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. 2022 Nov 29;21(3):274–289. doi: 10.1158/1541-7786.MCR-22-0772

Figure 2.

Figure 2. MUC1-C regulates PBRM1 expression and function in activating the STAT1 and IRF1 genes. A, Genome browser snapshots of ATAC-seq data from the PBRM1 gene in BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results are expressed as the percentage of undigested chromatin (mean±SD and individual values). B, BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for the indicated days were analyzed for PBRM1 mRNA levels by qRT-PCR (left). The results (mean±SD and individual values) are expressed as relative mRNA levels as compared with that obtained in control vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). C, Schema of the PBRM1 gene with localization of a PLS downstream to the TSS. Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with a control IgG, anti–MUC1-C and anti-IRF1. The DNA samples were amplified by qPCR with primers for the PBRM1 PLS region. The results (mean±SD and individual values) are expressed as relative fold enrichment as compared with that obtained with IgG (assigned a value of 1). D, BT-549/CshRNA, BT-549/IRF1shRNA and BT-549/IRF1shRNA#2 cells were analyzed for PBRM1 mRNA levels by qRT-PCR (left). The results (mean±SD and individual values) are expressed as relative mRNA levels as compared with that obtained in control vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). E, Nuclear lysates from BT-549 cells were precipitated with a control IgG and anti–MUC1-C. Input proteins and precipitates were immunoblotted with antibodies against the indicated proteins. F, Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with a control IgG and anti-PBRM1. The DNA samples were amplified by qPCR with primers for the indicated IRF1 and STAT1 regions. The results (mean±SD and individual values) are expressed as fold-enrichment as compared with that obtained from control IgG-precipitated chromatin (assigned a value of 1). G, Genome browser snapshots of ATAC-seq data from the indicated IRF1 regions in BT-549/CshRNA and BT-549/PBRM1shRNA cells. Chromatin from the indicated IRF1 regions was analyzed for accessibility by nuclease digestion. The results are expressed as the percentage of undigested chromatin (mean±SD and individual values). H, Genome browser snapshots of ATAC-seq data from the STAT1 PLS in BT-549/CshRNA and BT-549/PBRM1shRNA cells. Chromatin was analyzed for accessibility by nuclease digestion. The results are expressed as the percentage of undigested chromatin (mean±SD and individual values).

MUC1-C regulates PBRM1 expression and function in activating the STAT1 and IRF1 genes. A, Genome browser snapshots of ATAC-seq data from the PBRM1 gene in BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results are expressed as the percentage of undigested chromatin (mean ± SD and individual values). B, BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for the indicated days were analyzed for PBRM1 mRNA levels by qRT-PCR (left). The results (mean ± SD and individual values) are expressed as relative mRNA levels as compared with that obtained in control vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). C, Schema of the PBRM1 gene with localization of a PLS downstream to the TSS. Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with a control IgG, anti–MUC1-C and anti-IRF1. The DNA samples were amplified by qPCR with primers for the PBRM1 PLS region. The results (mean ± SD and individual values) are expressed as relative fold enrichment as compared with that obtained with IgG (assigned a value of 1). D, BT-549/CshRNA, BT-549/IRF1shRNA and BT-549/IRF1shRNA#2 cells were analyzed for PBRM1 mRNA levels by qRT-PCR (left). The results (mean ± SD and individual values) are expressed as relative mRNA levels as compared with that obtained in control vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). E, Nuclear lysates from BT-549 cells were precipitated with a control IgG and anti–MUC1-C. Input proteins and precipitates were immunoblotted with antibodies against the indicated proteins. F, Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with a control IgG and anti-PBRM1. The DNA samples were amplified by qPCR with primers for the indicated IRF1 and STAT1 regions. The results (mean ± SD and individual values) are expressed as fold-enrichment as compared with that obtained from control IgG-precipitated chromatin (assigned a value of 1). G, Genome browser snapshots of ATAC-seq data from the indicated IRF1 regions in BT-549/CshRNA and BT-549/PBRM1shRNA cells. Chromatin from the indicated IRF1 regions was analyzed for accessibility by nuclease digestion. The results are expressed as the percentage of undigested chromatin (mean ± SD and individual values). H, Genome browser snapshots of ATAC-seq data from the STAT1 PLS in BT-549/CshRNA and BT-549/PBRM1shRNA cells. Chromatin was analyzed for accessibility by nuclease digestion. The results are expressed as the percentage of undigested chromatin (mean ± SD and individual values). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.