Publications by authors named "Amanda G Fisher"

92 Publications

The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus.

Nat Commun 2021 01 4;12(1):99. Epub 2021 Jan 4.

MRC London Institute of Medical Sciences, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4Cd8a selection intermediates appear before Cd4Cd8a selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.
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http://dx.doi.org/10.1038/s41467-020-20306-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782583PMC
January 2021

Illuminating Epigenetics and Inheritance in the Immune System with Bioluminescence.

Trends Immunol 2020 11 6;41(11):994-1005. Epub 2020 Oct 6.

Lymphocyte Development Group, MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK. Electronic address:

The remarkable process of light emission by living organisms has fascinated mankind for thousands of years. A recent expansion in the repertoire of catalytic luciferase enzymes, coupled with the discovery of the genes and pathways that encode different luciferin substrates, means that bioluminescence imaging (BLI) is set to revolutionize longitudinal and dynamic studies of gene control within biomedicine, including the regulation of immune responses. In this review article, we summarize recent advances in bioluminescence-based imaging approaches that promise to enlighten our understanding of in vivo gene and epigenetic control within the immune system.
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http://dx.doi.org/10.1016/j.it.2020.09.001DOI Listing
November 2020

Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction.

Nat Commun 2020 08 17;11(1):4118. Epub 2020 Aug 17.

Lymphocyte Development Group, MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.
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http://dx.doi.org/10.1038/s41467-020-17823-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431861PMC
August 2020

Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.

Nat Immunol 2019 10 26;20(10):1372-1380. Epub 2019 Aug 26.

Lymphocyte Development Group, MRC London Institute for Medical Sciences, London, UK.

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.
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http://dx.doi.org/10.1038/s41590-019-0471-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754753PMC
October 2019

Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation.

PLoS Biol 2019 04 12;17(4):e2006506. Epub 2019 Apr 12.

Unit of Computational Medicine, Department of Medicine, Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.

The differentiation of self-renewing progenitor cells requires not only the regulation of lineage- and developmental stage-specific genes but also the coordinated adaptation of housekeeping functions from a metabolically active, proliferative state toward quiescence. How metabolic and cell-cycle states are coordinated with the regulation of cell type-specific genes is an important question, because 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 expression requires a feedforward circuit whereby Ikaros down-regulates the expression of Myc. Our findings show how coordination between differentiation and housekeeping states can be achieved by interconnected regulators. Similar principles likely coordinate differentiation and housekeeping functions during progenitor cell differentiation in other cell lineages.
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http://dx.doi.org/10.1371/journal.pbio.2006506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481923PMC
April 2019

Neuronatin deletion causes postnatal growth restriction and adult obesity in 129S2/Sv mice.

Mol Metab 2018 12 15;18:97-106. Epub 2018 Sep 15.

MRC London Institute of Medical Sciences, Du Cane Road, London, W12 0NN, UK; Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, Du Cane Road, London, W12 0NN, UK. Electronic address:

Objective: Imprinted 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.

Methods: To 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.

Results: Here 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.

Conclusions: The 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 is dependent upon genetic background.
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http://dx.doi.org/10.1016/j.molmet.2018.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6308027PMC
December 2018

Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation.

Nat Immunol 2018 09 20;19(9):932-941. Epub 2018 Aug 20.

Lymphocyte Development Group, Epigenetics Section, MRC London Institute of Medical Sciences, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1038/s41590-018-0184-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195188PMC
September 2018

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

Nucleic Acids Res 2017 Dec;45(21):e174

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

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.
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http://dx.doi.org/10.1093/nar/gkx811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716182PMC
December 2017

Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

Philos Trans R Soc Lond B Biol Sci 2017 Nov;372(1733)

Lymphocyte Development, MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN, UK

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 or by pluripotent reprogramming Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells 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 ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses 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 re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.
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http://dx.doi.org/10.1098/rstb.2016.0358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627160PMC
November 2017

Reconciling Epigenetic Memory and Transcriptional Responsiveness.

Cell Syst 2017 04;4(4):373-374

Lymphocyte Development Group, MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, UK.

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.
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http://dx.doi.org/10.1016/j.cels.2017.04.005DOI Listing
April 2017

A high-resolution map of transcriptional repression.

Elife 2017 03 20;6. Epub 2017 Mar 20.

Lymphocyte Development Group, MRC London Institute of Medical Sciences, Faculty of Medicine, Imperial College London, London, United Kingdom.

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.
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http://dx.doi.org/10.7554/eLife.22767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373822PMC
March 2017

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults.

