Methylation Silencing Research Articles

Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis

Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression.

Promoter methylation of the SALL2 tumor suppressor gene in ovarian cancers

The SALL2 gene product and transcription factor p150 were first identified in a search for tumor suppressors targeted for inactivation by the oncogenic mouse polyoma virus. SALL2 has also been identified as a cellular quiescence factor, essential for cells to enter and remain in a state of growth arrest under conditions of serum deprivation. p150 is a transcriptional activator of p21Cip1/Waf1 and BAX, sharing important growth arrest and proapoptotic properties with p53. It also acts as a repressor of c-myc. Restoration of SALL2 expression in cells derived from a human ovarian carcinoma (OVCA) suppresses growth of the cells in immunodeficient mice. Here we examine the pattern of p150 expression in the normal human ovary, in OVCA-derived cell lines and in primary ovarian carcinomas. Immunohistochemical staining showed that p150 is highly expressed in surface epithelial cells of the normal human ovary. Expression is exclusively from the P2 promoter governing the E1A splice variant of p150. The P2 promoter is CpG-rich and susceptible to methylation silencing. p150 expression was restored in OVCA cell lines following growth in the presence of 5-azacytidine. In a survey of 210 cases of OVCA, roughly 90% across major and minor histological types failed to show expression of the protein. Immunological and biochemical approaches were used to show hypermethylation of the SALL2 P2 promoter in OVCA-derived cell lines and in a majority of primary tumors. These results bring together molecular biological and clinical evidence in support of a role of SALL2 as a suppressor of ovarian cancers.

Targeted Methylation and Gene Silencing of VEGF-A in Human Cells by Using a Designed Dnmt3a–Dnmt3L Single-Chain Fusion Protein with Increased DNA Methylation Activity

The C-terminal domain of the Dnmt3a de novo DNA methyltransferase (Dnmt3a-C) forms a complex with the C-terminal domain of Dnmt3L, which stimulates its catalytic activity. We generated and characterized single-chain (sc) fusion proteins of both these domains with linker lengths between 16 and 30 amino acid residues. The purified sc proteins showed about 10-fold higher DNA methylation activities than Dnmt3a-C in vitro and were more active in bacterial cells as well. After fusing the Dnmt3a-3L sc enzyme to an artificial zinc-finger protein targeting the vascular endothelial cell growth factor A (VEGF-A) promoter, we demonstrate successful targeting of DNA methylation to the VEGF-A promoter in human cells and observed that almost complete methylation of 12 CpG sites in the gene promoter could be achieved. Targeted methylation by the Dnmt3a-3L sc enzymes was about twofold higher than that of Dnmt3a-C, indicating that Dnmt3a-3L sc variants are more efficient as catalytic modules in chimeric DNA methyltransfeases than Dnmt3a-C. Targeted methylation of the VEGF-A promoter with the Dnmt3a-3L sc variant led to a strong silencing of VEGF-A expression, indicating that the artificial DNA methylation of an endogenous promoter is a powerful strategy to achieve silencing of the corresponding gene in human cells.

Epigenetic changes in patients with multiple sclerosis

Epigenetic changes influence gene expression without altering the DNA sequence. DNA methylation, histone modification and microRNA-associated post-transcriptional gene silencing are three key epigenetic mechanisms. Multiple sclerosis (MS) is a disease of the CNS with both inflammatory and neurodegenerative features. Although studies on epigenetic changes in MS only began in the past decade, a growing body of literature suggests that epigenetic changes may be involved in the development of MS, possibly by mediating the effects of environmental risk factors, such as smoking, vitamin D deficiency and Epstein-Barr virus infection. Such studies are also beginning to deliver important insights into the pathophysiology of MS. For example, inflammation and demyelination in relapsing-remitting MS may be related to the increased differentiation of T cells toward a T-helper 17 phenotype, which is an important epigenetically regulated pathophysiological mechanism. In progressive MS, other epigenetically regulated mechanisms, such as increased histone acetylation and citrullination of myelin basic protein, might exacerbate the disease course. In this Review, we summarize current knowledge on the role of epigenetic changes in the pathophysiology of MS.

