Polycomb Target Research Articles

Ring1B Compacts Chromatin Structure and Represses Gene Expression Independent of Histone Ubiquitination

How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.

Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program

The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ectoderm-derived neural tissues. In contrast, the beta-cell signature of trimethylated H3K27, a mark of Polycomb-mediated repression, clusters with pancreatic progenitors, acinar cells and liver, consistent with the epigenetic transmission of this mark from endoderm progenitors to their differentiated cellular progeny. We also identified two H3K27 methylation events that arise in the beta-cell lineage after the pancreatic progenitor stage. One is a wave of cell-selective de novo H3K27 trimethylation in non-CpG island genes. Another is the loss of bivalent and H3K27me3-repressed chromatin in a core program of neural developmental regulators that enables a convergence of the gene activity state of beta-cells with that of neural cells. These findings reveal a dynamic regulation of Polycomb repression programs that shape the identity of differentiated beta-cells.

Polycomb complexes act redundantly to repress genomic repeats and genes.

Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, we find that genomic repeats are Polycomb targets and show that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation.

Widespread and tissue specific age-related DNA methylation changes in mice

Aberrant methylation of promoter CpG islands in cancer is associated with silencing of tumor-suppressor genes, and age-dependent hypermethylation in normal appearing mucosa may be a risk factor for human colon cancer. It is not known whether this age-related DNA methylation phenomenon is specific to human tissues. We performed comprehensive DNA methylation profiling of promoter regions in aging mouse intestine using methylated CpG island amplification in combination with microarray analysis. By comparing C57BL/6 mice at 3-mo-old versus 35-mo-old for 3627 detectable autosomal genes, we found 774 (21%) that showed increased methylation and 466 (13%) that showed decreased methylation. We used pyrosequencing to quantitatively validate the microarray data and confirmed linear age-related methylation changes for all 12 genomic regions examined. We then examined 11 changed genomic loci for age-related methylation in other tissues. Of these, three of 11 showed similar changes in lung, seven of 11 changed in liver, and six of 11 changed in spleen, though to a lower degree than the changes seen in colon. There was partial conservation between age-related hypermethylation in human and mouse intestines, and Polycomb targets in embryonic stem cells were enriched among the hypermethylated genes. Our findings demonstrate a surprisingly high rate of hyper- and hypomethylation as a function of age in normal mouse small intestine tissues and a strong tissue-specificity to the process. We conclude that epigenetic deregulation is a common feature of aging in mammals.

Alternative Epigenetic Chromatin States of Polycomb Target Genes

Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX) and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT–PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a “balanced” state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a “void” state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the “bivalent” chromatin state of embryonic stem cells, and gene expression in development.

Histone Acetylation and the Maintenance of Chromatin Compaction by Polycomb Repressive Complexes

Mechanisms controlling higher-order chromatin structure or chromatin compaction and linking this to gene regulation are poorly understood. Previously, we had shown that the PRC1 Polycomb repressive complex is required to maintain a compact chromatin state at Polycomb target loci in embryonic stem cells (ESCs) of the mouse and that this activity, together with the ability to repress target gene expression, is surprisingly independent of the histone ubiquitination activity of the Ring1B component of PRC1. Here we investigate and discuss the role of another histone modification--histone acetylation--in Polycomb function. We show that inhibition of histone deacetylases leads to some decompaction of Hox loci and suggest that histone deacetylation has a role in the pathway of PRC1-mediated chromatin compaction. We discuss whether PRC1 and histone hypoacetylation function together to establish a chromatin template at which stable nucleosomes act to antagonize transcriptional elongation.

Array-based DNA methylation profiling of primary lymphomas of the central nervous system

BackgroundAlthough primary lymphomas of the central nervous system (PCNSL) and extracerebral diffuse large B-cell lymphoma (DLBCL) cannot be distinguished histologically, it is still a matter of debate whether PCNSL differ from systemic DLBCL with respect to their molecular features and pathogenesis. Analysis of the DNA methylation pattern might provide further data distinguishing these entities at a molecular level.MethodsUsing an array-based technology we have assessed the DNA methylation status of 1,505 individual CpG loci in five PCNSL and compared the results to DNA methylation profiles of 49 DLBCL and ten hematopoietic controls.ResultsWe identified 194 genes differentially methylated between PCNSL and normal controls. Interestingly, Polycomb target genes and genes with promoters showing a high CpG content were significantly enriched in the group of genes hypermethylated in PCNSL. However, PCNSL and systemic DLBCL did not differ in their methylation pattern.ConclusionsBased on the data presented here, PCNSL and DLBCL do not differ in their DNA methylation pattern. Thus, DNA methylation analysis does not support a separation of PCNSL and DLBCL into individual entities. However, PCNSL and DLBCL differ in their DNA methylation pattern from non- malignant controls.

Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target

Transcription in eukaryotic genomes generates an extensive array of non-protein-coding RNA, the functional significance of which is mostly unknown. We are investigating the link between non-coding RNA and chromatin regulation through analysis of FLC - a regulator of flowering time in Arabidopsis and a target of several chromatin pathways. Here we use an unbiased strategy to characterize non-coding transcripts of FLC and show that sense/antisense transcript levels correlate in a range of mutants and treatments, but change independently in cold-treated plants. Prolonged cold epigenetically silences FLC in a Polycomb-mediated process called vernalization. Our data indicate that upregulation of long non-coding antisense transcripts covering the entire FLC locus may be part of the cold-sensing mechanism. Induction of these antisense transcripts occurs earlier than, and is independent of, other vernalization markers and coincides with a reduction in sense transcription. We show that addition of the FLC antisense promoter sequences to a reporter gene is sufficient to confer cold-induced silencing of the reporter. Our data indicate that cold-induced FLC antisense transcripts have an early role in the epigenetic silencing of FLC, acting to silence FLC transcription transiently. Recruitment of the Polycomb machinery then confers the epigenetic memory. Antisense transcription events originating from 3' ends of genes might be a general mechanism to regulate the corresponding sense transcription in a condition/stage-dependent manner.

Gene Regulation and Epigenetic Remodeling in Murine Embryonic Stem Cells by c-Myc

BackgroundThe Myc oncoprotein, a transcriptional regulator involved in the etiology of many different tumor types, has been demonstrated to play an important role in the functions of embryonic stem (ES) cells. Nonetheless, it is still unclear as to whether Myc has unique target and functions in ES cells.Methodology/Principal FindingsTo elucidate the role of c-Myc in murine ES cells, we mapped its genomic binding sites by chromatin-immunoprecipitation combined with DNA microarrays (ChIP-chip). In addition to previously identified targets we identified genes involved in pluripotency, early development, and chromatin modification/structure that are bound and regulated by c-Myc in murine ES cells. Myc also binds and regulates loci previously identified as Polycomb (PcG) targets, including genes that contain bivalent chromatin domains. To determine whether c-Myc influences the epigenetic state of Myc-bound genes, we assessed the patterns of trimethylation of histone H3-K4 and H3-K27 in mES cells containing normal, increased, and reduced levels of c-Myc. Our analysis reveals widespread and surprisingly diverse changes in repressive and activating histone methylation marks both proximal and distal to Myc binding sites. Furthermore, analysis of bulk chromatin from phenotypically normal c-myc null E7 embryos demonstrates a 70–80% decrease in H3-K4me3, with little change in H3-K27me3, compared to wild-type embryos indicating that Myc is required to maintain normal levels of histone methylation.Conclusions/SignificanceWe show that Myc induces widespread and diverse changes in histone methylation in ES cells. We postulate that these changes are indirect effects of Myc mediated by its regulation of target genes involved in chromatin remodeling. We further show that a subset of PcG-bound genes with bivalent histone methylation patterns are bound and regulated in response to altered c-Myc levels. Our data indicate that in mES cells c-Myc binds, regulates, and influences the histone modification patterns of genes involved in chromatin remodeling, pluripotency, and differentiation.

An Embryonic Stem Cell–Like Signature Identifies Poorly Differentiated Lung Adenocarcinoma but not Squamous Cell Carcinoma

An embryonic stem cell (ESC) profile correlates with poorly differentiated breast, bladder, and glioma cancers. In this article, we assess the correlation between the ESC profile and clinical variables in lung cancer. Microarray gene expression analysis was done using Affymetrix Human Genome U133A on 443 samples of human lung adenocarcinoma and 130 samples of squamous cell carcinoma (SCC). To identify gene set enrichment patterns, we used the Genomica software. Our analysis showed that an increased expression of the ESC gene set and a decreased expression of the Polycomb target gene set identified poorly differentiated lung adenocarcinoma. In addition, this gene expression signature was associated with markers of poor prognosis and worse overall survival in lung adenocarcinoma. However, there was no correlation between this ESC gene signature and any histologic or clinical variable assessed in lung SCC. This work suggests that not all poorly differentiated non-small cell lung cancers exhibit a gene expression profile similar to that of ESC, and that other characteristics may play a more important role in the determination of differentiation and survival in SCC of the lung.

