123 results found
Merkenschlager M, Ferreirós-Vidal I, Carroll T, et al., Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation, PLoS Biology, ISSN: 1544-9173
The differentiation of self-renewingprogenitor cells requires not only the regulation of lineage-and developmental stage-specific genes, but also the coordinated adaptation of housekeeping functionsfrom a metabolically active, proliferative state towards quiescence. How metabolic and cell cycle states are coordinated with the regulation of cell type-specific genes is an important question, as dissociation between differentiation, cell cycle, and metabolic states is a hallmark of cancer. Here we use a model system to systematically identify key transcriptional regulators of Ikaros-dependent B cell progenitor differentiation. We find that the coordinated regulation of housekeeping functions and tissue-specific gene expressionrequires afeedforward circuit whereby Ikarosdownregulates the expression of Myc. Our findings show how coordination between differentiation and housekeeping statescan be achieved by interconnected regulators. Similar principles likely coordinate differentiation and housekeeping functions during progenitor cell differentiation in other cell lineages.
Calderon L, Weiss FD, Carroll T, et al., 2019, Cohesin is continuously required to sustain neuronal gene expression, 29th Mammalian Genetics and Development Workshop of the Genetics-Society, Publisher: CAMBRIDGE UNIV PRESS, ISSN: 0016-6723
Millership S, Tunster SJ, Van de Pette M, et al., 2018, Neuronatin deletion causes postnatal growth restriction and adult obesity in 129S2/Sv mice, Molecular Metabolism, Vol: 18, Pages: 97-106, ISSN: 2212-8778
ObjectiveImprinted genes are crucial for the growth and development of fetal and juvenile mammals. Altered imprinted gene dosage causes a variety of human disorders, with growth and development during these crucial early stages strongly linked with future metabolic health in adulthood. Neuronatin (Nnat) is a paternally expressed imprinted gene found in neuroendocrine systems and white adipose tissue and is regulated by the diet and leptin. Neuronatin expression is downregulated in obese children and has been associated with stochastic obesity in C57BL/6 mice. However, our recent studies of Nnat null mice on this genetic background failed to display any body weight or feeding phenotypes but revealed a defect in glucose-stimulated insulin secretion due to the ability of neuronatin to potentiate signal peptidase cleavage of preproinsulin. Nnat deficiency in beta cells therefore caused a lack of appropriate storage and secretion of mature insulin.MethodsTo further explore the potential role of Nnat in the regulation of body weight and adiposity, we studied classical imprinting-related phenotypes such as placental, fetal, and postnatal growth trajectory patterns that may impact upon subsequent adult metabolic phenotypes.ResultsHere we find that, in contrast to the lack of any body weight or feeding phenotypes on the C57BL/6J background, deletion of Nnat in mice on 129S2/Sv background causes a postnatal growth restriction with reduced adipose tissue accumulation, followed by catch up growth after weaning. This was in the absence of any effect on fetal growth or placental development. In adult 129S2/Sv mice, Nnat deletion was associated with hyperphagia, reduced energy expenditure, and partial leptin resistance. Lack of neuronatin also potentiated obesity caused by either aging or high fat diet feeding.ConclusionsThe imprinted gene Nnat plays a key role in postnatal growth, adult energy homeostasis, and the pathogenesis of obesity via catch up growth effects, but this role
Merkenschlager M, Cuartero S, Weiss F, et al., 2018, Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation, Nature Immunology, Vol: 19, Pages: 932-941, ISSN: 1529-2908
Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.
Cantone I, Fisher AG, 2017, Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease, Philosophical Transactions of the Royal Society B: Biological Sciences, Vol: 372, ISSN: 1471-2970
X chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro. Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability andheterogeneity of human ESCs and iPSCs, hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ESCs to reprogram female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to reexpress human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naïve' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.
Fisher CL, Marks H, Cho LT-Y, et al., 2017, An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers., Nucleic Acids Research, Vol: 45, Pages: e174-e174, ISSN: 0305-1048
Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.
Fisher AG, Stumpf MPH, Merkenschlager M, 2017, Reconciling Epigenetic Memory and Transcriptional Responsiveness, CELL SYSTEMS, Vol: 4, Pages: 373-374, ISSN: 2405-4712
The molecular basis of cellular memory is important but poorly understood. Using estimates of histone dynamics, Martin Howard and colleagues construct a mathematical model that helps to explain both the stability and flexibility of Polycomb-mediated gene regulation in cellular memory.
