p300; and m Zaclb possesses putative transactivation activity. Also, m Zaclb did not require the presence o f GRIP1 or any other coactivators. However, unlike common coactivators for NR functions, the LXXLL motif o f m Zaclb is not the major functional domain responsible for the interaction with NRs. 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, mZac 1 b sometimes acted as a synergistic coactivator with p 160 coactivators or sometimes repressed the coactivator function o f p i 60 coactivators. Actually, the mechanism(s) o f coactivator or repressor function by m Zaclb in NR function are poorly understood. Based on results in this thesis, the role o f coactivator or repressor by m Zaclb in NR function, at least in part, depends on the type o f nuclear receptor, promoter context, and cell-type context. These three factors may determine the final functional role o f m Zaclb in NR functions, but we should reconsider three characteristics of m Zaclb with the fact that m Zaclb is not a member o f p i 60 coactivators. First, m Zaclb has the DNA binding property which may recognize specific elements around NR responsive element and thereby have cross talk with the NR complex. Second, the LXXLL m otif is not a necessary interaction domain for m Zaclb in its NR co-regulator function. The findings provide ideas to look for mZac lb-binding sites which should be distinct from the p i 60 coactivator binding site in NRs. Furthermore, it is a good issue to determine what controls the discrimination of binding sites for m Zaclb and p i 60 coactivators in NRs. Finally, m Zaclb interacts with NRs in a hormone-independent manner. This provides m Zaclb more chances to affect NRs functions without conformation changes by their ligands and may regulate other coactivators involved in NRs functions. 5.2 The coactivator function of Zacl in p53-dependent activation Intriguingly, Zacl was identified along with p53 in a functional screening system by virtue o f their common ability to induce expression o f type I PACAP receptor gene. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Furthermore, Zacl exhibited an anti-proliferative activity characterized by induction of extensive apoptosis and G1 arrest. The original idea to study the functions o f Zacl in apoptosis and cell cycle arrest was derived from the fact that Zacl was isolated in the same functional screen with p53. These common abilities of Zacl and p53 led me to test the possible role of Zac 1 in p53 transcriptional activation, although the previous study suggested that the mechanisms of apoptosis and cell cycle arrest by Zac I were different from those o f p53. The previous conclusion was from p53-null cell lines. However, it is still an interesting issue to study the relationship between Zacl and p53. In this thesis, I showed that Zacl had a dramatic coactivator effect (several hundred folds) on p53 by monitoring a p53 responsive element reporter system in HeLa cells. In other tested cell lines (H CT116 and MEF), the coactivator effect o f Zacl for p53 was much lower than in HeLa cells. My further studies have shown that Zacl dramatically and specifically enhanced the activity of p53-dependent promoters in HeLa cells through at least two pathways. First, Zacl bound to p53 and acted as a coactivator for p53. Second, Zacl bound to HPV-E6.18 protein and disrupted the E6-p53 complex, which leads to inactivation and degradation o f p53. Thus, the enhancement by Zacl was apparently due to a complex combination o f Zacl actions, including but possibly not limited to: direct coactivator effects of Zacl on p53; stabilization o f p53; and blocking the inactivation of p53 and CBP/p300 by E6.18 protein. The fact that Zacl is a coactivator for p53 suggests that Zacl could potentially induce cell cycle arrest and apoptosis through the p53- dependent pathway. However, this conflicts with the conclusions from other previous studies which indicate that the mechanism(s) o f cell cycle arrest and apoptosis by Zacl is 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. independent of p53-dependent pathways. p53-independent apoptosis induced by HPV E2 is similar to that observed with Z acl, but the mechanism o f p53 reactivation by mZacl is distinct from that of HPV E2. A comparison of the mechanisms o f p53, HPV E2 and Z acl in cell cycle arrest and apoptosis will help us understand more about cell cycle regulation and apoptosis. Such studies may also be relevant to the effect o f reactivation of p53 in the carcinogenic progression of HPV-associated cancers. 5.3 The future studies of Zacl Zacl is a multi-functional protein that can interact with and influence the activities of many other cellular proteins and signaling pathways; furthermore, the activity of Zacl is also subject to regulation by other cellular proteins and signaling pathways. It should be an attractive topic to further study the interactions o f p53 and Zac I in other transcriptional pathways, such as NRs, AP-l, NF-kB, and CREB. The repression by p53 and YY1 has been demonstrated to occur through recruitment o f TATA-binding protein or mSin3a-HDAC complex (Farmer et al. 1996; Murphy et al. 1999). It should be an open question whether the repression by p53 in NR functions also needs the corepressor complex or if the HDAC-mSin3a complex is involved in the repression effect by Zac 1 in NR functions. It will be an important step to link the structural domain(s) of Zacl with its functions in versatile transcriptional pathways. Therefore, potential Zacl- interacting proteins also can help us to discover new functions o f Zac 1 in cells, as shown by the roles of the latest discovered Zacl binding proteins in this thesis: NR coactivators, 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. p53, and E6 protein. The ability o f Zacl to interact with HPV E6 protein suggests the question whether Zacl, similar to p53, also interacts with other DNA viral proteins (such as SV40 large T antigen and E lB ). This issue remains to be addressed in further studies. In conclusion, the study on Zacl functions is a relatively new research field and all studies about Zac 1 or its family are not limited currently to any specific fields. These future advances will, in turn, allow us to understand the cross-talk mechanisms among different cellular functional pathways, for example, NRs, p53, AP-1, NF-kB, and cell cycle regulation, etc. However, the answers to many o f these issues await further study. Therefore, Zac 1 also is a good target for my future research career. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bibliography Abdollahi A., Roberts D., Godwin A.K., Schultz D.C., Sonoda G., Testa J.R. and Hamilton T.C. 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(1999) The human papillomavirus type 16 E6 oncoprotein can down-regulate p53 activity by targeting the transcriptional coactivator CBP/p300. J. Virol. 73, 6209-6219. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of th e copy subm itted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send U M I a complete manuscript and there are missing pages, these will be noted. 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Huang, Shih-Ming
(author)
Core Title
Isolation and characterization of mouse Zac1, a novel co-regulator for transcriptional activation by nuclear receptors and p53
School
Graduate School
Degree
Doctor of Philosophy
Degree Program
Biochemistry
Degree Conferral Date
2000-12
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University of Southern California
(original),
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Tag
biology, molecular,OAI-PMH Harvest
Language
English
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Stallcup, Michael R. (
committee chair
), Johnson, Deborah L. (
committee member
), Lai, Michael M.C. (
committee member
)
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Huang, Shih-Ming
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biology, molecular