Project description:Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency or developmentally related gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming. Gene expression was measured by Affymetric microarrays during the initial phase of the reprogramming of mouse embryonic fibroblasts.
Project description:Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency or developmentally related gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming. Genome-scale DNA methylation was measured by reduced representation bisulfite sequencing (RRBS) during the initial phase in the reprogramming of mouse embryonic fibroblasts.
Project description:Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency or developmentally related gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming. Covalent histone modifications were measured by ChIP-Seq during the initial phase in the reprogramming of mouse embryonic fibroblasts.
Project description:Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somatic cells into an embryonic-like state. Post-translational modifications of histones play an important role in this process. We previously used ChIP-seq to profile the distribution of specific histone H3 marks during the entire reprogramming process and found widespread loss of H3K27me3 in the initial stages[1]. Here, using an unbiased middle-down proteomics approach we have identified 74 unique isoforms of histone H4 and quantified them across nine stages of reprogramming. Our data showed substantial differences between the precursor cells and late-phase reprogramming cells. Also, ESCs and iPSCs displayed higher levels of H4 acetylation and tri-methylation concomitantly with lower levels of mono- and di-methylation when compared to cells undergoing reprogramming. These results suggest that during reprogramming, the histone H4 “PTM code” experiences dynamic changes in multiple waves, with a final reset taking place during the transition to an ESC-like state, in perfect agreement with the in-parallel generated multi-omics data[1]. Together, these data represent an invaluable resource to study PTMs of H4 and their potential cross-talk and extend our knowledge on the epigenetic mechanisms involved in acquisition of induced pluripotency.
Project description:We explored the potential reprogramming of Leishmania infantum proteome during its stationary phase after an initial, single-dose exposure to EVs released by drug-resistant parasites
Project description:The reprogramming of fibroblasts into pluripotent stem cells by Oct4, Sox2, Myc and Klf4 represents a unique system to understand the logic of cell fate decisions. The reprogramming process can be divided into initial, middle and maturation phases that are coupled to the ordered execution of multiple biological processes, of which one of the earliest is a mesenchymal to epithelial transition (MET). Yet, despite these critical insights, less is known about how the reprogramming factors alter the chromatin state to convert somatic cells back to pluripotent cells. Here we report the global chromatin accessibility dynamics as cells reprogram from a somatic to a pluripotent state as assessed by assay for transposase-accessible chromatin with sequencing (ATAC-seq). We show that chromatin changes during reprogramming are defined by a fast initial wave of chromatin changes from accessible/open to inaccessible/closed (OC), which is followed by a slower opening up of chromatin from closed to open (CO), and ends with a climactic wave of CO. The initial OC wave includes loci enriched with motifs for AP-1, TEAD, RUNX and ETS-family transcription factors. Consistently, over-expression of one of the AP-1-family members c-Jun, c-Fos and Fra1blocked reprogramming. c-Jun inhibits reprogramming by preventing OSK-driven OC and skewing the overall OC-CO dynamics. Paradoxically, we show that Sox2 and to a less extent Oct4 and Klf4 are CO-, not OC-, mediators , suggesting that they must first activate potent OC factors to remodel chromatin. Indeed, we show that Sap30, a component of the Sin3 complex8, functions as such an OC factor. Together, our results reveal the chromatin accessibility logic during reprogramming and identify Sap30 as one of the first responders that help silence somatic genes.