CLL Subsets with Distinct Stereotyped B Cell Receptors Have Distinct Epigenetic Make-up, Even Beyond IGHV Gene Mutational Status: DNA Methylation Profiling of IGHV-Unmutated CLL Stereotyped Subsets #6 and #8

AbstractAbstract 3869In CLL, subsets of patients with stereotyped B cell receptors (BcRs) account for one-third of the cohort. Increasing evidence suggests that cases assigned to the same subset can share similar biological and clinical features independently of IGHV gene mutational status, at least for selected major subsets. Consequently, the study of BcR stereotypy has important implications for refining patient stratification with the ultimate aim of implementing targeted therapy and, eventually, improving outcome. CLL stereotyped subsets #6 (IGHV1–69/IGKV3–20) and #8 (IGHV4–39/IGKV1(D)-39) both express unmutated BCRs yet exhibit different clinical behavior. In particular, subset #6 is less aggressive compared to subset #8, which exhibits the highest risk for development of Richter’s syndrome among all CLL. So far, no explanation is available for this difference, thus prompting investigation into the underlying mechanisms. Aberrant DNA methylation is increasingly recognized as relevant for CLL with strong correlations between promoter methylation and transcriptional silencing for critical genes, often depending on IGHV gene mutational status. With this in mind, we profiled the DNA methylation in CLL stereotyped subsets #6 and #8 and searched for epigenetic modifications that could be linked with their distinct clinicobiological features. DNA from CD19+ peripheral blood B cells of 10 subset #6 and 10 subset #8 cases was bisulfite-converted and analyzed with the Infinium HumanMethylation450 BeadChip array which allows interrogation of approximately 485,000 CpG sites. The methylation level of each CpG site was calculated in GenomeStudio Methylation module, while differential methylation analysis was performed using the Genomestudio software. Overall, 118 differentially methylated CpG sites (DMCpGs) were identified, of which 70 were hypomethylated while 48 were hypermethylated in subset #8 in comparison to subset #6. Hierarchical clustering of these DMCpGs enabled the discrimination of the two subsets. Seventy of the 118 DMCpGs were related to 61 genes, being preferentially located in regions of the corresponding locus associated with RNA transcripts, whereas for the remaining 48 DMCpGs no links with annotated transcripts could be identified. Of note, 14/48 of the latter DMCpGs were located in a CpG island on chromosome 14q32 and were hypermethylated in subset #8 versus subset #6, likely as a consequence of the t(14;19)(q32;q13) (BCL3/IGH) chromosomal translocation, which is frequently found in subset #8. In order to search for distinctive gene patterns among differentially methylated genes, we performed separate Gene Ontology (GO) analysis using the WebGestalt bioinformatics tool for (i) genes hypermethylated (n=19) and (ii) genes hypomethylated (n=42) in subset #8 versus subset #6. Interestingly, the most pronounced differences in methylation status concerned 10 genes in the latter category (HOXα5, PAX3, HLA-C, TP63, ZNF300, PRDM16, MGRN1, EMX2, PP1R14A, CCDC140). For genes hypermethylated in subset #8, GO analysis did not reveal any significant biological processes or molecular functions. In contrast, among genes hypomethylated in subset #8, we noted a significant overrepresentation of genes involved in developmental and metabolic biological processes, in particular, transcription factor and transcription regulator activity and DNA binding. In conclusion, we found distinct DNA methylation profiles between two CLL stereotyped subsets, both carrying unmutated BcRs yet showing different clinical behavior. The observed differential DNA methylation may be implicated in differences in gene expression programs that shape the biologic make-up and eventual clinical behavior of each subset. Disclosures:No relevant conflicts of interest to declare.

In Vitro Transcription Activities of Pol IV, Pol V, and RDR2 Reveal Coupling of Pol IV and RDR2 for dsRNA Synthesis in Plant RNA Silencing

In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases Pol IV and Pol V and the putative RNA-dependent RNA polymerase RDR2. Here we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to α-amanitin, and differ in their ability to displace the nontemplate DNA strand during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs), which guide cytosine methylation to corresponding sequences, requires both Pol IV and RDR2, which physically associate in vivo. Whereas Pol IV does not require RDR2 for activity, RDR2 is nonfunctional in the absence of associated Pol IV. These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis.

