With the advent of genome-wide approaches for probing gene expression, protein occupancy of DNA sites and nucleosome positioning, a molecular framework for understanding the mechanism that underlies differentiation is emerging. ESCs have become the model for generating a unified map of the network of mechanisms that controls pluripotency, self-renewal and differentiation. Master regulatory transcription factors such as OCT4, SOX2 and Nanog work together with miRNAs and chromatin-regulatory proteins to maintain a transcription circuitry that allows both self-renewal and pluripotent lineage commitment (see ref. 88
for a review). Early genetic studies indicated that BAF complexes have an essential role in pluripotency. More recent screens for chromatin-related proteins that are required for ESC morphology identified components of NURD complexes, the TIP60-p400 complex, CHD1 and, as expected, BAF complexes67,87
. BAF complexes are the only ATP-dependent remodellers that have been studied by genome-wide ChIP–Seq analysis, but the functions of TIP60 and CHD1 have been studied by using promoter microarrays, providing global mechanistic insights into the functions of chromatin remodellers32,87
As described earlier, the SWI/SNF-like complex in ESCs, esBAF, has a functionally and biochemically distinct composition that is required for self-renewal and pluripotency28,29
. High-resolution genome-wide ChIP–Seq studies showed that the esBAF complex is present at about one-quarter of all promoters in mouse ESCs, with the intensity of binding correlating positively with the expression level of a gene. The targets of the esBAF complex are enriched for genes that are expressed highly and selectively by ESCs, and these overlap extensively with the targets of the transcription factors OCT4, SOX2, Nanog, STAT3 and SMAD1, suggesting functional interplay between the esBAF complex and these pluripotency factors in regulating genes involved in maintaining ‘stemness’30,32
. A small number of genes have been studied in ESCs cultured in the prolonged absence of BRG1 or BAF components, and the findings suggest that the esBAF complex maintains ESC fate simply by activating ‘ESC genes’ and repressing genes involved in differentiation29–31
. However, a genome-wide analysis carried out after acute depletion of BRG1 points to the esBAF complex having additional, more complex, modes of action32
. Reduction of esBAF levels by RNAi-mediated knockdown of the core ATPase Brg1
causes a large number of esBAF targets to be both upregulated and downregulated32
. Surprisingly, the upregulated genes include ESC-enriched genes that were already being actively transcribed, suggesting that the esBAF complex refines the expression levels of some ESC genes to keep them within the correct boundaries32
. The de-repression of several pluripotency genes, including Oct4
, in blastocysts treated with Brg1
-directed short interfering RNAs30
suggests that this refinement might also occur in vivo
. Nonetheless, it is unclear whether the activating or repressing functions of the esBAF complex are more crucial, and it is similarly unclear how these different outcomes of remodelling (activation versus repression) are achieved.
In addition to esBAF, the TIP60-p400 complex is also required for maintaining the self-renewal potential and pluripotency of ESCs87
. Using promoter microarrays, p400 was found at more than one-half of all promoters across the genome in mouse ESCs, and the intensity of binding was similarly correlated with the activity of the gene. The complex seems to be recruited to its targets in two ways, directly by the H3K4me3 mark and indirectly by Nanog. Although the TIP60-p400 complex has histone-acetyltransferase activity, which is generally associated with gene activation, this complex functions mainly to repress developmental genes. Hence, TIP60-p400 might deposit H4 acetylation marks that function in an unconventional manner to mediate gene repression.
CHD1 was also recently implicated in pluripotency, through its ability to maintain the open chromatin configuration that is characteristic of mouse ESCs67
. Mouse ESCs in which Chd1
has been knocked down using RNAi maintain many of the characteristics of self-renewing ESCs, but they are defective in multilineage differentiation. CHD1 associates with the promoters of active genes and prevents the accumulation of heterochromatin through an unknown mechanism. The conversion of euchromatin to heterochromatin is presumably the cause of the impaired lineage commitment of CHD1-deficient ESCs, because the induction of differentiation transcription programs potentially requires all genes to be generally accessible, including transcriptionally silent developmental genes in ESCs. Although it is not known how CHD1 maintains open chromatin structure, one intriguing possibility is that it is involved in incorporating H3.3 into the chromatin. Because the phenotypes observed in RNAi studies are sometimes unreliable, the implication that CHD1 is involved in pluripotency will need to be confirmed by analysing embryos with null mutations of Chd1
These studies illustrate the non-overlapping roles and different modes of action of the various ATP-dependent chromatin remodellers in a single cell type, and they show that these remodellers have genetically non-redundant and programmatic roles in pluripotency, perhaps as a result of their coordinated action with the master regulatory transcription factors. Given the crucial roles of chromatin remodellers in pluripotency, it is curious that they were not identified in the search for factors capable of generating induced pluripotent stem (iPS) cells. The reason for this might be the strict stoichiometry of the complexes, the requirement for which is shown by the observation that overexpression of just one subunit often results in a dominant-negative phenotype.