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Epithelial to mesenchymal transition (EMT) is a key trans-differentiation process, which plays a critical role in physiology and pathology. Although gene expression changes in EMT have been scrutinized, study of epigenome is in its infancy. To understand epigenetic changes during TWIST-driven EMT, we used the AcceSssIble assay to study DNA methylation and chromatin accessibility in human mammary epithelial cells (HMECs). The DNA methylation changes were found to have functional significance in EMT - i.e. methylated genes were enriched for E-box motifs that can be recognized by TWIST, at the promoters suggesting a potential targeting phenomenon, whereas the demethylated regions were enriched for pro-metastatic genes, supporting the role of EMT in metastasis. TWIST-induced EMT triggers alterations in chromatin accessibility both independent of and dependent on DNA methylation changes, primarily resulting in closed chromatin conformation. By overlapping the genes, whose chromatin structure is changed during early EMT and a known "core EMT signature", we identified 18 driver candidate genes during EMT, 14 upregulated and 4 downregulated genes with corresponding chromatin structure changes. Among 18 genes, we focused on TRIM29 as a novel marker of EMT. Although loss of TRIM29 is insufficient to suppress CDH, it is enough to induce CDH2 and VIM. Gene functional annotation analysis shows the involvement of TRIM29 in epidermal development, cell differentiation and cell migration. Taken together, our results provide a robust snapshot of chromatin state during human EMT and identify TRIM29 as a core mediator of EMT.
RESUMO
Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation-independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954-64. ©2016 AACR.
Assuntos
Carcinoma de Células Renais/genética , Metilação de DNA/genética , Epigenômica/métodos , Carcinoma de Células Renais/patologia , Expressão Gênica , HumanosRESUMO
Epigenetic gene silencing, mediated by aberrant promoter DNA hypermethylation and repressive histone modifications, is a hallmark of cancer. Although heritable, the dynamic nature and potential reversibility through pharmacological interventions make such aberrations attractive targets. Since cancers contain multiple epigenetic abnormalities, combining therapies that target different defects could potentially enhance their individual efficacies. 5-Aza-2'-deoxycytidine (5-Aza-CdR), FDA-approved drug for the treatment of myelodysplastic syndrome, can inhibit DNA methyltransferases (DNMTs) upon incorporation into the DNA of dividing cells, resulting in global demethylation. More recently, the first histone demethylase, lysine specific demethylase 1 (LSD1), which demethylates both histone and non-histone substrates, has become a new target for epigenetic therapy. Using, clorgyline, an LSD1 inhibitor (LSD1i) to treat cancer cell lines, we show that clorgyline employs two mechanisms of action depending on the cell type: it can either induce global DNA demethylation or inhibit LSD1-driven H3K4me2 and H3K4me1 demethylation to establish an active chromatin configuration. We also investigate the therapeutic efficacy of combining 5-Aza-CdR with clorgyline and determine that this combinatorial treatment has synergistic effects on reactivating aberrantly silenced genes by enriching H3K4me2 and H3K4me1. Many of the reactivated genes are categorized as cancer testis antigens or belong to the interferon-signaling pathway, suggesting potential implications for immunotherapy. Together, our results demonstrate that combinatorial treatment consisting of a DNMT inhibitor (DNMTi) and an LSD1i have enhanced therapeutic values and could improve the efficacy of epigenetic therapy.
Assuntos
Azacitidina/análogos & derivados , Clorgilina/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inativação Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Azacitidina/farmacologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/efeitos dos fármacos , Decitabina , Sinergismo Farmacológico , Histonas/metabolismo , Humanos , Regulação para Cima/efeitos dos fármacosRESUMO
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.
