Abstract
The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF–Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb–Trithorax axis.
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Data availability
All sequencing data have been deposited in the GEO (accession no. GSE145016). Source data are provided with this paper.
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Acknowledgements
We thank members of the Crabtree laboratory for insightful comments and discussions over the course of the study. We are grateful to A. Krokhotin, S. Ramachandran (CU Anschutz) and V. Ramani (UCSF) for providing critical comments on the manuscript and for helpful discussions. This study was supported by NIH grants R01CA163915 (G.R.C.), R37NS046789 (G.R.C.) and DP2GM132935 (A.N.B.); the Howard Hughes Medical Institute (G.R.C.); a Swiss National Science Foundation (SNSF) postdoctoral fellowship (S.M.G.B.); the Walter V. and Idun Berry Postdoctoral fellowship program (C.M.W. and A.H.); and the Sir James Black postdoctoral fellowship (C.M.W. and S.M.G.B.).
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C.M.W. conceived the project and carried out experiments and analysis. A.H. performed ORCA experiments and analysis. S.M.G.B., J.G.K. and B.Z.S. performed experiments and analysis. G.R.C. and A.N.B. designed experiments, provided valuable insight and supervised the project. C.M.W. wrote the manuscript with assistance from all authors.
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G.R.C. is founder and stockholder of Foghorn Therapeutics. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Extended characterization of Brg1-AID degron, related to Fig. 1.
(a, Genotyping PCR for three homozygous knock-in clones (AID*20 was used for most experiments in the manuscript). b, Western blot of whole-cell extracts from knock-in lines showing 8.8kDa shift in migration from c-terminal tag (G4S)3-AID*-G4S-3×FLAG. Line 20 and 26 have homozygous insertions in frame that were also confirmed by Sanger sequencing. c, Representative western blot and quantitation of Brg1 abundance in parent line compared to Brg1-AID* ± osTIR1 induction (-auxin). Error bars represent mean ± standard deviation from five biological replicates. d, Comparison of Brg1 depletion kinetics between AID and Cre-lox genetic deletion by western blot and changes to colony morphology that occur at the 24-hour time-point. Cells were only grown on the same dish for 72 h to illustrate timing for Brg1fl/fl colony morphology changes. Experiment was repeated at least 3 times with similar results. Scale bar = 50 µm. e, Alkaline phosphatase staining of Brg1 AID* 20 line on gelatin coated dishes, showing that changes to colony morphology occur ~24 h. Top scale bar = 200 µm, bottom scale bar = 50 µm. Experiment was repeated at least 3 times with similar results.
Extended Data Fig. 2 Extended summary of RNA-seq results, related to Fig. 1.
a, Volcano plots depicting gene expression changes at 0.5, 1, 2, 4, and 8 hours of Brg1 degradation. Differentially expressed genes are colored red (FDR-corrected P < 0.05) from four biological replicates for each time-point. b, Pbrm1 degradation with auxin, does not derepress Hox genes, revealing that pBAF is not required for their repression and controls for auxin, Tir1 expression, and proteasomal degradation in the Brg1 degron. Error bars are mean ± standard error for n = 3 replicates. c, Gene set enrichment analysis for differentially downregulated genes at 4 and 8-hour timepoints. d, Overlap of differentially expressed genes in 72 h Brg1 fl/fl from12 and 8 h Brg1 AID calculated by Fisher’s exact test (p-value = 1.1e-46). e, Strong lentiviral Bmi1 overexpression doesn’t derepress Hox genes or other genes derepressed by Brg1 degron. Error bars depict standard error of the mean from three biological replicates.
