University of Southern California Dissertations and Theses

Mechanism of repression of RNA polymerase III-dependent transcription under hypoxia

(USC Thesis Other)

Mechanism of repression of RNA polymerase III-dependent transcription under hypoxia

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content MECHANISM OF REP PRESSION OF RNA POLYMERASE Ill-DEPE NDENT TRANSCRIPTION Copyright 2012 UNDER HYPOXIA by Qiu-yu Guo A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (BIOCHEMISTRY AND MOLECULAR BIOLOGY) December 2012 Qiu-yu Guo ACKNOWLEDGEMENTS The completion of this thesis would not be made possible without the support of several important people. I would like to express my gratitude first to my advisor of research, Dr. Debbie Johnson. She taught me the way to think as a scientist, offering me enough space to develop my own thoughts as well as critical scientific opinions when I was confounded. She has also witnessed my maturation with all her patience and encouragement, making my experience in her lab an illuminating as well as a happy one. The other equivalently critical support was from my family, both financially and mentally. Without this, I would never be able to come here and appreciate the education at USC. My father, especially, has done all he could to help me in every aspect of life just in order to make it easier for me to concentrate on my study. I would also like to thank my committee members, Dr. Michael Stallcup and Dr . Keigo Machida, for their time and energy spent on my training. Dr. Stallcup gave me an interview at the PIBBS admission, offering many brilliant ideas about my research and science in a broader aspect. Dr . Mach ida works in the same building with me and cares a lot about both my study and my life here. Both of them are very brilliant and kind people, 11 and I am so proud for being able to bring them in my committee. My thanks also goes to Dr. Zoltan Tokes, who is the director of the program I am in. Dr . Tokes meets with me regularly and helped me a lot in different kinds of issues during my study here. At last but not least, I would like to appreciate the efforts spent by my labmates in training and helping me to carry out my research. Dr. Sandra Johnson performed cell infection for me, which served as a foundation to all the experiments I have done. She also trained me in molecular cloning and helped me to tackle a lot of technical problems I encountered. Mr. Eric Min-Chuan Wang, a once Master student in our lab, carried out some preliminary experiments for my project, and helped me to initiate my research at this lab. I was trained by Dr. Beth Palian, Dr. Aarti Rohira, Dr. Elliot Chun-yun Chen and Mr. Abraham Kaslow in different aspects of experimental techniques. They also offered me important professional opinions in tackling the technical problems I've encountered. 111 TABLE OF CONTENTS Acknowledgements .......................................................................................................... ii list of Tables .......................................................................................................................... vi list of Figures ..................................................................................................................... vii Abstract .......................................................................................................................... viii Chapter 1: Introduction 1.1 Regulation of RNA Polymerase Ill activity and its link to cancer ......... 1 1.2 Tumor hypoxia and its effect on RNA Pol Ill activity .............................. 3 3.3 Chromatin modulation under hypoxia .................................................... 4 1.4 Scope of this thesis .................................................................................... S Chapter 2: Materials and Methods 2.1 Cell culture .................................................................................................. 7 2.2 Western immunoblotting analysis ........................................................... 7 2.3 Quantitative Real-time RT-PCR ................................................................. 9 Chapter 3: Results 3.1 RNA Pol Ill-mediated transcription of tRNA, but not U6 RNA genes, is repressed under hypoxia ............................................................................... 13 3.2 Repression of tRNA gene transcription under hypoxia is mediated by HIF-1a .............................................................................................................. 14 3.3 Brf1 expression is repressed under hypoxia and it is HIF-1a -dependent .................................................................................................... 16 3.4 Decreased expression of JARID1C alleviated the hypoxia-mediated reduction in Brfl expression, but not repression of RNA Pol Ill-mediated tra nsc ri ption .................................................................................................... 18 Chapter 4: Discussion 4.1 Repression of tRNA gene transcription under hypoxia is a common phenomenon in different cell types ............................................................. 21 !V 4.2 Multiple mechanisms are contributing to hypoxia-induced repression of tRNA gene transcription ................................................................................ 21 4.3 Summary ................................................................................................... 24 4.4 Future work and directions .................................................................... 26 v LIST OF TABLES Table 1. Antibodies for Western blots ...................................................................................... ll Table 2. Primers Sets for Quantitative PCR .............................................................................. ll vi LIST OF FIGURES Figure 1: RNA Pol Ill -mediated transcription of tRNA, but not U6 RNA genes, is repressed under hypoxia .................................................................................... 14 Figure 2: Repression of tRNA gene transcription under hypoxia is mediated by HIF-1a .................................................................................................................... 15 Figure 3: Brf1 expression is repressed under hypoxia and it is HI F-1a-dependent ................................................................................................ 17 Figure 4: Decreased expression of JARID1C alleviated the hypoxia-mediated reduction in Brfl expression ............................................................................... 