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Epigênese Genética , Fator 3 de Transcrição de Octâmero , Animais , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Fator 3 de Transcrição de Octâmero/genéticaRESUMO
DNA methylation inhibitors such as 5-aza-2'-deoxycytidine (5-Aza-CdR) are currently used for the treatment of myelodysplastic syndrome. Although global DNA demethylation has been observed after treatment, it is unclear to what extent demethylation induces changes in nucleosome occupancy, a key determinant of gene expression. We use the colorectal cancer cell line HCT116 as a model to address this question and determine that <2% of regions demethylated by 5-Aza-CdR treatment assume an open configuration. Consolidating our findings, we detect nucleosome retention at sites of global DNA methylation loss in DKO1, an HCT116-derived non-tumorigenic cell-line engineered for DNA methyltransferase disruption. Notably, regions that are open in both HCT116 cells after treatment and in DKO1 cells include promoters belonging to tumor suppressors and genes under-expressed in colorectal cancers. Our results indicate that only a minority of demethylated promoters are associated with nucleosome remodeling, and these could potentially be the epigenetic drivers causing the loss of tumorigenicity. Furthermore, we show that the chromatin opening induced by the histone deacetylase inhibitor suberoylanilide hydroxamic acid has strikingly distinct targets compared with those of 5-Aza-CdR, providing a mechanistic explanation for the importance of combinatorial therapy in eliciting maximal de-repression of the cancer epigenome.
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Cromatina/química , Metilação de DNA , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Decitabina , Genes Supressores de Tumor , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Regiões Promotoras Genéticas , VorinostatRESUMO
The ability to induce pluripotent stem cells from committed, somatic human cells provides tremendous potential for regenerative medicine. However, there is a defined neoplastic potential inherent to such reprogramming that must be understood and may provide a model for understanding key events in tumorigenesis. Using genome-wide assays, we identify cancer-related epigenetic abnormalities that arise early during reprogramming and persist in induced pluripotent stem cell (iPS) clones. These include hundreds of abnormal gene silencing events, patterns of aberrant responses to epigenetic-modifying drugs resembling those for cancer cells, and presence in iPS and partially reprogrammed cells of cancer-specific gene promoter DNA methylation alterations. Our findings suggest that by studying the process of induced reprogramming, we may gain significant insight into the origins of epigenetic gene silencing associated with human tumorigenesis, and add to means of assessing iPS for safety.
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Neoplasias/genética , Células-Tronco Pluripotentes/fisiologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cromatina/genética , Metilação de DNA , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Inativação Gênica , Genoma Humano , Humanos , Camundongos , Neoplasias/patologia , Células-Tronco Pluripotentes/patologiaRESUMO
Human embryonic stem cells (hESCs) have recently demonstrated the potential for differentiation into germ-like cells in vitro. This provides a novel model for understanding human germ cell development and human infertility. Mouse embryonic fibroblast (MEF) feeders and basic fibroblast growth factor (bFGF) are two sources of signaling that are essential for primary culture of germ cells, yet their role has not been examined in the derivation of germ-like cells from hESCs. Here protein and gene expression demonstrated that both MEF feeders and bFGF can significantly enrich germ cell differentiation from hESCs. Under enriched differentiation conditions, flow cytometry analysis proved 69% of cells to be positive for DDX4 and POU5F1 protein expression, consistent with the germ cell lineage. Importantly, removal of bFGF from feeder-free cultures resulted in a 50% decrease in POU5F1- and DDX4-positive cells. Quantitative reverse transcription-polymerase chain reaction analysis established that bFGF signaling resulted in an upregulation of genes involved in germ cell differentiation with or without feeders; however, feeder conditions caused significant upregulation of premigratory/migratory (Ifitm3, DAZL, NANOG, and POU5F1) and postmigratory (PIWIL2, PUM2) genes, along with the meiotic markers SYCP3 and MLH1. After further differentiation, >90% of cells expressed the meiotic proteins SYCP3 and MLH1. This is the first demonstration that signaling from MEF feeders and bFGF can induce a highly enriched population of germ-like cells derived from hESCs, thus providing a critically needed model for further investigation of human germ cell development and signaling. Disclosure of potential conflicts of interest is found at the end of this article.