BAF directly antagonizes polycomb-mediated repression from flies to humans (i.e. Trithorax Group). Yet, BAF somehow also promotes polycomb-mediated repression. How can BAF do both things? What’s the mechanism? 2/n
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Traditional approaches (i.e. cKO/RNAi) take days to deplete proteins and cannot distinguish primary from secondary effects. To address this question directly, we created an mESC line where the ATPase subunit Brg1 can be rapidly degraded (T1/2 ~30m, near-complete ~2h). 3/npic.twitter.com/iuLl96rBFp
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Brg1 degradation rapidly derepresses many highly polycomb-bound genes in a time-dependent manner, such as Hox clusters and neurodevelopmental genes. So what happens to polycomb? 4/npic.twitter.com/SxKtnSJ989
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PRC1 and PRC2 are redistributed from highly bound domains to weakly bound sites genome-wide, consistent with driving the transcriptional response. 5/npic.twitter.com/RBIiAlbVDn
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Is this loss sufficient to decompact Hox clusters? We collaborated with the Boettiger lab to trace the DNA paths at HoxA and HoxD clusters with ORCA. Indeed, the polycomb loss is actually sufficient to physically decompact Hox! 6/npic.twitter.com/nAIpoGQPbW
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We next looked at features associated with active chromatin (K4me3, K27ac, and ATAC) and find that they negatively correlated with polycomb changes. BAF doesn’t generate accessibility to load polycomb and is excluded from repressed domains. BAF isn't working bidirectionally. 7/npic.twitter.com/GchaW2K6hF
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Is BAF controlling polycomb dosage? We made dTAG mESCs to degrade PRC1, PRC2, and both. We titrate polycomb protein dosage at the same time-point as our Brg1 degron. Voila, we find that intermediate degradation mirrors Brg1 derepression, but maximal is stronger! 8/npic.twitter.com/R2plM2941V
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Finally, if our model is correct then polycomb overexpression should block Hox derepression when Brg1 is degraded. We overexpressed a minimal vPRC1 complex (most catalytic activity) and indeed find that it suppressed derepression! 9/npic.twitter.com/DvEDGR2ERW
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Collectively, our data supports the model that BAF activity promotes distal repression by titrating polycomb dosage. We’re sending this out soon, so please let me know if you have any comments, suggestions, or criticism! Thanks for following along! 10/npic.twitter.com/d7wtaz07T9
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Excited to read this in detail! Looks tremendous - well done.
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Thanks Gabe, I think the mechanism is similar to your result with Mot1.
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