Fig. 4: The SMARCA4-R1157W mutation enhances ATPase activity and chromatin remodeling ability.

a Coimmunoprecipitation assay with lysates prepared from HCT116 cells transfected with SMARCA4-WT or SMARCA4-R1157W plasmids. Anti-SMARCA4 antibody was used for the immunoprecipitation and lysates were immunoblotted for indicated proteins. b Time-dependent ATPase activity of SMARCA4-WT and SMARCA4-R1157W were assayed in vitro using γ-³²P-labeled ATP combined with thin-layer chromatography (left panel). Quantitative analysis of the ATPase activity assay (right panel). **P < 0.01. c Nucleosome sliding assay for establishing the remodeling activity of SMARCA4-WT and SMARCA4-R1157W at indicated time points (0, 30, 60, and 90 min) by native gel electrophoresis (left panel) for centrally positioned nucleosomes (P.N.) and laterally positioned nucleosomes. Quantitative analysis of the nucleosome repositioning (right panel). The vertical axis shows the percentage of [the amount of laterally P.N. DNA/(the amount of laterally P.N. DNA + the amount of centrally P.N. DNA)]. *P < 0.05. d DNase I chromatin accessibility assay was used to detect the effects of SMARCA4-WT and SMARCA4-R1157W on the accessibility of EGFR and TNS4 genomic regions. Primers were designed on the EGFR promoter region (−301 to −132) and exon (4727–4823) to detect the accessibility of EGFR loci (left panel). Primers were designed on the TNS4 promoter region (−400 to −228) and exon (2339–2491) to detect the accessibility of TNS4 loci (right panel). *P < 0.05, **P < 0.01. e Metaplot of ATAC-seq signals around TSSs in SMARCA4-WT or SMARCA4-R1157W HCT116 cells within a ±1 kb window. f Genomic tracks of ATAC in the vicinity of the TNS4 and EGFR loci in SMARCA4-R1157W versus SMARCA4-WT HCT116 cells. Track height is normalized to the relative number of mapped reads. In the graphs, the error bars are the standard deviation of the mean.