19 Vll ABSTRACT Deregulated RNA Polymerase Ill ( Pol 111)-dependent genes transcription is linked to oncogenesis. In this study, we investigated the response of RNA Pol Ill-dependent gene transcription to hypoxia, a common situation encountered by many solid tumors during their development. We demonstrate in several cancer cell lines that RNA Pol Ill dependent transcription of tRNAs, but not U6 RNAs, is repressed under hypoxia. Using gene-silencing techniques, we show that hypoxia-mediated repression of tRNA gene transcription is dependent on the Hypoxia-Inducible Factor ( HIF)-la, a transcription factor that is induced only in oxygen-deficient environment. Furthermore, the expression of Brfl, a critical transcription factor that is required for initiation of tRNA, but not U6 RNAs genes transcription, is repressed under hypoxia in a HIF-la-dependent manner, which may explain the HIF-la-dependent repression of tRNA gene transcription. Finally, the histone H3K4 demethylase JARIDlC, a downstream target gene of HIF, is shown to be required for repression of Brfl expression under hypoxia; however, simply decreasing the expression of JARIDlC is not sufficient to restore tRNA gene transcription under hypoxia, suggesting the existence of multiple mechanisms downstream of HIF through which RNA Pol Ill-dependent gene transcription is repressed. viii CHAPTER 1 INTRODUCTION 1.1 Regulation of RNA Polymerase Ill activity and its link to cancer RNA polymerase (Pol) Ill synthesizes a series of small non-coding RNA transcripts including tRNA and 55 ribosomal RNA, which are essential components of the translation machinery. Other important RNA Pol Ill products, such as U6 RNA, HRP RNA and Hl RNA, are required for RNA processing (Dieci et al., 2007). Elevated RNA Pol Ill- dependent transcription is associated with oncogenesis. More recent evidence revealed that enhanced RNA Pol Ill activity is indeed required for oncogenic transformation (Johnson and Johnson, 2008). Transcriptional initiation by RNA Pol Ill requires the assembly of a complex machinery of transcription factors on the target promoter. The exact repertoire of transcription factors utilized depends on the type of RNA Poll II target promoters. tRNA promoters are type 2 promoters, characterized by a A box and a B box within the coding region. General transcription factors TFIIIB and TFIIIC are recuited to tRNA promoters via A and B boxes to initiate transcription. TFIIIB consists of Brfl, lATA binding protein (TBP) and Bdpl, among which Brfl and Bdpl are unique to the TFIIIB complex. In contrast, U6 RNA promoters are type 3 promoters, which consists of a distal sequence element (DSE), a proximal sequence element (PSE) at the 5' region out of the gene, and a lATA box in the coding region. SNAPe and TFIIIB-Iike complexes are recruited to U6 promoters 1 through the PSE and TATA box, respectively, to initiate transcription. Brf2, instead of its homolog Brfl, is required for transcriptional initiation U6 RNAs; TBP is required for transcription of both tRNAs and U6 RNAs (Schramm and Hernandez, 2002). TFIIIB is a critical point of regulation by a number of cellular signaling pathways involved in oncogenesis. The extracellular signal-regulated kinase (ERK), which is activated in several signaling pathways, phosphorylates Brfl, enhancing TFIIIB-TFIIIC interaction (Felton-Edkins et al., 2003). The tumor suppressor PTEN, on the other hand, disrupts the association of TBP and Brfl by negatively regulating the PI3 K-Akt pathway leading to the dephosphorylation of Brfl (Woiwode et al., 2008). The expression of TB P was shown to be stimulated through epidermal growth factor (EGF)-activated Ras signaling (Zhong et al., 2004). In contrast, Mafl (Johnson et al., 2007) and c-Jun N terminal kinase 2 (Zhong et al., 2004) repress TB P expression. TFIII B is also regulated through its direct interaction with tumor suppressor or oncogenic proteins. p53 directly binds to TBP, therefore inhibiting its occupancy of TFIIIB on RNA Pol Ill target genes by TFIIIB (Crighton et al., 2003). Another tumor suppressor, Rb, also asscociates with TFIIIB but not TFIIIC and disrupts its promoter binding (Larminie et al., 1997). On the other hand, the oncogenic protein c-Myc binds to Brfl and activates Pol Ill-mediated transcription (Gomez-Roman et al., 2003). Even more strikingly, overexpression of TBP is sufficient to induce oncogenic transformation (Johnson et al., 2003). 2 1.2 Tumor hypoxia and its effect on RNA Pol Ill activity Hypoxia is a feature of most solid tumors. The growth speed of tumor cells is determined by the environment in which it resides in. Condensed tumor cell populations results in low oxygen availability, especially when it outgrows the available blood vessels in the lesion, thus ultimately leads to a low oxygen state or hypoxia. The hypoxic state is refractory for anti-tumor therapy, because of its multiple contributions to chemoresistance, radioresistance, angiogenesis, vasculogenesis, invasiveness, metastasis, resistance to cell death, altered metabolism and genomic instability (Wilson and Hay, 201 1). Hypoxia initiates a vast series of cellular responses. At the molecular level, one of the major mediators of such responses is hypoxia-inducible facto r-1 (HIF- 1). A functional H IF-1 is a dimer of two transcription factors, an oxygen-regulated a and a constitutively- expressed i3 subunit (Wang et al., 1995). HIF-1a associates with HIF- 113, allowing the dimer to occupy the target promoters at a typical HIF-reponsive element (HRE). HIF-1a is degraded at normal oxygen concentrations, as it is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (P HD2), which uses 02 and a-ketoglutarate as substrates. Prolylhydroxylated HIF-1a is bound by the von Hippei-Lindau tumor suppressor protein (VH L), which recruits an E3-ubiquitin ligase that targets HIF- 1a for proteasomal degradation (Kaelin Jr, 2008). HIF-1 transcriptionally activates the expression of hundreds of genes involved in many aspect of cancer biology, 3 including angiogenesis, erythropoiesis, glucose metabolism, cell survival, proliferation, apoptosis, and cell motility, etc (Semenza, 2003). Some evidence suggests that RNA Pol Ill-dependent gene transcription is suppressed under hypoxia in cardiomyocytes (Ernens et al., 2006), but whether this is a general effect on different types of cells has not been examined. 