Promoter methylation of tumor-related genes in gastric carcinogenesis.

Aberrant promoter methylation and subsequent silencing of cancer-related genes has been recognized as an important pathway involved in gastric carcinogenesis. In fact, several factors are believed to contribute to its induction in gastric epithelia, including aging, diet, chronic inflammation and infection of Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV). However, the underling mechanisms are not completely identified, despite the belief that increased expression or activity of DNA methyltransferases (DNMTs), or decreased demethylation activity may contribute to the excessive methylation. A great number of genes with promoter methylation have been observed in gastric cancer (GC), among which p16INK4A (p16), Mut L homologue 1 (MLH1), Epithelial-cadherin (E-cadherin), Runt-related transcription factor 3 (RUNX3), adenomatous polyposis coli (APC), O(6)-methylguanine-DNA methyltransferase (MGMT), Ras association domain family 1A (RASSF1A) and Death-associated protein kinase (DAPK) have been extensively studied. Unlike the distinct methylation characterization in single genes, methylation analysis of multiple genes may provide more information in risk prediction, early detection, prognosis assessment and chemotherapy choice for GC. Specifically, particular monitoring and screening should be performed on those over 45 years old, with precancerous gastric disease or infection of H. pylori or EBV. As an alternative to tumor tissues, methylation detection in patient sera or gastric washes may also be used in risk prediction and early detection. However, what still poses a great challenge as well as a puzzle is the determination of the very genes that should be used in methylation analysis. Because epigenetic alterations are normally reversible, drugs or chemical compounds with demethylating activity, such as 5-aza-2'-deoxycytidine (5-aza-dC) could be used in the treatment of patients with multiple gene methylation. In view of the adverse effects of 5-aza-dC, DNMT-targeted strategy has been proposed and may prove to be more effective than demethylating agents.

Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

Long noncoding RNA-mediated maintenance of DNA methylation and transcriptional gene silencing

Establishment of silencing by noncoding RNAs (ncRNAs) via targeting of chromatin remodelers is relatively well investigated; however, their role in the maintenance of silencing is poorly understood. Here, we explored the functional role of the long ncRNA Kcnq1ot1 in the maintenance of transcriptional gene silencing in the one mega-base Kcnq1 imprinted domain in a transgenic mouse model. By conditionally deleting the Kcnq1ot1 ncRNA at different stages of mouse development, we suggest that Kcnq1ot1 ncRNA is required for the maintenance of the silencing of ubiquitously imprinted genes (UIGs) at all developmental stages. In addition, Kcnq1ot1 ncRNA is also involved in guiding and maintaining the CpG methylation at somatic differentially methylated regions flanking the UIGs, which is a hitherto unknown role for a long ncRNA. On the other hand, silencing of some of the placental-specific imprinted genes (PIGs) is maintained independently of Kcnq1ot1 ncRNA. Interestingly, the non-imprinted genes (NIGs) that escape RNA-mediated silencing are enriched with enhancer-specific modifications. Taken together, this study illustrates the gene-specific maintenance mechanisms operational at the Kcnq1 locus for tissue-specific transcriptional gene silencing and activation.

Long noncoding RNA-mediated maintenance of DNA methylation and transcriptional gene silencing

Establishment of silencing by noncoding RNAs (ncRNAs) via targeting of chromatin remodelers is relatively well investigated; however, their role in the maintenance of silencing is poorly understood. Here, we explored the functional role of the long ncRNA Kcnq1ot1 in the maintenance of transcriptional gene silencing in the one mega-base Kcnq1 imprinted domain in a transgenic mouse model. By conditionally deleting the Kcnq1ot1 ncRNA at different stages of mouse development, we suggest that Kcnq1ot1 ncRNA is required for the maintenance of the silencing of ubiquitously imprinted genes (UIGs) at all developmental stages. In addition, Kcnq1ot1 ncRNA is also involved in guiding and maintaining the CpG methylation at somatic differentially methylated regions flanking the UIGs, which is a hitherto unknown role for a long ncRNA. On the other hand, silencing of some of the placental-specific imprinted genes (PIGs) is maintained independently of Kcnq1ot1 ncRNA. Interestingly, the non-imprinted genes (NIGs) that escape RNA-mediated silencing are enriched with enhancer-specific modifications. Taken together, this study illustrates the gene-specific maintenance mechanisms operational at the Kcnq1 locus for tissue-specific transcriptional gene silencing and activation.

