Heather M. O'Hagan

Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island.

Chronic exposure to inducers of DNA base oxidation and single and double strand breaks contribute to tumorigenesis. In addition to the genetic changes caused by this DNA damage, such tumors often contain epigenetically silenced genes with aberrant promoter region CpG island DNA hypermethylation. We herein explore the relationships between such DNA damage and epigenetic gene silencing using an experimental model in which we induce a defined double strand break in an exogenous promoter construct of the E-cadherin CpG island, which is frequently aberrantly DNA hypermethylated in epithelial cancers. Following the onset of repair of the break, we observe recruitment to the site of damage of key proteins involved in establishing and maintaining transcriptional repression, namely SIRT1, EZH2, DNMT1, and DNMT3B, and the appearance of the silencing histone modifications, hypoacetyl H4K16, H3K9me2 and me3, and H3K27me3. Although in most cells selected after the break, DNA repair occurs faithfully with preservation of activity of the promoter, a small percentage of the plated cells demonstrate induction of heritable silencing. The chromatin around the break site in such a silent clone is enriched for most of the above silent chromatin proteins and histone marks, and the region harbors the appearance of increasing DNA methylation in the CpG island of the promoter. During the acute break, SIRT1 appears to be required for the transient recruitment of DNMT3B and subsequent methylation of the promoter in the silent clones. Taken together, our data suggest that normal repair of a DNA break can occasionally cause heritable silencing of a CpG island–containing promoter by recruitment of proteins involved in silencing. Furthermore, with contribution of the stress-related protein SIRT1, the break can lead to the onset of aberrant CpG island DNA methylation, which is frequently associated with tight gene silencing in cancer.

RPA and ATR link transcriptional stress to p53

The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcription-blocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)- and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis.

Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Epigenetic silencing of genes in association with aberrant promoter DNA hypermethylation has emerged as a significant mechanism in the development of human cancers. Such genes are also often targets of the polycomb group repressive complexes in embryonic cells. The polycomb repressive complex 2 (PRC2) has been best studied in this regard. We now examine a link between PRC1 and cancer-specific gene silencing. Here, we show a novel and direct association between a constituent of the PRC1 complex, CBX7, with gene repression and promoter DNA hypermethylation of genes frequently silenced in cancer. CBX7 is able to complex with DNA methyltransferase (DNMT) enzymes, leading us to explore a role for CBX7 in maintenance and initiation of gene silencing. Knockdown of CBX7 was unable to relieve suppression of deeply silenced genes in cancer cells; however, in embryonal carcinoma (EC) cells, CBX7 can initiate stable repression of genes that are frequently silenced in adult cancers. Furthermore, we are able to observe assembly of DNMTs at CBX7 target gene promoters. Sustained expression of CBX7 in EC cells confers a growth advantage and resistance to retinoic acid-induced differentiation. In this setting, especially, there is increased promoter DNA hypermethylation for many genes by analysis of specific genes, as well as through epigenomic studies. Our results allow us to propose a potential mechanism through assembly of novel repressive complexes, by which the polycomb component of PRC1 can promote the initiation of epigenetic changes involving abnormal DNA hypermethylation of genes frequently silenced in adult cancers.

Induction of ser15 and lys382 modifications of p53 by blockage of transcription elongation

Blockage of transcription has been shown to induce the tumor suppressor p53 in human cells. We here show that RNA synthesis inhibitors blocking the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II, such as DRB and H7, induced rapid nuclear accumulation of p53 proteins that were not phosphorylated at ser15 or acetylated at lys382. In contrast, agents that inhibit the elongation phase of transcription, such as UV light, camptothecin or actinomycin D, induced the accumulation of nuclear p53 proteins that were modified at both of these sites. Furthermore, using a panel of DNA repair-deficient cells we show that persistent DNA lesions in the transcribed strand of active genes are responsible for the induction of the ser15 and lys382 modifications following UV-irradiation. We conclude that inhibition of transcription is sufficient for the accumulation of p53 in the nucleus regardless of whether the ser15 site of p53 is phosphorylated or not. Importantly, blockage of the elongation phase of transcription triggers a distinct signaling pathway leading to p53 modifications on ser15 and lys382. We propose that the elongating RNA polymerase complex may act as a sensor of DNA damage and as an integrator of cellular stress signals.

