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Title: Combinatorial Binding in Human and Mouse Embryonic Stem Cells Identifies Conserved Enhancers Active in Early Embryonic Development}
Article:
Abstract:
Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem( ES) cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas “gene regulatory hotspots” which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints.
Introduction:
Embryonic stem( ES) cells are derived from the inner cell mass of the blastocyst[1]. During the course of normal development, implantation of the blastocyst results in further differentiation into distinct cell types of the three primary germ layers that will later form the tissues and organs of the developing embryo[2]. This pluripotent state makes ES cells a unique in vitro cellular model system to study early embryogenesis. At the core of the regulatory network that maintains this state is a set of transcription factors amongst which OCT4 seems to play a key role[3],[4]. OCT4 has been shown to co-occupy many regulatory sites together with SOX2, NANOG and the co-activator p300[5]. The potency of these transcription factors is demonstrated by their ability to induce pluripotency in mouse and human somatic cells. This was achieved by the ectopic expression of OCT4 and SOX2 together with either KLF4 and c-MYC, or NANOG and LIN28[6],[7],[8]. The pivotal step in inducing and maintaining the pluripotent state occurs at the level of genomic DNA by the binding of transcription factors and co-factors that activate and repress gene expression. The largest fraction of the genome is non-coding with many non-coding elements being highly conserved. Even though it is expected that many of these elements harbor transcription factor binding sites and may act as enhancers, current understanding of the interplay between transcription factors and regulatory elements within the genome is limited. ChIP-Seq data pinpoints transcription factor binding sites not only in predefined regions such as promoters but in an unbiased way genome-wide. However, the high sensitivity comes along with a low specificity that makes identification of functional sites challenging. Nevertheless, in order to understand self renewal and pluripotency at the level of transcriptional regulation, it is crucial to identify a reliable set of regulatory elements that actively contribute to the regulation of gene expression in pluripotent cells such as embryonic stem cells and induced pluripotent stem cells. ES cells reflect a very early time point of development. Many genes which are important for early embryogenesis have a conserved function in mouse and human. OCT4, SOX2 and NANOG for example are essential for maintaining the pluripotent state both in mouse and human ES cells[3],[9]. However, despite their conserved function, where these transcription factors bind seems to be highly species-specific: A comparison of mouse and human ES cells revealed that only about 5% of binding events of the key pluripotency factors OCT4 and NANOG are conserved at orthologous genomic locations in both species[10]. A study of genome-wide binding in liver tissue reported the same with only about 7% conserved binding events for the liver transcription factors CEBP and HNF4 between mouse and human[11]. These data show how fast cis-regulatory elements can evolve compared to coding sequence, yet we do not know what discriminates conserved from non-conserved binding events. Genome-wide comparisons give average values over all observed binding events. These numbers are influenced by many factors such as the choice of control, processing of the data, and p-value and false discovery rate cutoffs. The biological impact of individual binding events may therefore be very different, ranging from non-functional binding to binding events which are essential for the regulation of associated genes and the survival of the individual[12]. For that reason, the fraction of conserved binding events is currently unknown for a highly confident set of enhancers. It is well known that transcription factors interact to regulate gene expression. It has been shown in mouse ES( mES) cells that combinatorial binding of Oct4, Sox2 and Nanog is associated with increased expression of nearby genes[5]. Here, we use combinatorial binding to increase the specificity of the ChIP-Seq technology in order to identify a highly confident set of regulatory elements. We investigate the association of these elements with the transcriptional co-activator Mediator, histone modifications and gene expression, to test whether the interactions of transcription factors provide a link between binding and activation of their target genes. Studies in Drosophila suggest that the combination of binding sites plays an important role during the evolution of gene regulatory elements[13], however the effect of combinatorial binding on evolution in mammals is currently unknown. Analysis of mammalian genome sequences suggested that developmentally active enhancers are highly conserved[14]. Integration of genome-wide binding data from mouse and human ES and embryonal carcinoma( EC/NCCIT) cells and mouse developmental tissues allows us for the first time to study the evolution of gene regulation in the light of combinatorial binding and developmental activity in mammalian cells using an in vitro system. Our analysis indicates that both characteristics contribute to the evolutionary constraint on regulatory elements and suggests that the integrated data represents an essential set of conserved enhancers that links pluripotency with early embryonal development.