Liang Z, Brown KE, Carroll T, et al., 2017, A high-resolution map of transcriptional repression, ELIFE, Vol: 6, ISSN: 2050-084X
Turning genes on and off is essential for development and homeostasis, yet little is known about the sequence and causal role of chromatin state changes during the repression of active genes. This is surprising, as defective gene silencing underlies developmental abnormalities and disease. Here we delineate the sequence and functional contribution of transcriptional repression mechanisms at high temporal resolution. Inducible entry of the NuRD-interacting transcriptional regulator Ikaros into mouse pre-B cell nuclei triggered immediate binding to target gene promoters. Rapid RNAP2 eviction, transcriptional shutdown, nucleosome invasion, and reduced transcriptional activator binding required chromatin remodeling by NuRD-associated Mi2beta/CHD4, but were independent of HDAC activity. Histone deacetylation occurred after transcriptional repression. Nevertheless, HDAC activity contributed to stable gene silencing. Hence, high resolution mapping of transcriptional repression reveals complex and interdependent mechanisms that underpin rapid transitions between transcriptional states, and elucidates the temporal order, functional role and mechanistic separation of NuRD-associated enzymatic activities.
Van de Pette M, Abbas A, Feytout A, et al., 2017, Visualizing changes in Cdkn1c expression links early life adversity to imprint mis-regulation in adults, Cell Reports, Vol: 31, Pages: 1090-1099, ISSN: 2211-1247
Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility.Here we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1cexpression in mice and showed that expression was modulated by environmental factors encounteredin utero.Acute exposure to chromatin modifyingdrugs resulted in de-repression of paternally inherited (silent) Cdkn1calleles in embryos that was temporary and resolved after birth.In contrast, deprivation of maternal dietary proteinin uteroprovoked permanent de-repression of imprinted Cdkn1cexpression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss.Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early life adversity to later-life outcomes.Furthermore,Cdkn1c-luciferasemice offer non-invasivetools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.
Cantone I, Dharmalingam G, Chan YW, et al., 2017, Allele-specific analysis of cell fusion-mediated pluripotent reprograming reveals distinct and predictive susceptibilities of human X-linked genes to reactivation, Genome Biology, Vol: 18, ISSN: 1474-760X
BackgroundInactivation of one X chromosome is established early in female mammalian development and can be reversed in vivo and in vitro when pluripotency factors are re-expressed. The extent of reactivation along the inactive X chromosome (Xi) and the determinants of locus susceptibility are, however, poorly understood. Here we use cell fusion-mediated pluripotent reprograming to study human Xi reactivation and allele-specific single nucleotide polymorphisms (SNPs) to identify reactivated loci.ResultsWe show that a subset of human Xi genes is rapidly reactivated upon re-expression of the pluripotency network. These genes lie within the most evolutionary recent segments of the human X chromosome that are depleted of LINE1 and enriched for SINE elements, predicted to impair XIST spreading. Interestingly, this cadre of genes displays stochastic Xi expression in human fibroblasts ahead of reprograming. This stochastic variability is evident between clones, by RNA-sequencing, and at the single-cell level, by RNA-FISH, and is not attributable to differences in repressive histone H3K9me3 or H3K27me3 levels. Treatment with the DNA demethylating agent 5-deoxy-azacytidine does not increase Xi expression ahead of reprograming, but instead reveals a second cadre of genes that only become susceptible to reactivation upon induction of pluripotency.ConclusionsCollectively, these data not only underscore the multiple pathways that contribute to maintaining silencing along the human Xi chromosome but also suggest that transcriptional stochasticity among human cells could be useful for predicting and engineering epigenetic strategies to achieve locus-specific or domain-specific human Xi gene reactivation.
Fisher AG, 2016, Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming, Nature Communications, Vol: 7, ISSN: 2041-1723
Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome.
Graham B, Marcais A, Dharmalingam G, et al., 2016, MicroRNAs of the miR-290-295 Family Maintain Bivalency in Mouse Embryonic Stem Cells., Stem Cell Reports, Vol: 6, Pages: 635-642, ISSN: 2213-6711
Numerous developmentally regulated genes in mouse embryonic stem cells (ESCs) are marked by both active (H3K4me3)- and polycomb group (PcG)-mediated repressive (H3K27me3) histone modifications. This bivalent state is thought to be important for transcriptional poising, but the mechanisms that regulate bivalent genes and the bivalent state remain incompletely understood. Examining the contribution of microRNAs (miRNAs) to the regulation of bivalent genes, we found that the miRNA biogenesis enzyme DICER was required for the binding of the PRC2 core components EZH2 and SUZ12, and for the presence of the PRC2-mediated histone modification H3K27me3 at many bivalent genes. Genes that lost bivalency were preferentially upregulated at the mRNA and protein levels. Finally, reconstituting Dicer-deficient ESCs with ESC miRNAs restored bivalent gene repression and PRC2 binding at formerly bivalent genes. Therefore, miRNAs regulate bivalent genes and the bivalent state itself.