Frequent inactivation of cysteine dioxygenase type 1 contributes to survival of breast cancer cells and resistance to anthracyclines.

Purpose: Genome-wide DNA methylation analyses have identified hundreds of candidate DNA-hypermethylated genes in cancer. Comprehensive functional analyses provide an understanding of the biologic significance of this vast amount of DNA methylation data that may allow the determination of key epigenetic events associated with tumorigenesis. Experimental Design: To study mechanisms of cysteine dioxygenase type 1 (CDO1) inactivation and its functional significance in breast cancer in a comprehensive manner, we screened for DNA methylation and gene mutations in primary breast cancers and analyzed growth, survival, and reactive oxygen species (ROS) production in breast cancer cells with restored CDO1 function in the context of anthracycline treatment. Results: DNA methylation-associated silencing of CDO1 in breast cancer is frequent (60%), cancer specific, and correlates with disease progression and outcome. CDO1 function can alternatively be silenced by repressive chromatin, and we describe protein-damaging missense mutations in 7% of tumors without DNA methylation. Restoration of CDO1 function in breast cancer cells increases levels of ROS and leads to reduced viability and growth, as well as sensitization to anthracycline treatment. Priming with 5-azacytidine of breast cancer cells with epigenetically silenced CDO1 resulted in restored expression and increased sensitivity to anthracyclines. Conclusion: We report that silencing of CDO1 is a critical epigenetic event that contributes to the survival of oxidative-stressed breast cancer cells through increased detoxification of ROS and thus leads to the resistance to ROS-generating chemotherapeutics including anthracyclines. Our study shows the importance of CDO1 inactivation in breast cancer and its clinical potential as a biomarker and therapeutic target to overcome resistance to anthracyclines. Clin Cancer Res; 19(12); 3201–11. ©2013 AACR.

Nuclear accumulation of p53 following inhibition of transcription is not due to diminished levels of MDM2

A common mechanism by which the tumor suppressor p53 accumulates in the nucleus following cellular stress is through the attenuation of its interaction with MDM2, a protein involved in the nuclear export and degradation of p53. This is accomplished by induced modifications of p53, MDM2 or both. We have previously found that the kinase and mRNA synthesis inhibitor DRB (5,6-dichloro-1-b-D-ribofuranosylbenzimidazole) induces the nuclear accumulation of p53 without concomitant phosphorylation of the ser15 site of p53, which is thought to be a modification important for the attenuation of p53–MDM2 interaction. It has been proposed that the mechanism by which p53 accumulates following blockage of transcription involves the downregulation of MDM2 expression. In this study, we tested this hypothesis and found that after DRB treatment, p53 accumulated despite the fact that MDM2 levels remained high in human cells. Furthermore, over expression of MDM2 did not prevent the accumulation of p53 following DRB treatment. In, addition, p53 accumulating in the nucleus after DRB treatment was able to interact with MDM2 and was ubiquitylated. These findings suggest that blockage of transcription induce the nuclear accumulation of p53 without breaking the p53–MDM2 regulation loop.

Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage

At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations. Previously, we demonstrated that oxidative damage causes epigenetic silencing proteins to become part of a large complex that is localized to GC-rich regions of the genome, including promoter CpG islands that are epigenetically silenced in cancer. However, whether these proteins were recruited directly to damaged DNA or during the DNA repair process was unknown. Here we demonstrate that the mismatch repair protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin. Hydrogen peroxide treatment induces the interaction of MSH2-MSH6 with DNMT1, suggesting that the recruitment is through a protein-protein interaction. Importantly, the reduction in transcription for genes with CpG island-containing promoters caused by oxidative damage is abrogated by knockdown of MSH6 and/or DNMT1. Our findings provide evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription, potentially preventing transcription from interfering with the repair process. This study uniquely brings together several factors that are known to contribute to colon cancer, namely inflammation, mismatch repair proteins, and epigenetic changes.

