Our studies indicate that esBAF determines the pattern of STAT3 binding across the pluripotent genome. STAT3 signalingactivates a very different group of genes in other cell types with distinct compositions of BAF complexes. We propose thatESC genome is conditioned by the esBAF complex, which is present on chromatin prior to STAT3binding, and can therefore contribute to determining which STAT3 target genesare activated in ESCs. In WT ESCs, Brg (and by inference esBAF complexes) mediates the accessibility of functional STAT3binding sites, thereby facilitating robust STAT3binding and STAT3-activated transcriptionin response to LIF (). Without esBAF, STAT3is still activated and localized to the nucleus but is unable to bind to the vast majority of its legitimate target sites in ES cells but is instead targeted to inappropriate sites. esBAF also prevents the inappropriate silencing of STAT3 target genes by Polycomb complexes, ensuring that ESCs maintain LIF competency required for pluripotency. Unexpectedly, esBAF also synergizes with Polycomb at a subset of genes, including all four Hox clusters, ensuring their firm repression in ESCs.
esBAF both antagonizes and synergizes with PRC2 to promote pluripotency
The role of SWI/SNF-like complexes in signaling is not surprising, since the yeast SWI/SNF complex was discovered in screens for regulators of signal-dependent gene activation. However, in yeast, a transcription factor SWI5 appears to recruit SWI/SNF to its target genes40
. In mammalian systems, STAT3 was shown in one case to be recruited to a target gene by an undefined Brg-containing complex41
, however other studies suggest the opposite,i.e. that STAT3 recruits Brg42
. Our studies provide genome-wide evidence that STAT3 recruitment is dependent upon the prior actions of Brg. In our proteomic analysis of esBAF complexes we did not detect STAT3 peptides indicating that it is unlikely that a stable interaction exist between esBAF and STAT3 in solution.However, esBAF occupancy also appears to be partially dependent on STAT3, indicating that the current models of unidirectional recruitment, either of STAT3 by Brg or of Brg by STAT3, are incomplete. Rather, we favor a model of interdependent recruitment. While STAT3 binding is dependent on Brg preassembly on target chromatin, steady-state occupancyof esBAF is reinforced by bound STAT3.
The nature of the antagonistic roles between PcG and Brg has been a matter of much debate 21–23
. Our studies demonstrate that genome-wide, Brg excludes PcG function because Brg deletion leads to the invasion of PcG at the sites we have tested and most Brg-activated genes show an increase in H3K27me3 and transcriptional silencing. Thus, we favor a mechanism of opposition in which Brg prevents PcG binding to a large group of genes including LIF/esBAF targets (). This is clearly not due to a loss of transcription, since removing LIF does not lead to PcG binding and H3K27me3 deposition. In human rabdhoid tumors caused by the deletion of BAF47 (Snf5),
re-expression of BAF47
leads to eviction of PcG and reduced H3K27Me3 at the Ink4a
, which is consistent with the genome-wide antagonism we report.
In contrast to textbook descriptions of the antagonism between Brm and Polycomb, we find that esBAF synergizes with PRC2 at all four Hox loci (), which must be effectively silenced in ESCs to prevent premature differentiation. Previous studies have not uncovered the presence of synergism between BAF and PcG complexesbecause only single genes were examined43
, and because synergism was not expected in light of early studies in Drosophila44
. This unexpected finding might be restricted to ESCs and could arise from the fact that ESCs contain a specialized SWI/SNF-like BAF complex (esBAF) not found in other cell types.
Hence, Brg appears to function in synergy with PcG to repress differentiation genes in ES cells, while preventing PcG-mediated repression of pluripotency-related genes activated by ESC transcription factors such as Stat3, Oct4 and Sox2. These findings imply that esBAF regulates the activity of Polycomb across the genome in both positive and negative modes. Clearly an important future goal will be to understand the biochemical basis of the ability of Brg to direct Polycomb function. This more complex view of Brg’s function is consistent with the observation that mutations in Brg, Brm or other components of mammalian BAF complexes have not shown homeotic-like defects in mice. Thus, during the course of evolution, the interplay between Brm-based chromatin remodeling complexes and PcG has expanded beyond mere antagonism. Nevertheless, the two complexes appear to use both their synergistic and antagonistic functions to facilitate and maintain the pluripotent state.