Cloning and characterization of the promoter of the cancer associated gene, FAT10

CLONING AND CHARACTERIZATION OF THE PROMOTER OF THE CANCER-ASSOCIATED GENE, FAT10 ZHANG DONGWEI NATIONAL UNIVERSITY OF SINGAPORE 2007 CLONING AND CHARACTERIZATION OF THE PROMOTER OF THE CANCER-ASSOCIATED GENE, FAT10 ZHANG DONGWEI (M. Sc.) Wuhan Institute of Virology Chinese Academy of Science A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to express my greatest gratitude to my supervisor Dr. Caroline Lee for her excellent supervision, encouragement, patience and unfailing support throughout the course of this work, and for her invaluable amendments to this thesis. I would like to thank the past and present members in CFG laboratory: Ren Jianwei, Wang Baoshuang, Tan Kun, Wang Jingbo, Wang Zihua, Alvin Lee, Xiao Peiyun, Wang Yu, Wang Lipeng and Alison Kan for their kind concern, helpful discussion, technical assistance, cooperation, and friendship. Specially thank Dr. Grace Pang for English correcting. My heartfelt and deepest appreciation goes to my wife, Tian Jing, for her love, patience, understanding, and support over these years. I also would like to thank my beloved daughter Esther, for the joy and happiness she brings me. Last, but certainly not the least, this thesis is dedicated to my parents, for their unwavering support, encouragement and belief in me always. Zhang Dongwei February 2007 Table of Contents ii TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF FIGURES vi LIST OF TABLES xiii LIST OF ABREVIATIONS ix LIST OF PUBLICATIONS xi SUMMARY xii CHAPTER I 1.1 INTRODUCTION Role of Ubiquitin in posttranslational modification 1 2 1.1.1 Ubiquitin and ubiquitylation 1.1.2 Ubiquitin-like protein family 1.1.3 The function of ubiquitin-like 2 3 4 1.2 6 General Information about FAT10 1.2.1 FAT10 as a new member of UBL family 1.2.2 FAT10 and immune response 1.2.3 FAT10 and tumorigenesis 1.2.3.1 FAT10 is overexpressed in tumour tissue 1.2.3.2 FAT10 and the chromosomal stability 1.3 Research objectives Specific Aim 1: Isolated and Characterize the Promoter of the FAT10 Gene Specific Aim 2: Determine if p53 plays a role in the regulation of FAT10 at the transcript level. Specific Aim 3: Evaluate if mutations/polymorphisms or differential methylation at the FAT10 promoter can account for the aberrant over-expression of FAT10 in the tumors of HCC patients. 6 8 10 10 11 13 13 15 16 Table of Contents CHAPTER II ISOLATION AND CHARACTERIZATION OF THE PROMOTER OF FAT10 GENE iii 18 2.1 Background 19 2.2 Materials and Methods 20 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 Cell lines and Transfection Reverse Transcript PCR DNA sequencing reaction Determination of FAT10, p53 and β-actin protein levels Identification of promoter region in the FAT10 gene Identification of IFN-g and TNF-a responsive domain in FAT10 promoter region 20 20 22 23 23 27 Results 28 2.3 2.3.1 FAT10 promoter resides at the 5’UTR 2.3.2 The responsive domain of the FAT10 promoter to TNF-α and IFN-γ resides in the region upstream of FAT10 promoter 2.3.3 FAT10 promoter activity is different in different cell lines 28 2.4 37 Discussion CHAPTER III EVALUATION OF THE ROLE OF P53 IN THE REGULATION OF FAT10 AT THE TRANSCRIPT LEVEL 32 35 40 3.1 Background 41 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 Materials and Methods Cell lines and Transfection Quantitation of FAT10, p53, p21 and MDM2 and β-actin transcript Generation of siRNA constructs against p53 Generation of construct pFATgal-CMVp53 Determination of the binding of p53 to FAT10 promoter region using DNA and chromatin immuno-precipitation (DIP and ChIP) 43 43 43 44 46 48 Table of Contents 3.3 Results iv 50 3.3.1 p53 negatively regulates FAT10 promoter activity. 