Fig 2: MUC1-C→BRG1 signaling integrates activation of the PBAF/PBRM1 and BAF/ARID1A pathways.A Lysates from DU-145/CshRNA and DU-145/ARID1AshRNA (left) or DU-145/CshRNA and DU-145/PBRM1shRNA (right) cells were immunoblotted with antibodies against the indicated proteins. B Lysates from DU-145/CshRNA and DU-145/BRG1shRNA (left) or DU-145/CshRNA and DU-145/ARID1AshRNA (right) cells were immunoblotted with antibodies against the indicated proteins. C Lysates from DU-145/CshRNA and DU-145/PBRM1shRNA cells were immunoblotted with antibodies against the indicated proteins. D Lysates from DU-145/CshRNA and DU-145/ARID1A (left) or DU-145/CshRNA and DU-145/PBRM1shRNA (right) cells were immunoblotted with antibodies against the indicated proteins. E DU-145/CshRNA and DU-145/PBRM1shRNA cells were assayed for tumorsphere formation (left). The results (mean ± SD of 3 biologic replicates) are expressed as relative tumorsphere number per field compared to the CshRNA control (assigned a value of 1) (right). Scale bar: 100 microns. F MUC1-C integrates activation of the esBAF and PBAF chromatin remodeling pathways in CRPC/NEPC progression. The MUC1-C cytoplasmic domain binds directly to the E2F1 DNA binding domain to activate expression of BRG1 and components of the esBAF and PBAF (PBRM1, ARID2, and BRD7) complexes. The MUC1-C→E2F1→esBAF/ARID1A pathway induces EMT and expression of NOTCH1, NANOG, and MYC. The present results demonstrate that the MUC1-C→E2F1→PBAF/PBRM1 pathway induces NRF2-mediated redox balance and expression of OSKM + NANOG. MUC1-C→E2F1→BRG1 signaling integrates cross-talk between the esBAF and PBAF pathways to drive the PC CSC state.

Fig 3: Altered ARID2 expression modulates miR-922 inhibitor-decreased malignant behavior of HepG2 cells. HepG2 cells were stably transfected with miR-922 inhibitor and transfected with plasmid for ARID2 expression or ARID2-specific shRNA for ARID2 silencing. Control cells were transfected with vehicle. (A) Cell Counting Kit-8 determined cell proliferation. (Bi and Bii) Number of apoptotic HepG2 cells was analyzed by flow cytometry. (Ci and Cii) Clonogenicity of each group of HepG2 cells was analyzed by colony formation assay. (Di and Dii) Wound healing analysis of each group of HepG2 cells. (Ei and Eii) Transwell assay analysis determined the invasion of each group of HepG2 cells. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001. ARID2, AT-rich interactive domain 2; miR, microRNA; sh, short hairpin; OD, optical density; NC, negative control; OE, over-expression.

Fig 4: Chromatin accessibility changes in the absence of ARID2 correlate with TF occupancy(A) Scatterplot displaying the absolute log p values of the TF motifs (de novo) enriched at SKmel147 ARID2 WT versus KO ATAC-altered regions.(B) Metagene profiles of SKmel147 ARID2 WT (parental and NTC) and KO (KO1.4 and KO3.20) enrichment of FOSL2 and TEAD4 ChIP-seq centered at ATAC decreased and increased regions that overlap with a TF peak (FOSL2 n = 4,548, TEAD4 n = 4,130).(C) Scatterplots of SKmel147 ARID2 WT versus KO log2FC of FOSL2 and TEAD4 with ATAC log2FC at ATAC increased and decreased regions that overlap with a TF peak, as in (B). SS18 log2FC was plotted as a color variable and Pearson correlations are shown.(D) Scatterplot displaying the absolute log p values of TF motifs (de novo) enriched at 501mel ARID2 WT versus KO ATAC-altered regions.(E) Metagene profiles of 501mel ARID2 WT (parental and NTC) and KO (KO1.8 and KO5.2) enrichment of MITF ChIP-seq intersected with ATAC decreased and increased regions (n = 4,105).(F) Scatterplot of 501mel ARID2 WT versus KO log2FC of MITF with ATAC log2FC at ATAC increased and decreased regions. SS18 is plotted as in (C).(G) RNA-sequencing normalized counts of MITF, FOLS2, and TEAD4 in SKmel147 and 501mel. See also Figure S4 and Tables S1, S2, and S6.

Fig 5: ARID2 depletion leads to melanoma metastasis in vivo(A) Schematic of the chicken CAM xenograft assay and regions of the chicken embryo where cells disseminate to, including the lower CAM and the liver.(B) FACS (fluorescence-activated cell sorting) quantification of SKmel147 ARID2 WT (NTC) and KO (KO1.4) cells of the primary tumor (n = 12 and n = 11, respectively), lower CAM (n = 19 and n = 16, respectively), and liver (n = 14 and n = 10, respectively) of the CAM xenografts 7 days post-injection. Nineteen independent replicates are aggregated, line represents the median. Welch’s t test. Primary tumor, p = 0.8580; lower CAM, p = 0.0019; liver, p = 0.0156. *p < 0.05; **p < 0.01; ns, p > 0.05.(C) Representative images of SKmel147 ARID2 WT (NTC) and KO (KO1.4) GFP+ cells that disseminated to the lower CAM.(D) Quantification of SKmel147 ARID2 WT (NTC, n = 5) and KO (KO1.4, n = 5) metastatic colonies in the lung 3 weeks post-tail-vein injection detected by fluorescence imaging. Line represents the median. Welch’s t test. p = 0.029. *p < 0.05.(E) Fluorescence images of lungs from mice with SKmel147 NTC and KO1.4 cells injected into the lateral tail vein.(F) Quantification of 4L ARID2 WT (NTC, n = 5) and KO (KO1.17, n = 5; KO5.11, n = 5) metastatic colonies in the lung 5 weeks post-tail-vein injection detected by fluorescence imaging. Line represents the median. Welch’s t test. NTC versus KO1.17, p = 0.0421; NTC versus KO.511, p = 0.0334. *p < 0.05.(G) Model of the mechanisms by which ARID2 loss regulates chromatin dynamics across the three types of SWI/SNF-bound regions, and gene expression pathways affected by PBAF loss. See also Figure S6.