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

Abstract

Recent epigenome-wide mapping studies describe nucleosomedepleted 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 highresolution 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. D uring development, each cell acquires its own epigenetic signature that provides guidelines to its cellular identity (1, 2). This epigenetic signature is accomplished by multiple epigenetic mechanisms, including DNA methylation, histone modifications, nucleosome positioning, and noncoding RNAs (3, 4). Pluripotent cells have a distinctive signature that is more dynamic compared with differentiated cells and allows for selfrenewal and pluripotency. Developmentally important genes are bivalent in embryonic stem cells, containing both active and repressive histone modifications (5, 6). The transcription factors OCT4, SOX2, and NANOG are known as core regulators of the transcription circuitry in pluripotent cells. The transcription autoregulatory loop ensures high levels of expression of these key stemness genes; they bind to their own regulatory regions, thereby maintaining expression patterns necessary for establishing and preserving pluripotent states (7).The nucleosome is the basic unit of chromatin and consists of DNA wound around a histone octamer protein core to achieve high compaction. Besides its role in packing the genome, pioneering studies have shown that nucleosome occupancy at gene promoters inhibits transcription initiation (8) and plays a critical role in epigenetic regulation (9). Genome-wide studies have shown that nucleosome-depleted regions (NDRs) are present at the transcription start sites of active genes and enhancers (10-14). More recently, genome-wide studies have begun to focus on the relationship between nucleosome positioning and gene expression (15, 16). Despite these observations, the role of dynamic nucleosome occupancy at gene regulatory regions and the relationship with transcription factors has not been well characterized at high resolution, particularly during the initial steps when embryonic stem cells lose their pluripotency.In this study, we used ...

The evidence for functional non-CpG methylation in mammalian cells

“…For example, nucleosome occlusion and hypermethylation of the distal enhancers of the NANOG/OCT4 and glucocorticoid receptor promoters prevents them from activating these genes. 9,10 The expression of the imprinted IGF2 gene is regulated by methylation at sites within the IGF2-H19 imprinted locus, which prevents binding of the CTCF insulator and allows interaction of the IGF2 gene promoter with its enhancer. 11 CpG…”

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma

“…formative than evaluating each epigenetic feature separately (Pardo et al 2011b;You et al 2011;Kelly et al 2012). Notably, the extent of cell-to-cell heterogeneity in chromatin accessibility at gene promoters in either disease-free or tumor cells remains ill-defined.…”

“…MAPit exploits exogenous addition of DNA methyltransferases (DNMTs), to probe accessibility of DNA in chromatin (Kladde et al 1996;Xu et al 1998b;Kilgore et al 2007;Pardo et al 2009). This technique has been used to simultaneously map DNA methylation and nucleosome positions on single molecules in many gene-specific studies (Kilgore et al 2007;Wolff et al 2010;Delmas et al 2011;Pardo et al 2011a;You et al 2011;Yang et al 2012;Darst et al 2013), and more recently, genome wide (Kelly et al 2012).…”

DNA Methylation and Histone Deacetylation: Interplay and Combined Therapy in Cancer

“…De novo DNA methylation and silencing of the pluripotency genes contributes to loss of pluripotency in differentiated cells (Farthing et al, 2008). Oct4 and Nanog are two such genes which are essential for pluripotency and early development and become methylated after differentiation (Hattori et al, 2004,Hattori et al, 2007,You et al, 2011. Thus, de novo methylation at promoter regions during cellular differentiation may in part control the loss of pluripotency by silencing stem cell-specific genes.…”