BATF3-dependent IRF4 binding inhibits FOXP3 in differentiating Tregs

BATF3 has been shown to inhibit FOXP3 expression in differentiating CD4 T cells, however, the role of IRF4 in this inhibition is unexplored. IRF4 binds DNA weakly on its own and requires interactions with other transcription factors. We investigated how BATF3/IRF4 interactions are necessary for IRF4 binding and BATF3-mediated FOXP3 inhibition. Specific BATF residues have been previously demonstrated to be necessary for BATF/IRF4. We cloned analogous mutations into BATF3 to create a BATF3Mut that does not interact with IRF4. We used a retroviral vector to overexpress the mutant and WT BATF3 in Batf/Batf3 DKO CD4 T cells, cultured the cells in iTreg-differentiating conditions for 24 hrs, and then collected cells for IRF4 ChIP-seq.