Safer, silencing-resistant lentiviral vectors: optimization of the ubiquitous chromatin-opening element through elimination of aberrant splicing.

Gammaretroviral and lentiviral vectors have been used successfully in several clinical gene therapy trials, although powerful enhancer elements have caused insertional mutagenesis and clonal dysregulation. Self-inactivating vectors with internal heterologous regulatory elements have been developed as potentially safer and more effective alternatives. Lentiviral vectors containing a ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 locus (A2UCOE), which allows position-independent, long-term transgene expression, are particularly promising. In a recently described assay, aberrantly spliced mRNA transcripts initiated in the vector A2UCOE sequence were found to lead to upregulation of growth hormone receptor gene (Ghr) expression in transduced murine Bcl-15 cells. Aberrant hybrid mRNA species formed between A2UCOE and a number of other cellular genes were also detected in transduced human PLB-985 myelomonocytic cells. Modification of the A2UCOE by mutation or deletion of recognized and potential cryptic splice donor sites was able to abrogate these splicing events and hybrid mRNA formation in Bcl-15 cells. This modification did not compromise A2UCOE regulatory activity in terms of resistance to CpG methylation and gene silencing in murine P19 embryonic carcinoma cells. These refined A2UCOE regulatory elements are likely to improve intrinsic biosafety and may be particularly useful for a number of clinical applications where robust gene expression is desirable.

Developmentally non-redundant SET domain proteins SUVH2 and SUVH9 are required for transcriptional gene silencing in Arabidopsis thaliana

In plants, RNA-directed DNA methylation (RdDM) and related transcriptional gene silencing (TGS) involve members of the suppressor of variegation 3-9-homologous (SUVH) group of putative histone methyltransferases. Utilizing a reverse genetic approach in Arabidopsis thaliana, we demonstrate that two closely related SUVH members, SUVH2 and SUVH9, act partially non-redundant in RdDM. DNA methylation, transcript accumulation and association with histone modifications were analyzed at the endogenous RdDM target AtSN1 (a SINE-like retroelement) in suvh2 and suvh9 single as well as suvh2 suvh9 double mutants. SUVH2 was found to be required for full DNA methylation at AtSN1 in early seed development and was also higher expressed in seeds than at later developmental stages. SUVH9 had its impact on RdDM later during vegetative development of the plant and was also higher expressed during that stage than at earlier developmental stages. The strongest reduction of RdDM at AtSN1 was found in suvh2 suvh9 double mutant plants. Histone 3-lysine 9-dimethylation (H3K9me2) associated with AtSN1 was reduced only in the simultaneous absence of functional SUVH2 and SUVH9. Thus, SUVH2 and SUVH9 functions in RdDM and TGS are overlapping in spite of some developmental specialization. Pol V specific transcripts were reduced in suvh2 suvh9 plants. This might indicate a role of these SUVH proteins in Pol V complex recruitment.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9934-x) contains supplementary material, which is available to authorized users.

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.

Abundant expression and hemimethylation of C19MC in cell cultures from placenta-derived stromal cells

MicroRNAs of the chromosome 19 microRNA cluster (C19MC) are known to be abundantly expressed in the placenta. Their genes are located on the long arm of chromosome 19 and seem to be part of a large imprinted region. Although the data available so far suggest important functions in the placenta, no data are available on their general expression patterns in cultures of placenta-derived mesenchymal stromal cells (PDMSC). Surprisingly, qRT-PCR on tissue cultures from first-trimester and term placenta mesenchymal stromal cells showed an abundant expression of the cluster members miR-517a-3p, miR-519a-3p, and miR-520c-3p. Accordingly, analyses of methylation patterns suggested that these cells had escaped methylation and epigenetic silencing, respectively, of the paternal allele. This was confirmed by the results of treatment of chorionic villous stromal cells by the demethylating agent 5-Aza-2′-deoxycytidine. Our results offer clear evidence that, in contrast to what is suggested in previous papers, members of C19MC are highly expressed in PDMSC indicating that their placenta-specific functions are not restricted to the trophoblast.