Talking to chromatin: post-translational modulation of polycomb group function

Polycomb Group proteins are important epigenetic regulators of gene expression. Epigenetic control by polycomb Group proteins involves intrinsic as well as associated enzymatic activities. Polycomb target genes change with cellular context, lineage commitment and differentiation status, revealing dynamic regulation of polycomb function. It is currently unclear how this dynamic modulation is controlled and how signaling affects polycomb-mediated epigenetic processes at the molecular level. Experimental evidence on regulation of polycomb function by post-translational mechanisms is steadily emerging: Polycomb Group proteins are targeted for ubiquitylation, sumoylation and phosphorylation. In addition, specific Polycomb Group proteins modify other (chromatin) associated proteins via similar post-translational modifications. Such modifications affect protein function by affecting protein stability, protein-protein interactions and enzymatic activities. Here, we review current insights in covalent modification of Polycomb Group proteins in the context of protein function and present a tentative view of integrated signaling to chromatin in the context of phosphorylation. Clearly, the available literature reveals just the tip of the iceberg, and exact molecular mechanisms in, and the biological relevance of post-translational regulation of polycomb function await further elucidation. Our understanding of causes and consequences of post-translational modification of polycomb proteins will gain significantly from in vivo validation experiments. Impaired polycomb function has important repercussions for stem cell function, development and disease. Ultimately, increased understanding of signaling to chromatin and the mechanisms involved in epigenetic remodeling will contribute to the development of therapeutic interventions in cell fate decisions in development and disease.

CBP-mediated acetylation of histone H3 lysine 27 antagonizesDrosophilaPolycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.

DNA hypermethylation accompanied by transcriptional repression in follicular lymphoma

High-throughput microarray technologies were used to study DNA methylation accompanied by transcriptional changes in follicular lymphoma (FL). Using Methylated CpG Island Amplification with Microarrays to study CpG Island DNA methylation in FL, we discovered widespread hypermethylation of homeobox genes and previously identified targets of polycomb repressive complex 2 (PRC2) in cell lines and primary tumors, but not in benign follicular hyperplasia (BFH). DNA methylation for HOXA11, HOXD10, HOXB7, HOXC12, PAX6, LHX9, SFMBT2, EN2, and PAX7 was independently validated in the RL cell line and HOXA11, HOXD10, PAX6, and EN2 in primary tumors. Combined Bisulfite Restriction Analysis (COBRA) also established DNA methylation for the previously identified PRC2 targets DCC, DES, GAD2, AQP5, GPR61, GRIA4, GJD2, and AMPH in FL but not in BFH. Gene expression analyses revealed 411 genes that were hypermethylated and transcriptionally repressed in RL, 74% of which were reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5-azaD) plus or minus the histone deacetylase inhibitor trichostatin A (TSA). Forty genes were also downregulated in primary FL. Our results suggest that extensive hypermethylation in promoters of polycomb target genes is a characteristic of FL and that loss of expression of certain SUZ12 target genes could be functionally relevant for lymphomagenesis.

The Synovial Sarcoma-Associated SYT-SSX2 Oncogene Antagonizes the Polycomb Complex Protein Bmi1

This study demonstrates deregulation of polycomb activity by the synovial sarcoma-associated SYT-SSX2 oncogene, also known as SS18-SSX2. Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation event that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. The role of the translocation products in this disease is poorly understood. We present evidence that the SYT-SSX2 fusion protein interacts with the polycomb repressive complex and modulates its gene silencing activity. SYT-SSX2 causes destabilization of the polycomb subunit Bmi1, resulting in impairment of polycomb-associated histone H2A ubiquitination and reactivation of polycomb target genes. Silencing by polycomb complexes plays a vital role in numerous physiological processes. In recent years, numerous reports have implicated gain of polycomb silencing function in several cancers. This study provides evidence that, in the appropriate context, expression of the SYT-SSX2 oncogene leads to loss of polycomb function. It challenges the notion that cancer is solely associated with an increase in polycomb function and suggests that any imbalance in polycomb activity could drive the cell toward oncogenesis. These findings provide a mechanism by which the SYT-SSX2 chimera may contribute to synovial sarcoma pathogenesis.

New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling

AbstractLymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype–specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell–like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

NA-Seq: A Discovery Tool for the Analysis of Chromatin Structure and Dynamics during Differentiation

It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements.

