Imperial College London

Professor Dame Amanda Fisher

Faculty of MedicineInstitute of Clinical Sciences

Visiting Professor
 
 
 
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Contact

 

amanda.fisher

 
 
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Assistant

 

Ms Alessandra Lisini +44 (0)20 3313 8236

 
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Location

 

CRB (Clinical Research Building)Hammersmith Campus

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Summary

 

Publications

Publication Type
Year
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138 results found

Al-Jibury E, King JWD, Guo Y, Lenhard B, Fisher AG, Merkenschlager M, Rueckert Det al., 2023, A deep learning method for replicate-based analysis of chromosome conformation contacts using Siamese neural networks, Nature Communications, Vol: 14, ISSN: 2041-1723

The organisation of the genome in nuclear space is an important frontier of biology. Chromosome conformation capture methods such as Hi-C and Micro-C produce genome-wide chromatin contact maps that provide rich data containing quantitative and qualitative information about genome architecture. Most conventional approaches to genome-wide chromosome conformation capture data are limited to the analysis of pre-defined features, and may therefore miss important biological information. One constraint is that biologically important features can be masked by high levels of technical noise in the data. Here we introduce a replicate-based method for deep learning from chromatin conformation contact maps. Using a Siamese network configuration our approach learns to distinguish technical noise from biological variation and outperforms image similarity metrics across a range of biological systems. The features extracted from Hi-C maps after perturbation of cohesin and CTCF reflect the distinct biological functions of cohesin and CTCF in the formation of domains and boundaries, respectively. The learnt distance metrics are biologically meaningful, as they mirror the density of cohesin and CTCF binding. These properties make our method a powerful tool for the exploration of chromosome conformation capture data, such as Hi-C capture Hi-C, and Micro-C.

Journal article

Dimond A, 2023, Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c, Scientific Reports, Vol: 13, Pages: 1-14, ISSN: 2045-2322

Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing.

Journal article

Fisher AGG, Merkenschlager M, 2023, Avrion Mitchison (1928-2022), NATURE IMMUNOLOGY, Vol: 24, Pages: 559-559, ISSN: 1529-2908

Journal article

Djeghloul D, Dimond A, Cheriyamkunnel S, Kramer H, Patel B, Brown K, Montoya A, Whilding C, Wang Y-F, Futschik ME, Veland N, Montavon T, Jenuwein T, Merkenschlager M, Fisher AGet al., 2023, Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis, NATURE STRUCTURAL & MOLECULAR BIOLOGY, Vol: 30, Pages: 489-+, ISSN: 1545-9993

Journal article

Gleneadie HJJ, Fernandez-Ruiz B, Sardini A, Van de Pette M, Dimond A, Prinjha RKK, McGinty J, French PMW, Bagci H, Merkenschlager M, Fisher AGGet al., 2023, Endogenous bioluminescent reporters reveal a sustained increase in utrophin gene expression upon EZH2 and ERK1/2 inhibition, COMMUNICATIONS BIOLOGY, Vol: 6

Journal article

Guo Y, Al-Jibury E, Garcia-Millan R, Ntagiantas K, King JWD, Nash AJ, Galjart N, Lenhard B, Rueckert D, Fisher AG, Pruessner G, Merkenschlager Met al., 2022, Chromatin jets define the properties of cohesin-driven in vivo loop extrusion, Molecular Cell, Vol: 82, Pages: 3769-3780.e5, ISSN: 1097-2765

Complex genomes show intricate organization in three-dimensional (3D) nuclear space. Current models posit that cohesin extrudes loops to form self-interacting domains delimited by the DNA binding protein CTCF. Here, we describe and quantitatively characterize cohesin-propelled, jet-like chromatin contacts as landmarks of loop extrusion in quiescent mammalian lymphocytes. Experimental observations and polymer simulations indicate that narrow origins of loop extrusion favor jet formation. Unless constrained by CTCF, jets propagate symmetrically for 1-2 Mb, providing an estimate for the range of in vivo loop extrusion. Asymmetric CTCF binding deflects the angle of jet propagation as experimental evidence that cohesin-mediated loop extrusion can switch from bi- to unidirectional and is controlled independently in both directions. These data offer new insights into the physiological behavior of in vivo cohesin-mediated loop extrusion and further our understanding of the principles that underlie genome organization.

