Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst 1
. Due to their ability to self-renew while retaining the potential to differentiate into most other cell types in the body, there has been growing interest to explore hESCs in regenerative medicine and as a model system to study early human development.
A prevailing theory holds that pluripotency is established and maintained by a network of transcription regulatory proteins 2, 3
. A core transcriptional regulatory network consisting of transcription factors OCT4, SOX2, NANOG and their regulatory target genes has been discovered to control the gene expression program to maintain self-renewal and pluripotency in human and mouse ESCs 3, 4, 5
. Oct4, Sox2 and Nanog bind to key cis
-regulatory sequences, including enhancers and promoters, recruiting co-activator complexes or transcriptional machinery to allow transcription and production of proteins 4, 5, 6
. Importantly, they regulate each other's transcription, through feed forward loops, and produce two bistable states: in the presence of appropriate levels of all three proteins, the network is able to maintain the stable expression of each gene; on the other hand, if any of these proteins is absent or inappropriately expressed, the cells would then exit from this state and take on another stable state where some or all the three proteins are repressed. Besides regulating themselves, Oct4, Sox2 and Nanog also control the expression of a large number of other pluripotency genes, many of which encode for additional transcription factors that work together with the three to further stabilize the pluripotent state.
While it is clear that the transcriptional regulatory network plays a critical role in establishing and maintaining pluripotency of ES cells, other factors have also been shown to be important. Among them are epigenetic factors, such as DNA methylation and histone modifications, cis
-regulating molecular marks that can exert long-lasting influences on gene activities in the cells 7, 8, 9
. Without DNA methylation, differentiation to certain lineages is severely blocked 10, 11
. Alterations that lead to abnormal chromatin modifications are also known to cause defects in either maintenance of pluripotency or cell fate specification 12, 13, 14
. Therefore, there has been considerable interest in the examination of the DNA methylation profiles and chromatin landscape in pluripotent stem cells and lineage-committed cell types.
Promoter bivalency, characterized by trimethylation of both histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) at gene promoters, has been proposed as an epigenetic mechanism for regulating development and proliferation 15, 16, 17, 18, 19, 20
. These promoters are considered poised for activation (or repression) at a later stage, since they contain both active and repressive modifications. These genes are not transcribed in ES cells, but could either become activated during differentiation with concurrent loss of the methylation mark on H3K27 or silenced as indicated by loss of H3K4me3 15, 21
. Given that not all repressed genes are targeted by H3K27me3, identifying which genes are influenced by this modification will be critical to understanding cell fate changes.
Regulation of chromatin state at specific enhancers may represent another epigenetic mechanism involved in pluripotency. We and others have demonstrated that transcriptional enhancers are characterized by the histone modification H3K4me1, and the presence of this mark is strongly correlated with tissue- and cell-type specific gene expression, suggesting a role for chromatin state in regulating enhancer activities 22, 23, 24
. Recent studies have further shown that transcription factor binding and function at enhancers are strongly influenced by preexisting chromatin structure 25, 26, 27
. More recently, Rada-Iglesias et al
. examined the chromatin state in hESCs differentiating along a neural epithelium lineage, and uncovered two classes of enhancers distinguished by the presence or absence of lysine 27 acetylation on histone H3 (H3K27ac). The presence of this mark is indicative of active state of enhancers and correlated with activation of stem cell genes in the hESCs, while its absence coupled with presence of H3K4me1 would suggest a poised state that could become activated upon differentiation 28
. Similar findings were also reported in mouse ES cells that undergo differentiation to neural progenitor cells 29
To further characterize the role of chromatin modifications in self-renewal, pluripotency and differentiation, we have employed a new model of ES cell differentiation, which involves the treatment of hESCs grown in defined medium by BMP4, inducing them to exit the pluripotent state and enter a mixture state of mesendodermal and trophectodermal cells. We mapped epigenetic differences in undifferentiated ES cell (hESC) and differentiated ES cell (denoted as DFC) genomes. We have identified potential enhancers using chromatin signature patterns, and examined the dynamics of chromatin state at both promoters and enhancers during the differentiation of these cells. We found remarkable differences of chromatin dynamics at human promoters and enhancers. The chromatin state at promoters is generally stable during differentiation, with a small fraction undergoing changes that primarily involve a switch between active acetylation and repressive methylation at H3K27, which further allowed us to define a set of genes that appears to be important for maintenance of ES cell pluripotency, and another set that is involved in differentiation. The use of epigenetic changes distinguishes them from the larger group of differentially expressed genes. Our experiments identified over 50 000 potential enhancers in the undifferentiated ES cell and differentiated ES cell genomes, with a majority of the enhancers displaying marked changes in chromatin states in a manner that correlates with differential expression of their predicted target genes. These enhancers, and the factors predicted to bind them based on motif analysis, now provide a basis for future investigation into the regulatory networks of hESCs and differentiation. In addition, we also identified a set of poised enhancers marked by a distinct chromatin signature near developmentally important genes induced early in differentiation, underscoring the importance of enhancer elements in regulating differentiation. More importantly, this unique set of enhancer elements likely provides one means by which stem cells could respond to stimuli and differentiate to various cell types, in part becoming a key characteristic of pluripotency.