1.3 Chromatin modulation under hypoxia The last decade has seen an inspiring series of discoveries to show how chromatin environment contributes to regulation of gene expression. A major contributor to chromatin modulation is the modification at the N terminal tail of histones, which leads to changes in the accessibility of chromatin to transcription factors and coactivators. Generally, high levels of histone acetylation and histone H3 lysine 4 (H3K4) methylation at the promoter regions and histone H3 lysine 36 (H3 K36) methylation within the coding region are associated with active genes, while high levels of histone H3 lysine 27 (H3K27) trimethylation at the promoters and the intragenic regions correlate with gene repression ( Barski et al., 2007). Methylation on histone H3K9 is generally thought to be associated with transcriptional silencing and maintenance of heterochromatin (Kouzarides, 2007). Several members of Jumonji (JMJ) family histone demethylases were shown to be induced under hypoxia through HIF-1 signaling (Xia et al., 2009). Hypoxia upregulates JMJD1A and JMJD2B through HIF-1 , therefore demethylating H3K9 and activating HIF- 1 4 target genes (Beyer et al., 2008). JARID1C, another member of the Jumonji family and a histone H3K4 demethylase, was also reported to be a target of HIF-1, but it mediates a repressive effect of HIF-1 on its target genes (Niu et al., 201 1). Mutation of JARID1C was first characterized to be associated with X-linked mental retardation (lwase et al., 2007). However, recent research demonstrated its possible involvement in cancer, as inactivation of JARID1C was identified in human renal cell carcinoma (RCC) cell lines (Dalgliesh et al., 20 10). 1.4 Scope of this thesis Elevated RNA Pol Ill-dependent gene transcription contributes to oncogenesis. Hypoxia is an important phase of tumor development, but how RNA Pol Ill-dependent transcription is regulated in cancer cells under hypoxia has never been investigated. Our results show that tRNA transcription is suppressed under hypoxia in several cancer cell lines. Further interrogation into the mechanism of hypoxia repression of Pol Ill-mediated transcription revealed that HIF-1 is an essential mediator in this process. Meanwhile, a recent global analysis indicated that H3K4 is highly methylated on most transcriptionally active tRNA promoters (Barski et al., 20 10). Given that JARID1C is a histone H3K4 demethylase that is induced under hypoxic conditions, this raised the question of whether chromatin modification is able to mediate the HI F-dependent repression of Pol Ill activity. Another question is how the RNA pol Ill-dependent transcription factors might be regulated under hypoxic conditions. Given these considerations and that 5 TFIIIB is the target of many regulatory responses; we investigated the role of HIF- 1 and JARIDlC on Poll II transcription and TFIIIB under hypoxia. 6 CHAPTER 2 MATERIALS AND METHODS 2.1 Cell Culture MCF7, Huh7 cells were used in this study. MCF7 cell line is a breast cancer cell line isolated in 1970 from a 69-year-old Caucasian woman. Huh-7 is a well differentiated hepatocyte derived cellular carcinoma cell line that was originally taken from a liver tumor in a 57-year-old Japanese male in 1982. Cells were grown under standard and hypoxic conditions in Dulbecco modified Eagle medium (Mediatech). Medium was supplemented with 10% of fetal bovine serum (Omega Scientific), 1% of penicillin (Mediatech) and 1% of glutamate (Mediatech). The cells were then planted onto 100-mm culture dishes at a density of 1x106 cells/dish and cultured for 48 hours in normal or hypoxic environments. For hypoxia, cultures were incubated in a hypoxia incubator with a gas mixture containing 1% oxygen and 5% carbon dioxide at 37°(, balanced with nitrogen. Prior to placing the cells in the incubator, the culture medium was replaced with fresh medium. Cells under norm oxic conditions were treated in the same way, but using a gas mixture of 20% oxygen and 5% carbon dioxide at 3JCC . 2.2 Western immunoblotting analysis After the cells were incubated in normal or hypoxic conditions for a certain period of time, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and 200 f.ll cell lysis buffer[20 mM Tris pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1mM EGTA, 0.1% (w/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM �-glycerolphosphate, 1 mM sodium vanadate, Protease Inhibitor Cocktail Set Ill (Calbiochem), and Pierce Halt Inhibitor (Pierce)] was 7 added onto each 100 mm dish. Cell lysates were then collected by scraping off each dish after 5 minutes incubation on ice, sonicated until the cell lysate was clear, and centrifuged at 10,000 g for 15 minutes. Supernatant which contained total protein of the cells was transferred into a new 1.5 ml tube. Bio-Rad protein assay reagent was used to measure the protein concentration of the cell lysates. A standard curve was made by using different concentration of Bovine serum albumin (BSA) at 0, 0.25, 0.38, 0.5, 0.75, 1, 1.5, and 2 mg/ml to determine the loading volumes of each sample. A combination of samples, cell lysis buffer, �-mercaptoethanol and bromophenol blue dye was prepared for Western blot analysis. Each mixture was well mixed and heated at 100°( for 10 minutes to ensure complete disruption of tertiary protein structure. Mixtures were then spun down and subjected to sodium dodecyl sulfate polyacrylami/de gel electrophoresis (50S PAGE) (100 f.lg/ lane). For �-actin blots, samples were electrophoresed in 10% gels; for Brfl, JARID1C and HIF-1a blots, samples were electrophoresed in 6% gels. Hybond ECL nitrocellulose membrane (GE Healthcare) was used for transfer of protein from SDS-PAGE gels. For �-actin blots, the protein was transferred at 70 Volts for one hour; for Brfl, JARID1C and HIF-1a blots, the protein was transferred at 35 Volts for 14 hours. After transfer, the membranes were blocked in 5% non-fat milk (dissolved in TBS) at room temperature for one hour. For 13-actin blots, bound primary antibody was visualized using horseradish peroxidase-conjugated secondary antibody (Pierce) before application of Western Lightening-ECL (PerkinEimer). For Brfl, JARID1C and HIF-1a blots, bound primary antibody was visualized using U-Cor fluorescent secondary antibodies followed by exposure with Odyssey infrared imaging system. Quantification of bands in Western blots was done with UNSCANIT or 8 with Odyssey infrared imaging system, respectively. The primary antibodies (all of which are anti-human) were used as indicated and are as following: rabbit monoclonal Brf-1 antibody (Bethyl Laboratories), mouse monoclonal HIF-la (BD Bioscience), and mouse monoclonal actin antibody (Sigma). The dilutions of the antibodies are listed in Table 1. 