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Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo
Joana Santos, C Filipe Pereira, Aida Di-Gregorio, Thomas Spruce, Olivia Alder, Tristan Rodriguez, Véronique Azuara, Matthias Merkenschlager, Amanda G Fisher
Epigenetics & Chromatin , 2010, DOI: 10.1186/1756-8935-3-1
Abstract: In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) stem cell lines that were derived from the inner cell mass (ICM), TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3). Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation.These data provide evidence that the diversification of defined embryonic and extra-embryonic lineages is accompanied by chromatin remodelling at specific loci. Stem cell lines from the ICM, TE and PrE can each dominantly reprogramme somatic cells but reset gene expression differently, reflecting their separate lineage identities and increasingly restricted d
Tracking the mechanical dynamics of human embryonic stem cell chromatin
Elizabeth Hinde, Francesco Cardarelli, Aaron Chen, Michelle Khine, Enrico Gratton
Epigenetics & Chromatin , 2012, DOI: 10.1186/1756-8935-5-20
Abstract: We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs) are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation.We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.Embryonic stem cells (ESCs) are pluripotent cells that are derived from the inner cell mass of the pre-implantation embryo at the blastocyst stage. They are characterized by their potential to self-renew indefinitely and to differentiate into any of the three germ layers - endoderm, mesoderm and ectoderm [1]. This dual capacity places opposing constraints on the organization of the ESC genome [2]. Self-renewal requires that the ESC genome maintains a cellular memory that specifies its pluripotent capacity, whilst pluripotency relies on the ESC genome being in a highly plastic state so as to have the ability to enter any one distinct differentiation pathway [3]. How these two key functional properties of ESCs are maintained is largely unknown. However, from a structural point of view it is thought that the open conformation of ESC chromatin and hyperdynamic association of chromatin structural proteins are in part responsible [2,4-6].Studies in several systems indicate that ESCs are rich in euchromatin and as differentiation progresses, undergo a rapid reorganization of large areas of the genome to accumulate highly condensed, tra
CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells  [PDF]
Martin Houlard,Soizik Berlivet equal contributor,Aline V Probst equal contributor,Jean-Pierre Quivy,Patrick Héry,Geneviève Almouzni,Matthieu Gérard
PLOS Genetics , 2006, DOI: 10.1371/journal.pgen.0020181
Abstract: During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.
The impact of chromatin modifiers on the timing of locus replication in mouse embryonic stem cells
Helle F J?rgensen, Véronique Azuara, Shannon Amoils, Mikhail Spivakov, Anna Terry, Tatyana Nesterova, Bradley S Cobb, Bernard Ramsahoye, Matthias Merkenschlager, Amanda G Fisher
Genome Biology , 2007, DOI: 10.1186/gb-2007-8-8-r169
Abstract: Here we used a panel of mutant mouse ES cell lines lacking important chromatin modifiers to dissect the relationship between chromatin structure and replication timing. We show that temporal control of satellite DNA replication is sensitive to loss of a variety of chromatin modifiers, including Mll, Eed, Dnmt1, Suv39h1/h2 and Dicer. The replication times of many single copy loci, including a 5 Mb contiguous region surrounding the Rex1 gene, were retained in chromatin modifier mutant ES cells, although a subset of loci were affected.This analysis demonstrates the importance of chromatin modifiers for maintaining correct replication of satellite sequences in pluripotent ES cells and highlights the sensitivity of some single copy loci to the influence of chromatin modifiers. Abundant histone acetylation is shown to correlate well with early replication. Surprisingly, loss of DNA methylation or histone methylation was tolerated by many loci, suggesting that these modifications may be less influential for the timing of euchromatin replication.DNA labeling experiments have shown that replication patterns are faithfully inherited through multiple cell divisions [1]. Individual genes replicate at similar times in each cell of a given type but locus replication timing often differs between cell types. In embryonic stem (ES) cells, the timing of DNA replication of several genes is altered in response to differentiation [2,3], which reflects changes in both gene expression and the decline in developmental potential that accompanies lineage commitment [4]. More generally, replication timing is influenced by both chromosome context [5,6] and underlying nucleotide composition [3]. Genome-wide and single gene analyses have shown that early replication timing correlates with transcriptional activity (reviewed in [7]) as well as with chromatin accessibility, or permissivity [8], and is often associated with enrichment of acetylated histones [9-11]. The exact relationship between chr
Global Chromatin Architecture Reflects Pluripotency and Lineage Commitment in the Early Mouse Embryo  [PDF]
Kashif Ahmed,Hesam Dehghani,Peter Rugg-Gunn,Eden Fussner,Janet Rossant,David P. Bazett-Jones
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0010531
Abstract: An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells.
