RESUMO
The Nucleosome Remodeling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. Whether these different complexes serve distinct functional activities during differentiation is not fully explored. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD2 and MBD3 variants for their potential to rescue the differentiation block observed for mouse embryonic stem cells (ESCs) lacking MBD3. While MBD3 is indeed crucial for ESC differentiation to neuronal cells, it functions independently of its MBD domain. We further identify that MBD2 isoforms can replace MBD3 during lineage commitment, however with different potential. Full-length MBD2a only partially rescues the differentiation block, while MBD2b, an isoform lacking an N-terminal GR-rich repeat, fully rescues the Mbd3 KO phenotype. In case of MBD2a, we further show that removing the methylated DNA binding capacity or the GR-rich repeat enables full redundancy to MBD3, highlighting the synergistic requirements for these domains in diversifying NuRD complex function.
Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Nucleossomos , Animais , Camundongos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Isoformas de Proteínas/genética , Diferenciação Celular , Células-Tronco Embrionárias Murinas , MamíferosRESUMO
Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.
Assuntos
Metilação de DNA , Impressão Genômica , Camundongos , Animais , Sequência de Bases , Metilação de DNA/genética , Epigenômica , Cromatina/genética , Proteínas Repressoras/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.
Assuntos
Cromatina , Histonas , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Proteômica/métodos , Animais , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Células-Tronco Embrionárias , Histonas/química , Histonas/genética , Histonas/metabolismo , CamundongosRESUMO
CD8+ T cells are key players in immunity against intracellular infections and tumors. The main cytokine associated with these protective responses is interferon-γ (IFN-γ), whose production is known to be regulated at the transcriptional level during CD8+ T cell differentiation. Here we found that microRNAs constitute a posttranscriptional brake to IFN-γ expression by CD8+ T cells, since the genetic interference with the Dicer processing machinery resulted in the overproduction of IFN-γ by both thymic and peripheral CD8+ T cells. Using a gene reporter mouse for IFN-γ locus activity, we compared the microRNA repertoires associated with the presence or absence of IFN-γ expression. This allowed us to identify a set of candidates, including miR-181a and miR-451, which were functionally tested in overexpression experiments using synthetic mimics in peripheral CD8+ T cell cultures. We found that miR-181a limits IFN-γ production by suppressing the expression of the transcription factor Id2, which in turn promotes the Ifng expression program. Importantly, upon MuHV-4 challenge, miR-181a-deficient mice showed a more vigorous IFN-γ+ CD8+ T cell response and were able to control viral infection significantly more efficiently than control mice. These data collectively establish a novel role for miR-181a in regulating IFN-γ-mediated effector CD8+ T cell responses in vitro and in vivo.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Interferon gama/imunologia , MicroRNAs/imunologia , Animais , Diferenciação Celular , Linhagem Celular , Cricetinae , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , RhadinovirusRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses.
Assuntos
Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide/genética , Transcriptoma/genética , Doença Aguda , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Linhagem da Célula/genética , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Decitabina/uso terapêutico , Doxorrubicina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologiaRESUMO
γδ T cells are major providers of proinflammatory cytokines. They are preprogrammed in the mouse thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon-γ (IFN-γ), which segregate with CD27 expression. In the periphery, CD27- γδ (γδ27-) T cells can be induced under inflammatory conditions to coexpress IL-17 and IFN-γ; the molecular basis of this functional plasticity remains to be determined. On the basis of differential microRNA (miRNA) expression analysis and modulation in γδ T cell subsets, we identified miR-146a as a thymically imprinted post-transcriptional brake to limit IFN-γ expression in γδ27- T cells in vitro and in vivo. On the basis of biochemical purification of Argonaute 2-bound miR-146a targets, we identified Nod1 to be a relevant mRNA target that regulates γδ T cell plasticity. In line with this, Nod1-deficient mice lacked multifunctional IL-17+ IFN-γ+ γδ27- cells and were more susceptible to Listeria monocytogenes infection. Our studies establish the miR-146a/NOD1 axis as a key determinant of γδ T cell effector functions and plasticity.