Cell Rep 2017 01;18(5):1090-1099

MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

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 Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression 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-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.
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http://dx.doi.org/10.1016/j.celrep.2017.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300902PMC
January 2017

Allele-specific analysis of cell fusion-mediated pluripotent reprograming reveals distinct and predictive susceptibilities of human X-linked genes to reactivation.

Genome Biol 2017 01 25;18(1). Epub 2017 Jan 25.

Lymphocyte Development Group, MRC London Institute of Medical Sciences, Hammersmith Campus, Imperial College London, Du Cane Road, London, W12 0NN, UK.

Background: Inactivation 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.

Results: We 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.

Conclusions: Collectively, 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.
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http://dx.doi.org/10.1186/s13059-016-1136-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264468PMC
January 2017

Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

Methods Mol Biol 2016 ;1480:289-99

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

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.
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http://dx.doi.org/10.1007/978-1-4939-6380-5_25DOI Listing
January 2018

Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming.

Nat Commun 2016 08 10;7:12354. Epub 2016 Aug 10.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

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.
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http://dx.doi.org/10.1038/ncomms12354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987517PMC
August 2016

MicroRNAs of the miR-290-295 Family Maintain Bivalency in Mouse Embryonic Stem Cells.

Stem Cell Reports 2016 05 14;6(5):635-642. Epub 2016 Apr 14.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK; Epigenetics Section, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2016.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939759PMC
May 2016

Cohesin's role in pluripotency and reprogramming.

Cell Cycle 2016 23;15(3):324-30. Epub 2015 Dec 23.

a Lymphocyte Development Group, MRC Clinical Sciences Center, Faculty of Medicine, Imperial College London , London , UK.

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.
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http://dx.doi.org/10.1080/15384101.2015.1128593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943700PMC
December 2016

Jarid2 Coordinates Nanog Expression and PCP/Wnt Signaling Required for Efficient ESC Differentiation and Early Embryo Development.

Cell Rep 2015 Jul 16;12(4):573-86. Epub 2015 Jul 16.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2015.06.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534826PMC
July 2015

microRNAs regulate cell-to-cell variability of endogenous target gene expression in developing mouse thymocytes.

PLoS Genet 2015 25;11(2):e1005020. Epub 2015 Feb 25.

MRC Clinical Sciences Centre, Imperial College London, London, United Kingdom.

The development and homeostasis of multicellular organisms relies on gene regulation within individual constituent cells. Gene regulatory circuits that increase the robustness of gene expression frequently incorporate microRNAs as post-transcriptional regulators. Computational approaches, synthetic gene circuits and observations in model organisms predict that the co-regulation of microRNAs and their target mRNAs can reduce cell-to-cell variability in the expression of target genes. However, whether microRNAs directly regulate variability of endogenous gene expression remains to be tested in mammalian cells. Here we use quantitative flow cytometry to show that microRNAs impact on cell-to-cell variability of protein expression in developing mouse thymocytes. We find two distinct mechanisms that 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 co-regulated with the target mRNA Cd69 to form an activation-induced incoherent feed-forward loop. Another microRNA, miR-181a, acts at least in part upstream of the target mRNA Cd69 to modulate cellular responses to activation. The ability of microRNAs to render gene expression more uniform across mammalian cell populations may be important for normal development and for disease.
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http://dx.doi.org/10.1371/journal.pgen.1005020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340958PMC
July 2015

Spatial enhancer clustering and regulation of enhancer-proximal genes by cohesin.

Genome Res 2015 Apr 12;25(4):504-13. Epub 2015 Feb 12.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, London W12 0NN, United Kingdom;

In addition to mediating sister chromatid cohesion during the cell cycle, the cohesin complex associates with CTCF and with active gene regulatory elements to form long-range interactions between its binding sites. Genome-wide chromosome conformation capture had shown that cohesin's main role in interphase genome organization is in mediating interactions within architectural chromosome compartments, rather than specifying compartments per se. However, it remains unclear how cohesin-mediated interactions contribute to the regulation of gene expression. We have found that the binding of CTCF and cohesin is highly enriched at enhancers and in particular at enhancer arrays or "super-enhancers" in mouse thymocytes. Using local and global chromosome conformation capture, we demonstrate that enhancer elements associate not just in linear sequence, but also in 3D, and that spatial enhancer clustering is facilitated by cohesin. The conditional deletion of cohesin from noncycling thymocytes preserved enhancer position, H3K27ac, H4K4me1, and enhancer transcription, but weakened interactions between enhancers. Interestingly, ∼ 50% of deregulated genes reside in the vicinity of enhancer elements, suggesting that cohesin regulates gene expression through spatial clustering of enhancer elements. We propose a model for cohesin-dependent gene regulation in which spatial clustering of enhancer elements acts as a unified mechanism for both enhancer-promoter "connections" and "insulation."
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http://dx.doi.org/10.1101/gr.184986.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381522PMC
April 2015

Initiation and maintenance of pluripotency gene expression in the absence of cohesin.

