Imperial College London

ProfessorMatthiasMerkenschlager

Faculty of MedicineInstitute of Clinical Sciences

Professor of Cell Biology
 
 
 
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Contact

 

+44 (0)20 3313 8239matthias.merkenschlager

 
 
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Location

 

4005CRB (Clinical Research Building)Hammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

150 results found

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

Calderon L, Weiss FD, Beagan JA, Oliveira MS, Wang Y-F, Carroll T, Dharmalingam G, Gong W, Tossell K, Vincenzo de Paola, Whilding C, Ungless MA, Fisher AG, Phillips-Cremins JE, Merkenschlager Met al., 2021, Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

<jats:title>Abstract</jats:title><jats:p>Cohesin and CTCF are major drivers of 3D genome organization. Even though human mutations underscore the importance of cohesin and CTCF for neurodevelopment, their role in neurons is only just beginning to be addressed. Here we conditionally ablate <jats:italic>Rad21</jats:italic> in cortical neurons, revealing a prominent role for cohesin in the expression of genes that facilitate neuronal maturation, homeostasis, and activation. In agreement with recent reports, activity-dependent genes were downregulated at baseline. However, in contrast to current models that attribute impaired activity-dependent gene expression to a role for cohesin and CTCF in anchoring enhancer-promoter contacts, we show that nearly all activity-dependent genes remain inducible in the absence of cohesin. While CTCF-based chromatin loops were substantially weakened, long-range contacts still formed robustly between activity-dependent enhancers and their target immediate early gene promoters. We suggest a model where neuronal cohesin facilitates the precise level of activity-dependent gene expression, rather than inducibility <jats:italic>per se</jats:italic>, and where inducibility of activity-dependent gene expression is linked to cohesin-independent enhancer-promoter contacts. These data expand our understanding of the importance of cohesin-independent enhancer-promoter contacts in regulating gene expression.</jats:p>

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

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

Bystricky K, Merkenschlager M, 2020, Editorial overview: Genome architecture and expression, CURRENT OPINION IN GENETICS & DEVELOPMENT, Vol: 61, Pages: III-VI, ISSN: 0959-437X

Journal article

Rojec M, Hocher A, Stevens KM, Merkenschlager M, Warnecke Tet al., 2019, Chromatinization of Escherichia coli with archaeal histones, eLife, Vol: 8, Pages: 1-23, ISSN: 2050-084X

Nucleosomes restrict DNA accessibility throughout eukaryotic genomes, withrepercussions for replication, transcription, and other DNA-templated processes. How this globallyrestrictive organization emerged during evolution remains poorly understood. Here, to betterunderstand the challenges associated with establishing globally restrictive chromatin, we expresshistones in a naive system that has not evolved to deal with nucleosomal structures: Escherichiacoli. We find that histone proteins from the archaeon Methanothermus fervidus assemble on the E.coli chromosome in vivo and protect DNA from micrococcal nuclease digestion, allowing us to mapbinding footprints genome-wide. We show that higher nucleosome occupancy at promoters isassociated with lower transcript levels, consistent with local repressive effects. Surprisingly,however, this sudden enforced chromatinization has only mild repercussions for growth unless cellsexperience topological stress. Our results suggest that histones can become established asubiquitous chromatin proteins without interfering critically with key DNA-templated processes.

Journal article

Jansen C, Ramirez RN, El-Ali NC, Gomez-Cabrero D, Tegner J, Merkenschlager M, Conesa A, Mortazavi Aet al., 2019, Building gene regulatory networks from scATAC-seq and scRNA-seq using Linked Self Organizing Maps, PLOS COMPUTATIONAL BIOLOGY, Vol: 15, ISSN: 1553-734X