Stage-Specific Germ-Cell Marker Genes Are Expressed in All Mouse Pluripotent Cell Types and Emerge Early during Induced Pluripotency  [PDF]
Xingbo Xu,D. V. Krishna Pantakani,Sandra Lührig,Xiaoying Tan,Tatjana Khromov,Jessica Nolte,Ralf Dressel,Ulrich Zechner,Wolfgang Engel
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0022413
Abstract: Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and found them to be comparable to the expression of cultured pluripotent cells originated from the germ line. Further, siRNA knockdown experiments have demonstrated the parallel maintenance and independence of pluripotent and GC/PrM networks in ESCs. Through chromatin immunoprecipitation experiments, we observed that pluripotent cells exhibit active chromatin states at GC marker genes and a bivalent chromatin structure at PrM marker genes. Moreover, gene expression analysis during the time course of iPS cells generation revealed that the expression of GC markers precedes pluripotency markers. Collectively, through our observations we hypothesize that the chromatin state and the expression of GC/PrM markers might indicate molecular parallels between in-vivo germ cell specification and pluripotent stem cell generation.
Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation  [PDF]
Yunzhe Zhang equal contributor,Marissa Cooke equal contributor,Shiraj Panjwani equal contributor,Kaixiang Cao,Beth Krauth,Po-Yi Ho,Magdalena Medrzycki,Dawit T. Berhe,Chenyi Pan,Todd C. McDevitt,Yuhong Fan
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002691
Abstract: Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.
RNA Polymerase III Transcriptomes in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells, and Relationships with Pluripotency Transcription Factors  [PDF]
Ravi K. Alla, Bradley R. Cairns
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0085648
Abstract: Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types, and that this variation is likely determined by a combination of the chromatin landscape, cell-specific DNA-binding transcription factors, and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells, there is presently little understanding of this aspect of the Pol III system in human ES cells. Here, we determine the occupancy profiles of Pol III components in human H1 ES cells, and also induced pluripotent cells, and compare to known profiles of chromatin, transcription factors, and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly, we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells, especially NANOG. Notably, in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases, a peak of H3K4me3 and/or RNA Pol II, between the H3K27me3 and Pol III binding peaks, suggesting that H3K4me3 and Pol II activity may “insulate” Pol III from neighboring repressive H3K27me3. Further, we find iPSCs have a larger Pol III repertoire than their precursors. Finally, the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together, our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity.
Epigenomics of human embryonic stem cells and induced pluripotent stem cells: insights into pluripotency and implications for disease
Alvaro Rada-Iglesias, Joanna Wysocka
Genome Medicine , 2011, DOI: 10.1186/gm252
Abstract: Embryonic stem cells (ESCs) and the early developmental stage embryo share a unique property called pluripotency, which is the ability to give rise to the three germ layers (endoderm, ectoderm and mesoderm) and, consequently, all tissues represented in the adult organism [1,2]. Pluripotency can also be induced in somatic cells during in vitro reprogramming, leading to the formation of so-called induced pluripotent stem cells (iPSCs; extensively reviewed in [3-7]). In order to fulfill the therapeutic potential of human ESCs (hESCs) and iPSCs, an understanding of the fundamental molecular properties underlying the nature of pluripotency and commitment is required, along with the development of methods for assessing biological equivalency among different cell populations.Functional complexity of the human body, with over 200 specialized cell types, and intricately built tissues and organs, arises from a single set of instructions: the human genome. How, then, do distinct cellular phenotypes emerge from this genetic homogeneity? Interactions between the genome and its cellular and signaling environments are the key to understanding how cell-type-specific gene expression patterns arise during differentiation and development [8]. These interactions ultimately occur at the level of the chromatin, which comprises the DNA polymer repeatedly wrapped around histone octamers, forming a nucleosomal array that is further compacted into the higher-order structure. Regulatory variation is introduced to the chromatin via alterations within the nucleosome itself - for example, through methylation and hydroxymethylation of DNA, various post-translational modifications (PTMs) of histones, and inclusion or exclusion of specific histone variants [9-15] - as well as via changes in nucleosomal occupancy, mobility and organization [16,17]. In turn, these alterations modulate access of sequence-dependent transcriptional regulators to the underlying DNA, the level of chromatin compaction, and
Regulatory Non-Coding RNAs in Pluripotent Stem Cells  [PDF]
Alessandro Rosa,Ali H. Brivanlou
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140714346
Abstract: The most part of our genome encodes for RNA transcripts are never translated into proteins. These include families of RNA molecules with a regulatory function, which can be arbitrarily subdivided in short (less than 200 nucleotides) and long non-coding RNAs (ncRNAs). MicroRNAs, which act post-transcriptionally to repress the function of target mRNAs, belong to the first group. Included in the second group are multi-exonic and polyadenylated long ncRNAs (lncRNAs), localized either in the nucleus, where they can associate with chromatin remodeling complexes to regulate transcription, or in the cytoplasm, acting as post-transcriptional regulators. Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), represent useful systems for modeling normal development and human diseases, as well as promising tools for regenerative medicine. To fully explore their potential, however, a deep understanding of the molecular basis of stemness is crucial. In recent years, increasing evidence of the importance of regulation by ncRNAs in pluripotent cells is accumulating. In this review, we will discuss recent findings pointing to multiple roles played by regulatory ncRNAs in ESC and iPSCs, where they act in concert with signaling pathways, transcriptional regulatory circuitries and epigenetic factors to modulate the balance between pluripotency and differentiation.
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