MicroRNA-181a regulates IFN-γ expression in effector CD8+ T cell differentiation

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.info:eu-repo/semantics/publishedVersio

ChromID identifies the protein interactome at chromatin marks

ISSN:1546-1696ISSN:1087-015

Lineage tracing of acute myeloid leukemia reveals the impact of hypomethylating agents on chemoresistance selection

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

DNA sequence and chromatin modifiers cooperate to confer epigenetic bistability at imprinting control regions

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

Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory gd T cell subsets

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

Epigenetic mechanisms of effector y&T cell differentiation : focus on histone modifications and microRNAs

Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2014T cell differentiation involves the establishment of distinct transcriptomes that are preserved throughout cell divisions. Many inheritable changes in gene expression are not due to alterations in the DNA sequence (genes and regulatory elements) but rather attributed to epigenetic mechanisms. There are several layers of epigenetic regulation, from histone modifications that control gene loci accessibility, to microRNAs (miRNAs) that act posttranscriptionally to induce mRNA decay. Effector T cell subsets can be functionally distinguished by the signature cytokines they produce, and interferon-gamma (IFN-γ) and interleukin-17 (IL-17) are critical pro-inflammatory mediators in contexts of infection, cancer and autoimmunity. Among the various cellular sources of these two cytokines, it is now clear that innate-like gamma-delta (γδ) T cells are major providers especially at early stages of the immune response. Our laboratory has recently shown that murine γδ T cells contain two thymic-derived subsets, segregated on the basis of CD27 expression, that produce either IFN-γδ (CD27+) or IL-17 (CD27-). While the molecular mechanisms that control the production of these cytokines have been extensively studied in CD4+ T cells, they remain poorly understood for γδ T cells. As this is particularly notable concerning epigenetic mechanisms, these have been the major focus of my thesis. In the first part of my thesis, 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 (γδIFNγ) or IL-17- producing (γδ17) γδ T cells. Given that these populations are present in lymphoid organs of naïve mice, we were able to analyse in vivo derived γδIFNγ (CD27+ ) or γδ17 (CD27-) cells. We found that CD27+ γδ cells are epigenetically committed to express Ifng but not Il17, whereas CD27- γδ cells spontaneously make IL-17 but can be induced to produce IFN-γ under specific inflammatory conditions in vitro and in vivo. This “plastic” behaviour of CD27- γδ cells associates with permissive histone H3 marks at loci encoding Ifng and upstream IFN-γ-driving transcription factors. By contrast, Il17 and related IL17-driving transcription factors are epigenetically and transcriptionally active in CD27- but silenced in CD27+ γδ cells. Hence, stable versus plastic behaviours of γδ cell subsets are controlled by integrated epigenetic and transcriptional mechanisms that regulate the expression of “master” transcription factors and effector cytokine genes. In the second part of my thesis, I explored another epigenetic level of gene regulation that is mediated by miRNAs. miRNAs are an evolutionarily conserved family of small non-coding RNAs that posttranscriptionally repress gene expression by targeting mRNA stability and/or blocking translation. Our analysis of T cell-specific miRNAdeficient mice (LckCre Dicer mice) revealed a global role of miRNAs in the differentiation of γδ T cells. Thus, we observed decreased numbers of γδ17 cells in the periphery (lymph node and spleen) and in the thymus of miRNA-deficient mice when compared to wild-type controls. To identify individual miRNAs implicated in γδ T cell differentiation, we undertook a transcriptome-wide analysis of miRNA expression in peripheral CD27+ and CD27- γδ T cell subsets and detected 35 differently expressed miRNAs. Based on the individual patterns of miRNA expression, we concentrated our analysis on miR-146a which was enriched in CD27- γδ T cells. We employed miR146a-deficient mice to investigate the impact of this miRNA on IL-17 and IFN-γ production by γδ T cells in naïve and parasite-infected mice. We detected a reduced frequency of γδ17 cells and an increased frequency of γδIFNγ cells in miR-146a-deficient mice during parasitic infections. Moreover, the retroviral-mediated over expression of miR-146a in wild-type γδ T cells led to a reduction in γδIFNγ cells. Therefore, miR-146a is the first miRNA shown to impact on γδ T cell differentiation. Future experiments will address the molecular mechanisms by which miR-146a controls IFN-γ and IL-17 expression in γδ T cells. Collectively, the data presented in this thesis provide novel insights into the epigenetic control of effector γδ T cell subsets that make key contributions to immune responses against infections and tumours, as well as to the pathogenesis of autoimmune diseases.A diferenciação de linfócitos T baseia-se no estabelecimento de transcriptomas específicos que se mantêm preservados através das várias divisões celulares. Muitas das alterações herdadas durante a expressão genética não são causadas por alterações ao nível da sequência do DNA (genes e