IDN2 and Its Paralogs Form a Complex Required for RNA–Directed DNA Methylation

IDN2/RDM12 has been previously identified as a component of the RNA–directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM.

Abstract 1049: DNMT3b increases transformation efficiency and accelerates carcinogen induced transformation of human bronchial epithelial cells

Abstract Malignant tumors and cell lines are characterized by increased expression of DNMT1, DNMT3a and DNMT3b. While evidence suggests that DNMT1, DNMT3a and DNMT3b co-operate in methylation and gene silencing during carcinogenesis, very little is known regarding gene targets for DNMT3a and DNMT3b and their impact on the development of adenocarcinoma. We developed an in vitro model using human telomerase/cyclin dependent kinase 4-immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to tobacco carcinogens. Carcinogen exposure (0.5mM MNU and 0.05µM BPDE) for 12 weeks induced transformation of HBEC2 cells and transformation efficiency was associated with DNA repair capacity. A significant increase in DNMT1 protein was observed during carcinogen exposure while moderate increases in DNMT3a and DNMT3b were observed in transformed cells. Increased DNMT3a and DNMT3b levels have been reported in several cancers. We observed 5-20 fold increased expression of DNMT3a and DNMT3b in tumor derived lung cancer cell lines. Therefore we tested the hypothesis that over expressing DNMT3a and DNMT3b would accelerate transformation by targeting specific genes for methylation and promote full malignancy in carcinogen-treated HBECs. Over expression of DNMT3a or DNMT3b did not affect DNA repair capacity and was not sufficient to drive spontaneous transformation as evidenced by inability to form colonies on soft agar. Transformation efficiency was increased 2-2.5 fold in the two HBEC2 cell lines over expressing DNMT3b after 12 weeks of carcinogen treatment relative to treated parental HBEC2. In addition, after 8 weeks, colony formation was evident in the DNMT3b over expressing, but not in parental HBECs indicating that increased expression of this de novo DNMT accelerates transformation. Over expressing DNMT3a had no effect on carcinogen-induced transformation. Current studies are evaluating global re-programming of the epigenome in transformed clones using the Illumina 450k promoter array and the effect of over expression of DNMT3b on stem cell markers. This study was supported by R01 ES008801 to S.A.B. and 1F32 CA157082 to I.T. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1049. doi:1538-7445.AM2012-1049

Abstract 992: Credentialing UHRF1 as a target for prostate cancer therapy

Abstract Alterations in DNA methylation are a hallmark of prostate carcinogenesis. UHRF1 is an essential component in the machinery required for the maintenance of DNA methylation in actively dividing cells. In S-phase, UHRF1 binds nascent hemimethylated DNA and recruits DNA methyltransferases to copy the methylation pattern to the daughter DNA strand in a manner analogous to semi-conservative replication. Additionally, it has been shown that UHRF1 interacts with methylated histones and histone methyltransferases, making it an important epigenetic regulator in proliferating cells. Because of its ties to epigenetic gene silencing and cell proliferation, we hypothesized that UHRF1 could serve as a target for prostate cancer treatment. In this study, we have taken steps to credential UHRF1 as a therapeutic target in prostate cancer and have developed a high-throughput UHRF1-DNA binding assay to screen for UHRF1 inhibitors. We show that UHRF1 is overexpressed in a panel of prostate cancer cell lines and it appears to track with proliferation in prostate cancer tissue. Through loss-of-function studies in prostate cancer cell lines PC3 and DU145, we show that long-term UHRF1 depletion leads to a decrease in clonogenic survival and an overall reduction in global levels of DNA methylation. Further, we find that UHRF1 is required to maintain DNA methylation and epigenetic silencing of hypermethylated genes in prostate cancer cells. Additionally, ChIP experiments revealed that UHRF1 can be bound at the promoter CpG islands of epigenetically silenced genes in prostate cancer cells. With the goal of ultimately developing UHRF1 inhibitors, we have developed a time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay, compatible with high-throughput screening, that can detect the binding of the SRA domain of UHRF1 to hemimethylated DNA. In summary, we have shown that UHRF1 plays a critical role in maintaining the DNA methylation of epigenetically silenced genes in prostate cancer and have developed an assay for high-throughput screening of UHRF1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 992. doi:1538-7445.AM2012-992