Genes Dev 2015 Jan;29(1):23-38

Lymphocyte Development Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London W12 ONN, United Kingdom;

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.
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http://dx.doi.org/10.1101/gad.251835.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281562PMC
January 2015

microRNA-mediated regulation of mTOR complex components facilitates discrimination between activation and anergy in CD4 T cells.

J Exp Med 2014 Oct 13;211(11):2281-95. Epub 2014 Oct 13.

Lymphocyte Development Group and Epigenetics Section, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London W12 0NN, England, UK Lymphocyte Development Group and Epigenetics Section, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London W12 0NN, England, UK

T cell receptor (TCR) signals can elicit full activation with acquisition of effector functions or a state of anergy. Here, we ask whether microRNAs affect the interpretation of TCR signaling. We find that Dicer-deficient CD4 T cells fail to correctly discriminate between activating and anergy-inducing stimuli and produce IL-2 in the absence of co-stimulation. Excess IL-2 production by Dicer-deficient CD4 T cells was sufficient to override anergy induction in WT T cells and to restore inducible Foxp3 expression in Il2-deficient CD4 T cells. Phosphorylation of Akt on S473 and of S6 ribosomal protein was increased and sustained in Dicer-deficient CD4 T cells, indicating elevated mTOR activity. The mTOR components Mtor and Rictor were posttranscriptionally deregulated, and the microRNAs Let-7 and miR-16 targeted the Mtor and Rictor mRNAs. Remarkably, returning Mtor and Rictor to normal levels by deleting one allele of Mtor and one allele of Rictor was sufficient to reduce Akt S473 phosphorylation and to reduce co-stimulation-independent IL-2 production in Dicer-deficient CD4 T cells. These results show that microRNAs regulate the expression of mTOR components in T cells, and that this regulation is critical for the modulation of mTOR activity. Hence, microRNAs contribute to the discrimination between T cell activation and anergy.
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http://dx.doi.org/10.1084/jem.20132059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203951PMC
October 2014

Getting rid of DNA methylation.

Trends Cell Biol 2014 Feb 9;24(2):136-43. Epub 2013 Oct 9.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

Methylation of cytosine within DNA is associated with transcriptional repression and genome surveillance. In plants and animals, conserved pathways exist to establish and maintain this epigenetic mark. Mechanisms underlining its removal are, however, diverse and controversial and can depend on DNA synthesis (passive) or be independent of it (active). Ten-eleven translocation (Tet)-mediated conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has recently been evoked as a possible mechanism in the initiation of active and passive DNA demethylation. This review discuses the recent progress in this exciting area.
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http://dx.doi.org/10.1016/j.tcb.2013.09.001DOI Listing
February 2014

Atypical heterochromatin organization and replication are rapidly acquired by somatic cells following fusion-mediated reprogramming by mouse ESCs.

Cell Cycle 2013 Oct 3;12(20):3253-61. Epub 2013 Sep 3.

Lymphocyte Development Group; MRC Clinical Sciences Centre; Imperial College London; London, UK.

We recently reported that mouse embryonic stem cells (ESCs) in S/G 2 are more efficient at reprogramming somatic cells than ESCs at other stages of the cell cycle. We also provided evidence that DNA replication is induced in the nuclei of somatic partners upon fusion with ESC partners, and showed that this was critical for their conversion toward a pluripotent state. (1) Here we have used counterflow centrifugal elutriation to enrich for ESCs at different cell cycle phases, so as to examine in detail the properties of S/G 2 phase cells. This revealed that the replication and organization of DAPI-intense heterochromatin in ESCs is unusual in two respects. First, replication of heterochromatin occurred earlier during S phase and was associated with precocious H3S10 phosphorylation. Second, heterochromatin protein 1 α (HP1α), which invariably marks DAPI-intense and H3K9me3-enriched pericentromeric domains in mouse somatic cells, (2) was not necessarily associated with these H3K9me3-enriched domains in undifferentiated ESCs. These data, which complement recent replication timing (3) and electron spectroscopic imaging (ESI) analyses, (4) suggest that heterochromatin is atypical in ESCs. Interestingly, as these unusual features were rapidly acquired by somatic nuclei upon ESC fusion-mediated reprogramming, our results suggest that fundamental changes in cell cycle structure and heterochromatin dynamics may be important for conferring pluripotency.
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http://dx.doi.org/10.4161/cc.26223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885636PMC
October 2013

DNA demethylation in pluripotency and reprogramming: the role of tet proteins and cell division.

