Abstract
The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.
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Acknowledgements
We would like to dedicate this paper to Joe Calarco, who died tragically during the course of this work. His energy, enthusiasm, and insight will continue to guide our work and his warmth brightens our memories.
We are grateful to N. Hathaway, O. Bell, L. Chen, and J. Ronan, for insightful comments and technical assistance. We thank M. Alhamadsheh for synthesis of FK1012. We also thank J. Buenrostro and B. Wu for helpful advice and technical assistance with accessibility studies and V. Petkova. C.K. was supported by the NIH Director's New Innovator Award DP2 (1DP2CA195762-01), the American Cancer Society Scholar Award (RSG-14-051-01-DMC), the Pew Scholar Award, the A.P. Giannini Foundation, the Alex's Lemonade Stand Foundation (ALSF) Young Investigator Award, and the NIH SARC Sarcoma SPORE Career Development and SPORE Project (5U54 CA168512-04) awards. G.R.C. is supported by NIH grants (CA163915, NS046789), CIRM RB4-05886, SFARI and the Howard Hughes Medical Institute.
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C.K. and G.R.C. conceived of the study and wrote the paper. C.K., R.T.W., J.P.C., C.M.W., E.L.M., S.M.G.B., and E.J.C. planned and performed the experiments. J.L.P. performed data analysis and statistics.
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C.K. and G.R.C. are scientific co-founders, shareholders, and consultants of Foghorn Therapeutics, Inc.
Integrated supplementary information
Supplementary Figure 1 Generation of a rapamycin-inducible recruitment system for mSWI/SNF (BAF) complexes to the Oct4 locus in MEFs.
(a) ChIP-seq tracks for Brg1 and Ezh2 over the Oct4 locus in ES cells and MEFs. (b) Lentiviral delivery vector design for Frb-V5-[BAF complex subunit, here shown, SS18] and direct fusion of FKBP to ZFHD1. (ZFHD1-FKBP) for tethering to binding array upstream of the modified Oct4 (Pou5f1) allele. (c) Enrichment of each mark over Oct4:WT and Oct4:CiA alleles in MEFs. The inserted allele and the wild-type allele show quantitatively similar chromatin landscapes. Error bars = Mean ± SD for n=3 experiments. (d) Baf47 and Baf57 Frb-V5 tagged complex subunits properly assemble into BAF complexes. (e) Density sedimentation analysis using 10-30% glycerol gradients indicate that introduced Frb-V5-Ss18 is stably incorporated as endogenous SS18 and is dedicated to the 2MDa BAF complexes. (f) Each Frb-V5 tagged BAF complex can be recruited by 20-40 fold upon 24 hours of rapamycin treatment. Error bars = Mean ± SD for n=3 experiments. (g) BAF complex recruitment (t=30’) to the Oct4:CiA locus in MEFs results in BAF complex occupancy levels similar to those in ES cells. Error bars = Mean ± SD for n=3 experiments. (h) BAF complex recruitment (fold enrichment by ChIP- qPCR) within the recruitment region at (left) +237bp, and (right)+377bp, from the ZHFD1 locus (-309bp and -169bp from the Oct4 TSS) using three different immunocapture antibodies.
Supplementary Figure 2 Chromatin marks, DNA accessibility, gene expression in BAF complex recruitment time course experiments.
(a) (left) Occupancy of PRC2 complexes and H3K27me3 is reduced at 60 minutes post-rapamycin treatment. (right) Recruitment of Frb-V5-Stop does not recruit BAF complexes, nor alter the occupancy of PRC2 or the H3K27me3 mark at t=60 minutes of rapamycin treatment. Error bars = Mean ± SD for n=3 experiments. (b) Total H3, H3K9me3, and H2A.Z are unchanged upon 60- minute BAF complex recruitment. Error bars = Mean ± SD for n=3 experiments. (c) Recruitment of Frb-V5-SS18 (BAF) to the Ascl1 locus. CATCH-IT results indicate nucleosomal turnover at 60’ post treatment with rapamycin. ChIP using V5, H3, H2AK119Ub shows site-specific decreases in histone mark enrichment upon BAF complex recruitment. Error bars = Mean ± SD for n=2 experiments for ChIP and n=4 experiments for CATCH-IT. (d) Schematic for modified ATAC-seq accessibility assays using Tn5 transposase. (e) Tn5 accessibility landscape over the CiA Oct4 locus shows enhanced accessibility within 60 minutes of BAF complex recruitment. (f) Overlap of Brg, Ezh2, and Ring1b MACS-called peaks displayed as Venn diagrams (published datasets). (g) Rapamycin addition (BAF complex recruitment) at various time points indicated does not result in increased percentage of GFP-positive cells (left) nor Oct4 gene expression (right) in CiA Oct4 MEFs. Error bars = Mean ± SD for n=3 experiments.
(h) RNA Pol II ChIP-qPCR over the CiA Oct4 locus indicates lack of recruitment upon BAF complex recruitment over a time course. Error bars = Mean ± SD for n=2 experiments.
Supplementary Figure 3 Structures and downstream reformation of heterochromatin in FK1012 competition (washout) experiments.
(A) Structures of (left) Rapamycin and (right) FK1012 showing the regions that bind FKBP, but not FRB, making FK1012 an effective competitor for rapamycin-induced recruitment of BAF complexes. (B) FK1012 competition (washout) triggers downstream removal of recruited BAF complexes at the +237bp from ZFHD1 site (-309bp from Oct4 TSS). (C-D) Reformation of downstream (C) PRC2 and (D) PRC1 repression upon FK1012 washout at the +237bp (from ZNFHD1 array) site. (E) DNA accessibility is lost and heterochromatin reforms downstream upon FK1012 washout. All experiments are a representative n=1 except Ring1b where error bars = Mean ± SD for n=3 experiments.
Supplementary Figure 4 SS18-SSX- containing (SS-like) BAF complexes display enhanced occupancy and PcG displacement at the Oct4 locus in MEFs.
(A) Recruitment of BAF complexes is similar at the recruitment site (+0bp; -443bp of the Oct4 TSS). (B-D) Eviction of (B) Ezh2, (C) Ring1b, and (D) H3K27me3 at the recruitment site (+0) is similar between WT BAF complexes and SS-like BAF complexes. (E) SS-like BAF complexes show enhanced occupancy downstream (+1034bp) of recruitment site. (F-H) Eviction of (F) Ezh2, (G) Ring1b, and (H) H3K27me3 downstream (+1034bp) of recruitment site is enhanced by SS-like BAF complexes as compared to wild type. Error bars represent Mean ± SEM for n=3 experiments.
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Kadoch, C., Williams, R., Calarco, J. et al. Dynamics of BAF–Polycomb complex opposition on heterochromatin in normal and oncogenic states. Nat Genet 49, 213–222 (2017). https://doi.org/10.1038/ng.3734
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DOI: https://doi.org/10.1038/ng.3734
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