1.1.1 p53 binds to the 5’ half consensus sequence of p53 binding site of the FAT10 promoter and plays a role in the responsiveness of FAT10 promoter to p53 58 3.4 66 Discussion 50 CHAPTER IV EVALUATION OF THE ROLE OF MUTATIONS/POLYMORPHISM OR DIFFERENIAL METHYLATION AT THE FAT10 PROMOTER IN ACCOUNTING FOR THE ABERRANT OVER-EXPRESSION OF FAT10 IN THE TUMORS OF HCC PATIENTS 69 4.1 Background 70 4.2 Materials and Methods 72 4.2.1 4.2.2 4.2.3 4.2.4 Patient samples Isolation of genomic DNA and RNA from tissue samples Identification of Mutations/Polymorphisms through DNA sequencing Determination of the Methylation Status at the FAT10 promoter using Methylation-Specific Sequencing 72 72 72 4.3 Results 75 4.3.1 The expression levels of FAT10 in tumour tissue are much higher than adjacent non-tumorous liver tissues Only polymorphisms, but not mutations were identified in the ~1.3kb region of FAT10 promoter Differential Methylation at the FAT10 promoter was observed between tumour and adjacent normal liver tissues of HCC patients 4.3.2 4.3.3 4.4 Discussion 4.4.1 No mutations at the FAT10 Promoter were observed. 4.4.2 Differential methylation may account for the differences in FAT10 gene expression between HCC tumor and adjacent non-tumorous tissues 73 75 77 84 89 89 91 Table of Contents v CONCLUSION 93 REFERENCES 94 PUBLICATION Table of Contents vi LIST OF FIGURES Figure 1 Genomic structure of FAT10 gene 14 Figure 2 The strategy for TA-cloning of FAT10 promoter 25 Figure 3 Strategy for mutating the TATA box in FAT10 promoter region 26 Figure 4 Characterization of FAT10 promoter region 29 Figure 5 The diagram of FAT10 promoter that confers the highest promoter activity with important putative binding sites 30 Induction of different truncated FAT10 promoter by IFN-γ and TNF-α. 33 FAT10 promoter can not be induced by IFN-γ and TNF-α Synergistically 34 Figure 8 FAT10 promoter is more active in some cell-lines than others 36 Figure 9 The strategy to construct the SiRNA specific to p53 45 Figure 10 Strategy for making construct pFAT/β-gal-CMV/p53 47 Figure 11 p53 repression of FAT10 promoter activity 51 Figure 12 Repression of FAT10 transcription level in Hep3B cells by p53 52 Figure 13 Release the repression of p53 to FAT10 promoter by pH1-Sip53 in Hep3B cells 54 FAT10 promoter activity is enhanced by the addition of siRNA against p53 in p53+/+ KB3-1 cells and 293 cells 56 Endogenous FAT10 transcriptional level is also related to endogenous p53, along with other p53 regulated genes 57 Delineation of regions in FAT10 promoter that is responsible for the responsiveness of the promoter to p53 61 FAT10 promoter region emcompassing the p53 binding half site binds p53 in vivo 64 p53 binding the chromatin of the FAT10 promoter region emcompassing the p53 binding half site 65 Figure 6 Figure 7 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Table of Contents Figure 19 vii Comparison of FAT10 transcript level in tumor tissues and adjacent non-tumorous tissues 76 Figure 20 SNPs in FAT10 promoter 79 Figure 21 FAT10 Promoter activity with different haplotype structures 82 Figure 22 The CG dinucleotides in FAT10 promoter region 86 Figure 23 The strategy for identifying the methylation status of CG dinucloetides in FAT10 promoter 87 Table of Contents viii LIST OF TABLES Table 1 Table 2 Table 3 Oligonucleotides for amplification of different length of fragments upstream of the transcription start site (TSS) or translation start site (TLSS) of FAT10 gene 24 Single nucleotide polymorphisms identified in the 5’-flanking region of FAT10 80 The methylation status of CG dinucleotides in FAT10 promoter in HCC patient tissues 88 Table of Contents LIST OF ABREVIATIONS aa amino acid Ab antibody β-gal β-galactosidase bp base pair ChIP Chromatin Immunoprecipitation CIN Chromosome Instability CMV Cytomegalovirus CPRG Chlorphenol red- β-D-galactopyranoside DEPC Diethyl pyrocarbonate DIP DNA immunoprecipitation EBV Epstein-Barr virus EGFP Enhanced Green Fluorescent Protein DMEM