Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement of the epigenetic landscape. To characterize this epigenomic roadmap, we have performed MethylC-seq, ChIP-seq (H3K4/K27/K36me3) and RNA-Seq on samples taken at several time points during murine secondary reprogramming as part of Project Grandiose. We find that DNA methylation gain during reprogramming occurs gradually, while loss is achieved only at the ESC-like state. Binding sites of activated factors exhibit focal demethylation during reprogramming, while ESC-like pluripotent cells are distinguished by extension of demethylation to the wider neighbourhood. We observed that genes with CpG-rich promoters demonstrate stable low methylation and strong engagement of histone marks, whereas genes with CpG-poor promoters are safeguarded by methylation. Such DNA methylation-driven control is the key to the regulation of ESC-pluripotency genes, including Dppa4, Dppa5a and Esrrb. These results reveal the crucial role that DNA methylation plays as an epigenetic switch driving somatic cells to pluripotency.

The properties of spin-labelled myosin, prepared from rabbit skeletal and scallop adductor muscle, on forming a long-lived complex with ADP and vanadate (M.ADP.Vi), have been investigated. In the case of an iodoacetamide-based label attached to rabbit myosin or subfragment 1, M.ADP.Vi formation is characterized by a marked increase in the mobility of the probe, similar to that seen during steady-state ATPase activity. Hence, this complex appears to be a good analogue of the M**ADP.Pi state. The kinetics of M.ADP.Vi formation were determined by following the electron paramagnetic resonance (e.p.r.) signal with time and were analysed according to the scheme: (formula; see text) After correction for Vi polymerization, K'4 = 3.2 X 10(-4)M, k'-3 = 8.7 X 10(-3) s-1 and k'3 = 1.5 X 10(-4) s-1. The major effect of spin-labelling the reactive SH1 thiol is to increase k'3, so that M.ADP.Vi dissociates over a period of hours rather than days. In contrast, a maleimide-based spin-label attached to rabbit myosin does not exhibit a large change in mobility, on formation of the M.ADP.Vi complex. However, the small change observed in both the conventional and saturation transfer spectra questions the assumption that this probe is completely insensitive to librational motion during ATPase activity. The immobilized spectrum of the iodoacetamide-based spin label attached to scallop myosin is insensitive to M.ADP.Vi formation in the presence or absence of Ca2+. Under these conditions, the label appears to reflect gross head motion and hence this observation lends no support to the idea that, in the myosin-linked regulatory system, Ca2+ operates by controlling the flexibility of the subfragment 1-subfragment 2 joint.

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.