2.3 Quantitative Real-time RT-PCR Total RNA was isolated from cells by using RNA STAT-60 (Tel-Test, Inc., Friendswood, Texas) according to the manufacturers' protocol, and the RNA concentration was determined using a spectrophotometer. The samples were then treated with Turbo DNA-free DNase (Ambion) to eliminate the DNA content. eDNA was synthesized using the SuperScript First Strand Synthesis System for RT-PCR (Invitrogen). RNA templates were mixed with 250 ng of random hexamers and 10 mM dNTPs in a total volume of 10 f.ll, and incubated at 65°C for 5 min. A master mix was then added to the RNA/random hexamers reaction for a total reaction volume of 20 f.ll containing 1" Strand buffer at lOX, 0.1 M DTT, 25 mM MgCI,, lOOU of RNase Out, and 100 U of SuperScript reverse transcriptase. The reaction mix was incubated at 25°C for 10 minutes, sooc for 50 minutes, and then at ssoc for 15 minutes. Real-time PCR analysis was performed on the Mx3000P QPCR System using brilliant SYBR Green QPCR Master Mix (Stratagene) or Kapa SYBR FAST qPCR kit according to the manufacturers' instructions. Primer sets for Pol Ill products, pre-tRNALeu, pre- tRNAiMet, U6, TBP, Brfl, Bdpl, VEGF and actin are listed in Table 2. Relative amounts of product transcripts were quantified using the comparative threshold cycle method (L:u'>Ct) with actin serving as the endogenous reference control. eDNA reverse transcribed as described above and derived from 9 human liver total RNA (Strategene) was used as a calibrator to control for differences between experiments. The results were then graphed. The sequences of the primers are listed in Table 2. 10 Table 1. Antibodies for Western Blot Target secondary dilution HIF-1 anti-rabbit 1:250 Brf1 anti-rabbit 1:1000 �-actin anti-mouse 1:5000 Table 2. Primers Sets for Quantitative RT-PCR Target Primers Annealing Temperature pre-tRNALeu (F) 5'-GTC AGG ATG GCC GAG TGG TCT AAG-3' 61°( (R) 5'-CCA CGC CTC CAT ACG GAG AAC CAG AAG ACC C-3' pre-tRNAiMet (F) 5'-CTG GGC CCA TAA CCC AGA G-3' 61°( (R) 5'-TGG TAG CAG AGG ATG GTT TC- 3' TBP (F) 5'-CTC AGG GTG CCA TGA CTC CCG-3' 55°( (R) 5'-TTG TTG TTG CTG CTG CTG CCT TTG-3' U6 (F) 5'GGA ATCTAG AAC ATA TAC TAAAAT TGG AAC-3' 61°( 11 (R) 5'-GGA ACT CGA GTT TGC GTG TCA TCC TTG CGC-3' Brf1 (F) 5'-CCT CGG GCC TCT GCG GAG CAG-3' 61°( (R) 5-TCA TCA ATG GTC AAC TGA CTG GTG G-3 Bdp1 (F) 5'-GCT GAT AGA GAT ACT CCT C-3' 61°( (R) 5'-CCA GAG ACA AGA ATC TTC TC-3' VEGF (F) 5'-TGC ACT GGA CCC TGG CTTTAC TGC-3' 61°( (R) 5'-GCA GCC CGC ACA CCG CAT T-3' �-actin (F) 5'-CGA CAA CGG CTC CGG CAT G -3' 55°( (R)5'-CTG-GGG-TGT-TGA-AGG-TCT-CAA-ACA-TG-3' 12 Chapter 3 Results 3.1 RNA Pol Ill-mediated transcription of tRNA, but not U6 RNA genes, is repressed under hypoxia To investigate how RNA polymerase Ill activity is regulated in hypoxic cancer cells, we incubated 2 cancer cell lines, MCF7 and Huh7 cells, in normoxia (21%02, 5%C02) or hypoxia (1%02, 5%C02) for 48 hours. HIF-1a was induced in cells incubated in hypoxia chamber (Figure 1), indicating the deficiency of oxygen in the cell culture. Both cell lines showed dramatic reduction in pre-tRNAi Met and pre-tRNA Le" transcription, but not U6 RNA (Figure 1 and data not shown). VEGF was used as a positive control, since it is a canonical hypoxia-inducible gene. Noticeably, tRNA tMet and tRNA Leo gene promoters are type 2 promoters, while the U6 RNA promoter is a type 3 promoter. These 2 types of promoters have different structure and utilize different transcription factors for transcriptional initiation, a fact that may serve as a possible explanation for the differential transcriptional responses of these promoters to hypoxic conditions. Overall, the data demonstrates that tRNA, but not U6 RNA synthesis is repressed under hypoxia. 13 A. Hypoxia + B. VEGF o Normoxic • Hypoxic Figure 1. RNA Po/ Ill-mediated transcription of tRNA, but not U6 RNA genes, is repressed under hypoxia. MCF7 cells were incubated under normoxia or hypoxia for 48 h. A). Protein isolated from the cells was subjected to immunoblot analysis with primary antibodies as indicated. B). Total RNA was isolated and reverse-transcribed, and qPCR was performed with primer sets as indicated {3 independent experiments). 3.2 Repression oftRNAgenetranscription under hypoxia is mediated by HIF-la HIF-1a is induced only under hypoxia, and it is one of the major transcription factors that mediate cellular response to hypoxia. Therefore, the question was raised whether HIF-1a mediates the repression of Pol Ill-dependent gene transcription under hypoxia. In order to explore this possible scenario, we infected Huh7 cells with shRNA 14 A. Hypoxia + + HIF-1a shRNA + + HIF-1a---- ] 13-actin� l B. Mel pre-tRNA1 Leu pre-tRNA .l!l 1 Q) 0.. a> ·c ffi 1il 1 £c: <..)� uf- -<( .f� Hypoxia + + + + HIF-1a shRNA + + + + VEGF U6RNA en «>.9. en ... c: 0 co en £ c: <..)� ul- -<( oz LJ...a::: Hypoxia + + + + HIF-1a shRNA + + + + Figure 2. Repression of tRNA gene transcription under hypoxia is mediated by HIF-1a. Huh7 cells were infected with lentivirus vectors encoding non-silencing RNA or shRNA targeting HIF-la, and then incubated under normoxia or hypoxia for 48 h. A). lmmunoblot analysis was performed using lysates prepared from Huh7 cells with primary antibodies as indicated. B). Total RNA isolated from those cells was subject to RT-qPCR, with primer sets as specified {3 independent experiments). 15 targeting HIF-la to block its induction, and then examined how this would affect RNA Pol Ill-dependent gene transcription under hypoxia. As shown in Figure 2B, tRNA transcription was repressed under hypoxia in control cells, but not in HIF-la-deficient cells. On the other hand, transcription of U6 RNA was not significantly affected by either hypoxia or HI F-la silencing. This data confirms that the repression of tRNA transcription under hypoxia is dependent on HI F-la. 3.3 Brfl expression is repressed under hypoxia and it is HIF-la-dependent Brfl is a key component of TFIIIB complex, which is recruited on tRNA promoters, but not on U6 RNA promoters, to initiate transcription by RNA Poll II. Brfl is also a critical regulatory point by a number of cellular signaling pathways that modulates RNA Pol Ill transcriptional activity. Given the fact that tRNAs but not U6 RNA transcription is subject to HIF-la-dependent repression under hypoxia, Brfl could be a downstream target of regulation in this process. Therefore, immunoblot analysis was performed to examine Brfl protein expression under normoxia or hypoxia. It turned out that the expression of Brfl, but not TBP or Bdpl, which are the other 2 components of TFIIIB, was repressed under hypoxia (Figure 3A). However, Brfl amounts did not change under hypoxia in cells infected with HIF-la-targeting shRNA (Figure 3B). This data shows that there is an HIF-la-dependent repression of Brfl expression under hypoxia, which is consistent with the fact that the transcription of tRNAs, but not U6 RNAs, are repressed in a HIF-la-dependent manner 16 A. Hypoxia B. + ,... 1- TBP 1- -j-. Brf1 L__ _: · � � - HIF-1 a 1- 1- Actin H IF-1 a shRNA Hypoxia + + 1.5 Cl) 0) c � 1.0 0 '0 0 0.5 LL. + + Brf1 TBP .__ ______ __,J I � Brf 1 .