KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency [ChIP-seq]

Summary: Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors (TFs), as is strikingly exemplified in somatic cell reprogramming to pluripotent stem cells (PSCs) by OCT4, KLF4, SOX2 and cMYC. How TFs orchestrate the complex molecular changes around their targets in a temporal manner remains largely elusive. Here, using KLF4 as a paradigm, we provide the first TF-centric view of chromatin reorganization and the association to 3D enhancer rewiring and the transcriptional changes of linked genes during reprogramming of mouse embryonic fibroblasts to (MEFs) to PSCs. Inducible depletion of KLF factors in PSC caused a genome-wide decrease in connectivity of enhancers and disruption of KLF4 binding site from PSC-specific enhancers was sufficient to reduce expression of genes within the enhancer hub partly by impairing long-range contacts. Our study provides an integrative view of the intricate activities of a master regulator during a controlled cell fate transition and offers novel insights into the order and nature of molecular events that follow TF binding. 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) inclusive of embryonic stem cells (ESCs) 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.