Journal article

Gomez-Cabrero D, Tarazona S, Ferreiros-Vidal I, Ramirez RN, Company C, Schmidt A, Reijmers T, von Saint Paul V, Marabita F, Rodriguez-Ubreva J, Garcia-Gomez A, Carroll T, Cooper L, Liang Z, Dharmalingam G, van der Kloet F, Harms AC, Balzano-Nogueira L, Lagani V, Tsamardinos I, Lappe M, Maier D, Westerhuis JA, Hankemeier T, Imhof A, Ballestar E, Mortazavi A, Merkenschlager M, Egner JT, Conesa Aet al., 2019, STATegra, a comprehensive multi-omics dataset of B-cell differentiation in mouse, Scientific Data, Vol: 6, Pages: 1-15, ISSN: 2052-4463

Multi-omics approaches use a diversity of high-throughput technologies to profile the different molecular layers of living cells. Ideally, the integration of this information should result in comprehensive systems models of cellular physiology and regulation. However, most multi-omics projects still include a limited number of molecular assays and there have been very few multi-omic studies that evaluate dynamic processes such as cellular growth, development and adaptation. Hence, we lack formal analysis methods and comprehensive multi-omics datasets that can be leveraged to develop true multi-layered models for dynamic cellular systems. Here we present the STATegra multi-omics dataset that combines measurements from up to 10 different omics technologies applied to the same biological system, namely the well-studied mouse pre-B-cell differentiation. STATegra includes high-throughput measurements of chromatin structure, gene expression, proteomics and metabolomics, and it is complemented with single-cell data. To our knowledge, the STATegra collection is the most diverse multi-omics dataset describing a dynamic biological system.

Journal article

Palmisano I, Danzi MC, Hutson TH, Zhou L, McLachlan E, Serger E, Shkura K, Srivastava PK, Hervera A, Neill NO, Liu T, Dhrif H, Wang Z, Kubat M, Wuchty S, Merkenschlager M, Levi L, Elliott E, Bixby JL, Lemmon VP, Di Giovanni Set al., 2019, Epigenomic signatures underpin the axonal regenerative ability of dorsal root ganglia sensory neurons, Nature Neuroscience, Vol: 22, Pages: 1913-1924, ISSN: 1097-6256

Axonal injury results in regenerative success or failure, depending on whether the axon lies in the peripheral or the CNS, respectively. The present study addresses whether epigenetic signatures in dorsal root ganglia discriminate between regenerative and non-regenerative axonal injury. Chromatin immunoprecipitation for the histone 3 (H3) post-translational modifications H3K9ac, H3K27ac and H3K27me3; an assay for transposase-accessible chromatin; and RNA sequencing were performed in dorsal root ganglia after sciatic nerve or dorsal column axotomy. Distinct histone acetylation and chromatin accessibility signatures correlated with gene expression after peripheral, but not central, axonal injury. DNA-footprinting analyses revealed new transcriptional regulators associated with regenerative ability. Machine-learning algorithms inferred the direction of most of the gene expression changes. Neuronal conditional deletion of the chromatin remodeler CCCTC-binding factor impaired nerve regeneration, implicating chromatin organization in the regenerative competence. Altogether, the present study offers the first epigenomic map providing insight into the transcriptional response to injury and the differential regenerative ability of sensory neurons.

Journal article

Cuartero S, Innes AJ, Merkenschlager M, 2019, Towards a better understanding of cohesin mutations in AML, Frontiers in Oncology, Vol: 9, ISSN: 2234-943X

Classical driver mutations in acute myeloid leukemia (AML) typically affect regulatorsof cell proliferation, differentiation, and survival. The selective advantage of increasedproliferation, improved survival, and reduced differentiation on leukemia progression isimmediately obvious. Recent large-scale sequencing efforts have uncovered numerousnovel AML-associated mutations. Interestingly, a substantial fraction of the mostfrequently mutated genes encode general regulators of transcription and chromatinstate. Understanding the selective advantage conferred by these mutations remains amajor challenge. A striking example are mutations in genes of the cohesin complex,a major regulator of three-dimensional genome organization. Several landmark studieshave shown that cohesin mutations perturb the balance between self-renewal anddifferentiation of hematopoietic stem and progenitor cells (HSPC). Emerging data nowbegin to uncover the molecular mechanisms that underpin this phenotype. Amongthese mechanisms is a role for cohesin in the control of inflammatory responses inHSPCs and myeloid cells. Inflammatory signals limit HSPC self-renewal and driveHSPC differentiation. Consistent with this, cohesin mutations promote resistance toinflammatory signals, and may provide a selective advantage for AML progression.In this review, we discuss recent progress in understanding cohesin mutations inAML, and speculate whether vulnerabilities associated with these mutations could beexploited therapeutically

