28 results found
Roman-Trufero M, Ito CM, Pedebos C, et al., 2020, Evolution of an amniote-specific mechanism for modulating ubiquitin signalling via phosphoregulation of the E2 enzyme UBE2D3, Molecular Biology and Evolution, ISSN: 0737-4038
Genetic variation in the enzymes that catalyse post-translational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signalling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as a ubiquitin donor for E3 ligases that catalyse ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologues to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse ES cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extra-embryonic primitive endoderm (PrE), levels of the PDGFRα and FGFR1 receptor tyrosine kinases (RTKs) were reduced and PreE differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the RTKs. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.
Bell E, Curry EW, Megchelenbrink W, et al., 2020, Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naïve pluripotency, Nature Communications, Vol: 11, ISSN: 2041-1723
Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation.
Cui W, Zhang S, Bell E, et al., 2019, OCT4 and PAX6 determine the dual function of SOX2 in human ESCs as a key pluripotent or neural factor, Stem Cell Research and Therapy, Vol: 10, ISSN: 1757-6512
BackgroundSox2 is a well-established pluripotent transcription factor that plays an essential role in establishing and maintaining pluripotent stem cells (PSCs). It is also thought to be a linage specifier that governs PSC neural lineage specification upon their exiting the pluripotent state. However, the exact role of SOX2 in human PSCs was still not fully understood. In this study, we studied the role of SOX2 in human embryonic stem cells (hESCs) by gain- and loss-of-function approaches and explored the possible underlying mechanisms.ResultsWe demonstrate that knockdown of SOX2 induced hESC differentiation to endoderm-like cells, whereas overexpression of SOX2 in hESCs enhanced their pluripotency under self-renewing culture conditions but promoted their neural differentiation upon replacing the culture to non-self-renewal conditions. We show that this culture-dependent dual function of SOX2 was probably attributed to its interaction with different transcription factors predisposed by the culture environments. Whilst SOX2 interacts with OCT4 under self-renewal conditions, we found that, upon neural differentiation, PAX6, a key neural transcription factor, is upregulated and shows interaction with SOX2. The SOX2-PAX6 complex has different gene regulation pattern from that of SOX2-OCT4 complex.ConclusionsOur work provides direct evidence that SOX2 is necessarily required for hESC pluripotency; however, it can also function as a neural factor, depending on the environmental input. OCT4 and PAX6 might function as key SOX2-interacting partners that determine the function of SOX2 in hESCs.
Tomaz RA, Harman JL, Karimlou D, et al., 2017, Jmjd2c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation, Development, Vol: 144, Pages: 567-579, ISSN: 0950-1991
Jmjd2/Kdm4 H3K9-demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we uncover that Jmjd2c facilitates this process by stabilizing the assembly of Mediator-Cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2c-knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a/Ehmt2, independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and furthermore destabilizes loading of the Mediator and Cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c-G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with impact on timely gene activation.
Rayon T, Menchero S, Rollan I, et al., 2016, Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells, Scientific Reports, Vol: 6, ISSN: 2045-2322
The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation.
Percharde M, Azuara V, 2013, Essential roles for the nuclear receptor coactivator Ncoa3 in pluripotency, CELL CYCLE, Vol: 12, Pages: 195-196, ISSN: 1538-4101
Percharde M, Lavial F, Ng J-H, et al., 2012, Ncoa3 functions as an essential Esrrb coactivator to sustain embryonic stem cell self-renewal and reprogramming, GENES & DEVELOPMENT, Vol: 26, Pages: 2286-2298, ISSN: 0890-9369
Lavial F, Bessonnard S, Ohnishi Y, et al., 2012, Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development, GENES & DEVELOPMENT, Vol: 26, Pages: 1445-1458, ISSN: 0890-9369
O'Loghlen A, Munoz-Cabello AM, Gaspar-Maia A, et al., 2012, MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation, CELL STEM CELL, Vol: 10, Pages: 33-46, ISSN: 1934-5909
Alder O, Lavial F, Helness A, et al., 2010, Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment, Development, Vol: 137, Pages: 2483-2492, ISSN: 0950-1991
Pluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. We show that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, our results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development.