Cell Stem Cell 2013 Sep;13(3):265-9

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.

Cytosine methylation is found in the genomes of many plants and animals and has been associated with transcriptional silencing in mammals. At critical stages in embryo development, when cellular potential is reset, DNA methylation is lost in a series of "sequential waves." The mechanism underlying this is controversial and complex. Several new reports now suggest that TET enzymes and cell division are important for these in vivo transitions as well as for experimentally induced reprogramming.
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http://dx.doi.org/10.1016/j.stem.2013.08.005DOI Listing
September 2013

Cohesin-based chromatin interactions enable regulated gene expression within preexisting architectural compartments.

Genome Res 2013 Dec 3;23(12):2066-77. Epub 2013 Sep 3.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, London W12 0NN, United Kingdom;

Chromosome conformation capture approaches have shown that interphase chromatin is partitioned into spatially segregated Mb-sized compartments and sub-Mb-sized topological domains. This compartmentalization is thought to facilitate the matching of genes and regulatory elements, but its precise function and mechanistic basis remain unknown. Cohesin controls chromosome topology to enable DNA repair and chromosome segregation in cycling cells. In addition, cohesin associates with active enhancers and promoters and with CTCF to form long-range interactions important for gene regulation. Although these findings suggest an important role for cohesin in genome organization, this role has not been assessed on a global scale. Unexpectedly, we find that architectural compartments are maintained in noncycling mouse thymocytes after genetic depletion of cohesin in vivo. Cohesin was, however, required for specific long-range interactions within compartments where cohesin-regulated genes reside. Cohesin depletion diminished interactions between cohesin-bound sites, whereas alternative interactions between chromatin features associated with transcriptional activation and repression became more prominent, with corresponding changes in gene expression. Our findings indicate that cohesin-mediated long-range interactions facilitate discrete gene expression states within preexisting chromosomal compartments.
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http://dx.doi.org/10.1101/gr.161620.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847776PMC
December 2013

Reprogramming and the pluripotent stem cell cycle.

Curr Top Dev Biol 2013 ;104:223-41

MRC Genome Damage and Stability Centre, University of Sussex, Falmer, United Kingdom.

Embryonic stem cells (ESCs) can self renew and retain the potential to differentiate into each of the cell types within the body. During experimental reprogramming, many of the features of ESCs can be acquired by differentiated target cells. One of these is the unusual cell division cycle that characterizes ESCs in which the Gap (G) phases are short and DNA Synthesis (S) phase predominates. Growing evidence has suggested that this atypical cell-cycle structure may be important for maintaining pluripotency and for enhancing pluripotent conversion. Here, we review current knowledge of cell-cycle regulation in ESCs and outline how this unique cell-cycle structure might contribute to successful reprogramming.
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http://dx.doi.org/10.1016/B978-0-12-416027-9.00007-3DOI Listing
March 2014

Epigenetic programming and reprogramming during development.

Nat Struct Mol Biol 2013 Mar 5;20(3):282-9. Epub 2013 Mar 5.

Lymphocyte Development Group, Medical Research Council Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, London, UK.

Cell identity is determined by specific gene expression patterns that are conveyed by interactions between transcription factors and DNA in the context of chromatin. In development, epigenetic modifiers are thought to stabilize gene expression and ensure that patterns of DNA methylation and histone modification are reinstated in cells as they divide. Global erasure of epigenetic marks occurs naturally at two stages in the mammalian life cycle, but it can also be artificially engineered using a variety of reprogramming strategies. Here we review some of the recent advances in understanding how epigenetic remodeling contributes to conversion of cell fate in vivo and in vitro. We summarize current models of epigenetic erasure and discuss the various enzymes and mechanisms that may operate in cellular reprogramming.
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http://dx.doi.org/10.1038/nsmb.2489DOI Listing
March 2013

Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion.

Mol Cell 2013 Mar 28;49(6):1023-33. Epub 2013 Feb 28.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.
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http://dx.doi.org/10.1016/j.molcel.2013.01.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613797PMC
March 2013

DNA synthesis is required for reprogramming mediated by stem cell fusion.

Cell 2013 Feb;152(4):873-83

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.

Embryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.
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http://dx.doi.org/10.1016/j.cell.2013.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605571PMC
February 2013