Colin Kenny (University of Iowa)| Rami Dilshat (University of Iceland)| Hannah Seberg (University of Iowa)| Eric VanOtterloo (University of Iowa)| Gregory Bonde (University of Iowa)| Eiríkur Steingrímsson (University of Iceland)| Robert Cornell (University of Iowa)
The phenotypic state of melanoma cells is determined by the activity of microphthalmia-associated transcription factor (MITF). Activating-enhancer binding protein 2 (TFAP2) family members co-regulate a subset of the genes regulated by MITF, but the respective functions of TFAP2 paralogs and MITF are unclear. Evidence that TFAP2 paralogs can bind nucleosomes, and that MITF binds to Brg1, support a model wherein TFAP2 paralogs bind closed chromatin and facilitate access to MITF which in turn recruits chromatin remodelers. To test this model we generated TFAP2A/TFAP2C double knockout SK-MEL-28 cells and monitored binding sites of TFAP2A and of MITF, and marks of open chromatin (ATAC-seq), active chromatin (H3K27Ac), repressed chromatin (H3K27me3) and active promoters (H3K4me3). Integration of the data revealed that, consistent with the model, at a large subset of loci bound by both MITF and TFAP2A in wild-type cells, MITF binding, H3K27AC and ATAC-seq signals were significantly reduced in TFAP2 knockout cells. Unexpectedly, at a subset of them, MITF binding but not H3K27AC or ATAC-seq signals were reduced, implying distinct mechanisms at such loci. RNA-seq profiles of TFAP2 knockout and MITF knockout SK-MEL-28 cells showed that the subset MITF-dependent genes that is also TFAP2-dependent is enriched for those regulating pigmentation, while the subset that is TFAP2-independent is not. Consistently, embryonic melanophores in zebrafish tfap2a/tfap2e double mutants had strongly reduced expression of genes in the first subset but not the latter. These findings illustrate how the presence of a specific pioneer factor affects the target genes of a pleiotropic transcriptional activator, and suggest how TFAP2 paralogs may influence melanoma progression.