Dulbecco’s modified Eagle’s medium dNTP deoxyribonucleotide triphosphate HCC Hepatocellular Carcinoma hr hour IFN-γ Interferon-γ Kb kilo base pair KDa kilo Dalton MHC Major histocompatibility complex mins minutes ng nanogram O/N overnight ix Table of Contents ORFs Open reading frames PBS phosphate-buffered saline PCR polymerase chain reaction RT room temperature RT-PCR reverse transcription polymerase chain reaction Sec Second SiRNA Small interfering RNA SNP Single Nucleotide Polymorphism TNF-α Tumour Necrosis Factor- α TLSS Translation Start Site TSS Transcription Start site UBL Ubiquitin-like modifiers UTR Untranslated Region x Table of Contents xi LIST OF PUBLICATION S Zhang DW, Jeang KT, Lee CG. p53 negatively regulates the expression of FAT10, a gene upregulated in various cancers. Oncogene. 2006 Apr 13;25(16):2318-27. Lim CB, Zhang D, Lee CG. FAT10, a gene up-regulated in various cancers, is cellcycle regulated. Cell Div. 2006 Sep 8;1:20 Table of Contents xii SUMMARY FAT10 is a member of the ubiquitin-like modifier (UBL) family of proteins and has been implicated to play important roles such as antigen presentation, cytokine response, apoptosis and mitosis. Recently, our laboratory reported that the FAT10 gene is up regulated in 90% of hepatocellular carcinomas and over-expression of FAT10 gene may lead to chromosomal stability. As part of the studies to elucidate the mechanism behind FAT10 gene regulation, we identified and characterized the promoter of the FAT10 gene. We found that the 5’UTR, from the transcription start site to 15 bases before the start codon, displayed significant promoter activity. Regions upstream of the 5’UTR (from +26 to -1997) did not confer any promoter activity. As FAT10 expression was reported to be induced by cytokines, we also explored the role of the FAT10 promoter in cytokine responsiveness. We found that the distal promoter region, -1716 to -975, was highly responsive to interferon- γ and tumour necrosis factor-α with 4~5 times higher expression upon treatment with cytokines. FAT10 promoter activity and expression is significantly repressed in KB3-1 and HepG2 cells, which have wild-type p53, but not in p53-negative Hep3B cells. The role of p53 in regulating FAT10 expression was evident by the significant downregulation (PG), SNP5 (e1 –143 A>G) and SNP3 (5’UR –616 T>C), have high major allele frequency of greater than 10%, while the others including the two novel SNPs (SNP4 5’UR -169 C>T and SNP2 5’UR –876 G>A) were of low allele frequency (T) were observed in both the DNA of HCC samples and normal samples (Table 2). However, the other two low frequency SNPs, SNP1 (5’UR –914 Chapter IV Evaluation of the role of mutations/polymorphisms and differential methylation Figure 20. SNPs in FAT10 promoter. (A) The location of SNPs in FAT10 promoter. There are six SNPs identified in ours and previous studies in FAT10 promoter, three common SNPs which are SNP3 (5’UR -616 T>C), SNP5 (e1 -143 A>G) and SNP6 (e1 -121 A>G) and three low frequency SNPs which are SNP1 (5’UR -914 G>C), SNP2 (5’UR –876 G>A) and SNP4 (5’UR – 169 C>T). Both SNP2 and SNP4 are two new SNPs (not reported before). (B) SNPs haplotype frequency of FAT10 promoter in HCC patient samples (tumor tissue and adjacent non-tumor tissue) and normal samples from healthy population. Haplotype frequencies were derived from genotype data using Arlequin. Tumor tissue and adjacent nontumor tissue show exactly the same SNPs and haplotype profiles. Haplotype 5, 6, 7 specifically belong to HCC patient group, while haplotype 8 was only fond in normal population. No significant difference was observed in the haplotype distribution of HCC samples and normal samples (the Fisher’s exact Test, p>0.05) 78 Chapter IV Evaluation of the role of mutations/polymorphisms and differential methylation 79 Chapter IV Evaluation of the role of