Extended Data Fig. 3 Brg1 degradation results in quick redistribution of PRC1&2 but doesn’t affect net dosage, related to Fig. 2.
a, Principal component analysis of Ring1b and Suz12 ChIP-seq (n = 4 replicates) b, Browser snapshot of the difference in normalized counts (8 h – 0 h) for Ring1b and Suz12 at Hox A, B, C, and D depicting net loss across all four clusters. c, Average metagene plots of normalized counts (to read-depth) at sites that were significantly increased or decreased by DESeq2 analysis (FDR-corrected P < 0.1) and normalized to peak intensity, providing additional confirmation that normalization is correct. d, ChIP qPCR timecourse at 4, 8, and 24 h Brg1 degradation for Ring1b at sites that were significantly decreased by ChIP-seq. Error bars depict standard error of the mean from four biological replicates. (* = p < 0.05, ** = p < 0.005, *** = p < 0.0005; two-sided t test with Holm-Sidak multiple comparison correction). e, (left) Representative western blot showing Brg1 degradation efficiency and that H2AK119ub and H3K27me3 marks don’t change at 8 h time-point (ChIP-seq time-point for the complexes that deposit these marks). (right) Representative western showing Brg1 degradation efficiency over a longer timecourse and that Suz12, Ring1b, and H3K27me3 marks don’t noticeably change even at 48 h. Experiment was repeated at least 2 times with similar results (f) Correlation of all Ring1b and Suz12 peaks that are ± 2kb from gene transcription start sites and expression changes at 8 h Brg1 degradation. Correlation and p-values were obtained from Pearson’s product moment correlation. g, chromatin state enrichment for all, increased, and decreased peak changes for Ring1b and Suz12. h, ChIP qPCR and qRT-PCR validation of genes that gain Ring1b and Suz12 and have reduced gene expression following 8 h Brg1 degradation. Error bars depict standard error of the mean from five biological replicates.
Extended Data Fig. 4 Comparison of ORCA to Hi-C, related to Fig. 3.
a, Average contact frequency as measured by high resolution Hi-C from serum grown cells33. b, Average contact frequency as measured by ORCA (200-nm threshold). c, Pearson’s correlation and Pearson’s r were computed using all unique pairwise combinations of X,Y barcodes measured. d, Histograms depicting the difference in median distance for barcode pair combinations at HoxA. e, Histograms depicting the difference in median distance for barcode pair combinations at HoxD.
Extended Data Fig. 5 Extended analysis related to Fig. 4.
a, Heatmap depicting the negative correlation between H3K4me3 and Ring1b and Suz12 as the difference in normalized counts (8 h – 0 h) at peaks (8,220) that intersect H3K4me3, Ring1b, and Suz12 at merged peak center, sorted by decreasing H3K4me3 signal. b, Heatmap depicting the negative correlation between H3K27ac and PRC1&2 as the difference in normalized counts (8 h – 0 h) at peaks (4,140) that intersect H3K27ac, Ring1b, and Suz12 at merged peak center, sorted by decreasing H3K27ac signal. c,d, Scatterplots showing negative correlation between ATAC-seq changes (KO/control)44 and Ring1b (8 h/0 h) or Suz12 (8 h/0 h).
Extended Data Fig. 6 Transcription is not required for polycomb redistribution.
a, Experimental design to test whether polycomb redistribution requires transcription. b, qRT-PCR depicting fold change by ΔΔCt method with indicated treatments from n = 3 biological replicates. c,d, Scatterplot showing that Ring1b and Suz12 changes following Brg1 degradation are highly correlated despite global inhibition of transcription for all peaks c, and peaks that were significantly changed by Brg1 degradation alone (d) (FDR < 0.1). e, Scatterplot of changes to Ring1b and Suz12 upon treatment with auxin (auxin / control) or triptolide (triptolide / control) showing distinct effects from the two treatments. Correlation and p-values were obtained from Pearson’s product moment correlation for c-e, from n = 4 biological replicates at the 8 h timepoint.
