Summary Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription-factors (TFs), and chromatin-states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic-enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic-enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic-TF Fra1. Pluripotency-enhancer selection is a step-wise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency-enhancers are selected later and require OS and other pluripotency TFs. Somatic and pluripotency-TFs modulate reprogramming efficiency when overexpressed by altering OSK-targeting, somatic-enhancer inactivation, and pluripotency-enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific-TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.
Summary Naïve human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X-chromosome state has remained unresolved. Here, we show that the inactive X-chromosome (Xi) of primed hESCs was reactivated in naïve culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X-chromosomes with XIST expression and chromosome-wide transcriptional dampening, and initiated XIST-mediated X-inactivation upon differentiation. Both establishment of and exit from the naïve state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X-chromosome dosage compensation processes in early human development: X-dampening and X-inactivation. However, remaining differences between naïve hESCs and embryonic cells related to monoallelic XIST expression and non-random X-inactivation highlight the need for further culture improvement. As the naïve state reset Xi abnormalities seen in primed hESCs it may provide cells better suited for downstream applications.
Reprogramming of somatic cells into iPSCs involves a dramatic reorganization of chromatin. To identify posttranslational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared to both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9methylation to reprogramming, we show that the H3K9methyltransferases Ehmt1, Ehmt2, and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, inhibition of heterochromatin-protein-1γ (Cbx3), a protein known to recognize H3K9methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in ESCs, Cbx3 associates with active transcriptional start sites, suggesting a developmentally-regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the association of Cbx3 with active transcription, the H3K9methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9methylation restrict late reprogramming events, and suggest that a dramatic change in global chromatin character is an epigenetic roadblock for reprogramming.
Summary The relationship between three-dimensional organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in embryonic stem cells (ESCs), iPSCs, and fibroblasts, and uncover a pluripotency-specific genome organization that is gradually re-established during reprogramming. Our data confirm that long-range chromatin interactions are primarily associated with the spatial segregation of open and closed chromatin, defining overall chromosome conformation. Additionally, we identified two further levels of genome organization in ESCs characterized by co-localization of regions with high pluripotency factor occupancy and strong enrichment for Polycomb proteins/H3K27me3, respectively. Underlining the independence of these networks and their functional relevance for genome organization, loss of the Polycomb protein Eed diminishes interactions between Polycomb-regulated regions without altering overarching chromosome conformation. Together, our data highlight a pluripotency-specific genome organization in which Nanog and H3K27me3 occupy distinct nuclear spaces and reveal a role for cell type-specific gene-regulatory networks in genome organization.
SUMMARY Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.
Functional changes in spatial genome organization during human development are poorly understood. Here we report a comprehensive profile of nuclear dynamics during human cardiogenesis from pluripotent stem cells by integrating Hi-C, RNA-seq and ATAC-seq. While chromatin accessibility and gene expression show complex on/off dynamics, large-scale genome architecture changes are mostly unidirectional. Many large cardiac genes transition from a repressive to an active compartment during differentiation, coincident with upregulation. We identify a network of such gene loci that increase their association inter-chromosomally, and are targets of the muscle-specific splicing factor RBM20. Genome editing studies show that TTN pre-mRNA, the main RBM20-regulated transcript in the heart, nucleates RBM20 foci that drive spatial proximity between the TTN locus and other inter-chromosomal RBM20 targets such as CACNA1C and CAMK2D . This mechanism promotes RBM20-dependent alternative splicing of the resulting transcripts, indicating the existence of a cardiac-specific trans -interacting chromatin domain (TID) functioning as a splicing factory.
Methylation of cytosines (5meC) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5meC levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo.DOI: http://dx.doi.org/10.7554/eLife.06205.001
SUMMARY Applications of ESCs require faithful chromatin changes during differentiation but the fate of each X-chromosome-state in differentiating ESCs is unclear. Female human ESC-lines either carry two active X-chromosomes (XaXa), an Xa and inactive-X-chromosome with or without XIST-RNA-coating (XiXIST+Xa;XiXa), or an Xa and an eroded-Xi (XeXa) where the Xi no longer expresses XIST-RNA and has partially reactivated. Here, we established XiXa, XeXa, and XaXa ESC-lines and followed their X-chromosome-state during differentiation. Surprisingly, we found that the X-state pre-existing in primed ESCs is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells lacked XIST, did not initiate X-inactivation, and displayed higher X-linked gene-expression than XiXa cells. These results demonstrate that X-chromosome-dosage-compensation is not required for ESC differentiation. Our data imply that XiXIST+Xa ESCs are most suited for downstream applications and show that all other X-states are abnormal byproducts of our ESC-derivation and propagation methods.
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