Genetic determinants and epigenetic effects of pioneer factor binding [ATAC-seq]

Transcription factors (TFs) are the core drivers of gene regulatory networks that control developmental transitions and a complete understanding of how they access, alter and maintain specific gene expression patterns remains an important goal. To begin a systematic dissection of the molecular components that either enable or constrain TF activity, we investigated the genomic occupancy of two distinct TFs, the pioneer factor FOXA2 and the pluripotency-associated factor OCT4 (POU5F1), in both endogenous and ectopic settings. We find that, while stable binding of FOXA2 is highly cell type specific and similar to what is observed for most TFs including OCT4, pioneer activity can be distinguished by notable sampling of additional loci that are occupied in alternative lineages. In our ectopic system, FOXA2 binding can be selectively stabilized at previously sampled sites by co-expressing the lineage specific regulator GATA4. Alternatively, we observe minimal influence of chromatin state on discrete, stabilized binding choices for FOXA2 but a strong bias towards open chromatin for ectopic OCT4 targets. Finally, we demonstrate that FOXA2 binding and nucleosome remodeling at silent loci can occur when the cell cycle is halted in G1, but surprisingly subsequent changes in DNA methylation require DNA replication. Taken together, our results provide several new molecular insights that contribute to our basic understanding of gene regulation and pave the way for a more rational use of ectopic TFs for cellular reprogramming. To combine the dynamics of FOXA2 binding with the accessibility of DNA, we assessed the chromatin accessibility using ATAC-seq. Tagmentation was performed on whole nuclei at 37C for 45 minutes as previously described in (Buenrostro et al., 2013).DNA was isolated on PCR min-elute columns (Qiagen) and a small amount of the DNA was amplified for 9, 12 and 15 cycles to determine optimal cycling conditions. The rest of the DNA was then amplified using the chosen cycle number and PCR libraries were purified using double sided Ampure clean up to remove high molecular weight fragments. .55x Ampure volume was added to the PCR, mixed and incubated. Supernatant was removed following magnet separation and cleaned-up with a 1X Ampure volume. Libraries were sequenced on a HiSeq 2500 at 8pmol. Please note that the processed data 'RPKM_Table.txt' was generated from all ATAC-seq and ChIP-seq studies (GSE90453, GSE90454, GSE92490, GSE92491) and is linked to the GSE90454 records.