Assuntos
MicroRNAs/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Proteínas de Ligação a DNA/genética , Listeria monocytogenes , Listeriose/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Proteína Adaptadora de Sinalização NOD1/genéticaRESUMO
Pro-inflammatory interleukin (IL)-17-producing γδ (γδ17) T cells are thought to develop exclusively in the thymus during fetal/perinatal life, as adult bone marrow precursors fail to generate γδ17 T cells under homeostatic conditions. Here, we employ a model of experimental autoimmune encephalomyelitis (EAE) in which hematopoiesis is reset by bone marrow transplantation and demonstrate unequivocally that Vγ4+ γδ17 T cells can develop de novo in draining lymph nodes in response to innate stimuli. In vitro, γδ T cells from IL-17 fate-mapping reporter mice that had never activated the Il17 locus acquire IL-17 expression upon stimulation with IL-1ß and IL-23. Furthermore, IL-23R (but not IL-1R1) deficiency severely compromises the induction of γδ17 T cells in EAE, demonstrating the key role of IL-23 in the process. Finally, we show, in a composite model involving transfers of both adult bone marrow and neonatal thymocytes, that induced γδ17 T cells make up a substantial fraction of the total IL-17-producing Vγ4+ T-cell pool upon inflammation, which attests the relevance of this novel pathway of peripheral γδ17 T-cell differentiation.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Interleucina-23/imunologia , Linfonodos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Células Th17/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/patologia , Transplante de Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/imunologia , Movimento Celular , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica , Hematopoese/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-1beta/farmacologia , Interleucina-23/genética , Interleucina-23/farmacologia , Linfonodos/patologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Transdução de Sinais , Células Th17/patologia , Timo/imunologia , Timo/patologiaRESUMO
γδ T lymphocytes are programmed into distinct IFN-γ-producing CD27(+) (γδ27(+)) and IL-17-producing CD27(-) (γδ27(-)) subsets that play key roles in protective or pathogenic immune responses. Although the signature cytokines are shared with their αß Th1 (for γδ27(+)) and Th17 (for γδ27(-)) cell counterparts, we dissect in this study similarities and differences in the transcriptional requirements of murine effector γδ27(+), γδ27(-)CCR6(-), and γδ27(-)CCR6(+) γδ T cell subsets and αß T cells. We found they share dependence on the master transcription factors T-bet and RORγt for IFN-γ and IL-17 production, respectively. However, Eomes is fully dispensable for IFN-γ production by γδ T cells. Furthermore, the Th17 cell auxiliary transcription factors RORα and BATF are not required for IL-17 production by γδ27(-) cell subsets. We also show that γδ27(-) (but not γδ27(+)) cells become polyfunctional upon IL-1ß plus IL-23 stimulation, cosecreting IL-17A, IL-17F, IL-22, GM-CSF, and IFN-γ. Collectively, our in vitro and in vivo data firmly establish the molecular segregation between γδ27(+) and γδ27(-) T cell subsets and provide novel insight on the nonoverlapping transcriptional networks that control the differentiation of effector γδ versus αß T cell subsets.
Assuntos
Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Th17/imunologia , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interferon gama/biossíntese , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-1beta/imunologia , Interleucina-23/imunologia , Interleucinas/metabolismo , Ativação Linfocitária , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/análise , Proteínas com Domínio T/genética , Subpopulações de Linfócitos T/fisiologia , Fatores de Transcrição/genética , Interleucina 22RESUMO
microRNA (miRNA) mediated regulation of protein expression has emerged as an important mechanism in T-cell physiology, from development and survival to activation, proliferation, and differentiation. One of the major classes of proteins involved in these processes are cytokines, which are both key input signals and major products of T-cell function. Here, we summarize the current data on the molecular cross-talk between cytokines and miRNAs: how cytokines regulate miRNA expression, and how specific miRNAs control cytokine production in T cells. We also describe the inflammatory consequences of deregulating the miRNA/cytokine axis in mice and humans. We believe this topical area will have key implications for immune modulation and treatment of autoimmune pathology.
Assuntos
Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , MicroRNAs/metabolismoRESUMO
γδ T cells are major providers of the pro-inflammatory cytokines interferon-γ (IFNγ) and interleukin-17 (IL-17) in protective or pathogenic immune responses. Notably, murine γδ T cells commit to either IFNγ or IL-17 production during development in the thymus, before any subsequent activation in the periphery. Here we discuss the molecular networks that underlie thymic γδ T cell differentiation, as well as the mechanisms that sustain or modify their functional properties in the periphery. We concentrate on recent findings on lymphoid and tissue-resident γδ T cell subpopulations, with an emphasis on genome-wide studies and their added value to elucidate the regulation of γδ T cell differentiation at the transcriptional and epigenetic (chromatin) levels.
Assuntos
Diferenciação Celular , Epigênese Genética , Linfócitos T/citologia , Linfócitos T/imunologia , Transcrição Gênica , Animais , Humanos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Timo/citologiaRESUMO
Two distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27(-) γδ T cells) or interferon-γ (IFN-γ) (CD27(+) γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27(+) γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27(-) γδ T cells displayed permissive chromatin configurations at loci encoding both cytokines and their regulatory transcription factors and differentiated into cells that produced both IL-17 and IFN-γ in a tumor microenvironment.
Assuntos
Diferenciação Celular/genética , Epigênese Genética , Perfilação da Expressão Gênica , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma , Animais , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Metilação , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Mucosal antibody responses play a major role in mediating homeostasis with the intestinal flora. It has been suggested that imbalance in the IgA+ and IgG+ intestinal B cell repertoire may be associated with the development of diseases such as inflammatory bowel disease. Despite this, little is known about the antibody specificity of human intestinal plasmablasts. Here, we have determined the reactivity profile of single isolated IgA+ and IgG+ plasmablasts from human terminal ileum using antibody cloning and in vitro expression. We found that approximately 25% of intestinal IgA and IgG plasmablast antibodies were polyreactive; the majority were antigen-specific. Antigen specificity was not only directed against enteropathogenic microbes but also against commensal microbes and self antigens. Regardless of their reactivity, all intestinal antibodies were somatically mutated and showed signs of antigen-mediated selection, suggesting that they developed from antigen-specific B cell responses. Together, our data indicate that antigen-specific immune responses to intestinal microbes are largely responsible for the maintenance of intestinal homeostasis and thus provide a basis for understanding the deregulated immune responses observed in patients with inflammatory bowel disease.