Cellular fusion is a key process in many fields ranging from historical gene mapping studies and monoclonal antibody production, through to cell reprogramming. Traditional methodologies for cell fusion rely on the random pairing of different cell types and generally result in low and variable fusion efficiencies. These approaches become particularly limiting where substantial numbers of bespoke one-to-one fusions are required, for example, for in-depth studies of nuclear reprogramming mechanisms. In recent years, microfluidic technologies have proven valuable in creating platforms where the manipulation of single cells is highly efficient, rapid and controllable. These technologies also allow the integration of different experimental steps and characterisation processes into a single platform. Although the application of microfluidic methodologies to cell fusion studies is promising, current technologies that rely on static trapping are limited both in terms of the overall number of fused cells produced and their experimental accessibility. Here we review some of the most exciting breakthroughs in core microfluidic technologies that will allow the creation of integrated platforms for controlled cell fusion at high throughput. © 2015 Society of Chemical Industry.
Malinowski AR, Fisher AG, 2016, Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion., Pages: 289-299
Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.
Cohesin is required for ES cell self-renewal and iPS-mediated reprogramming of somatic cells. This may indicate a special role for cohesin in the regulation of pluripotency genes, perhaps by mediating long-range chromosomal interactions between gene regulatory elements. However, cohesin is also essential for genome integrity, and its depletion from cycling cells induces DNA damage responses. Hence, the failure of cohesin-depleted cells to establish or maintain pluripotency gene expression could be explained by a loss of long-range interactions or by DNA damage responses that undermine pluripotency gene expression. In recent work we began to disentangle these possibilities by analyzing reprogramming in the absence of cell division. These experiments showed that cohesin was not specifically required for reprogramming, and that the expression of most pluripotency genes was maintained when ES cells were acutely depleted of cohesin. Here we take this analysis to its logical conclusion by demonstrating that deliberately inflicted DNA damage - and the DNA damage that results from proliferation in the absence of cohesin - can directly interfere with pluripotency and reprogramming. The role of cohesin in pluripotency and reprogramming may therefore be best explained by essential cohesin functions in the cell cycle.
Landeira D, Bagci H, Malinowski AR, et al., 2015, Jarid2 Coordinates Nanog Expression and PCP/Wnt Signaling Required for Efficient ESC Differentiation and Early Embryo Development, Cell Reports, Vol: 12, Pages: 573-586, ISSN: 2211-1247
Jarid2 is part of the Polycomb Repressor complex 2 (PRC2) responsible for genome-wide H3K27me3 deposition. Unlike other PRC2-deficient embryonic stem cells (ESCs), however, Jarid2-deficient ESCs show a severe differentiation block, altered colony morphology, and distinctive patterns of deregulated gene expression. Here, we show that Jarid2−/− ESCs express constitutively high levels of Nanog but reduced PCP signaling components Wnt9a, Prickle1, and Fzd2 and lowered β-catenin activity. Depletion of Wnt9a/Prickle1/Fzd2 from wild-type ESCs or overexpression of Nanog largely phenocopies these cellular defects. Co-culture of Jarid2−/− with wild-type ESCs restores variable Nanog expression and β-catenin activity and can partially rescue the differentiation block of mutant cells. In addition, we show that ESCs lacking Jarid2 or Wnt9a/Prickle1/Fzd2 or overexpressing Nanog induce multiple ICM formation when injected into normal E3.5 blastocysts. These data describe a previously unrecognized role for Jarid2 in regulating a core pluripotency and Wnt/PCP signaling circuit that is important for ESC differentiation and for pre-implantation development.
Ing-Simmons E, Seitan VC, Faure AJ, et al., 2015, Spatial enhancer clustering and regulation of enhancer-proximal genes by cohesin, GENOME RESEARCH, Vol: 25, Pages: 504-513, ISSN: 1088-9051
Blevins R, Bruno L, Carroll T, et al., 2015, microRNAs Regulate Cell-to-Cell Variability of Endogenous Target Gene Expression in Developing Mouse Thymocytes, Plos Genetics, Vol: 11, ISSN: 1553-7404
The development and homeostasis of multicellular organisms relies on gene regulationwithin individual constituent cells. Gene regulatory circuits that increase the robustness ofgene expression frequently incorporate microRNAs as post-transcriptional regulators.Computational approaches, synthetic gene circuits and observations in model organismspredict that the co-regulation of microRNAs and their target mRNAs can reduce cell-to-cellvariability in the expression of target genes. However, whether microRNAs directly regulatevariability of endogenous gene expression remains to be tested in mammalian cells. Herewe use quantitative flow cytometry to show that microRNAs impact on cell-to-cell variabilityof protein expression in developing mouse thymocytes. We find two distinct mechanismsthat control variation in the activation-induced expression of the microRNA target CD69.First, the expression of miR-17 and miR-20a, two members of the miR-17-92 cluster, is coregulatedwith the target mRNA Cd69 to form an activation-induced incoherent feed-forwardloop. Another microRNA, miR-181a, acts at least in part upstream of the target mRNA Cd69to modulate cellular responses to activation. The ability of microRNAs to render gene expressionmore uniform across mammalian cell populations may be important for normal developmentand for disease.