mutations/polymorphisms and differential methylation 80 Chapter IV Evaluation of the role of mutations/polymorphisms and differential methylation 81 G>C) and SNP2 (5’UR –876 G>A) in the FAT10 promoter were observed only in the HCC patient samples (Table 2). From the genotype data of these six SNPs, a total of seven haplotypes were inferred from HCC patients using an expectation maximisation (EM) algorithm in the ArlequinTM software program (Fig. 20B). As there was no difference in the sequence between tumor and adjacent non-tumorous tissue, no difference was observed in the haplotype distribution of tumor versus adjacent normal tissues from HCC patients. Although no significant difference in the haplotype distribution was observed between HCC patients and normal individuals (Fisher’s Exact Test, P>0.05) (Fig. 20B), there were three low frequency haplotypes that were only found in HCC patients (CGTCAG, GATCAG and GGTTGG) and one (GGCTAA) that is only observed in normal individuals (Fig. 20B). More samples may need to be examined before any conclusions can be drawn. An in silico strategy using the web-based program, MatInspector, was employed to evaluate whether the SNPs at the FAT10 promoter can potentially affect transcription factor binding. As shown in Table 2, the putative transcription factor binding sites were abolished in 2 SNPs identified in the 5’ untranslated region of FAT10 gene. The major G allele of SNP2 (5'UR-876G>A) was predicted to be bound by GATA-binding factor 2 and hepatic nuclear factor 1, whereas the alternative minor A allele was predicted not to bind any factor. Similarly, the major T allele of SNP3 (5'UR -616T>C) predicts a myocyte enhancer factor-binding site, whereas the alternative minor C allele does not. The creation of putative binding sites was predicted for two SNPs (SNPs 1 and 6) (Table 2). Finally, for the last 2 SNPs (SNPs 4 and 5), the putative binding sites predicted for the major allele not longer exist when Chapter IV Evaluation of the role of mutations/polymorphisms and differential methylation 0.5 ** ** 0.4 0.35 ** 0.3 0.25 ** 0.2 ** ** 0.15 ** 0.1 0.05 GGCTAA GGTTGG GATCAG CGTCAG GGTCGG GGTCAG GGCCAA SNP6 SNP5 SNP4 SNP3 SNP2 SNP1 GGTCAA 0 Haplotypes (OD.sec-1)βgal / RFUegfp Normalized βgal Activity 0.45 Figure 21. FAT10 Promoter activity with different haplotype structures. Promoter activity of FAT10 promoter with different haplotype structures were determined as β-gal activity (normalized against EGFP). Experiment was carried out in hep3B cells and performed in four independent occasions. The order of SNPs in a haplotype structure is from 5’ to 3’ (Figure20). The hapolotypes with high frequency were boxed. ** denotes significant difference (P0.05). 4.4.2 Differential methylation may account for the differences in FAT10 gene expression between HCC tumor and adjacent non-tumorous tissues CG dinucleotides are present in the regulatory regions of many genes (Bird, 1996). In normal cells, the cytosines in the CG dinucleotides generally remain unmethylated (Bird, 1996). However, in the promoter sequences of genes associated with certain cancers or inherited diseases, more cytosines at the promoter region were found to be methylated (Herman et al., 1996). The methylation status in the DNA of humans and other mammals play an important role in determining whether some genes are or are not expressed. Abnormal DNA methylation plays an important role in other developmental diseases as well. Abnormal increases or decreases in DNA Chapter IV Evaluation of the role of mutations/polymorphisms and differential methylation 92 methylation are often observed in human cancers and may contribute to their development (Baylin et al., 1998; Herman et al., 1996). Seven CG sites were observed to reside in FAT10 promoter region. In order to study the correlation between the methylation status of these