Search

Paternal and maternal epigenomes undergo marked changes after fertilization1. Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications2–4 and differences in chromosome organization and accessibility, both in gametes5–8 and after fertilization5,8–10. However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo8,9 versus the pre-existence of TADs and loops in the zygote5,11. The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allele-specific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell high-throughput chromosome conformation capture (HiC) protocol12,13, during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression—including in recently described, transiently imprinted loci14. We also find TADs that arise in a non-parental-specific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos15. We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development. Single-cell allelic HiC analysis, combined with allelic gene expression and chromatin states, reveals parent-of-origin-specific dynamics of chromosome organization and gene expression during mouse preimplantation development.

Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis, and the derivation of induced pluripotent stem cells (iPSCs). X-Chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease. ispartof: Genome Research vol:29 issue:10 pages:1659-1672 ispartof: location:United States status: published

Sub-nuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments, where transcription and epigenetic factors are either concentrated or depleted. The inactive X chromosome offers a paradigm for studying sub-nuclear compartmentalization. When the non-coding Xist RNA coats the X chromosome, it recruits repressors and chromatin factors that trigger gene silencing, and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in X-chromosome inactivation (XCI) and might explain exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during initiation of XCI, and that its diffusion is not prevented by biophysical constraints. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin bound fraction. These findings demonstrate that initial exclusion of RNA Pol2 from the inactive X is a consequence of its reduced binding rate at the chromatin and gene level, rather than the biophysical compartmentalization of the inactive X heterochromatin domain. The Xist silent compartment is thus a biochemical rather than a biophysical compartment, at least during initiation of XCI.

Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN1-3, the loss of which has been associated with deficient XCI at multiple loci2-6. Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.

The transcription factor C/EBPa is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, approximate to 100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPa expression. This sequence is present in the genes encoding C/EBPa in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPa expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPa in myeloid cells.

International audience; Blood cells are derived from a common set of hematopoietic stem cells, which differentiate into more specific progenitors of the myeloid and lymphoid lineages, ultimately leading to differentiated cells. This developmental process is controlled by a complex regulatory network involving cytokines and their receptors, transcription factors, and chromatin remodelers. Using public data and data from our own molecular genetic experiments (quantitative PCR, Western blot, EMSA) or genome-wide assays (RNA-sequencing, ChIP-sequencing), we have assembled a comprehensive regulatory network encompassing the main transcription factors and signaling components involved in myeloid and lymphoid development. Focusing on B-cell and macrophage development, we defined a qualitative dynamical model recapitulating cytokine-induced differentiation of common progenitors, the effect of various reported gene knockdowns, and the reprogramming of pre-B cells into macrophages induced by the ectopic expression of specific transcription factors. The resulting network model can be used as a template for the integration of new hematopoietic differentiation and transdifferentiation data to foster our understanding of lymphoid/myeloid cell-fate decisions.

Short-term and long-term transcriptional memory is the phenomenon whereby the kinetics or magnitude of gene induction is enhanced following a prior induction period. Short-term memory persists within one cell generation or in postmitotic cells, while long-term memory can survive multiple rounds of cell division. We have developed a tissue culture model to study the epigenetic basis for long-term transcriptional memory (LTTM) and subsequently used this model to better understand the epigenetic mechanisms that enable heritable memory of temporary stimuli. We find that a pulse of transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) induces LTTM on a subset of target genes that survives nine cell divisions. The chromatin landscape at genes that acquire LTTM is more repressed than at those genes that do not exhibit memory, akin to a latent state. We show through chromatin immunoprecipitation (ChIP) and chemical inhibitor studies that RNA polymerase II (Pol II) elongation is important for establishing memory in this model but that Pol II itself is not retained as part of the memory mechanism. More generally, our work reveals that a transcription factor involved in lineage specification can induce LTTM and that failure to rerepress chromatin is one epigenetic mechanism underlying transcriptional memory.

Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alteration in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations. Using a highly efficient reprogramming system, Sardina et al. examined the dynamics of DNA methylation and hydroxymethylation. They found that throughout the process several transcription factors can recruit Tet2 to specific sites, leading to demethylation. Some of these sites became demethylated before chromatin opening.

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is lengthy and inefficient. The development of a reprogramming system that allows rapid and synchronous reprogramming to pluripotency is imperative for understanding the mechanism of iPSC formation and for future therapeutic applications. We have recently reported that a short expression in mouse primary B cells of the transcription factor C/EBPα before the induction of pluripotency factors increases the iPSC reprogramming efficiency >100-fold, involving 95% of the cells within a week. Here we present a dataset containing the time course of gene expression during this process as determined by microarray and RNA-seq techniques.

A pulse of C/EBPα followed by overexpression of the transcription factors Oct4, Sox2, Klf4 and Myc leads to fast and very efficient reprogramming of B cell precursors to induced pluripotent stem cells; C/EBPα facilitates transient chromatin accessibility and accelerates expression of pluripotency genes through a mechanism that involves activation of the Tet2 enzyme. A new study by Thomas Graf and colleagues describes how a pulse of C/EBPα (the transcription factor CCAAT/enhancer binding protein-α) followed by overexpression of the Yamanaka 'OSKM' reprogramming factors leads to fast and very efficient reprogramming of B-cell precursors to induced pluripotent stem (iPS) cells. The authors found that C/EBPα facilitates chromatin accessibility and accelerates expression of pluripotency genes through a mechanism that involves activation of the Tet2 enzyme. This demonstration of highly efficient and fast reprogramming of B cells into iPS cells provides a model for the study of the reprogramming process and may also have clinical relevance. CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM)1,2. However, how C/EBPα accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPα expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial–mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a ‘path-breaker’ as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPα also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM-induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPα-induced immune cell conversion3, our data indicate that Tet2 provides a mechanistic link between iPS cell reprogramming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications.

Summary Transcription-factor-induced somatic cell conversions are highly relevant for both basic and clinical research yet their mechanism is not fully understood and it is unclear whether they reflect normal differentiation processes. Here we show that during pre-B-cell-to-macrophage transdifferentiation, C/EBPα binds to two types of myeloid enhancers in B cells: pre-existing enhancers that are bound by PU.1, providing a platform for incoming C/EBPα; and de novo enhancers that are targeted by C/EBPα, acting as a pioneer factor for subsequent binding by PU.1. The order of factor binding dictates the upregulation kinetics of nearby genes. Pre-existing enhancers are broadly active throughout the hematopoietic lineage tree, including B cells. In contrast, de novo enhancers are silent in most cell types except in myeloid cells where they become activated by C/EBP factors. Our data suggest that C/EBPα recapitulates physiological developmental processes by short-circuiting two macrophage enhancer pathways in pre-B cells., Graphical Abstract, Highlights • C/EBPα activates two classes of prospective myeloid enhancers in B cells • Pre-existing enhancers are bound by PU.1 and become hyper-activated by C/EBPα • C/EBPα acts as a pioneer factor with delayed kinetics on de novo enhancers • The two types of enhancers direct myeloid cell fate in B cells and hematopoiesis, In this study, Graf and colleagues show that, during B-cell-to-macrophage transdifferentiation, C/EBPα activates two types of myeloid enhancers in B cells. The two enhancer types are differentially activated, dictating gene expression levels of macrophage genes during the conversion of B cells into macrophages and normal hematopoiesis.