Reprogramming of a somatic nucleus to an induced pluripotent state can be achieved in vitro through ectopic expression of Oct4 (Pou5f1), Sox2, Klf4 and c-Myc. While the ability of these factors to regulate transcription in a pluripotent context has been studied extensively, their ability to interact with and remodel a somatic genome remains underexplored. Several recent studies have begun to provide mechanistic insights that will eventually lead to a more rational design and improved understanding of nuclear reprogramming.
"A large body of evidence shows that transcriptional and epigenetic mechanisms govern the generation of iPSCs (Gifford and Meissner, 2012; Jaenisch and Young, 2008). The incorporation of a 5 0 guanine cap analog (an antireverse diguanosine cap "
[Show abstract][Hide abstract] ABSTRACT: Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process.
[Show abstract][Hide abstract] ABSTRACT: In all known cases of transcription factor (TF)-based reprogramming, the process is relatively slow and inefficient. For example, it takes about a month for the ectopic expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) to fully reprogram human somatic cells to pluripotency. Furthermore, recent studies indicate that there is an initial stochastic phase, whereby random cells in the converting population begin to express a few genes of the new fate, followed by a so-called deterministic phase, whereby activation of a network for the new fate leads to homogeneous changes in gene expression patterns within a subset of the cell population. We recently mapped the initial interactions between OSKM factors and the human genome during the first 48 h of human fibroblast conversion to pluripotency. Unlike that reported in ES and iPS cells, distal enhancer sites in closed chromatin dominate the initial O, S, K and M binding distribution, showing that promoter occupancy is a later event in reprogramming. O, S and K act as pioneer factors for c-Myc, and c-Myc enhances the engagement of O, S and K. Despite the ability of OSKM to access closed chromatin, megabase-scale chromatin regions in somatic cells, referred to as "differentially bound regions" (DBRs), are remarkably refractory to OSKM binding at 48 h, though they become bound in pluripotent cells. These DBRs are highly enriched for the repressive H3K9me3 mark and span genes at the top of the deterministic hierarchy. Transient knockdown of the relevant chromatin modifiers allows access of OSKM to DBRs and a more rapid and efficient conversion to pluripotency. Thus, overcoming DBR barriers helps explain the conversion from a stochastic to a deterministic phase of transcription factor-mediated cell type conversion.
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