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. 2017 Apr;24(4):344-352.
doi: 10.1038/nsmb.3384. Epub 2017 Feb 27.

TOP2 Synergizes With BAF Chromatin Remodeling for Both Resolution and Formation of Facultative Heterochromatin

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Free PMC article

TOP2 Synergizes With BAF Chromatin Remodeling for Both Resolution and Formation of Facultative Heterochromatin

Erik L Miller et al. Nat Struct Mol Biol. .
Free PMC article

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.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. TOP2 is required for transcription-independent BAF-mediated resolution of facultative heterochromatin to accessible chromatin
(a) Strategy for BAF recruitment to the CiA:Oct4 locus in fibroblasts. The recruitment site is −638 to −232 bp from the TSS. The two primer sets upstream of the recruitment sites target both the CiA:Oct4 and unmodified Oct4 alleles, whereas the other primer sets target only the CiA:Oct4 locus. BRG1 ChIP (b) or TOP2A etoposide-ChIP (c) in fibroblasts with the BAF recruitment system treated with 3 nM rapamycin (Rap) for 1 hour. (d) ATAC-qPCR in cells treated with rapamycin and 1 μM ICRF-193 for 1 hour to inhibit TOP2. (e) eGFP flow cytometry of cells treated with rapamycin for 1 hour. 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. Lines represent means and error bars represent s.e.m. from 4 (b,d) or 6 (c) cell passages.
Figure 2
Figure 2. Active TOP2 is recruited immediately upon BAF recruitment and is required for the initial stage of accessibility induction
(a) Strategy for timecourse of active TOP2/strand-cleaved decatenation intermediate detection during BAF recruitment. (b) TOP2A etoposide-ChIP in cells treated with 3 nM rapamycin for various times before fixation with 100 μM etoposide. (c) Strategy for timecourse of TOP2 inhibition during BAF recruitment. (d) ATAC-qPCR in cells treated with rapamycin and 1 μM ICRF-193 after various delays. Significance assessed by t-tests as before. Bars represent means and error bars represent s.e.m. from 6 (b) or 3 (d) cell passages.
Figure 3
Figure 3. TOP2 and BAF promote accessibility of enhancers and promoters genome-wide
DNA gel electrophoresis of MNase digests of ES cells treated with 1 μM ICRF-193 for 24 hours (a) or Brg1fl/fl; actin-CreER (b) ES cells treated with tamoxifen (Tax) to knockout Brg1 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. (c) Fold-change densitometry of MNase digests in log-scale. Significance of treatment assessed by t-tests as before. Overall significance of the interaction effect of treatment and size range assessed by three-way ANOVA. Bars represent actual values from 2 cell passages (ICRF) or means (Brg1fl/fl) and error bars represent s.e.m. from 4 cell passages. (d) Row-wise z-score heatmaps of ATAC-seq fragment density at ATAC-seq peaks sorted by edgeR-adjusted fold-change. (e) Percentage of ATAC-seq peaks altered upon ICRF-193 or tamoxifen treatment. (f) Cumulative probability distributions of specified log2 fold-change ATAC-seq fragment density over specified ATAC-seq peaks. Significance between “Decreased” and “Unchanged” distributions assessed by Kolmogorov-Smirnov tests. (g) Classification of ATAC-seq peaks by genome element. (h) log2 observed/expected overlap of ATAC-seq peaks by genome elements.
Figure 4
Figure 4. TOP2 is required for optimal BAF-mediated recruitment of OCT4
(a) Venn diagram of overlapping BRG1 and OCT4 ChIP-seq peaks in ES cells. (b) Diagram of OCT4 motifs at the CiA:Oct4 locus. Red arrowhead indicates the OCT4 motif in the DNA binding 19 bp downstream from the zinc-finger recruitment site. (c) Strategy for testing BAF/TOP2 pioneering for OCT4 using BAF recruitment to the CiA:Oct4 locus in fibroblasts while expressing exogenous OCT4. OCT4 ChIP in fibroblasts with the BAF recruitment system over-expressing OCT4 treated with 3 nM rapamycin (d) in the presence of 1 μM ICRF-193 (e). (f) eGFP flow cytometry of fibroblasts over-expressing OCT4 treated with rapamycin for 1 hour. Significance assessed by t-tests as before. Lines represent means and error bars represent s.e.m. from 3 cell passages (d,e).
Figure 5
Figure 5. TOP2 and BAF are required for recruitment of pluripotency factors genome-wide
(a) Cumulative probability distributions of log2 fold-change STAT3 ChIP-seq fragment density in Brg1fl/fl ES cells at STAT3 peaks overlapping specified ATAC-seq peaks. (b) MNase-seq nucleosome dyad density across specified subsets of linker-bound STAT3 sites in Brg1fl/fl ES cells. Vertical lines indicate nucleosome positions and values are changes in nucleosome positioning (bp, tamoxifen – EtOH). (c) Heatmap of log2 observed/expected change in accessibility of accessible ChIP-seq peaks from various datasets, sorted by enrichment for decreased accessibility upon ICRF-193 treatment. Top 20 ChIP-seq datasets are shown. Changes in Brg1fl/fl MNase-seq mean flanking nucleosome positions (d) and nucleosome dyad density profiles (e) around specified linker-bound ChIP-seq peaks or 38,864 randomly shuffled sites that exclude TSSs, TESs, or enhancers. (f) Model for loss accessibility, nucleosome spacing, and transcription factor binding.
Figure 6
Figure 6. TOP2 is required for reformation of facultative heterochromatin
(a) Strategy for BAF recruitment and rapamycin washout using 100 nM FK506 with TOP2 inhibition using 1 μM ICRF-193. ATAC-qPCR in fibroblasts treated with 3 nM rapamycin for 1 hour and subsequently washed out with FK506 (b) in the presence of ICRF-193 (c). (d) TOP2A etoposide-ChIP in cells treated with rapamycin and subsequently washed out with FK506. (e) Heatmap of log2 observed/expected change in accessibility of various ChIP-seq peaks, sorted by enrichment for increased accessibility upon ICRF-193 treatment. Top 10 ChIP-seq datasets are shown. *: does not include bivalent peaks. (f) Model for the role of TOP2 and BAF in resolution and reformation of heterochromatin. Significance of individual primer sets assessed by t-tests as specified (b), versus control washout (c), or versus no rapamycin control (d). Overall significance of the effect of washout (b,d) or ICRF-193 (c) assessed by three-way ANOVA. Lines and bars represent means and error bars represent s.e.m. from 5 (b,c) or 9 (d) cell passages.

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