Using Mouse Embryonic Fibroblast to test the pioneer factor activity of POU4F3

Previous unbiased screening for the nucleosome binding affinity of transcription factors predicts that POU domain transcription factors have strong nucleosome binding potential. Here using reprogramming strategy we found that POU4F3 has pioneer factor activity at ATOH1 target elements that require POU4F3 for their accessibility. Overall design: We overexpressed ATOH1, POU4F3, ATOH1+POU4F3 and a GFP control in mouse embryonic fibroblasts (MEFs), and used CUT&RUN and µATACseq to assess the transcription factor binding and the resulting chromatin structure at the 1,458 ATOH1 targets that required POU4F3 for their accessibility in hair cells. We found that POU4F3 alone was able to bind most of these distal elements within closed chromatin in MEFs, demonstrating pioneer factor activity. The presence of POU4F3 also enabled ATOH1 to recognize and bind these targets in MEFs. Surprisingly, although overexpression of ATOH1+POU4F3 resulted in the opening of a small number of these distal elements in MEFs (233 / 1,458), the majority remained closed (963 / 1,458), but were nevertheless bound by both transcription factors, suggesting that additional hair cell-specific transcription factors / co-factors, which are not present in MEFs, are required for the selective opening and activation of this group of distal elements.