elementos reguladores), surgindo na sequência de mecanismos epigenéticos. Existem várias etapas envolvidas na regulação epigenética, desde modificações de histonas que controlam a acessibilidade aos loci genéticos a microRNAs (miRNAs) que atuam a nível pós-transcricional, induzindo a degradação do mRNA. As subpopulações de células T efetoras distinguem-se funcionalmente pelas citocinas específicas que produzem. O interferão-gama (IFN-γ) e a interleucina-17 (IL-17) constituem exemplos de duas citocinas pro-inflamatórias, considerados mediadores críticos em contextos de infeção, cancro e autoimunidade. Sendo produzidas por vários tipos de células, tornou-se evidente que os linfócitos T gamma-delta (γδ) de tipo inato, constituem os principais produtores destas duas citocinas em estadios iniciais da resposta imunitária. O nosso laboratório mostrou recentemente que os linfócitos T γδ de ratinho possuem duas subpopulações distintas, segregadas com base na expressão de CD27, sendo uma produtora de de IFNγ (CD27+ ) e outra de IL-17 (CD27- ). Apesar dos mecanismos moleculares responsáveis pela regulação da produção destas citocinas se encontrarem bem descritos para os linfócitos T CD4 produzidos in vitro, permanecem ainda pouco caracterizados nos linfócitos T γδ. A minha tese incide sobretudo no estudo dos mecanismos epigenéticos subjacentes a estes processos, uma vez que a lacuna de conhecimentos moleculares é maior neste domínio específico. Na primeira parte da minha tese, efetuou-se a caracterização, através de estudos de larga escala, dos padrões de metilação da histona H3 bem como dos níveis de mRNA dos fatores de transcrição reguladores da expressão de IFNγ e IL-17. Estas experiências tiveram como objetivo identificar vias de regulação da produção de linfócitos T γδ produtores de IFNγ (γδIFNγ) ou IL-17 (γδ17). Tendo em conta que estas populações estão presentes nos órgãos linfoides de ratinhos “naïve”, foi possível analisar linfócitos T γδIFNγ (CD27+ ) ou γδ17 (CD27- ) in vivo. Chegou-se à conclusão que os linfócitos T γδ CD27+ estão condicionados, do ponto de vista epigenético, à expressão da citocina IFNγ mas não da IL-17, contrariamente aos linfócitos T γδ CD27- que produzem espontâneamente IL-17 mas podem também ser induzidos a produzir IFN-γ em condições inflamatórias específicas tanto in vitro como in vivo. Este comportamento “plástico” dos linfócitos γδ CD27- está associado à presença de marcas permissivas da histona H3 nos loci que codificam o Ifnγ e os fatores de transcrição responsáveis pela regulação da produção desta citocina. Contrariamente, o Il17 e os fatores de transcrição reguladores da expressão de IL-17 encontram-se epigenética e transcricionalmente ativos nos linfócitos T γδ CD27- mas silenciados nos linfócitos T γδ CD27+ . Pode então concluir-se que os fenótipos “estável” versus “plástico” das subpopulações de linfócitos T γδ são controlados por mecanismos epigenéticos e transcricionais que se encontram integrados e são responsáveis pela regulação da expressão de fatores de transcrição principais e de citocinas efectoras. Na segunda parte da minha tese, explorei a regulação da expressão genética por miRNAs que constitui um outro nível de regulação epigenética. Os miRNAs são, do ponto de vista evolutivo, uma família conservada de pequenos RNAs não codificantes que medeiam a inibição pós transcricional da expressão genética através do diminuição da estabilidade e/ou bloqueio da tradução de um dado mRNA. A análise de ratinhos deficientes para a produção de miRNAs especificamente em linfócitos T (ratinhos LckCre Dicer), demonstrou um papel global dos miRNAs sobre a diferenciação de linfócitos T γδ. Verificou-se uma diminuição da quantidade de linfócitos T γδ17 na periferia (nódulos linfáticos e baço) e no timo de ratinhos deficientes para a produção de miRNAs quando comparados com ratinhos controlo “wild-type”. Com o objectivo de identificar os miRNAs implicados na diferenciação dos linfócitos T γδ, efectuamos microarrays que permitiram a deteção de 35 miRNAs diferencialmente expressos nas subpopulações de linfócitos T γδ CD27- e CD27+ . Após análise dos padrões individuais de expressão destes miRNAs, optamos por incidir os estudos subsequentes num miRNA específico, o miR-146a, enriquecido nos linfócitos T γδ CD27- . A utilização de um modelo de ratinhos deficientes em miR-146a permitiu-nos investigar o impacto deste miRNA na produção de IL17 e IFN-γ pelos linfócitos T γδ em ratinhos naïve e ratinhos infetados com parasitas. Detetou-se uma redução da frequência de linfócitos T γδ17 e um aumento da frequência de linfócitos T γδIFNγ em ratinhos deficientes em miR-146a aquando da infeção por parasitas. Adicionalmente, estudos funcionais de sobre-expressão, mediada por retrovírus, do miR-146a em linfócitos T γδ “wild-type” demonstraram que este miRNA promove a diminuição do número de linfócitos T γδIFNγ. Identificou-se assim, neste estudo, o primeiro miRNA, miR-146a, envolvido no controlo da diferenciação de linfócitos T γδ. Serão ainda necessárias mais experiências a fim de estabelecer os mecanismos moleculares pelos quais o miR-146a controla a expressão do IFN-γ e IL-17 pelos linfócitos T γδ. No seu conjunto, os dados descritos nesta tese apresentam novas perspetivas ao nível do controlo epigenético de subpopulações de células T γδ efetoras, possuidoras de funções relevantes nas respostas imunes a infeções e tumores, bem como na patogénese de doenças autoimunes.European Research Council (ERC