__ _______ _, I .._ HIF-1 a ._l- _______ - __,1 � t3-acti n Fold ll Brf1: 1.00 0.84 0.66 0.95 o Normoxic • Hypoxic Cl) 0) 1.5 ; 1.0 .s::; 0 '0 0.5 0 LL. Bdp1 Figure 3. Br/1 expression is repressed under hypoxia and it is HIF-1 a-dependent. A). MCF7 cells were incubated under normoxia or hypoxia for 48 h. The lysates were subject to immunoblot analysis using primary antibodies as indicated. B). Huh7 cells infected with mismatched RNA or shRNA tar geting HIF-la were incubated under normoxia or hypoxia for 48 h period. The lysates were subject to immunoblot analysis with primary antibodies as indicated. 17 under hypoxia. This suggests that Brfl repression under hypoxia contributes to the reduced transcription of tRNA. 3.4 Decreased expression of JARIDlC alleviated the hypoxia-mediated reduction in Brfl expression, but not repression of RNA Pol Ill-mediated transcription A recent genome-wide study reported the association of H3K4me3 markers on only active tRNA promoters (Barski. 2010). Coincidentally, a histone H3K4 demethylase, JARID1C, was found to be induced by HIF-1 signaling, and is required to repress a subset of the HIF- 1 target genes (Niu, 201 1). These results provide evidence to support a hypothesis that JARID1C is induced under hypoxia, removing the H3 K4me3 marks on tRNA promoters and repressing tRNA gene transcription. Therefore, we silenced the expression of JARID1C in Huh7 cells with shRNA targeting JARID1C and examined RNA Pol Ill-dependent gene transcription in those cells. As shown in Figure 4B, JARID1C was expressed at a low level under normoxia, and was dramatically induced under hypoxia. The shRNA silenced the expression of JARID1C in both normoxia and hypoxia. Quantitative RT-PCR results showed that consistent with previous data, pre-tRNAi Met and pre-tRNA Leo synthesis was repressed under hypoxia, but knockdown of JARID1C expression did not restore it to its level under nomoxia (Figure 4A). However, surprisingly, Brfl expression, which is repressed under hypoxia, was shown to be rescued in the JARID1C knockdown cells (Figure 4B). This indicates JARID1C, a downstream target of HIF-1a, is also required to repress Brf1 expression 18 A. pre-tRNA� B. JARID1C shRNA - + Hypoxia I I· 1- - c 0 Q) ·u; 0>1/1 c � "'a. .C X (.)., :2 � 0 .fro .s 0 JARID1 C shRNA + Hypoxia + - - + Leo pre-IRNA - + + + + + + ,.._JARID1C -1�Brfl -I� {3-actln + + Figure 4. Decreased expression of JARID1C alleviated the hypoxia-mediated reduction in Brf1 expression, but not repression of RNA Pol Ill-mediated transcription. Huh7 cells infected with empty vector or shRNA targeting JARID1C were incubated under normoxia or hypoxia for 48 h. A). Total RNA was isolated and reverse-transcribed; the subsequent eDNA was subjected to qPCR with primer sets as indicated {3 independent experiments); B). The lysates were subject to immunoblot analysis with primary antibodies as indicated. Quantification of bands Brfl bands is displayed on the bottom {3 independent experiments). 19 under hypoxia, but simply reducing JARIDlC expression is not sufficient to abrogate the repression of tRNA gene transcription observed under hypoxia. 20 Chapter 4 Discussion 4.1 Repression of tRNA gene transcription under hypoxia is a common phenomenon in different cell types RNA Polymerase Ill (RNA Pol Ill) products are essential for protein synthesis, protein trafficking and RNA processing, and its deregulation leads to oncogenesis and neoplastic transformation (Dieci et al., 2007; Johnson and Johnson, 2008). tRNA synthesis is elevated in cancer cells. However, how RNA Pol Ill is regulated under hypoxia, a common phase of solid tumor development, has never been examined. An initial study in cardiomyocytes suggested that RNA Pol Ill-dependent gene transcription is suppressed under hypoxia (Ernens et al., 2006). Our study demonstrated that synthesis of tRNAs, but not U6 RNAs, is repressed under hypoxia in two cancer cell lines (Figure 1), implying that hypoxia-mediated repression of tRNA gene transcription also occurs in cancer cells. 4.2 Multiple mechanisms are contributing to hypoxia-induced repression of tRNA gene transcription Hypoxia has a wide effect on cell behavior and intracellular signaling. Therefore, multiple hypoxia-induced events could contribute to the repression of tRNA gene transcription. 21 We observed that HIF-la is required for the repression of Brfl under hypoxia, and that neither TBP nor Bdpl expression is changed under hypoxia. Given the fact that Brfl is involved in tRNAs but not U6 RNAs gene transcription, while TBP is required for both tRNAs and U6 RNAs gene transcription, this result is consistent with our data that only the transcription of tRNA genes, but not U6 RNA genes, is repressed under hypoxia. Therefore, the regulation of Brfl could be critical in hypoxia-mediated effect on RNA Pol Ill transcription. However, the other results in this study did not support this deduction. JARIDlC is a histone demethylase targeting H3 K4me3. JARIDlC is induced by HIF signaling and is required for the suppression of several HIF-downstream target genes (Niu et al., 201 1). Since high levels of H3K4me3 markers cluster on promoter regions of active tRNA genes (Barski et al., 2010), we proposed that under hypoxia, JARIDlC is induced through HIF signaling, and therefore contributes to decreased H3 K4me3 levels on tRNA promoters and a reduction in tRNA gene transcription. Our data showed that JARIDlC is induced under hypoxia, contributing to repression of Brfl expression (Figure 4). It is possible that JARIDlC is recruited to Brfl promoter regions and demethylates the local trimethyl markers on Histone H3K4 residues, therefore inhibiting the transcription of Brfl mRNA. However, silencing of JARIDlC under hypoxia is not sufficient to restore the tRNA level, suggesting other events that take place under hypoxia are attributable to hypoxic repression of tRNA gene transcription, and this is why silencing JARIDlC is not sufficient to restore tRNA 22 gene transcription under hypoxia. How JARIDlC affect the chromatin environment on tRNA promoters remains to be examined. A possible mechanism that may play a role in HIF-la-mediated response of Pol Ill-dependent gene transcription has to do with the interaction of HIF and c-Myc. It was shown that c-Myc binds to Brfl and thus activates RNA Pol Ill transcription (Gomez Roman et al., 2003). HIF-la was reported to bind to and repress the activity of c-Myc protein, resulting in inhibition of mitochondrial biogenesis in VHL-deficient renal cell carcinoma cell lines (Zhang et al., 2007). Our data indicated that HIF-la is required for repression of tRNA synthesis under hypoxia (Figure 2), and that HIF-la also inhibits expression of Brfl under hypoxia (Figure 3). Therefore, a reasonable inference is that under hypoxia, due