Discussion:
The enormous amount of genome-wide binding data produced in recent years has improved our understanding of the self-renewing and pluripotent state of embryonic stem cells[3],[5],[27],[40]. By integrating data from ES cells with developmental enhancers we discovered that the very same regulatory elements bound by key pluripotency factors in ES cells frequently act as enhancers during early development. This finding provides an unknown link between the gene regulatory networks of ES cells and early development at the level of transcriptional regulation. The finding that binding at these developmental enhancers is highly conserved in mouse and human ES cells suggests that these elements are crucial for the maintenance of the pluripotent state. Nevertheless, some questions remain. Based on our data we cannot explain why in pluripotent cells OCT4, SOX2 and NANOG bind to enhancers which are also active at developmental stages when neither NANOG nor OCT4 are expressed. It is likely that these elements are poised for activation[23],[27], and an open chromatin state might be maintained throughout development to enable recruitment of transcription factors, co-activators, or histone modification proteins throughout cellular specification. These enhancers bound in multiple developmental stages by multiple factors show properties of gene regulatory hotspots, elements that influence gene expression in numerous cell types from pluripotent cells to at least cells of the mouse embryo at day 11. 5. The existence of such gene regulatory hotspots could explain the extraordinarily high level of binding conservation observed in ES cells, since mutations of these elements would influence a major part of early embryogenesis. In contrast to these hotspots, loss of individual binding events can more easily be substituted by nearby binding events, and is likely to influence only a limited number of cell types. Our analysis suggests that the fast evolutionary rewiring of regulatory networks indeed mainly affects individual binding events, while combinatorial binding at these regulatory hotspots is under stronger evolutionary constraint. The definition of combinatorial binding in this study relies on ChIP-Seq technology. We define combinatorial bound loci as genomic regions were we observe binding of different transcription factors in similar cell types. These experiments are independent of each other and reflect measures from a mixed population of cells. Co-localization could therefore be observed without direct physical interactions( for example through indirect interaction or competitive binding). However, based on the results of this and other studies[35],[41] we believe that co-localization as observed by the ChIP-Seq technology indeed reflects combinatorial binding. One of the difficulties in analyzing genome-wide datasets is how to discriminate true binding sites from false positive binding sites. It is impossible to identify a set of exclusively true binding sites, due to technical limitations, but also due to biological variation since many binding events will only be important under specified developmental cues. A more stringent p-value cutoff decreases the fraction of false binding sites in the data while at the same time true positive binding events will be lost. Combinatorial binding is likely to select for true binding sites as well, since non-functional binding events are unlikely to be detected in multiple experiments( supplementary Figure S10). However, combinatorial binding is different from a stringent control of false positives as can be seen by Mediator co-localization and binding conservation( Figures 3 and 6). It has been shown that groups of transcription factor binding sites are more likely to be conserved than isolated sites[42] which supports the value of combinatorial binding for transcriptional regulation. This is an important insight for future studies, which should consider the combination of transcription factors for defining regulatory networks. One limitation of the ChIP-Seq technology is that we cannot exclude combinatorial binding at isolated binding events. Weak or sporadic combinatorial binding events might be missed and therefore wrongly assigned as individual binding events( false negatives). We compared the results using two different cutoffs, a loose cutoff( full data set) with few false negatives and a very stringent cutoff( stringent data set) with many false negatives. The results largely agree: Combinatorial binding events( OCT4, SOX2, NANOG) consistently show the strongest association with Mediator( supplementary Figure S2) and highest levels of binding conservation( supplementary Figures S4 and S9). This suggests that the influence of the p-value cutoff and false negative binding events is limited on this analysis. Most of the binding data in this study is obtained from embryonic stem cells. We integrated data sets from two mouse cell lines( V6. 5 and E14). Interestingly, loci bound in both cell lines are much more likely to show co-localization with Mediator( supplementary Figure S8A). In human, we extended the available data by OCT4 ChIP-Seq from embryonal carcinoma cells to obtain data from different cell lines. We observe that, loci bound in EC and ES cells are much more likely to show combinatorial binding( supplementary Figure S8B). Therefore employing closely related cell lines is a biologically relevant approach for identifying important binding sites when data on combinatorial binding is not available. Developmental cues that lead to differentiation of cells during early embryogenesis involves binding of transcription factors at regulatory sequences in the genome. We have demonstrated that in ES cells, the combination of transcription factors that bind to regulatory elements is important for transcriptional activation. Combinatorial binding of OCT4, SOX2 and NANOG identifies enhancers characterized by H3K27ac and Mediator co-localization. Many of these combinatorially bound enhancers are active during early development. The comparison of mouse and human ES cells shows that both combinatorial binding and multiple activity of enhancers in ES cells and development increase the evolutionary constraint. The set of conserved combinatorially bound embryonic stem cell enhancers is available( supplementary Table S2) and might be helpful as a set of putative human developmental enhancers. Our analysis suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events. In contrast to these events, there is a group of conserved enhancers in the genome which recruit multiple interacting factors and are active in multiple tissues of the developing embryo( Figure 9). Many of these “gene regulatory hotspots” are under strong evolutionary constraints and seem to play a major role by linking the regulatory networks of cellular differentiation during early mammalian development.
The mammalian body is composed of hundreds of distinct cell types . During embryogenesis , this diversity is created by multiple cell fate decisions and differentiation events . Embryonic stem ( ES ) cells provide the in vitro model to study differentiation and early development . Their pluripotent state is maintained by transcription factors such as OCT4 , SOX2 and NANOG which bind to regulatory elements within the genome . Understanding the interplay between transcription factor binding , gene expression and cellular differentiation is key to understanding the development of the mammalian embryo . In this study we find that combinatorial binding of OCT4 , SOX2 and NANOG in ES cells identifies enhancers which are associated with active transcription . We observe that these enhancers also frequently show activity at later developmental stages . Using data from mouse and human ES cells we find that these combinatorially bound enhancers which are active in pluripotent cells and development show extraordinarily high levels of binding conservation ( >50% ) . Our analysis suggests that these conserved “gene regulatory hotspots” integrate the transcriptional network that promotes pluripotency into the gene regulatory networks that promote cell fate decisions and differentiation during early embryonic development .