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: CAMBRIDGE UNIV PRESS, ISSN: 0016-6723

Conference paper

Jansen C, Ramirez RN, El-Ali NC, Gomez-Cabrero D, Tegner J, Merkenschlager M, Conesa A, Mortazavi Aet al., 2018, Building gene regulatory networks from scATAC-seq and scRNA-seq using Linked Self-Organizing Maps, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>Rapid advances in single-cell assays have outpaced methods for analysis of those data types. Different single-cell assays show extensive variation in sensitivity and signal to noise levels. In particular, scATAC-seq generates extremely sparse and noisy datasets. Existing methods developed to analyze this data require cells amenable to pseudo-time analysis or require datasets with drastically different cell-types. We describe a novel approach using self-organizing maps (SOM) to link scATAC-seq and scRNA-seq data that overcomes these challenges and can generate draft regulatory networks. Our SOMatic package generates chromatin and gene expression SOMs separately and combines them using a linking function. We applied SOMatic on a mouse pre-B cell differentiation time-course using controlled Ikaros over-expression to recover gene ontology enrichments, identify motifs in genomic regions showing similar single-cell profiles, and generate a gene regulatory network that both recovers known interactions and predicts new Ikaros targets during the differentiation process. The ability of linked SOMs to detect emergent properties from multiple types of highly-dimensional genomic data with very different signal properties opens new avenues for integrative analysis of single-cells.</jats:p>

Working paper

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

Cuartero S, Merkenschlager M, 2018, Three-dimensional genome organization in normal and malignant haematopoiesis., Current Opinion in Hematology, Vol: 25, Pages: 323-328, ISSN: 1065-6251

PURPOSE OF REVIEW: The three-dimensional organization of the genome inside the nucleus impacts on key aspects of genome function, including transcription, DNA replication and repair. The chromosome maintenance complex cohesin and the DNA binding protein CTCF cooperate to drive the formation of self-interacting topological domains. This facilitates transcriptional regulation via enhancer-promoter interactions, controls the distribution and release of torsional strain, and affects the frequency with which particular translocations arise, based on the spatial proximity of translocation partners. Here we discuss recent insights into the mechanisms of three-dimensional genome organization, their relationship to haematopoietic differentiation and malignant transformation. RECENT FINDINGS: Cohesin mutations are frequently found in myeloid malignancies. Significantly, cohesin mutations can drive increased self-renewal of haematopoietic stem and progenitor cells, which may facilitate the accumulation of genetic lesions and leukaemic transformation. It is therefore important to elucidate the mechanisms that link cohesin to pathways that regulate the balance between self-renewal and differentiation. Chromosomal translocations are key to lymphoid malignancies, and recent findings link three-dimensional genome organization to the frequency and the genomic position of DNA double strand breaks. SUMMARY: Three-dimensional genome organization can help explain genome function in normal and malignant haematopoiesis.

Journal article

Harmston N, Ing-Simmons E, Tan G, Perry M, Merkenschlager M, Lenhard Bet al., 2017, Topologically associating domains are ancient features that coincide with Metazoan clusters of extreme noncoding conservation., Nature Communications, Vol: 8, ISSN: 2041-1723

Developmental genes in metazoan genomes are surrounded by dense clusters of conserved noncoding elements (CNEs). CNEs exhibit unexplained extreme levels of sequence conservation, with many acting as developmental long-range enhancers. Clusters of CNEs define the span of regulatory inputs for many important developmental regulators and have been described previously as genomic regulatory blocks (GRBs). Their function and distribution around important regulatory genes raises the question of how they relate to 3D conformation of these loci. Here, we show that clusters of CNEs strongly coincide with topological organisation, predicting the boundaries of hundreds of topologically associating domains (TADs) in human and Drosophila. The set of TADs that are associated with high levels of noncoding conservation exhibit distinct properties compared to TADs devoid of extreme noncoding conservation. The close correspondence between extreme noncoding conservation and TADs suggests that these TADs are ancient, revealing a regulatory architecture conserved over hundreds of millions of years.Metazoan genomes contain many clusters of conserved noncoding elements. Here, the authors provide evidence that these clusters coincide with distinct topologically associating domains in humans and Drosophila, revealing a conserved regulatory genomic architecture.