Journal article

Robles-Rebollo I, Cuartero S, Canellas-Socias A, Wells S, Karimi MM, Mereu E, Chivu AG, Heyn H, Whilding C, Dormann D, Marguerat S, Rioja I, Prinjha RK, Stumpf MPH, Fisher AG, Merkenschlager Met al., 2022, Cohesin couples transcriptional bursting probabilities of inducible enhancers and promoters, Nature Communications, Vol: 13, ISSN: 2041-1723

Innate immune responses rely on inducible gene expression programmes which, in contrast to steady-state transcription, are highly dependent on cohesin. Here we address transcriptional parameters underlying this cohesin-dependence by single-molecule RNA-FISH and single-cell RNA-sequencing. We show that inducible innate immune genes are regulated predominantly by an increase in the probability of active transcription, and that probabilities of enhancer and promoter transcription are coordinated. Cohesin has no major impact on the fraction of transcribed inducible enhancers, or the number of mature mRNAs produced per transcribing cell. Cohesin is, however, required for coupling the probabilities of enhancer and promoter transcription. Enhancer-promoter coupling may not be explained by spatial proximity alone, and at the model locus Il12b can be disrupted by selective inhibition of the cohesinopathy-associated BET bromodomain BD2. Our data identify discrete steps in enhancer-mediated inducible gene expression that differ in cohesin-dependence, and suggest that cohesin and BD2 may act on shared pathways.

Journal article

Calderon L, Weiss FD, Beagan JA, Oliveira MS, Georgieva R, Wang Y-F, Carroll TS, Dharmalingam G, Gong W, Tossell K, de Paola V, Whilding C, Ungless MA, Fisher AG, Phillips-Cremins JE, Merkenschlager Met al., 2022, Cohesin-dependence of neuronal gene expression relates to chromatin loop length, eLife, Vol: 11, ISSN: 2050-084X

Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here, we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in mouse primary cortical neurons. Immediate early genes (IEGs) remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the IEGs Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterized by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend on cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts.Editor's

Journal article

Brown KE, Fisher AG, 2021, Reprogramming lineage identity through cell-cell fusion, CURRENT OPINION IN GENETICS & DEVELOPMENT, Vol: 70, Pages: 15-23, ISSN: 0959-437X

Journal article

Weiss FD, Calderon L, Wang Y-F, Georgieva R, Guo Y, Cvetesic N, Kaur M, Dharmalingam G, Krantz ID, Lenhard B, Fisher A, Merkenschlager Met al., 2021, Neuronal genes deregulated in Cornelia de Lange Syndrome respond to removal and reexpression of cohesin, Nature Communications, Vol: 12, ISSN: 2041-1723

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterize the transcriptional profile of cortical neurons from CdLS patients and find deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupts 3D genome organization and transcriptional control in post-mitotic cortical mouse neurons, demonstrating that cohesin is continuously required for neuronal gene expression. The genes affected by acute depletion of cohesin belong to similar gene ontology classes and show significant numerical overlap with genes deregulated in CdLS. Interestingly, reconstitution of cohesin function largely rescues altered gene expression, including the expression of genes deregulated in CdLS.

Journal article

Karimi MM, Guo Y, Cui X, Pallikonda HA, Horkova V, Dore MH, Wang Y-F, Ruiz Gil S, Rodriguez-Eteban G, Robles Rebollo I, Bruno L, Georgieva R, Patel B, Elliott J, Dauphars D, Krangel MS, Lenhard B, Heyn H, Fisher AG, Stepanek O, Merkenschlager Met al., 2021, The order and logic of CD4 CD8 lineage choice and differentiation in mouse thymus, Nature Communications, Vol: 12, ISSN: 2041-1723

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, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ 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.

Journal article

Dimond A, Van de Pette M, Fisher AG, 2020, Illuminating epigenetics and inheritance in the immune system with bioluminescence, Trends in Immunology, Vol: 41, Pages: 994-1005, ISSN: 0167-5699

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.

Journal article

Djeghloul D, Patel B, Kramer H, Dimond A, Whilding C, Brown KE, Kohler A-C, Feytout A, Veland N, Elliott J, Bharat T, Tarafder A, Loewe J, Ng B, Guy J, Huseyin M, Klose RJ, Merkenschlager M, Fisher AGet al., 2020, Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction, Nature Communications, Vol: 11, Pages: 1-15, ISSN: 2041-1723

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.

