Abstract
The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures.
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Acknowledgements
This work was supported by funds awarded to G.R.C. from the Howard Hughes Medical Institute, the Simons Foundation Autism Research Initiative, and the NIH (grants NS046789 and CA163915). E.L.M. was supported by the Lucille P. Markey Stanford Graduate Fellowship in Biomedical Research and by the Stanford University Genetics & Developmental Biology Training Program (NIH-NIGMS T32 GM007790). D.C.H. was supported by NCI career transition award K99CA184043. C.H. is supported by NCI career transition award K99CA187565. ATAC-seq libraries were prepared with advice from B. Wu. We used the BioX3 cluster, which is supported by NIH S10 Shared Instrumentation Grant 1S10RR02664701, for sequencing analysis. Many thanks to A. Koh and C. Weber for technical advice and support.
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The studies of TOP2/BAF function were designed and conducted by E.L.M., G.R.C., and D.C.H. The CiA system was conceived by G.R.C., adapted for analysis of BAF mechanisms by C.K., and adapted for analysis of LSH and INO80 mechanisms by J.P.C. ATAC-seq experiments were conceived by W.J.G. and J.D.B., performed by E.L.M. and C.-Y.C., and analyzed by E.L.M. MNase assays and sequencing were performed by E.L.M., K.Z., and K.C. and analyzed by E.L.M. Binding kinetics were calculated by C.H. E.L.M. and G.R.C. wrote the manuscript, with input from the other authors.
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Integrated supplementary information
Supplementary Figure 1 Recruitment of other chromatin remodelers does not induce accessibility, and ICRF-193 does not affect BAF recruitment.
(a) Western blots of CiA:Oct4 fibroblasts over-expressing V5-tagged SS18-FRB and/or HA-tagged ZF-FKBP. Uncropped blot images are shown in Supplementary Data Set 1. V5 (SS18-FRB) ChIP (b) or RNA Pol. II ChIP (e) in BAF recruitment system treated with 3 nM rapamycin (Rap) for 1 hour. (c) ATAC-qPCR in LSH or INO80 recruitment system. (d) V5 (LSH-FRB) ChIP in LSH recruitment system. (f) BAF155 ChIP in cells with BAF recruitment system treated with rapamycin and 1 μM ICRF-193 for 1 hour. (g) ATAC-qPCR in cells treated with rapamycin and 1 μM topotecan for 1 hour to inhibit TOP1. Significance assessed by two-tailed t-tests versus no rapamycin control or as specified: n.s.: p ≥ 0.1, •: p < 0.1, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Data are actual values from 2 cell passages (b,c) or means ± s.e.m. from 3 cell passages (d,e,f,g).
Supplementary Figure 2 Comparison of the effects of TOP2 inhibition with those of Brg1 deletion on chromatin accessibility.
Western blots of ES cells treated with 1 μM ICRF-193 for 24 hours (a), or Brg1fl/fl; actin-CreER (b) or Baf53afl/–; actin-CreER (c) ES cells treated with tamoxifen (Tax) to knockout Brg1 or Baf53a, respectively. (d) DNA gel electrophoresis of MNase digests of Baf53afl/– cells using 6, 8, 10, or 12 units of MNase. Arrowheads point to different nucleosome species. M: DNA marker. Uncropped gel images are shown in Supplementary Data Set 1. (e) Fold-change densitometry of MNase digests in log-scale. (f) ATAC-qPCR of Brg1fl/fl cells. Significance assessed by t-tests as before. Overall significance of effect of tamoxifen treatment assessed by three-way ANOVA. Data are mean ± s.e.m. from 3 cell passages. (g) Contingency heatmaps/tables of ATAC-seq peak overlap counts. (h) ATAC-seq fragment dyad density across regulatory regions.
Supplementary Figure 3 OCT4 recruitment without BAF recruitment does not co-recruit BAF or resolve heterochromatin.
(a) Western blots of CiA:Oct4 ES cells and fibroblasts over-expressing V5-tagged SS18-FRB and OCT4. Uncropped blot images are shown in Supplementary Data Set 1. (b) BRG1 ChIP in fibroblasts with the BAF recruitment system and over-expressing OCT4 treated with 3 nM rapamycin. (c) Strategy for direct OCT4 recruitment to the CiA:Oct4 locus in fibroblasts. ATAC-qPCR (d) and BRG1/OCT4 ChIP (e) in fibroblasts over-expressing OCT4-GAL4 in the absence of BAF recruitment. Significance assessed by t-tests as before. Overall significance of the effect of OCT4-GAL4 expression on ATAC-qPCR assessed by three-way ANOVA. Data are mean ± s.e.m. from 3 (b) or 4 cell passages (d,e).
Supplementary Figure 4 esBAF promotes accessibility at pluripotency factor binding sites.
ATAC-seq dyad density across transcription factor binding sites in ES cells treated with 1 μM ICRF-193 for 24 hours (a) or Brg1fl/fl ES cells (b).
Supplementary Figure 5 esBAF promotes nucleosome spacing at pluripotency factor binding sites.
(a) MNase-seq phasogram analysis of Brg1fl/fl ES cells. MNase-seq nucleosome (b,c,e) or ATAC-seq (d) dyad density across linker-bound ChIP-seq peaks or 38,864 randomly shuffled sites that exclude TSSs, TESs, or enhancers (b,c) or accessible motifs (d,e) in Brg1fl/fl ES cells. Vertical lines indicate nucleosome positions and values are changes in nucleosome positioning (bp, tamoxifen – EtOH). (f) OCT4 (left) and SOX2 (right) ChIP in ES cells treated with 1 μM ICRF-193 for 24 hours or Brg1fl/fl ES cells. Significance assessed by t-tests as before. Overall significance of treatment effect assessed by three-way ANOVA. Data are actual values from 2 cell passages.
Supplementary Figure 6 BAF recruitment can be washed out with FK506.
(a) V5 (SS18-FRB) ChIP in fibroblasts with the BAF recruitment system treated with 3 nM rapamycin (Rap) for 1 hour with subsequent washout of rapamycin and concurrent addition of 100 nM FK506. Data are mean ± s.e.m. from 4 cell passages. (b) ATAC-seq dyad density across non-bivalent histone modification sites in ES cells treated with 1 μM ICRF-193 for 24 hours.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 2531 kb)
Supplementary Table 1
ATAC-seq peaks with decreased, unchanged, or increased accessibility after 24 h of ICRF-193 treatment or conditional knockout of Brg1 or Baf53a (XLSX 16292 kb)
Supplementary Table 2
List of public ChIP-seq data sets used (XLSX 49 kb)
Supplementary Data Set 1
Uncropped gels (PDF 12146 kb)
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Miller, E., Hargreaves, D., Kadoch, C. et al. TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin. Nat Struct Mol Biol 24, 344–352 (2017). https://doi.org/10.1038/nsmb.3384
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DOI: https://doi.org/10.1038/nsmb.3384
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