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TOP2 synergizes with BAF chromatin remodeling for both resolution and formation of facultative heterochromatin

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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Figure 1: TOP2 is required for transcription-independent BAF-mediated resolution of facultative heterochromatin to accessible chromatin.
Figure 2: Active TOP2 is recruited immediately after BAF recruitment and is required for the initial stage of accessibility induction.
Figure 3: TOP2 and BAF promote the accessibility of enhancers and promoters genome-wide.
Figure 4: TOP2 is required for optimal BAF-mediated recruitment of OCT4.
Figure 5: TOP2 and BAF are required for the recruitment of pluripotency factors genome-wide.
Figure 6: TOP2 is required for the re-formation of facultative heterochromatin.

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References

  1. Chen, T. & Dent, S.Y. Chromatin modifiers and remodellers: regulators of cellular differentiation. Nat. Rev. Genet. 15, 93–106 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Boettiger, A.N. et al. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529, 418–422 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ghirlando, R. & Felsenfeld, G. Chromatin structure outside and inside the nucleus. Biopolymers 99, 225–232 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hathaway, N.A. et al. Dynamics and memory of heterochromatin in living cells. Cell 149, 1447–1460 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kadoch, C. et al. Dynamics of BAF-Polycomb complex opposition on heterochromatin in normal and oncogenic states. Nat. Genet. 49, 213–222 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Ho, L. et al. An embryonic stem cell chromatin remodeling complex, esBAF, is an essential component of the core pluripotency transcriptional network. Proc. Natl. Acad. Sci. USA 106, 5187–5191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bao, X. et al. A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63. Genome Biol. 16, 284 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Morris, S.A. et al. Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions. Nat. Struct. Mol. Biol. 21, 73–81 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Ho, L. et al. esBAF facilitates pluripotency by conditioning the genome for LIF/STAT3 signalling and by regulating polycomb function. Nat. Cell Biol. 13, 903–913 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kadoch, C. & Crabtree, G.R. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. Cell 153, 71–85 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wilson, B.G. et al. Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer Cell 18, 316–328 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Stanton, B.Z. et al. Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin. Nat. Genet. 49, 282–288 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Ho, L. & Crabtree, G.R. Chromatin remodelling during development. Nature 463, 474–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ho, L. et al. An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency. Proc. Natl. Acad. Sci. USA 106, 5181–5186 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kidder, B.L., Palmer, S. & Knott, J.G. SWI/SNF-Brg1 regulates self-renewal and occupies core pluripotency-related genes in embryonic stem cells. Stem Cells 27, 317–328 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Kleger, A. et al. Increased reprogramming capacity of mouse liver progenitor cells, compared with differentiated liver cells, requires the BAF complex. Gastroenterology 142, 907–917 (2012).

    Article  PubMed  Google Scholar 

  18. Singhal, N. et al. Chromatin-remodeling components of the BAF complex facilitate reprogramming. Cell 141, 943–955 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Yoo, A.S. et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Staahl, B.T. & Crabtree, G.R. Creating a neural specific chromatin landscape by npBAF and nBAF complexes. Curr. Opin. Neurobiol. 23, 903–913 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Kadoch, C. et al. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nat. Genet. 45, 592–601 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shain, A.H. & Pollack, J.R. The spectrum of SWI/SNF mutations, ubiquitous in human cancers. PLoS One 8, e55119 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Hodges, C., Kirkland, J.G. & Crabtree, G.R. The many roles of BAF (mSWI/SNF) and PBAF complexes in cancer. Cold Spring Harb. Perspect. Med. 6, a026930 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Kadoch, C. & Crabtree, G.R. Mammalian SWI/SNF chromatin remodeling complexes and cancer: mechanistic insights gained from human genomics. Sci. Adv. 1, e1500447 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Ronan, J.L., Wu, W. & Crabtree, G.R. From neural development to cognition: unexpected roles for chromatin. Nat. Rev. Genet. 14, 347–359 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Santen, G.W., Kriek, M. & van Attikum, H. SWI/SNF complex in disorder: SWItching from malignancies to intellectual disability. Epigenetics 7, 1219–1224 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hainer, S.J. & Fazzio, T.G. Regulation of nucleosome architecture and factor binding revealed by nuclease footprinting of the ESC genome. Cell Rep. 13, 61–69 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hu, G. et al. Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1. Genome Res. 21, 1650–1658 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dykhuizen, E.C. et al. BAF complexes facilitate decatenation of DNA by topoisomerase IIα. Nature 497, 624–627 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wijdeven, R.H. et al. Genome-wide identification and characterization of novel factors conferring resistance to topoisomerase II poisons in cancer. Cancer Res. 75, 4176–4187 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Mertz, J.E. & Miller, T.J. In vivo catenation and decatenation of DNA. Mol. Cell. Biol. 3, 126–131 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Vos, S.M., Tretter, E.M., Schmidt, B.H. & Berger, J.M. All tangled up: how cells direct, manage and exploit topoisomerase function. Nat. Rev. Mol. Cell Biol. 12, 827–841 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fernández, X., Díaz-Ingelmo, O., Martínez-García, B. & Roca, J. Chromatin regulates DNA torsional energy via topoisomerase II-mediated relaxation of positive supercoils. EMBO J. 33, 1492–1501 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Salceda, J., Fernández, X. & Roca, J. Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA. EMBO J. 25, 2575–2583 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Naughton, C. et al. Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures. Nat. Struct. Mol. Biol. 20, 387–395 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kouzine, F. et al. Transcription-dependent dynamic supercoiling is a short-range genomic force. Nat. Struct. Mol. Biol. 20, 396–403 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. King, I.F. et al. Topoisomerases facilitate transcription of long genes linked to autism. Nature 501, 58–62 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Thakurela, S. et al. Gene regulation and priming by topoisomerase IIα in embryonic stem cells. Nat. Commun. 4, 2478 (2013).