Journal article

Bruno L, Ramlall V, Studer RA, Sauer S, Bradley D, Dharmalingam G, Carroll T, Ghoneim M, Chopin M, Nutt SL, Elderkin S, Rueda DS, Fisher AG, Siggers T, Beltrao P, Merkenschlager Met al., 2019, Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system, Nature Immunology, Vol: 20, Pages: 1372-1380, ISSN: 1529-2908

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 non-equivalence 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 towards sub-maximal 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.

Journal article

Ferreirós-Vidal I, Carroll T, Zhang T, Lagani V, Ramirez RN, Ing-Simmons E, Garcia A, Cooper L, Liang Z, Papoutsoglou G, Dharmalingam G, Guo Y, Tarazona S, Fernandes SJ, Noori P, SIlberberg G, Fisher AG, Tsamardinos I, Mortazavi A, Lenhard B, Conesa A, Tegner J, Merkenschlager M, Gomez-Cabrero Det al., 2019, Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation, PLoS Biology, Vol: 17, 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.

Journal article

Calderon L, Weiss FD, Carroll T, Irvine EE, Dharmalingam G, Tossell K, De Paola V, Whilding C, Ungless MA, Withers DJ, Fisher AG, Merkenschlager Met al., 2019, Cohesin is continuously required to sustain neuronal gene expression, 29th Mammalian Genetics and Development Workshop of the Genetics-Society, Publisher: HINDAWI LTD, ISSN: 0016-6723

Conference paper

Millership S, Tunster SJ, Van de Pette M, Choudhury A, Irvine E, Christian M, Fisher AG, John RM, Scott J, Withers DJet 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

Journal article

Merkenschlager M, Cuartero S, Weiss F, Dharmalingam G, Guo Y, Ing-Simmons E, Masella S, Robles-Rebollo I, Xiao X, Barozzi I, Djeghloul D, Amano M, Niskanen H, Petretto E, Dowell R, Tachibana K, Kaikkonen M, Nasmyth K, Lenhard B, Natoli G, Fisher Aet 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.

Journal article

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.

Journal article

Fisher CL, Marks H, Cho LT-Y, Andrews R, Wormald S, Carroll T, Iyer V, Tate P, Rosen B, Stunnenberg HG, Fisher AG, Skarnes WCet 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.

Journal article

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.

Journal article

Liang Z, Brown KE, Carroll T, Taylor B, Vidal IF, Hendrich B, Rueda D, Fisher AG, Merkenschlager Met 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.

Journal article

Van de Pette M, Abbas A, Feytout A, McNamara G, Bruno L, To WK, Dimond A, Sardini A, Webster Z, McGinty J, Paul EJ, Ungless MA, French PMW, Withers DJ, Uren A, Ferguson-Smith AC, Merkenschlager M, John RM, Fisher AGet 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.

Journal article

Cantone I, Dharmalingam G, Chan YW, Kohler AC, Lenhard B, Merkenschlager M, Fisher AGet 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.

Journal article

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.

Journal article

Graham B, Marcais A, Dharmalingam G, Carroll T, Kanellopoulou C, Graumann J, Nesterova TB, Bermange A, Brazauskas P, Xella B, Kriaucionis S, Higgs DR, Brockdorff N, Mann M, Fisher AG, Merkenschlager Met 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.

Journal article

Chiu FWY, Bagci H, Fisher AG, deMello AJ, Elvira KSet al., 2016, A microfluidic toolbox for cell fusion, Journal of Chemical Technology and Biotechnology, Vol: 91, Pages: 16-24, ISSN: 1097-4660

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.

Journal article

Malinowski AR, Fisher AG, 2016, Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion, POLYCOMB GROUP PROTEINS: METHODS AND PROTOCOLS, Editors: Lanzuolo, Bodega, Publisher: HUMANA PRESS INC, Pages: 289-299, ISBN: 978-1-4939-6378-2

Book chapter

Gupta P, Lavagnolli T, Mira-Bontenbal H, Fisher AG, Merkenschlager Met al., 2015, Cohesin's role in pluripotency and reprogramming., Cell Cycle, Vol: 15, Pages: 324-330, ISSN: 1551-4005

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.

Journal article

Landeira D, Bagci H, Malinowski AR, Brown KE, Soza-Ried J, Feytout A, Webster Z, Ndjetehe E, Cantone I, Asenjo HG, Brockdorff N, Carroll T, Merkenschlager M, Fisher AGet 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.

Journal article

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