Santos J, Pereira CF, Di-Gregorio A, et al., 2010, 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, Epigenetics & Chromatin, Vol: 3, ISSN: 1756-8935
BackgroundDuring early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE) and the primitive endoderm (PrE). Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression.ResultsIn 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.ConclusionsThese data p
Banito A, Rashid S, Acosta JC, et al., 2009, Senescence impairs successful reprogramming to pluripotent stem cells, Genes Dev
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16(INK4a), and p21(CIP1). Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.
Takousis P, Johonnett P, Williamson J, et al., 2007, Replication timing profile reflects the distinct functional and genomic features of the MHC class II region, CELL CYCLE, Vol: 6, Pages: 2393-2398, ISSN: 1538-4101
Jorgensen HF, Azuara V, Amoils S, et al., 2007, The impact of chromatin modifiers on the timing of locus replication in mouse embryonic stem cells, Genome Biology, Vol: 8, ISSN: 1474-7596
BackgroundThe time of locus replication during S-phase is tightly regulated and correlates with chromatin state. Embryonic stem (ES) cells have an unusual chromatin profile where many developmental regulator genes that are not yet expressed are marked by both active and repressive histone modifications. This poised or bivalent state is also characterized by locus replication in early S-phase in ES cells, while replication timing is delayed in cells with restricted developmental options.ResultsHere 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.ConclusionThis 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.
Williams RRE, Azuara V, Perry P, et al., 2006, Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus, JOURNAL OF CELL SCIENCE, Vol: 119, Pages: 132-140, ISSN: 0021-9533
Azuara V, 2006, Profiling of DNA replication timing in unsynchronized cell populations, NATURE PROTOCOLS, Vol: 1, Pages: 2171-2177, ISSN: 1754-2189
Perry P, Sauer S, Billon N, et al., 2004, A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction, CELL CYCLE, Vol: 3, Pages: 1645-1650, ISSN: 1538-4101
Azuara V, Brown KE, Williams RRE, et al., 2003, Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication, NATURE CELL BIOLOGY, Vol: 5, Pages: 668-U49, ISSN: 1465-7392
Azuara V, Fisher AG, 2003, Maintaining Transcriptional States Through DNA Replication, CELL CYCLE, Vol: 2, Pages: 521-524, ISSN: 1538-4101
Skok JA, Brown KE, Azuara V, et al., 2001, Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes, NATURE IMMUNOLOGY, Vol: 2, Pages: 848-854, ISSN: 1529-2908
Azuara V, Grigoriadou K, Lembezat MP, et al., 2001, Strain-specific TCR repertoire selection of IL-4-producing Thy-1(dull) gamma delta thymocytes, EUROPEAN JOURNAL OF IMMUNOLOGY, Vol: 31, Pages: 205-214, ISSN: 0014-2980
Pereira P, Hermitte V, Lembezat MP, et al., 2000, Developmentally regulated and lineage-specific rearrangement of T cell receptor V alpha/delta gene segments, EUROPEAN JOURNAL OF IMMUNOLOGY, Vol: 30, Pages: 1988-1997, ISSN: 0014-2980
Azuara V, Pereira P, 2000, Genetic mapping of two murine loci that influence the development of IL-4-producing Thy-1(dull) gamma delta thymocytes, JOURNAL OF IMMUNOLOGY, Vol: 165, Pages: 42-48, ISSN: 0022-1767
Gerber DJ, Azuara V, Levraud JP, et al., 1999, IL4-producing gamma delta T cells that express a very restricted TCR repertoire are preferentially localized in liver and spleen, JOURNAL OF IMMUNOLOGY, Vol: 163, Pages: 3076-3082, ISSN: 0022-1767
Azuara V, Lembezat MP, Pereira P, 1998, The homogeneity of the TCR delta repertoire expressed by the Thy-1(dull) gamma delta T cell population is due to cellular selection, EUROPEAN JOURNAL OF IMMUNOLOGY, Vol: 28, Pages: 3456-3467, ISSN: 0014-2980
Azuara V, Levraud JP, Lembezat MP, et al., 1997, A novel subset of adult gamma delta thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire, EUROPEAN JOURNAL OF IMMUNOLOGY, Vol: 27, Pages: 544-553, ISSN: 0014-2980
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