Hiwi Mediated Tumorigenesis Is Associated with DNA Hypermethylation

Expression of Piwi proteins is confined to early development and stem cells during which they suppress transposon migration via DNA methylation to ensure genomic stability. Piwi's genomic protective function conflicts with reports that its human ortholog, Hiwi, is expressed in numerous cancers and prognosticates shorter survival. However, the role of Hiwi in tumorigenesis has not been examined. Here we demonstrate that (1) over-expressing Hiwi in sarcoma precursors inhibits their differentiation in vitro and generates sarcomas in vivo; (2) transgenic mice expressing Hiwi (mesodermally restricted) develop sarcomas; and (3) inducible down-regulation of Hiwi in human sarcomas inhibits growth and re-establishes differentiation. Our data indicates that Hiwi is directly tumorigenic and Hiwi-expressing cancers may be addicted to Hiwi expression. We further show that Hiwi associated DNA methylation and cyclin-dependent kinase inhibitor (CDKI) silencing is reversible along with Hiwi-induced tumorigenesis, via DNA-methyltransferase inhibitors. Our studies reveal for the first time not only a novel oncogenic role for Hiwi as a driver of tumorigenesis, but also suggest that the use of epigenetic agents may be clinically beneficial for treatment of tumors that express Hiwi. Additionally, our data showing that Hiwi-associated DNA hyper-methylation with subsequent genetic and epigenetic changes favoring a tumorigenic state reconciles the conundrum of how Hiwi may act appropriately to promote genomic integrity during early development (via transposon silencing) and inappropriately in adult tissues with subsequent tumorigenesis.

Functional Consequences of Subunit Diversity in RNA Polymerases II and V

Multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V.

CpG-island methylation of GATA-family members GATA3 and GATA5 in renal cell carcinoma and association with clinicopathological parameters and progression-free survival.

370 Background: Transcriptional inactivation and CGI methylation of GATA3 and −5 has been reported to be involved in mammary carcinoma, pancreatic cancer, colorectal and gastric carcinogenesis. A recent study demonstrated that a loss of GATA-3 expression due to partially methylation silencing in several renal cell carcinoma (RCC) patients. We quantitatively investigated GATA3 and −5 CGI methylation in RCC and analyzed its association with clinical characteristics as well as progression free survival of patients. Methods: Methylation data were obtained from a quantitative methylation-specific polymerase chain reaction assay (QMSP) for both genes. We investigated 108 RCC and 77 paired tissue samples as well as six RCC cell lines. Statistical analyses were carried out using the paired t-test for matched tumor (TU) and adjacent normal (adN) samples, logistic regression for comparisons of independent samples and cox regression for survival analysis. Results: In paired samples we found a significant higher methylation in TU compared to adN for GATA3 (P=0.007) and for GATA5 (P=3.6*10−9) for all RCCs. GATA5 showed also strong correlations between methylation and status of metastasis (P=0.05) and advanced (pT≥3 and/or N+, M+) tumor samples compared to localized (pT≤2, N0, M0) tumors (P=4.7*10−9). A decreased progression free survival in cox proportional hazard model analysis could be demonstrated for patients with a high GATA5 methylation (P=0.0006, HR=6.5) and a trend could also be seen for GATA3 methylated patients (P=0.06). Conclusions: GATA3 and −5 were identified to demonstrate tumor-specific CGI hypermethylation in renal cell cancer patients. The association of GATA5 CGI methylation with metastasis, advanced disease and progression free survival of patients indicates that epigenetic alterations of both genes are involved in renal cell carcinogenesis. GATA5 methylation could serve as a biomarker for tumor progression. Further prospective and functional investigations are necessary to clarify whether CGI methylation of GATA family members can provide independent information for future clinical management of patients with RCC.