CG dinucleotides and the aberrant expression of FAT10 in HCC patients (Lee et al., 2003), we performed methylation-specific sequencing to screen the methylation status of these CG dinucleotides at the FAT10 promoter in 11 HCC patients. We found that generally, higher FAT10 expression is correlated significantly with reduced methylation of the CGs at the FAT10 promoter (Kappa value=0.468) (Table 3). The exceptions are patients 6, 10 and 11 whereby although the HCC tumor tissues has higher FAT10 expression, the methylation status of the tumor is either more in the tumor (P6) or no different from the adjacent normal tissues (P10 and 11). It is possible that differential methylation of other unexamined sites correlates with the FAT10 expression. A previous report demonstrated that methylation status in the coding region may also play a role in the regulation of gene expression (Irvine et al., 2002). Hence, it would be worthwhile to determine the methylation status of the coding region as well as regions further upstream the 1.5 kb promoter that we examined. In summary, although no mutations were identified at the FAT10 promoter in the tumor of HCC patients, polymorphisms at this promoter was identified which facilitated differential FAT10 promoter activities. Importantly, the methylation status at this promoter was found to correlate significantly with FAT10 expression levels. Conclusion 93 Conclusion In order to elucidate the mechanism of the aberrant expression of FAT10 in liver tissues of HCC patients, we initially cloned and characterized FAT10 promoter. We found that the core promoter of FAT10 gene resides at the 5’ UTR and regions upstream of the 5’UTR did not confer any promoter activity. Interestingly, we found that FAT10 may be a downstream gene of p53 as it can be significantly repressed by p53. Results from Chromatin immunoprecipitation suggests that p53 represses FAT10 by directly binding to its consensus site which resides upstream of the transcriptional start site in FAT10 promoter. We also found that the region between -975 and -1997 bp upstream of the transcription start site may play a role in the response of FAT10 promoter to TNF-α and IFN-γ cytokines. To elucidate the mechanism behind the aberrant expression of FAT10 in the tumor tissues of HCC patients, we sequenced the ~1.3 kb of the FAT10 promoter to screen for mutations and determined the methylation status of this promoter in HCC patients. No mutations can be found in the tumor tissue of the FAT10 promoter that can account for the aberrant FAT10 expression. However, 6 polymorphisms were identified in this promoter. Haplotypes of these polymorphisms were found to mediate different FAT10 promoter activity. Significantly we found that the methylation status at the FAT10 promoter inversely correlated significantly with FAT10 expression. Reference 94 Reference Aguiar, J., Santurlidis, S., Nowok, J., Alexander, C., Rudnicki, D., Gispert, S., Schulz, W. and Auburger, G. (1999). 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Isolation and Characterization of the Promoter of the FAT10 Gene CHAPTER II ISOLATION AND CHARACTERIZATION OF THE PROMOTER OF FAT10 GENE Adapted from Zhang et al (2006 Apr 13;25(16):2318-27, Oncogene) 18 Chapter II 2.1 Isolation and Characterization of the Promoter of the FAT10 Gene 19 Background FAT10, a new member of UBL family, contains two ubiquitin-like domains joined by a short linker and is 29%... methylation at the FAT10 promoter can account for the aberrant over-expression of FAT10 in the tumors of HCC patients Our lab has previously reported that the expression of the FAT10 gene is upregulated in greater than 90% of HCC patients (Lee et al., 2003) To determine whether there are mutations within the FAT10 promoter that can account for the aberrant over-expression of the FAT10 gene in the tumors of HCC... account