KLF4 binding during reprogramming is linked to enhancer rewiring and is critical for the architecture and regulation of enhancer hubs [4C-Seq]

Purpose: We captured on a genome-wide scale the binding of KLF4 during iPSC formation and its effects on chromatin state, transcriptional activity and chromatin topology around its targets. Methods: We used a well-characterized reprogramming system to apply genome-wide assays that map KLF4 binding (ChIP-seq), chromatin accessibility (ATAC-seq), enhancer and gene activity (H3K27ac ChIP-seq and RNA-seq), enhancer connectivity (H3K27ac Hi-ChIP) as well as KLF4-centric chromatin looping (KLF4 Hi-ChIP) at different stages during acquisition of pluripotency. Results: Integrative analysis of our results generated a reference map of stage-specific chromatin changes around KLF4 bound loci and established strong links with enhancer. rewiring and concordant transcriptional changes. Genetic manipulation of KLF4 binding from a PSC enhancer further supported the ability of KLF4 to function both as a transcriptional regulator and a chromatin organizer. Conclusions: Our study offers novel insights into the intricate roles of a master regulator during cell fate transition. Mouse embryonic fibroblasts (MEFs) (Rosa26-M2rtTA/Col1a1-OKSM) induced with doxycycline (dox) in the presence of ascorbic acid. We collected bulk populations on day 3 after dox treatment, whereas at later stages, on day 6 and day 9, we sorted SSEA1 positive cells to enrich for cells on the trajectory towards induced pluripotency. Finally, we used pluripotent stem cells (PSCs) as a reference point for established pluripotency. Biological replicates were collected to assay for chromatin accessibility (paired-end ATAC-seq), gene activity (single-end RNA-seq), H3K27ac ChIP-seq (single-end) in MEFs, day3, 6, 9 and PSCs. KLF4 binding (single-end ChIP-seq) was assessed in day3, 6, 9 and PSCs. KLF4/H3K27ac centric looping (paired-end Hi-ChIP) was studied in either day3, day6 and PSCs for KLF4 or MEFs and PSCs for H3K27ac. 4C-Seq was conducted for individual viewpoints (Mycn, Ets1, Tbx3) to validate findings from HiChIP/Hi-C assays.