Methylation of homeobox genes is a frequent and early epigenetic event in breast cancer

IntroductionAberrant methylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. However, its impact on tumor development is not fully determined, and its potential as a diagnostic biomarker remains to be validated. Methylation profiling of invasive breast carcinoma has been largely explored. Conversely, very little and sparse information is available on early-stage breast cancer. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, we have analyzed the DNA methylation profile of ductal carcinoma in situ, a premalignant breast lesion with a great potential to progress toward invasive carcinoma.MethodsWe have utilized a comprehensive and sensitive array-based DNA mapping technique, the methylated-CpG island recovery assay, to profile the DNA methylation pattern in ductal carcinoma in situ. Differential methylation of CpG islands was compared genome-wide in tumor DNA versus normal DNA utilizing a statistical linear model in the LIMMA software package.ResultsUsing this approach, we have identified 108 significant CpG islands that undergo aberrant DNA methylation in ductal carcinoma in situ and stage I breast tumors, with methylation frequencies greater than or comparable with those of more advanced invasive carcinoma (50% to 93%). A substantial fraction of these hypermethylated CpG islands (32% of the annotated CpG islands) is associated with several homeobox genes, such as the TLX1, HOXB13, and HNF1B genes. Fifty-three percent of the genes hypermethylated in early-stage breast cancer overlap with known Polycomb targets and include homeobox genes and other developmental transcription factors.ConclusionsWe have identified a series of new potential methylation biomarkers that may help elucidate the underlying mechanisms of breast tumorigenesis. More specifically, our results are suggestive of a critical role of homeobox gene methylation in the insurgence and/or progression of breast cancer.

Methylation of Polycomb Target Genes in Intestinal Cancer Is Mediated by Inflammation

Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently, it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb (PcG) proteins. However, specific mechanisms responsible for methylation of PcG target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging, and cancer. We found that inflammation leads to aberrant DNA methylation in PcG target genes, with 70% of the approximately 250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wild-type mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. Sixty percent of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.

Stability and Dynamics of Polycomb Target Sites in Drosophila Development

Polycomb-group (PcG) and Trithorax-group proteins together form a maintenance machinery that is responsible for stable heritable states of gene activity. While the best-studied target genes are the Hox genes of the Antennapedia and Bithorax complexes, a large number of key developmental genes are also Polycomb (Pc) targets, indicating a widespread role for this maintenance machinery in cell fate determination. We have studied the linkage between the binding of PcG proteins and the developmental regulation of gene expression using whole-genome mapping to identify sites bound by the PcG proteins, Pc and Pleiohomeotic (Pho), in the Drosophila embryo and in a more restricted tissue, the imaginal discs of the third thoracic segment. Our data provide support for the idea that Pho is a general component of the maintenance machinery, since the majority of Pc targets are also associated with Pho binding. We find, in general, considerable developmental stability of Pc and Pho binding at target genes and observe that Pc/Pho binding can be associated with both expressed and inactive genes. In particular, at the Hox complexes, both active and inactive genes have significant Pc and Pho binding. However, in comparison to inactive genes, the active Hox genes show reduced and altered binding profiles. During development, Pc target genes are not simply constantly associated with Pc/Pho binding, and we identify sets of genes with clear differential binding between embryo and imaginal disc. Using existing datasets, we show that for specific fate-determining genes of the haemocyte lineage, the active state is characterised by lack of Pc binding. Overall, our analysis suggests a dynamic relationship between Pc/Pho binding and gene transcription. Pc/Pho binding does not preclude transcription, but levels of Pc/Pho binding change during development, and loss of Pc/Pho binding can be associated with both stable gene activity and inactivity.

RUNX3 Methylation Reveals that Bladder Tumors Are Older in Patients with a History of Smoking

Exposure to tobacco smoke is associated with increased DNA methylation at certain genes in both lung and bladder tumors. We sought to identify interactions in bladder cancer between DNA methylation and a history of smoking, along with any possible effect of aging. We measured DNA methylation in 342 transitional cell carcinoma tumors at BCL2, PTGS2 (COX2), DAPK, CDH1 (ECAD), EDNRB, RASSF1A, RUNX3, TERT, and TIMP3. The prevalence of methylation at RUNX3, a polycomb target gene, increased as a function of age at diagnosis (P = 0.031) and a history of smoking (P = 0.015). RUNX3 methylation also preceded methylation at the other eight genes (P < 0.001). It has been proposed that DNA methylation patterns constitute a "molecular clock" and can be used to determine the "age" of normal tissues (i.e., the number of times the cells have divided). Because RUNX3 methylation increases with age, is not present in normal urothelium, and occurs early in tumorigenesis, it can be used for the first time as a molecular clock to determine the age of a bladder tumor. Doing so reveals that tumors from smokers are "older" than tumors from nonsmokers (P = 0.009) due to tumors in smokers either initiating earlier or undergoing more rapid cell divisions. Because RUNX3 methylation is acquired early on in tumorigenesis, then its detection in biopsy or urine specimens could provide a marker to screen cigarette smokers long before any symptoms of bladder cancer are present.