Lavagnolli T, Gupta P, Hörmanseder E, et al., 2015, Initiation and maintenance of pluripotency gene expression in the absence of cohesin., Genes Dev, Vol: 29, Pages: 23-38
Cohesin is implicated in establishing and maintaining pluripotency. Whether this is because of essential cohesin functions in the cell cycle or in gene regulation is unknown. Here we tested cohesin's contribution to reprogramming in systems that reactivate the expression of pluripotency genes in the absence of proliferation (embryonic stem [ES] cell heterokaryons) or DNA replication (nuclear transfer). Contrary to expectations, cohesin depletion enhanced the ability of ES cells to initiate somatic cell reprogramming in heterokaryons. This was explained by increased c-Myc (Myc) expression in cohesin-depleted ES cells, which promoted DNA replication-dependent reprogramming of somatic fusion partners. In contrast, cohesin-depleted somatic cells were poorly reprogrammed in heterokaryons, due in part to defective DNA replication. Pluripotency gene induction was rescued by Myc, which restored DNA replication, and by nuclear transfer, where reprogramming does not require DNA replication. These results redefine cohesin's role in pluripotency and reveal a novel function for Myc in promoting the replication-dependent reprogramming of somatic nuclei.
Marcais A, Blevins R, Graumann J, et al., 2014, microRNA-mediated regulation of mTOR complex components facilitates discrimination between activation and anergy in CD4 T cells, JOURNAL OF EXPERIMENTAL MEDICINE, Vol: 211, Pages: 2281-2295, ISSN: 0022-1007
Piccolo FM, Fisher AG, 2014, Getting rid of DNA methylation, TRENDS IN CELL BIOLOGY, Vol: 24, Pages: 136-143, ISSN: 0962-8924
Seitan VC, Faure AJ, Zhan Y, et al., 2013, Cohesin-based chromatin interactions enable regulated gene expression within preexisting architectural compartments, GENOME RESEARCH, Vol: 23, Pages: 2066-2077, ISSN: 1088-9051
Brown KE, Bagci H, Soza-Ried J, et al., 2013, Atypical heterochromatin organization and replication are rapidly acquired by somatic cells following fusion-mediated reprogramming by mouse ESCs, CELL CYCLE, Vol: 12, Pages: 3253-3261, ISSN: 1538-4101
Bagci H, Fisher AG, 2013, DNA Demethylation in Pluripotency and Reprogramming: The Role of Tet Proteins and Cell Division, CELL STEM CELL, Vol: 13, Pages: 265-269, ISSN: 1934-5909
Piccolo FM, Bagci H, Brown KE, et al., 2013, Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion, MOLECULAR CELL, Vol: 49, Pages: 1023-1033, ISSN: 1097-2765
Piccolo FM, Bagci H, Brown KE, et al., 2013, Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion (vol 49, pg 1023, 2013), MOLECULAR CELL, Vol: 49, Pages: 1176-1176, ISSN: 1097-2765
Ferreiros-Vidal I, Carroll T, Taylor B, et al., 2013, Genome-wide identification of Ikaros targets elucidates its contribution to mouse B-cell lineage specification and pre-B-cell differentiation, BLOOD, Vol: 121, Pages: 1769-1782, ISSN: 0006-4971
Cantone I, Fisher AG, 2013, Epigenetic programming and reprogramming during development, NATURE STRUCTURAL & MOLECULAR BIOLOGY, Vol: 20, Pages: 282-289, ISSN: 1545-9993
Tsubouchi T, Soza-Ried J, Brown K, et al., 2013, DNA Synthesis Is Required for Reprogramming Mediated by Stem Cell Fusion, CELL, Vol: 152, Pages: 873-883, ISSN: 0092-8674
Tsubouchi T, Fisher AG, 2013, Reprogramming and the Pluripotent Stem Cell Cycle, EPIGENETICS AND DEVELOPMENT, Editors: Heard, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 223-241, ISBN: 978-0-12-416027-9
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