Genome wide SOX4 specific binding sites in BT549 human TNBC cells

We identified the SOX4 transcription factor and integrin alpha V encoded by the ITGAV gene as important resistance mechanisms to T cell-mediated cytotoxicity of TNBC cells. Here, we performed RNA seq. analysis of SOX4 and ITGAV deficient (CRISPR knockout) BT549 human and 4T1 murine TNBC cells as compared to control edited counterparts. Additionally, we performed SOX4 specific ChIP-seq studies in BT549 human TNBC cells. The objective of the study was to identify the molecular regulators and signaling pathways that mediate resistance to T cell mediated immunity. GSEA analysis of RNA-seq data showed that the 'interferon response' represented one of the top pathways for genes upregulated in SOX4 or ITGAV edited compared to control TNBC cells. In contrast, gene sets associated with TGF beta and TNF alpha/NF kappa b were negatively enriched in both Sox4 and Itgav edited TNBC cells. Further analysis of RNA-seq data showed that SOX4 or ITGAV edited TNBC cells contained higher mRNA levels of many interferon-stimulated genes (ISGs), including genes associated with important innate immune pathways such as RIG-I/MDA-5, cGAS - STING and the AIM2 inflammasome Chromatin from 10x10^6 cells was used for each ChIP. Nuclei/cells were fixed with 2 mM DSG (Pierce) for 45 min at RT (shaking) prior to formaldehyde fixation for 10min at RT. The reaction was quenched with glycine (0.125M). Nuclei/cells were then washed twice with ice-cold PBS, lysed in ChIP sonication buffer (50mM HEPES pH7.9, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.2% SDS) supplemented with protease inhibitors, and were subjected to sonication to obtain DNA fragments of 300-800 bp. Immunoprecipitation was done using 5 ul of antibody and 40 ug of chromatin. The soluble chromatin (40 µg) was immunoprecipitated with 10 μg of SOX4 antibody (ab86809 Abcam). Chromatin immunoprecipitation (ChIP) experiments in human BT549 TNBC cells with a SOX4 specific antibody identified SOX4 specific peaks in the regulatory regions of innate immune genes (such as IFIH1, encoding MDA-5), interferon pathway genes (such as IRF7 and ISG15) and MHC-I pathway genes (including TAP1, TAP2, PSMB9, HLA-B and HLA-C). These data are consistent with the hypothesis that SOX4 regulates the expression of multiple genes in the MHC-I and innate immunity pathways.