Chromatin modifications during repair of environmental exposure-induced DNA damage: A potential mechanism for stable epigenetic alterations

Exposures to environmental toxicants and toxins cause epigenetic changes that likely play a role in the development of diseases associated with exposure. The mechanism behind these exposure‐induced epigenetic changes is currently unknown. One commonality between most environmental exposures is that they cause DNA damage either directly or through causing an increase in reactive oxygen species, which can damage DNA. Like transcription, DNA damage repair must occur in the context of chromatin requiring both histone modifications and ATP‐dependent chromatin remodeling. These chromatin changes aid in DNA damage accessibility and signaling. Several proteins and complexes involved in epigenetic silencing during both development and cancer have been found to be localized to sites of DNA damage. The chromatin‐based response to DNA damage is considered a transient event, with chromatin being restored to normal as DNA damage repair is completed. However, in individuals chronically exposed to environmental toxicants or with chronic inflammatory disease, repeated DNA damage‐induced chromatin rearrangement may ultimately lead to permanent epigenetic alterations. Understanding the mechanism behind exposure‐induced epigenetic changes will allow us to develop strategies to prevent or reverse these changes. This review focuses on epigenetic changes and DNA damage induced by environmental exposures, the chromatin changes that occur around sites of DNA damage, and how these transient chromatin changes may lead to heritable epigenetic alterations at sites of chronic exposure. Environ. Mol. Mutagen. 55:278–291, 2014.

Reduction of Murine Colon Tumorigenesis Driven by Enterotoxigenic Bacteroides fragilis Using Cefoxitin Treatment

BACKGROUND Chronic inflammation and composition of the colon microbiota have been associated with colorectal cancer in humans. The human commensal enterotoxigenic Bacteroides fragilis (ETBF) is linked to both inflammatory bowel disease and colorectal cancer and, in our murine model, causes interleukin 17A (IL-17A)-dependent colon tumors. In these studies, we hypothesized that persistent colonization by ETBF is required for tumorigenesis. METHODS We established a method for clearing ETBF in mice, using the antibiotic cefoxitin. Multiple intestinal neoplasia mice were colonized with ETBF for the experiment duration or were cleared of infection after 5 or 14 days. Gross tumors and/or microadenomas were then evaluated. In parallel, IL-17A expression was evaluated in wild-type littermates. RESULTS Cefoxitin treatment resulted in complete and durable clearance of ETBF colonization. We observed a stepwise increase in median colon tumor numbers as the duration of ETBF colonization increased before cefoxitin treatment. ETBF eradication also significantly decreased mucosal IL-17A expression. CONCLUSIONS The timing of ETBF clearance profoundly influences colon adenoma formation, defining a period during which the colon is susceptible to IL-17A-dependent tumorigenesis in this murine model. This model system can be used to study the microbiota-dependent and molecular mechanisms contributing to IL-17A-dependent colon tumor initiation.

Mismatch repair proteins initiate epigenetic alterations during inflammation-driven tumorigenesis

Aberrant silencing of genes by DNA methylation contributes to cancer, yet how this process is initiated remains unclear. Using a murine model of inflammation-induced tumorigenesis, we tested the hypothesis that inflammation promotes recruitment of epigenetic proteins to chromatin, initiating methylation and gene silencing in tumors. Compared with normal epithelium and noninflammation-induced tumors, inflammation-induced tumors gained DNA methylation at CpG islands, some of which are associated with putative tumor suppressor genes. Hypermethylated genes exhibited enrichment of repressive chromatin marks and reduced expression prior to tumorigenesis, at a time point coinciding with peak levels of inflammation-associated DNA damage. Loss of MutS homolog 2 (MSH2), a mismatch repair (MMR) protein, abrogated early inflammation-induced epigenetic alterations and DNA hypermethylation alterations observed in inflammation-induced tumors. These results indicate that early epigenetic alterations initiated by inflammation and MMR proteins lead to gene silencing during tumorigenesis, revealing a novel mechanism of epigenetic alterations in inflammation-driven cancer. Understanding such mechanisms will inform development of pharmacotherapies to reduce carcinogenesis. Cancer Res; 77(13); 3467-78. ©2017 AACR.