for the aberrant over-expression of FAT10 in the tumors of HCC patients The following are the specific aims that help address the primary objective of this study Specific Aim 1: Isolate and Characterize the Promoter of the FAT10 Gene FAT10 gene comprises only two exons with an intervening 3.6kb intron and an upstream putative promoter region (Fig 1) The 5’UTR resides in exon 1 To isolate and characterize... cytokine response, apoptosis and mitosis Recently, our laboratory reported that the FAT10 gene is up regulated in 90% of hepatocellular carcinomas and over-expression of FAT10 gene may lead to chromosomal stability As part of the studies to elucidate the mechanism behind FAT10 gene regulation, we identified and characterized the promoter of the FAT10 gene We found that the 5’UTR, from the transcription start... tissues of these HCC patients Two of these polymorphisms were novel We computationally inferred the haplotype of these six polymorphisms and recapitulated these SNP haplotypes in vitro in the promoter- reporter (β-gal) system to determine the effects of the different SNP haplotypes on FAT10 promoter activity We proceeded to explore if the distribution of these SNPs in HCC was different from that of non-HCC... wt p53 was introduced To demonstrate the reversal of the effect of p53 on FAT10 promoter activity, siRNA against p53 or siRNA against a random sequence with no known homology to human, mouse or rat was also introduced and FAT10 promoter activity was determined We also attempted to delineate the region of the FAT10 promoter that confers responsiveness of the FAT10 promoter to p53 In silico analyses were... in the 5’ flanking region (-616(T/C)) and two at the 5’UTR (+82(A/G) and +104(A/G)), occurred at high frequency in both the normal and HCC patients With the current data, we did not find obvious correlation between the polymorphisms at the FAT10 promoter region and the relative FAT10 expression levels in HCC patients Nonetheless, we recapitulated various combinations of these three polymorphisms and. .. enhanced FAT10 expression and promoter activity P53 was found to bind in vivo to the 5’ half-consensus sequence of Table of Contents xiii the p53 binding site located in the FAT10 promoter Hence, we propose that FAT10 is a downstream target of p53 We proceeded to investigate if the up-regulation of FAT10 expression in the tumors of HCC patients can be accounted for by mutations or aberrant methylation at the. .. methylation at the FAT10 promoter region Through sequencing of approximately 37 HCC individuals and 39 normal individuals, we did not find any mutations at the FAT10 promoter region in the tumor of the patients that could account for the differential expression of the tumor and adjacent normal liver tissues in HCC patients Nonetheless, we identified six polymorphisms, two of which were novel Three of these six... role of FAT10 in immune response To elucidate the mechanism of FAT10 gene regulation, we isolated and characterized the promoter of FAT10 Interestingly, we found significant promoter activity in the 5’ untranslated region (UTR) (+1 bp to +209 bp) of the FAT10 gene but no promoter activity in regions upstream of the 5’UTR alone (from +26 bp to -1997 bp) Region -975 to +209 conferred maximum promoter ... II Isolation and Characterization of the Promoter of the FAT10 Gene 32 2.3.2 The responsive domain of the FAT10 promoter to TNF-α and IFN-γ resides in the region upstream of FAT10 promoter To.. .CLONING AND CHARACTERIZATION OF THE PROMOTER OF THE CANCER -ASSOCIATED GENE, FAT10 ZHANG DONGWEI (M Sc.) Wuhan Institute of Virology Chinese Academy of Science A THESIS SUBMITTED FOR THE DEGREE... Chapter II 2.3 Isolation and Characterization of the Promoter of the FAT10 Gene 28 Results 2.3.1 FAT10 promoter resides at the 5’UTR To better understand the regulation of FAT10 gene expression,