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1.
Nat Immunol ; 20(10): 1372-1380, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451789

RESUMO

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.

2.
PLoS Biol ; 17(4): e2006506, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30978178

RESUMO

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.


Assuntos
Linfócitos B/citologia , Genes myc , Células Precursoras de Linfócitos B/citologia , Animais , Linfócitos B/metabolismo , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Linhagem da Célula , Bases de Dados Genéticas , Regulação para Baixo , Regulação da Expressão Gênica , Genes Essenciais , Humanos , Fator de Transcrição Ikaros/metabolismo , Ativação Linfocitária , Camundongos , Células Precursoras de Linfócitos B/metabolismo , Fatores de Transcrição/metabolismo
3.
Mol Metab ; 18: 97-106, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30279096

RESUMO

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.


Assuntos
Transtornos do Crescimento/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Obesidade/genética , Adiposidade/genética , Animais , Peso Corporal/genética , Metabolismo Energético , Deleção de Genes , Impressão Genômica , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Obesidade/metabolismo
4.
Nat Immunol ; 19(9): 932-941, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30127433

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Autorrenovação Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide Aguda/genética , Macrófagos/fisiologia , Proteínas Nucleares/genética , Fosfoproteínas/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Inflamação/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Mutação/genética
5.
Nucleic Acids Res ; 45(21): e174, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28981838

RESUMO

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.


Assuntos
Alelos , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Técnicas de Inativação de Genes/métodos , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Expressão Gênica , Vetores Genéticos , Histona-Lisina N-Metiltransferase/genética , Camundongos , Complexo Repressor Polycomb 2/genética
6.
Philos Trans R Soc Lond B Biol Sci ; 372(1733)2017 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-28947657

RESUMO

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 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 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 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'.


Assuntos
Reprogramação Celular , Cromossomos Humanos X/genética , Predisposição Genética para Doença/genética , Inativação do Cromossomo X/genética , Animais , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Feminino , Fibroblastos , Humanos , Camundongos , RNA Longo não Codificante
7.
Cell Syst ; 4(4): 373-374, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28448796

RESUMO

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.


Assuntos
Cromatina , Proteínas de Drosophila , Regulação da Expressão Gênica , Histonas/genética , Proteínas do Grupo Polycomb/genética
8.
Elife ; 62017 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-28318487

RESUMO

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.


Assuntos
Regulação para Baixo , Inativação Gênica , Transcrição Genética , Animais , Células Cultivadas , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Histona Desacetilases/metabolismo , Fator de Transcrição Ikaros/metabolismo , Camundongos , Células Precursoras de Linfócitos B/fisiologia , RNA Polimerase II/metabolismo , Fatores de Tempo
9.
Cell Rep ; 18(5): 1090-1099, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28147266

RESUMO

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.


Assuntos
Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Impressão Genômica/fisiologia , Alelos , Animais , Cromatina/fisiologia , Metilação de DNA/fisiologia , Epigênese Genética/fisiologia , Camundongos
10.
Genome Biol ; 18(1): 2, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-28118853

RESUMO

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.


Assuntos
Alelos , Fusão Celular , Reprogramação Celular , Genes Ligados ao Cromossomo X , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ativação Transcricional , Cromatina/genética , Cromatina/metabolismo , Análise por Conglomerados , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos , Expressão Gênica , Perfilação da Expressão Gênica , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo Único , Inativação do Cromossomo X/genética
11.
Methods Mol Biol ; 1480: 289-99, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27659994

RESUMO

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.


Assuntos
Fusão Celular/métodos , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/genética , Células-Tronco Embrionárias/citologia , Animais , Núcleo Celular/genética , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia
12.
Nat Commun ; 7: 12354, 2016 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-27507283

RESUMO

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.


Assuntos
Reprogramação Celular/genética , Cromatina/genética , Cromossomos Humanos X/genética , Histonas/genética , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Animais , Fusão Celular/métodos , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Epigênese Genética , Feminino , Fibroblastos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/metabolismo
13.
Stem Cell Reports ; 6(5): 635-642, 2016 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-27150236

RESUMO

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.


Assuntos
RNA Helicases DEAD-box/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 2/genética , Ribonuclease III/genética , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/genética , Camundongos , Regiões Promotoras Genéticas , Ativação Transcricional/genética
14.
Cell Cycle ; 15(3): 324-30, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26701823

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reprogramação Celular , Proteínas Cromossômicas não Histona/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/genética , Dano ao DNA , Proteínas de Ligação a DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Interferência de RNA
15.
Cell Rep ; 12(4): 573-86, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26190104

RESUMO

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 ß-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 ß-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.


Assuntos
Blastocisto/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Proteína Homeobox Nanog , Complexo Repressor Polycomb 2/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
16.
Genome Res ; 25(4): 504-13, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25677180

RESUMO

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."


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Família Multigênica/genética , Proteínas Repressoras/metabolismo , Timócitos/citologia , Animais , Sítios de Ligação/genética , Fator de Ligação a CCCTC , Células Cultivadas , Histonas/genética , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética
17.
PLoS Genet ; 11(2): e1005020, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25714103

RESUMO

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.


Assuntos
Sobrevivência Celular/genética , MicroRNAs/genética , Biossíntese de Proteínas/genética , Timócitos/metabolismo , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Camundongos , RNA Mensageiro/biossíntese
18.
Genes Dev ; 29(1): 23-38, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25561493

RESUMO

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.


Assuntos
Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Reprogramação Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Xenopus
19.
J Exp Med ; 211(11): 2281-95, 2014 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-25311506

RESUMO

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.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Anergia Clonal/genética , Anergia Clonal/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , MicroRNAs/genética , Serina-Treonina Quinases TOR/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica , Humanos , Interleucina-2/biossíntese , Camundongos , Camundongos Transgênicos , MicroRNAs/química , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/genética
20.
Trends Cell Biol ; 24(2): 136-43, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24119665

RESUMO

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.


Assuntos
Metilação de DNA , DNA/metabolismo , Animais , DNA/biossíntese , DNA/genética , Humanos
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