    Article  PubMed  Google Scholar 

  39. Tiwari, V.K. et al. Target genes of Topoisomerase IIβ regulate neuronal survival and are defined by their chromatin state. Proc. Natl. Acad. Sci. USA 109, E934–E943 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sano, K., Miyaji-Yamaguchi, M., Tsutsui, K.M. & Tsutsui, K. Topoisomerase IIβ activates a subset of neuronal genes that are repressed in AT-rich genomic environment. PLoS One 3, e4103 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Madabhushi, R. et al. Activity-induced DNA breaks govern the expression of neuronal early-response genes. Cell 161, 1592–1605 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Puc, J. et al. Ligand-dependent enhancer activation regulated by topoisomerase-I activity. Cell 160, 367–380 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Erdel, F., Schubert, T., Marth, C., Längst, G. & Rippe, K. Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites. Proc. Natl. Acad. Sci. USA 107, 19873–19878 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Johnson, T.A., Elbi, C., Parekh, B.S., Hager, G.L. & John, S. Chromatin remodeling complexes interact dynamically with a glucocorticoid receptor-regulated promoter. Mol. Biol. Cell 19, 3308–3322 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Banaszynski, L.A., Liu, C.W. & Wandless, T.J. Characterization of the FKBP.rapamycin.FRB ternary complex. J. Am. Chem. Soc. 127, 4715–4721 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Tanabe, K., Ikegami, Y., Ishida, R. & Andoh, T. Inhibition of topoisomerase II by antitumor agents bis(2,6-dioxopiperazine) derivatives. Cancer Res. 51, 4903–4908 (1991).

    CAS  PubMed  Google Scholar 

  47. Iwafuchi-Doi, M. et al. The pioneer transcription factor FoxA maintains an accessible nucleosome configuration at enhancers for tissue-specific gene activation. Mol. Cell 62, 79–91 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Mieczkowski, J. et al. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility. Nat. Commun. 7, 11485 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Rodriguez, J. & Tsukiyama, T. ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress. Genes Dev. 27, 74–86 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    Article  CAS  PubMed  Google Scholar 

  51. John, S. et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat. Genet. 43, 264–268 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zaret, K.S., Lerner, J. & Iwafuchi-Doi, M. Chromatin scanning by dynamic binding of pioneer factors. Mol. Cell 62, 665–667 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. de Dieuleveult, M. et al. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells. Nature 530, 113–116 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Hainer, S.J. et al. Suppression of pervasive noncoding transcription in embryonic stem cells by esBAF. Genes Dev. 29, 362–378 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Orvis, T. et al. BRG1/SMARCA4 inactivation promotes non-small cell lung cancer aggressiveness by altering chromatin organization. Cancer Res. 74, 6486–6498 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Hu, G. et al. H2A.Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation. Cell Stem Cell 12, 180–192 (2013).

    Article  CAS  PubMed  Google Scholar 

  57. Wapinski, O.L. et al. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell 155, 621–635 (2013).

    Article  CAS  PubMed  Google Scholar 

  58. Krasnow, M.A. & Cozzarelli, N.R. Catenation of DNA rings by topoisomerases. Mechanism of control by spermidine. J. Biol. Chem. 257, 2687–2693 (1982).

    CAS  PubMed  Google Scholar 

  59. Huang, H.S. et al. Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature 481, 185–189 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Soufi, A., Donahue, G. & Zaret, K.S. Facilitators and impediments of the pluripotency reprogramming factors' initial engagement with the genome. Cell 151, 994–1004 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Cui, K. & Zhao, K. Genome-wide approaches to determining nucleosome occupancy in metazoans using MNase-Seq. Methods Mol. Biol. 833, 413–419 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    Article  CAS  PubMed  Google Scholar 

  64. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

  66. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  67. Leinonen, R., Sugawara, H. & Shumway, M. The sequence read archive. Nucleic Acids Res. 39, D19–D21 (2011).

    Article  CAS  PubMed  Google Scholar 

  68. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Meyer, L.R. et al. The UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids Res. 41, D64–D69 (2013).

    Article  CAS  PubMed  Google Scholar 

  71. Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Stadler, M.B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).

    Article  CAS  PubMed  Google Scholar 

  73. Whyte, W.A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  75. Zang, C. et al. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 25, 1952–1958 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Zhang, Y., Shin, H., Song, J.S., Lei, Y. & Liu, X.S. Identifying positioned nucleosomes with epigenetic marks in human from ChIP-Seq. BMC Genomics 9, 537 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Valouev, A. et al. Determinants of nucleosome organization in primary human cells. Nature 474, 516–520 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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Authors

Contributions

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.

Corresponding author

Correspondence to Gerald R Crabtree.

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The authors declare no competing financial interests.

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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