to the induction of HI F-la, c-Myc activity is inhibited, leading to the abrogation of c-Myc-stimulated Pol Ill transcription. In contrast, HIF-2a, which is also induced under hypoxia, is reported to interact with c-Myc, too, but resulting in enhancing of c-Myc activity (Gordan et al., 2007). Balancing of the counter effects of HIF-la and HIF-2a may be cell-type specific and has to do with the pathological background of different cell lines. A second prospect that may contribute to the hypoxia-mediated repression of RNA Pol Ill-dependent gene transcription observed concerns the regulation of RNA Pol Ill-dependent transcription by the PI3K pathway. The tumor suppressor PTEN is a negative regulator of PI3K pathway as well as Pol Ill-dependent transcription (Woiwode 23 et al., 2008). PTEN decreases the phosphorylation state of Brfl and disrupts the interaction of Brfl with TBP. Intriguingly, in TSC-/-cells, PTEN expression was shown to be controlled by HIF-la, with HRE elements present on the PTEN promoter (Mahimainathan et al., 2009). These results raise the possibility that under hypoxia, HIF la is induced, increasing the level of PTEN, resulting in inhibition of Brfl activity by dephosphorylating it. This scenario is especially interesting given our data that restoration of Brfl expression by silencing JARIDlC expression under hypoxia is not sufficient to bring back tRNA transcription to its level found under normoxic conditions. Hypoxia might lead to dephosphorylation as well as reduced expression of Brfl, with abrogation of both events required for restoration of t RNA synthesis. 4.3 Summary In summary, we have confirmed the repression of RNA Pol Ill-dependent gene transcription under hypoxia in several cancer cell lines. We identified a critical role of HIF-la in mediating this effect. We provided evidence that HIF-la regulates RNA Pol Ill dependent gene transcription, and that it represses the expression of Brfl, a critical transcription factor for tRNA transcription, under hypoxia. We also investigated the role of JARIDlC, a downstream target of HIF-la, in hypoxia-mediated repression of RNA Pol Ill-dependent gene transcription, and elucidated it as a regulator of Brfl expression. Although abrogation of the induction of JARIDlC is sufficient restore Brfl expression, it does not restore RNA Pol Ill-dependent tRNA gene transcription under hypoxia. It 24 suggests that it requires signaling events other than JARIDlC downstream of HIF-la to repress tRNA gene transcription, and that overexpression of Brfl might not be sufficient to restore tRNA gene transcription under hypoxia. In a broader sense, elevated RNA Pol Ill-dependent activity is usually associated with cell growth and proliferation, and vice versa. Tissue hypoxia, whether as a result of physiological or pathological changes, always poses stress on cells. Therefore, it is natural for the cells to respond with a regression of growth and proliferation in the form of repression of RNA Pol Ill-dependent gene transcription. As indicated in our results, HIF-la, likely through its downstream targets, plays a critical role in the cells' response to hypoxia. In the dynamic process of tumor hypoxia, angiogenesis and vasculagenesis is enhanced by the HIF-stimulated expression of VEGF. However, in the initial phase of hypoxia, HIF-la-mediated repression of RNA Pol Ill-dependent gene transcription might serve as a pro-survival adaption to the adverse nutrient conditions. On the other hand, in cells with elevated HIF signaling under normoxia, blockade of HIF or its downstream signaling could lead to elevated RNA Pol Ill-dependent gene transcription, therefore poising the cells toward oncogenic transformation. For example, in clear cell VHL-deficient renal cell carcinoma (ccRCC), HIF is induced under normoxia in the absence of VHL. Interestingly, inactivating mutations of JARIDlC were identified in a sub-group of ccRCC cases (Dalgliesh et al., 20 10), raising the idea that those cases might display elevated tRNA transcription, facilitating oncogenic transformation. In our study, 25 blocking of JARIDlC is not sufficient to restore the activity of RNA Pol Ill-dependent gene transcription under hypoxia, but this might not be always true for other cancer cells with different genetic backgrounds. Since our results suggest multiple signaling events downstream of HIF-la are very likely to be involved in the hypoxia-mediated repression of RNA Pol Ill-dependent gene transcription, blockade of any of these events might partially elevate tRNA transcription. Given all these considerations, how HIF-la-mediated repression of RNA Pol Ill transcription under hypoxia affects the fate of cancer cells generally is very likely to be dependent on distinct genetic background and environmental factors of individual cell types. In vivo studies are required to further elaborate these mechanisms. 4.4 Future work and directions The future work for this study will focus on elucidating the mechanism of how RNA Pol Ill-dependent transcription is repressed under hypoxia. We have shown that HIF-la is required for hypoxia-mediated repression of tRNA transcription, and that silencing of HIF-la alone is sufficient to restore tRNA gene expression level under hypoxia (Figure 2). Based on this piece of information, two important aspects of the mechanistic details need to be investigated. The first is whether HIF-la regulates the transcription of tRNAs directly, e.g., whether HIF-la is recruited on the promoters of tRNA genes. This is an especially interesting question given that HI F-la is also required for the repression of Brfl expression under hypoxia, 26 suggesting that HIF-1a might also be able to regulate RNA Pol Ill activity through regulating Brfl transcription. Another important aspect for future studies is to identify the downstream targets of HIF- 1 signaling that represses RNA Pol Ill transcription under hypoxia. JARID1C, as a downstream target of HIF, was shown not to be responsible for repression of RNA Pol Ill-dependent transcriptioin. However, it remains possible that chromatin environment at tRNA promoter regions are changed under hypoxia, which contributes to the inhibition of transcription. As JARID1C was shown to be required for repression of Brfl under hypoxia, this raises the idea that JARID1C might partially regulate tRNA transcription indirectly through regulating Brfl expression. In either case, the H3K4 methylation status markers need to be examined on tRNA or Brf1 promoter regions under hypoxia to confirm the involvement of chromatin remodeling in regulating RNA Pol Ill-dependent gene transcription. In addition, the possibility of JARID1C is directly recruited on those promoter regions also needs to be examined. Another