TFAP2 paralogs facilitate chromatin access for MITF at 1 pigmentation genes but inhibit expression of cell-cell adhesion 2 genes independently of MITF 3

26 In developing melanocytes and in melanoma cells, multiple paralogs of the 27 Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) 28 contribute to expression of genes encoding pigmentation regulators, but their 29 interaction with Microphthalmia transcription factor (MITF), a master regulator of 30 these cells, is unclear. Supporting the model that Tfap2 facilitates MITF’s ability to 31 activate expression of pigmentation genes, single-cell seq analysis of zebrafish 32 embryos revealed that pigmentation genes are only expressed in the subset of mitfa - 33 expressing cells that also express Tfap2 paralogs. To test this model in SK-MEL-28 34 melanoma cells we deleted the two TFAP2 paralogs with highest expression, 35 TFAP2A and TFAP2C, creating TFAP2 knockout ( TFAP2 -KO) cells . We then 36 assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, 37 and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active 38 enhancers and silenced chromatin, respectively . Integrated analyses of these 39 datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched 40 for roles in pigmentation and proliferation, and directly repress enhancers near 41 genes enriched for roles in cell adhesion. Consistently, compared to WT cells, 42 TFAP2 -KO cells proliferate less and adhere to one another more. TFAP2 paralogs 43 and MITF co-operatively activate a subset of enhancers, with the latter necessary for 44 MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do 45 not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by 46 which TFAP2 profoundly influences the set of genes activated by MITF, and thereby 47 the phenotype of pigment cells and melanoma cells.