Extended Data Fig. 7 PRC1 does not restrict BAF binding despite extensive overlap.
a, Heatmaps depicting Ring1b, Suz12, BAF155, and Brg1 ChIPseq signal from79 at peaks (8,228) that overlap Ring1b and Suz12, sorted by decreasing Ring1b signal (left) or Brg1 peaks (97,987) sorted by decreasing Brg1 signal (right). b, High overlap between Brg1 and BAF155 despite different number of peaks called due to antibody quality and read depth. c, BAF155 ChIP-seq in Ring1bfl/fl x Ring1a-/- x ActinCreER ESCs treated with Tamoxifen or EtOH for 72 h. MA plot (TAM/EtOH) highlights large number of unchanged peaks (>95% in blue) (n = 2). d, Analysis of the chromatin features that characterize BAF155 binding sites reveal that Ring1a/b double KO does not alter BAF complex binding, as the complex remains mainly bound to enhancers and bivalent promoters in both control and mutant ESCs. e, Representative genome tracks showing similar BAF155 levels across the Lefty1/2 locus in Tamoxifen and EtOH treated cells. f, Metagene plot and heatmaps showing that BAF155 ChIPseq signal is minimally affected by Ring1b deletion (g) Characterization of BAF155 peaks in EtOH and Tamoxifen conditions. Of the 49,000 detected BAF155 peaks in this dataset, less than 5% were significantly changed upon Ring1a/b double KO in ESCs and the small number of gained and decreased sites was similar. Despite 56% of Ring1b peaks overlapping with a BAF peak, only 1% of differentially bound peaks are within a PRC1 domain (bottom).
Extended Data Fig. 8 Characterization of EED/Ring1b degron and extended analysis related to Fig. 5.
a, Schematic depicting EED and Ring1b dTAG targeted protein degradation strategy in mESCs, where EED is tagged at the C-terminus and Ring1b is tagged at the N-terminus with FKBPF36V to enable targeted degradation with dTAG13 PROTAC. b, Representative western blot and quantitation of EED and Ring1b abundance in parent line compared to lines tagged with FKBPF36V. Error bars depict mean ± standard error of the mean from three tagged EED lines and four tagged Ring1b lines. c, Genotyping PCR and western from whole cell extract showing 14.6kDa shift in migration from C-terminus (G4S)3-FKBPF36V-G4S-V5 tag on three different EED clones used for experiments. d, Genotyping PCR and western from whole cell extract with 14.2kDa shift in migration from N-terminus FKBPF36V-G4S-HA-(G4S)3. Experiment was repeated at least 2 times with similar results (e) Brg1 ChIPseq showing localization to the TSS of differentially expressed genes, ChIP from79. f, Brg1 MNase ChIPseq showing localization to the TSS of differentially expressed genes, ChIP from80. g, Brg1 MNase ChIPseq showing higher occupancy at PRC bound genes repressed by Brg1 (independent of polycomb). Compare to Fig. 5f. h, Brg1 represses accessibility at PRC bound genes derepressed by Brg1 degron and Brg1/PRC degron. Accessibility change (Brg1 KO / control) by overlap (Fig. 6f) for peaks ± 2kb from gene promoter, ATAC data from44 with two-sided t-test p-value above. (i,top) Higher and broader polycomb domains over genes derepressed by both Brg1 and PRC degron than genes derepressed by Brg1 degron alone, with Suz12 occupancy shown here. (i, bottom) Broad domains over genes derepressed by both Brg1 and PRC degron show substantially more redistribution in the Brg1 degron. Compare to Ring1b signal in Fig. 6. (j) Expression difference (Brg1 – PRC Log2 Fold Change) for genes derepressed by Brg1 and PRC degron, over dosage titration (Most dosage sensitive genes are shown in Fig. 6g), from n = 4 biological replicates. Boxplot bounds depict quartile 1, median, and quartile 3 with whiskers at 1.5× interquartile range.
Extended Data Fig. 9 Histone modifications deposited by PRC1 and PRC2 are higher in 2i growth conditions and results in weaker derepression, related to Figs. 2 and 6.
a, Western blot and relative quantification of H2AK119ub and H3K27me3 normalized to H3 in cells grown in Serum/LIF or 2i growth conditions from n = 3 biological replicates. b, Hox gene derepression measured by qRT-PCR for cells grown in serum, 2i, and the ratio of serum/2i from n = 2 biological replicates.
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Weber, C.M., Hafner, A., Kirkland, J.G. et al. mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution. Nat Struct Mol Biol 28, 501–511 (2021). https://doi.org/10.1038/s41594-021-00604-7
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DOI: https://doi.org/10.1038/s41594-021-00604-7
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