Fig 2: PARP1 co-occurs with BRG1 at the promoters of transcriptionally active genes in MDA-MB-231 cells. (A) An example computeMatrix/plotProfile plot of ChIP-Seq data for BRG1. BRG1 peaks are centered at PARP1-enriched regions in the genome of the MDA-MB-231 cell. (B) Association of PARP1/BRG1 peaks with gene regulatory regions shows a prevailing occurrence at the active gene promoters. Enhancers emerged based on H3K27ac/H3K4me1 status, whereas promoters were assumed as TSS ± 2 kbp. PARP1/BRG1-enriched regions were assigned to particular groups and quantified by bedtools Intersect intervals. (C) BRG1 distribution at the PARP1 peaks reveals a relatively strong correlation with histone markers and with POLR2A, which is typical for actively transcribed genes (multiBamSummary/plotCorrelation; PARP1 peaks in bed for scoring). (D) Venn diagram showing that a high proportion of PARP1 positive gene promoters are characterized by the presence of BRG1 and strong acetylation of H3K27. (E) Triple-positive gene promoters identified in (D) represent a functional association with numerous intracellular processes. GO-enriched terms were identified in AmiGO2. (F) Differential expression of genes from two selected GOs, i.e., DNA repair and positive regulation of the cell cycle (marked in red in (E)) in cancer cells (DCIS – ductal carcinoma in situ, pre-invasive malignancy of the breast and two breast cancer cell lines: MCF7 and MDA-MB-231) versus normal cells, as quantified by Cuffdiff. The heatmap shows Log2 of the calculated fold change (Log2FC; cancer versus normal cells). (G) UCSC Genome browser visualization of PARP1, BRG1, and H3K27ac enrichment using Model-based Analysis of ChIP-Seq data (MACS) (bigwig) at the NEIL3 promoter.

Fig 3: SMARCA4def cells are vulnerable to KDM6s inhibition.a Distribution and mean of half-maximal effective concentration (EC50) values for GSK-J4 (MTT assays. Supplementary Fig. 6a) in the indicated groups of cells. Values, from each cell line and from two independent experiments are represented. Two-sided unpaired Student’s t-test. ****P < 0.0001. In box-whisker plots, the horizontal band inside box indicates the median, the bottom and top edges of the box 25th–75th percentiles and the whiskers indicate the min to max. b Representative clonogenic assays for the indicated cells and treatments. c Viability of the indicated cells infected with a short hairpin non-target (shNT) control or with two shRNAs targeting SMARCA4 (#1 and #4), measured using MTT assays, after treatment with increasing concentrations of GSK-J4 for 5 days. Lines, number of viable cells relative to untreated cells. Data are presented as mean and SD from three replicates and two experiments. d Representative clonogenic assays for the indicated cells infected with shNT, shKDM6A (#63, #64) or shKDM6B (#676, #976). e Viability of the indicated cell lines, measured using MTT assays, infected with a non-target (shNT) control or with two shRNAs targeting KDM6A or KDM6B after treatment with increasing concentrations of GSK-J4 for 5 days. Lines, number of viable cells relative to the untreated cells. Error bars, mean ± SD from triplicates. f Distribution and mean of the EC50 from two independent experiments per each of the three LC cell lines. Lines show mean ± SD. P-values were calculated using paired two-tailed Student’s t test. *P = 0.015; ****P < 0.0001; ns, not significant. A Source Data file is available for this figure.

Fig 4: Role of Zscan4-associated GABF complex in MT2/MERVL activation. (A) Western blot analysis of Flag-Zscan4c/Brg1/Brd9 co-immunoprecipitation in Flag-Zscan4c overexpressed ESCs or in control vector overexpressed ESCs. IP, immunoprecipitation. IP was done with anti-Flag antibody. 1.25% input was loaded as control. (B–D) Western blot analysis of Brg1/Bd9 co-immunoprecipitation with HA-zinc finger domains (B), HA-SCAN domain (C), and HA-internal domain (D) of Zscan4c. IP was done with anti-HA antibody and separated by SDS/PAGE. 1.25% input was loaded as control. (E) Enrichment heatmap of Brd9 and Brg1 around the binding regions of Zscan4c. The ChIP-seq signal was calculated as the ratio of normalized reads relative to input. (F) ChIP-qPCR analysis of Brg1 and Brd9 enrichment on MT2 in control ESCs (Control OE) and Zscan4c-overexpressed (Zscan4c OE) ESCs. Biological triplicate data were presented as mean ± s.e.m. (G) Relative Luciferase activity of vector containing MT2 in control ESCs (Control OE) and Zscan4c-overexpressed (Zscan4c OE) ESCs treated with inhibitor of Brd9 (BI7273). DMSO treated samples were included as control. Biological triplicate data were presented as mean ± s.e.m. (H) Schematic plot of Zscan4c function in activating MT2/MERVL and 2-cell/4-cell cleavage genes. In the absence of Zscan4c, MT2 enhancer activity is not activated and MT2 nearby 2-cell/4-cell genes are silenced. In the presence of Zscan4c, Zscan4c binds to MT2, interacts with GBAF complex member Brg1 and Brd9, and augments H3K4me1, H3K27ac, and H3K14ac deposition on MT2/MERVL, thus activates MT2 which drives the expression of MERVL and enhances the expression of 2-cell/4-cell genes.

Fig 5: Sequential salt extraction analysis highlights diverse chromatin binding profiles. (A) Western blot (WB) analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 sequential salt extraction (SSE) extracts visualizing FET‐FOPs FUS‐DDIT3 (DDIT3 antibody) or EWSR1‐FLI1 (● FLI1 antibody), as well as EWSR1 and FUS. (B) SSE binding profiles, visualizing the average amount of protein (n = 3) extracted in the different salt fractions in three MLS cell lines quantified from WB signals in A. FUS‐DDIT3 displayed a distinct binding profile compared to EWSR1 and FUS (adjusted P‐value 0.0016 and 0.0018, respectively). (C) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing SWI/SNF core subunits: BAF170, BAF155, BAF60A, BAF57, BAF47, BRG1, BRM, BAF53A and SS18. (D) SSE binding profiles for SWI/SNF core components (n = 4), quantified from WB signals in C. Two SWI/SNF core proteins had a slightly different binding profile as illustrated in the two smaller graphs. (E) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing SWI/SNF subtype‐specific subunits: ARID1A and ARID1B (cBAF), PBRM1, ARID2 and BRD7 (PBAF), and GLTSCR1, GLTSCR1L, and BRD9 (GBAF). (F) SSE binding profiles for cBAF‐, PBAF‐ and GBAF‐components (n = 4), quantified from WB signals in E. PBRM1 displayed a distinct binding profile compared to BRD7 (P = 0.0036). (G) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing PRC2‐component EZH2 and control Histone H4. (A, C, E, G) Loading WB: Nuclear SSE extracts (250 mm, 500 mm and 1000 mm), corresponding to equal initial volume of each salt fraction, were loaded on the gel to directly compare the amount of protein extracted in each fraction. (B, D, F) Binding profiles: Mean +/‐ SD (standard deviation) is shown, n = 3‐4. Statistical significance was determined by 2‐way ANOVA, ** P < 0.01. Detailed plots for each cell line is available in Fig. S2.