Deterministic direct reprogramming of somatic cells to pluripotency [ChIP-Seq]. Mus musculus

Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Overall design: Samples include Mbd3+/+, Mbd3flox/- and Mbd3-/- cells from mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors). Three histone modifications are given: H3K4me3, H3K27me3 and H3K27ac. In addition, binding data of Mbd3, Mi2B and Oct4 is given in various stages. Please note that 20 independent clonal series generated in our study were carrying either the GOF18 deltaPE-Oct4-GFP transgenic reporter (Addgene plasmid 52382) or the complete GOF18 Oct4-GFP transgenic reporter (Addgene plasmid 60527), the clonal series selected for genomic analysis included an Mbd3+/+ clone that carries the GOF18 deltaPE-Oct4-GFP transgenic reporter, andMbd3flox/-and Mbd3-/- cells that carry the GOF18 Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements). These reporters can be identified when analyzing the Oct4 locus in genomic DNA input datasets. As we do not use Oct4–GFP or any other selection for sorting cells before conducting genomic experiments, the difference in transgene reporters would not influence the interpretation of our genomic analysis data in any way. In these genomics studies, the endogenous Nanog and Oct4 loci are not manipulated and are identical between all cell lines because the Oct4–GFP reporters were introduced via random transgenesis and validated for specificity. Notable, for the mouse induced pluripotent stem (iPS) cell efficiency results presented in our study (for example, in Figs 1 and 2, and Supplementary Videos 1–4), cell lines carried the matched Nanog–GFP knock-in reporter or Oct4– GFP transgenic reporter (containing both distal and proximal enhancer elements as delineated in Extended Data Fig. 3a). Thus, all iPS cell efficiency and kinetic comparisons were conducted by using matched and validated pluripotency reporter systems.