Pioneer factor GAF cooperates with PBAP and NURF to regulate transcription [ATAC-seq]

The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promoters which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation. Overall design: We treated two independent replicates of Drosophila S2 cells with dsRNA to LACZ (control), GAF, NURF301, BAP170 (the unique subunits of the NURF and PBAP complexes, respectively), and NURF301+BAP170. After 5 days, we harvested cells, validated knockdowns, and performed PRO-seq, ATAC-seq and 3'RNA-seq. We also performed CUT&RUN for both GAF and NURF301 in untreated S2 cells.