Daniel D. De Carvalho

Epigenetic modifications as therapeutic targets

Epigenetic modifications work in concert with genetic mechanisms to regulate transcriptional activity in normal tissues and are often dysregulated in disease. Although they are somatically heritable, modifications of DNA and histones are also reversible, making them good targets for therapeutic intervention. Epigenetic changes often precede disease pathology, making them valuable diagnostic indicators for disease risk or prognostic indicators for disease progression. Several inhibitors of histone deacetylation or DNA methylation are approved for hematological malignancies by the US Food and Drug Administration and have been in clinical use for several years. More recently, histone methylation and microRNA expression have gained attention as potential therapeutic targets. The presence of multiple epigenetic aberrations within malignant tissue and the abilities of cells to develop resistance suggest that epigenetic therapies are most beneficial when combined with other anticancer strategies, such as signal transduction inhibitors or cytotoxic treatments. A key challenge for future epigenetic therapies will be to develop inhibitors with specificity to particular regions of chromosomes, thereby potentially reducing side effects.

DNA methylation screening identifies driver epigenetic events of cancer cell survival

Cancer cells typically exhibit aberrant DNA methylation patterns that can drive malignant transformation. Whether cancer cells are dependent on these abnormal epigenetic modifications remains elusive. We used experimental and bioinformatic approaches to unveil genomic regions that require DNA methylation for survival of cancer cells. First, we surveyed the residual DNA methylation profiles in cancer cells with highly impaired DNA methyltransferases. Then, we clustered these profiles according to their DNA methylation status in primary normal and tumor tissues. Finally, we used gene expression meta-analysis to identify regions that are dependent on DNA methylation-mediated gene silencing. We further showed experimentally that these genes must be silenced by DNA methylation for cancer cell survival, suggesting these are key epigenetic events associated with tumorigenesis.

DNA methylation and cellular reprogramming

The recent discovery that a small number of defined factors are sufficient to reprogram somatic cells into pluripotent stem cells has significantly expanded our knowledge of the plasticity of the epigenome. In this review we discuss some aspects of cell fate plasticity and epigenetic alterations, with emphasis on DNA methylation during cellular reprogramming. Recent data suggest that DNA methylation is a major barrier to induced pluripotent stem (iPS) cell reprogramming. The demethylating agent 5-azacytidine can enhance the efficiency of iPS cells generation and the putative DNA demethylase protein activation-induced cytidine deaminase (AID/AICDA) can erase DNA methylation at pluripotency gene promoters, thereby allowing cellular reprogramming. Elucidation of the epigenetic changes taking place during cellular reprogramming will enhance our understanding of stem cell biology and facilitate therapeutic applications.

Polycomb-Repressed Genes Have Permissive Enhancers that Initiate Reprogramming

Key regulatory genes, suppressed by Polycomb and H3K27me3, become active during normal differentiation and induced reprogramming. Using the well-characterized enhancer/promoter pair of MYOD1 as a model, we have identified a critical role for enhancers in reprogramming. We observed an unexpected nucleosome-depleted region (NDR) at the H3K4me1-enriched enhancer at which transcriptional regulators initially bind, leading to subsequent changes in the chromatin at the cognate promoter. Exogenous Myod1 activates its own transcription by binding first at the enhancer, leading to an NDR and transcription-permissive chromatin at the associated MYOD1 promoter. Exogenous OCT4 also binds first to the permissive MYOD1 enhancer but has a different effect on the cognate promoter, where the monovalent H3K27me3 marks are converted to the bivalent state characteristic of stem cells. Genome-wide, a high percentage of Polycomb targets are associated with putative enhancers in permissive states, suggesting that they may provide a widespread avenue for the initiation of cell-fate reprogramming.

OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes

Recent epigenome-wide mapping studies describe nucleosome-depleted regions (NDRs) at transcription start sites and enhancers. However, these static maps do not address causality or the roles of NDRs in gene control, and their relationship to transcription factors and DNA methylation is not well understood. Using a high-resolution single-molecule mapping approach to simultaneously investigate endogenous DNA methylation and nucleosome occupancies on individual DNA molecules, we show that the unmethylated OCT4 distal enhancer has an NDR, whereas NANOG has a clear NDR at its proximal promoter. These NDRs are maintained by binding of OCT4 and are required for OCT4 and NANOG expression. Differentiation causes a rapid loss of both NDRs accompanied by nucleosome occupancy, which precedes de novo DNA methylation. NDRs can be restored by forced expression of OCT4 in somatic cells but only when there is no cytosine methylation. These data show the central role of the NDRs, established by OCT4, in ensuring the autoregulatory loop of pluripotency and, furthermore, that de novo methylation follows the loss of NDRs and stabilizes the suppressed state.

Nucleosomes Containing Methylated DNA Stabilize DNA Methyltransferases 3A/3B and Ensure Faithful Epigenetic Inheritance

How epigenetic information is propagated during somatic cell divisions is still unclear but is absolutely critical for preserving gene expression patterns and cellular identity. Here we show an unanticipated mechanism for inheritance of DNA methylation patterns where the epigenetic mark not only recruits the catalyzing enzyme but also regulates the protein level, i.e. the enzymatic product (5-methylcytosine) determines the level of the methylase, thus forming a novel homeostatic inheritance system. Nucleosomes containing methylated DNA stabilize de novo DNA methyltransferases, DNMT3A/3B, allowing little free DNMT3A/3B enzymes to exist in the nucleus. Stabilization of DNMT3A/3B on nucleosomes in methylated regions further promotes propagation of DNA methylation. However, reduction of cellular DNA methylation levels creating more potential CpG substrates counter-intuitively results in a dramatic decrease of DNMT3A/3B proteins due to diminished nucleosome binding and subsequent degradation of the unstable free proteins. These data show an unexpected self-regulatory inheritance mechanism that not only ensures somatic propagation of methylated states by DNMT1 and DNMT3A/3B enzymes but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.

SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation

Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.

Abstract B56: DNA demethylation in gene bodies is of therapeutic significance

Reversal of aberrant promoter hypermethylation and associated gene silencing has been the primary focus of epigenetic therapy. Recently, gene body methylation has been implicated as having a functional role in the regulation of gene expression; however it is unclear how gene body methylation changes might contribute to the therapeutic effects of epigenetic therapy. To investigate the role of DNA demethylation in inducing gene expression changes in a comprehensive way, we treated the HCT116 colon cancer cell line with 5-Aza-2′-deoxycytidine (5-Aza-CdR) transiently for 24 hours and monitored changes in gene expression and DNA methylation levels at gene bodies and promoters for up to 42 days after treatment using 450K Infinium DNA methylation arrays and expression arrays. We observed that probes that became demethylated and subsequently regained methylation did at different rates. As we expected, demethylation of methylated promoters is negatively correlated with expression (R2≤ -0.75) and considerable demethylation of promoters (485) and the associated gene reactivation could be maintained for up to 42 days after drug treatment. Most of these genes are de novo methylated and their expression is down-regulated in primary colon cancers in the TCGA database. Strikingly, a group of genes shows a positive correlation between demethylation of gene bodies and down-regulation of gene expression after treatment (R2≥ 0.75). Four hundred ninety eight have fast rebound methylation kinetics and decreased expression and they are enriched for genes involved in metabolic process which are over-expressed in primary colon cancer tissues. Furthermore, 494 genes with sustained down-regulation of gene expression maintained their demethylated status and those are enriched for de novo methylation targets and over-expressed genes in colon cancer. Our results demonstrate that DNA demethylation at gene bodies induced by 5-Aza-CdR can be of therapeutic significance by resulting in the down-regulation of genes that are overexpressed in cancers. Our findings also confirm that demethylation treatment could potentially cause prolonged reactivation of genes that are silenced by promoter methylation while simultaneously down-regulating genes that are overexpressed and have body methylation. Together, demethylation of gene bodies may confer therapeutic benefit in addition to demethylation of gene promoters. Citation Format: Han Han Zhang, Xiaojing Yang, Daniel D. De Carvalho, Peter A. Jones, Gangning Liang. DNA demethylation in gene bodies is of therapeutic significance. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B56.

Abstract 677: Transient exposure to decitabine results in sustained cell growth inhibition and long term DNA demethylation at specific loci.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnGene silencing mediated by aberrant promoter DNA hypermethylation is one of the key features of cancer. Although such modification is heritable, its dynamic nature and reversibility through pharmacological interventions make it an attractive therapeutic target. Over the past few decades, various DNA methyltransferase inhibitors have been developed with the goal of reactivating aberrantly silenced genes. Two FDA-approved demthylating agents, decitabine and azacitidine are efficacious for the treatment of myeloid malignancies (MDS). It has been shown that transient exposure of tumor cells to low dose decitabine and azacitidine results in reduced tumorigenecity. However, the mechanism underlying clinical efficacies of these DNA methyltransferase inhibitors remains unclear. Here we investigate the long-term effect (up to 10 weeks) on population doubling time and the returning of DNA methylation (rebound methylation) after transient exposure to decitabine in three cancer cell lines, HCT116, T24 and HL-60. We show that transient exposure results in inhibition of cell growth and reduction of their colony formation capacity (up to 42 days). Furthermore, we also show that the rebound methylation occurs at different rates depending on the region and the type of genes. The discrepancy in population doubling time between the vehicle and decitabine treated cells disappears when rebound methylation fully restores, suggesting the presence of a correlation between cell doubling and rebound methylation. The majority of probes, which exhibit fast rebound methylation, are located in gene bodies. We show a positive correlation between gene body methylation and expression. However, there is a small group of fast rebound probes located at the promoters. Many of them belong to cancer-testis antigens, which get reactivated and quickly re-silenced. On the other hand, the majority of probes, which exhibit slow rebound methylation are located at promoters and show an inverse correlation between methylation and expression. The presence of a nucleosome-depleted region and enrichments of active histone marks, H3K4me3 and H2A.Z are observed at the promoters of those genes up to 42 days after drug treatment. Interesting, this group is enriched for genes down-regulated in colon adenocarcinoma when compared to normal colon (p=3.3e−6). Taken together, the sustained reactivation of those genes may be the driving force in reducing tumorigenecity of cancer cells. Our study elucidates the mechanism of decitabine, thus advancing our understanding of epigenetic therapy.nnCitation Format: Gangning Liang, Han Han, Daniel D. De Carvalho, Xiaojing Yang, Peter A. Jones. Transient exposure to decitabine results in sustained cell growth inhibition and long term DNA demethylation at specific loci. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 677. doi:10.1158/1538-7445.AM2013-677nnNote: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract PR07: SNF5 is an essential executor of epigenetic regulation during differentiation

Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosomedepleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4 activated target genes such as OCT4 itself and NANOG. Furthermore, using loss and gain of function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.nnThis abstract is also presented as Poster A39 .nnCitation Format: Jueng Soo You, Daniel D. De Carvalho, Chao Dai, Minmin Liu, Kurinji Pandiyan, Xianghong Zhou, Gangning Liang, Peter A. Jones. SNF5 is an essential executor of epigenetic regulation during differentiation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR07.