Dissecting the roles of MBD2 isoforms in regulating NuRD complex function during cellular differentiation

The Nucleosome Remodelling 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. If these different complexes serve distinct biochemical and/or functional activities during differentiation is not completely understood. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD3 and MBD2 variants for their potential to rescue the differentiation block observed in mouse embryonic stem cells (ESCs) lacking MBD3. Our study reveals that while MBD3 is indeed crucial for ESC differentiation to neuronal cells, this function is independent of its MBD domain or binding to methylated DNA. While MBD3 isoforms are highly redundant, we identify that MBD2 isoforms vary in their potential to fully rescue the absence of MBD3 during lineage commitment. Full-length MBD2a only partially rescues the differentiation block; MBD2b, which lacks the N-terminal GR-rich repeat, fully rescues the differentiation block in MBD3 KO ES cells, and cells expressing the testis-specific isoform MBD2t that lacks the coiled-coil domain required for NuRD interactions are not able to generate any differentiated cells. In case of MBD2a, we further show that removing the m-CpG DNA binding capacity or the GR-rich repeat renders the protein fully redundant to MBD3, highlighting the requirements for these domains in diversifying NuRD complex function. In sum, our results highlight a partial redundancy of MBD2 and MBD3 during cellular differentiation and point to specific functions of distinct MBD2 isoforms and specific domains within the NuRD complex

Epigenetic and transcriptional regulation of γδ T cell differentiation: Programming cells for responses in time and space

International audienceγδ 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

Author Correction: Lineage tracing of acute myeloid leukemia reveals the impact of hypomethylating agents on chemoresistance selection

An amendment to this paper has been published and can be accessed via a link at the top of the pape 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

The majority of intestinal IgA+ and IgG+ plasmablasts in the human gut are antigen-specific

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