Fig 1: CUT&Tag of RNAPIIS5P and BRG1 at bivalent promoters.a, K-means clustering of RNAPII-S5P and H3K27me3 CUT&Tag reads relative to RefSeq annotated gene promoter TSSs to group promoters as active (I, RNAPII-S5P enriched) and bivalent (II, H3K27me3 enriched), and not enriched for either (III). b, c, Heatmaps (bottom) and average plots (top) comparing RNAPII-S5P and BRG1 occupancy by spike-in calibrated CUT&Tag relative to bivalent promoter TSSs in untreated cells (DMSO) versus cells treated with Flavopiridol or Actinomycin D at indicated time points post drug treatment. d, Heatmaps (bottom) and average plots (top) comparing H3K27me3 occupancy by spike-in calibrated CUT&Tag relative to bivalent promoter TSSs in untreated cells (DMSO) and cells treated with Flavopiridol. e, Heatmaps (bottom) and average plots (top) comparing H3K9me3 occupancy (CUT&Tag) with RNAPII-S5P and BRG1 relative to H3K9me3 peaks in untreated cells (DMSO) versus cells treated with Flavopiridol or Actinomycin D. RNAPII-S5P and BRG1 CUT&Tag reads were spike-in calibrated and plotted to the same scales as in panels b and c, respectively. Datasets are representative of at least two biological replicates.
Fig 2: CUT&RUN.ChIP of BRG1 at promoters.a, Heatmaps (bottom) and average plots (top) of RNAPII-S5P CUTAC separated by fragment size, relative to primary peaks (summits) of RNAPII-S5P CUTAC. b, Comparison of RNAPII-S5P CUTAC fragment size distribution over peaks (promoter and enhancer NDR spaces) in cells treated with DMSO (control) and Flavopiridol; same data as used for Fig. 2a. c, d, Enrichment of nucleosomal (=150 bp, solid lines) and subnucleosomal (= 120 bp, broken lines) reads from BRG1 CUT&RUN and CUT&RUN.ChIP experiments, relative to gene promoter TSSs, in DMSO and Flavopiridol treated cells. CUT&RUN.ChIP data were plotted as enrichment in histone ChIP over IgG isotype control. Datasets are representative of at least two biological replicates.
Fig 3: Enrichment of BAF increases nucleosome eviction.a, Comparison of promoter chromatin structure and chromatin accessibility by means of S5P CUTAC fragment size distribution over peaks (promoter and enhancer NDR spaces) in cells treated with DMSO (control) and Flavopiridol. Peaks were called with DMSO control. b, Heatmaps comparing nucleosomal (=150 bp reads) and subnucleosomal (=120 bp reads) protection by BAF (BRG1 CUT&RUN) and BAF-associated histones (BRG1 CUT&RUN.ChIP) in untreated (DMSO control) cells. Heatmaps were plotted relative to S5P CUTAC summits and sorted by decreasing accessibility (CUTAC signal). CUT&RUN.ChIP heatmaps show enrichment over IgG isotype control (for ChIP). c, d, Enrichment of nucleosomal (=150 bp, solid lines) and subnucleosomal (= 120 bp, broken lines) reads from BRG1 CUT&RUN and CUT&RUN.ChIP experiments, relative to S5P CUTAC summits, in DMSO (c) and Flavopiridol treated (d) cells. e, Flavopiridol treatment causes eviction of partially unwrapped nucleosomes through enrichment of BAF at promoters and enhancers, leading to NDR persistence. Datasets are representative of at least two biological replicates.
Fig 4: Pluripotency TF binding drives NDR establishment in mESCs.a, Heatmaps (bottom) and average plots (top) of pluripotency TF CUT&RUN reads relative to RNAPII-S5P CUTAC summits, showing TF binding at sites of DNA accessibility. Heatmaps were sorted by decreasing accessibility (CUTAC signal). b, Representative genomic tracks comparing occupancy of TFs (CUT&RUN), BRG1 (CUT&Tag), and RNAPII-S5P (CUT&Tag) in SL and 2i conditions. All datasets were spike-in calibrated. c, Violin plots of spike-in calibrated CUT&RUN (TF) and CUT&Tag (BRG1 and RNAPII-S5P) signal distribution comparing factor occupancy over RNAPII-S5P CUTAC peaks in 2i versus SL. Median value (solid line), upper- and lower quartiles (broken lines) and outliers were calculated using the Tukey method. d, e, Enrichment of nucleosomal (=150 bp, solid lines) and subnucleosomal (= 120 bp, broken lines) reads from BRG1 CUT&RUN and CUT&RUN.ChIP experiments, relative to NANOG foci (smallest fragment within primary peaks called in SL condition), in SL (d) and 2i (e). f, Heatmaps (bottom) and average plots (top) of RNAPII S5P CUTAC (20–120 bp reads only) relative to NANOG foci, comparing chromatin accessibility in 2i versus SL. Datasets are representative of at least two biological replicates.
Fig 5: CUT&RUN of pluripotency TFs in SL versus 2i culture conditions.a, Heatmaps (bottom) and average plots (top) comparing pluripotency TF occupancy by CUT&RUN at RNAPII-enriched (active) and H3K27me3-enriched (bivalent) promoters. Promoters were grouped based on K-means clustering of RNAPII-S5P and H3K27me3 CUT&Tag reads mapping to a 5 kb window around the TSSs of RefSeq-annotated mESC genes, see Extended Data Fig. 4a. b, Immunofluorescent staining comparing pluripotency TF and BRG1 expression in SL versus 2i culture conditions. Cy5-conjugated secondary antibodies were used in all experiment except for KLF4, where Rhodamine red-conjugated antibody was used. DAPI (blue) was used to stain the nucleus in cells. c, Heatmaps (bottom) and average plots (top) comparing pluripotency TF occupancy by spike-in calibrated CUT&RUN in SL versus 2i culture conditions.
Supplier Page from Abcam for BRG1 peptide