downstream target of HIF- 1 that might be involved in repressing tRNA transcription is PTEN. In order to explore this scenario, we first need to confirm that PTEN is overexpressed under hypoxia. Then, we may utilize some PTEN-null cancer cell lines to investigate if PTEN is required for repression of tRNA synthesis under hypoxia. In addition, the phosphorylation status of Brf1 under hypoxia or normoxia should also be examined. We have shown that JARID1C is crucial in controlling Brfl expression under 27 hypoxia (Figure 4); however, restoration of Brfl under hypoxia in JARIDlC-silenced cells is not sufficient to restore tRNA transcription. An explanation for this phenomena is that Brfl phosphorylation, as well as its expression, is inhibited under hypoxia, due to the overexpression of PTEN. Therefore, simply restoring the amount of Brfl is not sufficient to restore its function. To this end, we might need to examine whether tRNAs gene transcription can be restored under hypoxia by silencing JARIDlC in PTEN-deficient cells. The final set of experiments that needs to be done is to explore the interaction between c-Myc and HIF-la under hypoxia. How HIF-la affects the recruitment c-Myc on tRNA promoters under hypoxia should be examined. Then how HIF-la interacts with c Myc on tRNA promoters would be explored. 28 References Barski, A., Chepelev, 1., Liko, D., Cuddapah, S., Fleming, A. B., Birch, J., Cui, K., White, R. J., and Zhao, K. (2010). Poll I and its associated epigenetic marks are present at Pol Ill transcribed noncoding RNA genes. Nature structural & molecular biology 17, 629-634. Barski, A., Cuddapah, S., Cui, K., Roh, T. Y., Schones, D. E., Wang, Z., Wei, G., Chepelev, 1., and Zhao, K. (2007). High-resolution profiling of histone methylations in the human genome. Cell 129, 823-837. Beyer, S., Kristensen, M. M., Jensen, K. S., Johansen, J. V., and Staller, P. (2008). The histone demethylases JMJD1A and JMJD2B are transcriptional targets of hypoxia inducible factor HI F. Journal of Biological Chemistry 283, 36542. Crighton, D., Woiwode, A., Zhang, C., Mandavia, N., Morton, J. P., Warnock, L. J., Milner, J., White, R. J., and Johnson, D. L. (2003). p53 represses RNA polymerase Ill transcription by targeting TB P and inhibiting promoter occupancy by TFIIIB. The EMBO journal 22, 2810-2820. Dalgliesh, G. L., Furge, K., Greenman, C., Chen, L., Bignell, G., Butler, A., Davies, H., Edkins, S., Hardy, C., and Latimer, C. (2010). Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 463, 360-363. Dieci, G., Fiorino, G., Castelnuovo, M., Teichmann, M., and Pagano, A. (2007). The expanding RNA polymerase Ill transcriptome. TRENDS in Genetics 23, 614-622. Ernens, 1., Goodfellow, S. J., Innes, F., Kenneth, N. S., Derblay, L. E., White, R. J., and Scott, P. H. (2006). Hypoxic stress suppresses RNA polymerase Ill recruitment and tRNA gene transcription in cardiomyocytes. Nucleic acids research 34, 286-294. Felton-Edkins, Z. A., Fairley, J. A., Graham, E. L., Johnston, I. M., White, R. J., and Scott, P. H. (2003). The mitogen-activated protein (MAP) kinase ER K induces tRNA synthesis by phosphorylating TFIIIB. The EMBO journal 22, 2422-2432. Gomez-Roman, N., Grandori, C., Eisenman, R. N., and White, R. J. (2003). Direct activation of RNA polymerase Ill transcription by c-Myc. Nature 421, 290-294. Gordan, J. D., Bertout, J. A., Hu, C. J., Diehl, J. A., and Simon, M. C. (2007). HIF-2 [alpha] promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity. Cancer cell 11, 335-347. 29 lwase, S., Lan, F., Bayliss, P., de Ia Torre-Ubieta, L., Huarte, M., Qi, H. H., Whetstine, J. R., Bonni, A., Roberts, T. M., and Shi, Y. (2007). The X- Linked Mental Retardation Gene< i> SMCX/JARIDlC</i> Defines a Family of Histone H3 Lysine 4 Demethylases. Cell 128, 1077- 1088. 29 Johnson, D., and Johnson, S. (2008). Cell biology. RNA metabolism and oncogenesis Science 320, 461-462. Johnson, S. A. S., Dubeau, L., Kawalek, M., Dervan, A., Schonthal, A. H., Dang, C. V., and Johnson, D. L. (2003). Increased expression of TATA-binding protein, the central transcription factor, can contribute to oncogenesis. Molecular and cellular biology 23, 3043-305 1. Johnson, S. S., Zhang, C., Fromm, J., Willis, I. M., and Johnson, D. L. (2007). Mammalian Mafl is a negative regulator of transcription by all three nuclear RNA polymerases. Molecular cell 26, 367-379. Kaelin Jr, W. G. (2008). The von Hippei-Lindau tumour suppressor protein: 02 sensing and cancer. Nature Reviews Cancer 8, 865-873. Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705. Larminie, C. G. C., Cairns, C. A., Mital, R., Martin, K., Kouzarides, T., Jackson, S. P., and White, R. J. (1997). Mechanistic analysis of RNA polymerase Ill regulation by the retinoblastoma protein. The EMBO journal 16, 2061-2071. Mahimainathan, L., Ghosh-Choudhury, N., Venkatesan, B., Das, F., Mandai, C. C., Dey, N., Habib, S. L., Kasinath, B. S., Abboud, H. E., and Choudhury, G. G. (2009). TSC2 deficiency increases PTEN via HI Fla. Journal of Biological Chemistry 284, 27790-27798. Niu, X., Zhang, T., Liao, L., Zhou, L., Lindner, D., Zhou, M., Rini, B., Van, Q., and Yang, H. (20 11). The von Hippei-Lindau tumor suppressor protein regulates gene expression and tumor growth through histone demethylase JARIDlC. Oncogene. Schramm, L., and Hernandez, N. (2002). Recruitment of RNA polymerase Ill to its target promoters. Genes & development 16, 2593-2620. Semenza, G. L. (2003). Targeting HIF- 1 for cancer therapy. Nature Reviews Cancer 3, 721-732. 30 Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-hel ix-PAS heterodimer regulated by cellular 02 tension. Proceedings of the National Academy of Sciences 92, 5510. Wilson, W. R., and Hay, M. P. (20 11). Targeting hypoxia in cancer therapy. Nature Reviews Cancer 11, 393-4 10. Woiwode, A., Johnson, S. A. S., Zhong, S., Zhang, C., Roeder, R. G., Teichmann, M., and Johnson, D. L. (2008). PTEN represses RNA polymerase Ill-dependent transcription by targeting the TFIIIB complex. Molecular and cellular biology 28, 4204-4214.30 Xi a, X., Lemieux, M. E., Li, W., Carroll, J. S., Brown, M., Liu, X. S., and Kung, A. L. (2009). Integrative analysis of HIF binding and transactivation reveals its role in maintaining histone methylation homeostasis. Proceedings of the National Academy of Sciences 106, 4260. Zhang, H., Gao, P., Fukuda, R., Kumar, G., Krishnamachary, B., Zeller, K. 1., Dang, C. V., and Semenza, G. L. (2007). HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer cell 11, 407-420. Zhong, S., Zhang, C., and Johnson, D. L. (2004). Epidermal growth factor enhances cellular TATA binding protein levels and induces RNA polymerase l-and Ill-dependent gene activity. Molecular and cellular biology 24, 51 19-5129. 31