Journal article

Perez MEF, Bloznelyte K, Merkenschlager M, Rueda Det al., 2017, Visualizing CTCF-mediated DNA looping at the single-molecule level, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S141-S141, ISSN: 0175-7571

Conference paper

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

Perez MEF, Bloznelyte K, Merkenschlager M, Rueda Det al., 2017, Visualizing CTCF mediated DNA looping at the single molecule level, 61st Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 169A-170A, ISSN: 0006-3495

Conference paper

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

Gupta P, Lavagnolli T, Mira-Bontenbal H, Merkenschlager Met al., 2016, Analysis of Cohesin Function in Gene Regulation and Chromatin Organization in Interphase., Methods Mol Biol, Vol: 1515, Pages: 197-216

Cohesin is essential for the maintenance of chromosomes through the cell cycle. In addition, cohesin contributes to the regulation of gene expression and the organization of chromatin in interphase cells. To study cohesin's role in gene expression and chromatin organization, it is necessary to avoid secondary effects due to disruption of vital cohesin functions in the cell cycle. Here we describe experimental approaches to achieve this and the methods applied to define cohesin's role in interphase.

Journal article

Merkenschlager M, Nora EP, 2016, CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation, Annual Review of Genomics and Human Genetics, Vol: 17, ISSN: 1545-293X

Genome function, replication, integrity, and propagation rely on the dynamic structural organization of chromosomes during the cell cycle.Genome folding in interphase provides regulatory segmentation for appropriate transcriptional control, facilitates ordered genome replication, and contributes to genome integrity by limiting illegitimate recombination. Here, we review recent high-resolution chromosome conformation capture and functional studies that have informed models of the spatial and regulatory compartmentalization of mammalian genomes, and discuss mechanistic models for how CTCF and cohesin control the functional architecture of mammalian chromosomes. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 17 is August 31, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Journal article

Bond J, Domaschenz R, Roman-Trufero M, Sabbattini P, Ferreiros-Vidal I, Gerrard G, Asnafi V, Macintyre E, Merkenschlager M, Dillon Net al., 2016, Direct interaction of Ikaros and Foxp1 modulates expression of the G protein-coupled receptor G2A in B-lymphocytes and acute lymphoblastic leukemia, Oncotarget, Vol: 7, Pages: 65923-65936, ISSN: 1949-2553

Ikaros and Foxp1 are transcription factors that play key roles in normal lymphopoiesis and lymphoid malignancies. We describe a novel physical and functional interaction between the proteins, which requires the central zinc finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion of this region, which corresponds to the IK6 isoform that is commonly associated with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132 gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and caused cell cycle changes, including G2 arrest. Co-expression of wild-type Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL samples revealed a significant increase in GPR132 expression in IKZF1-deleted BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may influence the regulation of G2A in B-ALL. Our results reveal a novel effect of Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential modulator of the cell cycle in Ikaros-deleted B-ALL.

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

Ing-Simmons E, Merkenschlager M, 2016, Oncometabolite Tinkers with Genome Folding, Boosting Oncogene Expression., Trends in Molecular Medicine, Vol: 22, Pages: 185-187, ISSN: 1471-4914

A recent article makes a compelling case for a new mechanism by which heterozygous mutations in isocitrate dehydrogenases (IDH1/2) - implicated in cancer - undermine gene regulation. 2-Hydroxyglutarate (2HG) produced by mutant IDH alters the binding of the chromosomal organizer protein CTCF, disrupting the spatial and regulatory organization of the genome.

Journal article

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

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