Abstract (if available)

Abstract Deregulated RNA Polymerase III (Pol III)-dependent genes transcription is linked to oncogenesis. In this study, we investigated the response of RNA Pol III-dependent gene transcription to hypoxia, a common situation encountered by many solid tumors during their development. We demonstrate in several cancer cell lines that RNA Pol III-dependent transcription of tRNAs, but not U6 RNAs, is repressed under hypoxia. Using gene-silencing techniques, we show that hypoxia-mediated repression of tRNA gene transcription is dependent on the Hypoxia-Inducible Factor (HIF)-1α, a transcription factor that is induced only in oxygen-deficient environment. Furthermore, the expression of Brf1, a critical transcription factor that is required for initiation of tRNA, but not U6 RNAs genes transcription, is repressed under hypoxia in a HIF-1α-dependent manner, which may explain the HIF-1α-dependent repression of tRNA gene transcription. Finally, the histone H3K4 demethylase JARID1C, a downstream target gene of HIF, is shown to be required for repression of Brf1 expression under hypoxia

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

RNA polymerase III-dependent transcription is repressed under prolonged hypoxic conditions

PDF

SUMOylation regulates RNA polymerase III -- dependent transcripton via MAF1

PDF

Positive regulation of RNA polymerase III-mediated transcription of tRNA genes by the Mediator kinase submodule

PDF

Repression of RNA polymerase III-dependent transcription by the tumor suppressors p53 and PTEN

PDF

Placental growth factor mediated transcriptional and post-transcriptional regulation of hemeoxygenase-1

PDF

Novel roles for Maf1 in embryonic stem cell differentiation and adipogenesis

PDF

A novel role for hypoxia-inducible factor-1alpha (HIF-1alpha) in the regulation of inflammatory chemokines and leukotriene expression in sickle cell disease

PDF

Transcriptional regulation of IFN-γ and PlGF in response to Epo and VEGF in erythroid cells

PDF

Study of the mechanism by which hypoxia-inducible-factor-1 alpha (HIF-1α) regulates heat shock protein-90 alpha (Hsp90α) secretion in breast cancer

PDF

Characterization of a fragment in secreted Hsp90α with potential therapeutic benefits in wound healing and cancer

PDF

Placenta growth factor-miRNAs-lncRNAs axis in the regulation of ET-1 gene involved in pulmonary hypertension in sickle cell disease

PDF

The role of miRNA and its regulation in pulmonary hypertension in sickle cell disease

PDF

Synthetic small molecules and protein secondary structure mimetics as modulators of hypoxia-inducible transcription and integrin receptors function

PDF

Creating a multiple micrornia expression vector to target GRP78, an ER chaperone and signaling regulator in cancer

PDF

Lnc-RNA and WNT signal synergistically promotes self-renewal of tumor initiating stem-like cells via epigenetic regulation

PDF

Do ZFX and ZNF711 regulate the same genes in HEK293T cells?

PDF

The mechanisms of somatic cell reprogramming

PDF

Transcriptional regulation in nephron progenitor cells

PDF

The relationship between DNA methylation and transcription factor binding in colon cancer cells

PDF

Modulation of transcription and receptor function with synthetic small molecules and multi-finctional integrin ligands

Asset Metadata

Creator Guo, Qiuyu (author)

Core Title Mechanism of repression of RNA polymerase III-dependent transcription under hypoxia

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 11/29/2012

Defense Date 07/19/2012

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

Tag cancer,HIF-1alpha,hypoxia,OAI-PMH Harvest,RNA polymerase III

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Johnson, Deborah L. (committee chair), Machida, Keigo (committee member), Stallcup, Michael R. (committee member)

Creator Email qiuyuguo@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-124902

Unique identifier UC11291218

Identifier usctheses-c3-124902 (legacy record id)

Legacy Identifier etd-GuoQiuyu-1371-1.pdf

Dmrecord 124902

Document Type Dissertation

Rights Guo, Qiuyu

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA