RESUMO
Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain repression of genes specific for other cell lineages. In vitro, RNA inhibits PRC2 activity, but the effect of RNA on PRC2 in cells is less clear, with studies concluding that RNA either antagonizes or promotes PRC2 chromatin association. The addition of RNase A to chromatin immunoprecipitation reactions has been reported to reduce detection of PRC2 target sites, suggesting the existence of RNA bridges connecting PRC2 to chromatin. Here, we show that the apparent loss of PRC2 chromatin association after RNase A treatment is due to non-specific chromatin precipitation. RNA degradation precipitates chromatin out of solution, thereby masking enrichment of specific DNA sequences in chromatin immunoprecipitation reactions. Maintaining chromatin solubility by the addition of poly-L-glutamic acid rescues detection of PRC2 chromatin occupancy upon RNA degradation. These findings undermine support for the model that RNA bridges PRC2 and chromatin in cells.
Assuntos
Cromatina , Complexo Repressor Polycomb 2 , Complexo Repressor Polycomb 2/metabolismo , RNA/metabolismo , Artefatos , Ribonuclease Pancreático/metabolismo , Estabilidade de RNARESUMO
Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.
Assuntos
Estruturas R-Loop , RNA , RNA/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina , Fator de Ligação a CCCTC/metabolismo , CoesinasRESUMO
Huge progress has been made in understanding the biology of innate lymphoid cells (ILC) by adopting several well-known concepts in T cell biology. As such, flow cytometry gating strategies and markers, such as CD90, have been applied to indentify ILC. Here, we report that most non-NK intestinal ILC have a high expression of CD90 as expected, but surprisingly a sub-population of cells exhibit low or even no expression of this marker. CD90-negative and CD90-low CD127+ ILC were present amongst all ILC subsets in the gut. The frequency of CD90-negative and CD90-low CD127+ ILC was dependent on stimulatory cues in vitro and enhanced by dysbiosis in vivo. CD90-negative and CD90-low CD127+ ILC were a potential source of IL-13, IFNγ and IL-17A at steady state and upon dysbiosis- and dextran sulphate sodium-elicited colitis. Hence, this study reveals that, contrary to expectations, CD90 is not constitutively expressed by functional ILC in the gut.
Assuntos
Colite , Imunidade Inata , Humanos , Colite/metabolismo , Citocinas/metabolismo , Disbiose/metabolismo , Linfócitos/metabolismo , Antígenos Thy-1/imunologiaRESUMO
BACKGROUND & AIMS: Resistance to single cytokine blockade, namely anti-tumor necrosis factor (TNF) therapy, is a growing concern for patients with inflammatory bowel disease (IBD). The transcription factor T-bet is a critical regulator of intestinal homeostasis, is genetically linked to mucosal inflammation and controls the expression of multiples genes such as the pro-inflammatory cytokines interferon (IFN)-γ and TNF. Inhibiting T-bet may therefore offer a more attractive prospect for treating IBD but remains challenging to target therapeutically. In this study, we evaluate the effect of targeting the transactivation function of T-bet using inhibitors of P-TEFb (CDK9-cyclin T), a transcriptional elongation factor downstream of T-bet. METHODS: Using an adaptive immune-mediated colitis model, human colonic lymphocytes from patients with IBD and multiple large clinical datasets, we investigate the effect of cyclin-dependent kinase 9 (CDK9) inhibitors on cytokine production and gene expression in colonic CD4+ T cells and link these genetic modules to clinical response in patients with IBD. RESULTS: Systemic CDK9 inhibition led to histological improvement of immune-mediated colitis and was associated with targeted suppression of colonic CD4+ T cell-derived IFN-γ and IL-17A. In colonic lymphocytes from patients with IBD, CDK9 inhibition potently repressed genes responsible for pro-inflammatory signalling, and in particular genes regulated by T-bet. Remarkably, CDK9 inhibition targeted genes that were highly expressed in anti-TNF resistant IBD and that predicted non-response to anti-TNF therapy. CONCLUSION: Collectively, our findings reveal CDK9 as a potential target for anti-TNF-resistant IBD, which has the potential for rapid translation to the clinic.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Colite/tratamento farmacológico , Quinase 9 Dependente de Ciclina , Citocinas/metabolismo , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Inibidores do Fator de Necrose TumoralRESUMO
Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain gene repression and is essential for cell differentiation. In low-grade endometrial stromal sarcoma (LG-ESS), the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. We show that JAZF1-SUZ12 dysregulates PRC2 composition, genome occupancy, histone modification, gene expression, and cell differentiation. Loss of the SUZ12 N terminus in the fusion protein abrogates interaction with specific PRC2 accessory factors, reduces occupancy at PRC2 target genes, and diminishes H3K27me3. Fusion to JAZF1 increases H4Kac at PRC2 target genes and triggers recruitment to JAZF1 binding sites during cell differentiation. In human endometrial stromal cells, JAZF1-SUZ12 upregulated PRC2 target genes normally activated during decidualization while repressing genes associated with immune clearance, and JAZF1-SUZ12-induced genes were also overexpressed in LG-ESS. These results reveal defects in chromatin regulation, gene expression, and cell differentiation caused by JAZF1-SUZ12 that may underlie its role in oncogenesis.
Assuntos
Proteínas Correpressoras , Proteínas de Ligação a DNA , Histonas , Proteínas de Neoplasias , Complexo Repressor Polycomb 2 , Fatores de Transcrição , Diferenciação Celular/genética , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3's sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways.
Assuntos
Fator de Transcrição GATA3 , Proteínas com Domínio T/metabolismo , Células Th2 , Animais , Linhagem da Célula , DNA/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Expressão Gênica , Camundongos , Proteínas com Domínio T/genética , Células Th2/citologia , Células Th2/metabolismoRESUMO
T-bet is the lineage-specifying transcription factor for CD4+ TH 1 cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarized to the TH 1 lineage, predisposed to produce IFN-γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T-helper response.
Assuntos
Proteínas com Domínio T , Células Th1 , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Ativação Linfocitária , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Células Th2RESUMO
Gene expression programs controlled by lineage-determining transcription factors are often conserved between species. However, infectious diseases have exerted profound evolutionary pressure, and therefore the genes regulated by immune-specific transcription factors might be expected to exhibit greater divergence. T-bet (Tbx21) is the immune-specific, lineage-specifying transcription factor for T helper type I (Th1) immunity, which is fundamental for the immune response to intracellular pathogens but also underlies inflammatory diseases. We compared T-bet genomic targets between mouse and human CD4+ T cells and correlated T-bet binding patterns with species-specific gene expression. Remarkably, we found that the majority of T-bet target genes are conserved between mouse and human, either via preservation of binding sites or via alternative binding sites associated with transposon-linked insertion. Species-specific T-bet binding was associated with differences in transcription factor-binding motifs and species-specific expression of associated genes. These results provide a genome-wide cross-species comparison of Th1 gene regulation that will enable more accurate translation of genetic targets and therapeutics from pre-clinical models of inflammatory and infectious diseases and cancer into human clinical trials.
Assuntos
Regulação da Expressão Gênica/genética , Proteínas com Domínio T/genética , Células Th1/fisiologia , Animais , Sítios de Ligação/genética , Bases de Dados Genéticas , Expressão Gênica/genética , Genoma/genética , Humanos , Camundongos , Ligação Proteica/genética , Proteínas com Domínio T/metabolismo , Células Th1/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcriptoma/genéticaRESUMO
A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.
Assuntos
Cromatina/metabolismo , RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Nucleossomos/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ligação Proteica/fisiologia , Proteômica/métodos , RNA Polimerase II/metabolismo , Transcrição Gênica/fisiologia , Fatores de Elongação da Transcrição/metabolismoRESUMO
BACKGROUND AND AIMS: Differential responsiveness to interleukin [IL]-2 between effector CD4+ T cells [Teff] and regulatory T cells [Treg] is a fundamental mechanism of immunoregulation. The single nucleotide polymorphism [SNP] rs61839660, located within IL2RA [CD25], has been associated with the development of Crohn's disease [CD]. We sought to identify the T cell immune phenotype of IBD patients who carry this SNP. METHODS: Teff and Treg were isolated from individuals homozygous [TT], heterozygous [CT], or wild-type [CC] for the minor allele at rs61839660, and used for phenotyping [flow cytometry, Cytometry Time Of Flight] functional assays or T cell receptor [TCR] sequencing. Phosphorylation of signal transducer and activator of transcription 5 [STAT5] was assessed in response to IL-2, IL-7, and in the presence of basiliximab, a monoclonal antibody directed against CD25. Teff pro-inflammatory cytokine expression levels were assessed by reverse transcription quantitative polymerase chain reaction after IL-2 and/or TCR stimulation. RESULTS: Presence of the minor T allele enhances CD25 expression, leading to increased STAT5 phosphorylation and pro-inflammatory cytokine transcript expression by Teff in response to IL-2 stimulation in vitro. Teff from TT individuals demonstrate a more activated gut homing phenotype. TCR sequencing analysis suggests that TT patients may have a reduced clonal capacity to mount an optimal regulatory T cell response. CONCLUSIONS: rs61839660 regulates the responsiveness of T cells to IL-2 signalling by modulating CD25 expression. As low-dose IL-2 is being trialled as a selective Treg modulator in CD, these findings highlight the potential for adverse effects in patients with this genotype.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença de Crohn/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Estudos de Casos e Controles , Doença de Crohn/imunologia , Bases de Dados Factuais , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Medicina Estatal , Reino UnidoRESUMO
Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is an invaluable method to profile of enrichment of histone modifications and transcription factor binding sites across the genome. However, standard ChIP-seq protocols require large numbers of cells (>107) as starting material, which are often impossible to obtain for rare immune populations. Here we describe a streamlined ChIP protocol optimised for small cell numbers in conjunction with transposon-tagging mediated sequencing library preparation (ChIPmentation) which allows the analysis of samples of as low as 105 cells.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Imunoprecipitação da Cromatina , DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , Projetos de Pesquisa , Fluxo de TrabalhoRESUMO
Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches. SIGNIFICANCE: Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.
Assuntos
Cordoma/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Proteínas Fetais/genética , Histona Desmetilases/antagonistas & inibidores , Proteínas com Domínio T/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cordoma/genética , Cordoma/patologia , Cromatina/genética , Cromatina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Epigênese Genética , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Terapia de Alvo Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas com Domínio T/metabolismoRESUMO
CD4+ T cell functional inhibition (exhaustion) is a hallmark of malaria and correlates with impaired parasite control and infection chronicity. However, the mechanisms of CD4+ T cell exhaustion are still poorly understood. In this study, we show that Ag-experienced (Ag-exp) CD4+ T cell exhaustion during Plasmodium yoelii nonlethal infection occurs alongside the reduction in mammalian target of rapamycin (mTOR) activity and restriction in CD4+ T cell glycolytic capacity. We demonstrate that the loss of glycolytic metabolism and mTOR activity within the exhausted Ag-expCD4+ T cell population during infection coincides with reduction in T-bet expression. T-bet was found to directly bind to and control the transcription of various mTOR and metabolism-related genes within effector CD4+ T cells. Consistent with this, Ag-expTh1 cells exhibited significantly higher and sustained mTOR activity than effector T-bet- (non-Th1) Ag-expT cells throughout the course of malaria. We identified mTOR to be redundant for sustaining T-bet expression in activated Th1 cells, whereas mTOR was necessary but not sufficient for maintaining IFN-γ production by Th1 cells. Immunotherapy targeting PD-1, CTLA-4, and IL-27 blocked CD4+ T cell exhaustion during malaria infection and was associated with elevated T-bet expression and a concomitant increased CD4+ T cell glycolytic metabolism. Collectively, our data suggest that mTOR activity is linked to T-bet in Ag-expCD4+ T cells but that reduction in mTOR activity may not directly underpin Ag-expTh1 cell loss and exhaustion during malaria infection. These data have implications for therapeutic reactivation of exhausted CD4+ T cells during malaria infection and other chronic conditions.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Malária/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Plasmodium yoelii/fisiologia , Proteínas com Domínio T/metabolismo , Células Th1/imunologia , Animais , Senescência Celular , Regulação da Expressão Gênica , Glicólise , Humanos , Tolerância Imunológica , Memória Imunológica , Interferon gama/metabolismo , Interleucina-27/metabolismo , Ativação Linfocitária , Malária/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas com Domínio T/genéticaRESUMO
Organ transplantation is often lifesaving, but the long-term deleterious effects of combinatorial immunosuppression regimens and allograft failure cause significant morbidity and mortality. Long-term graft survival in the absence of continuing immunosuppression, defined as operational tolerance, has never been described in the context of multiple major histocompatibility complex (MHC) mismatches. Here, we show that miR-142 deficiency leads to indefinite allograft survival in a fully MHC mismatched murine cardiac transplant model in the absence of exogenous immunosuppression. We demonstrate that the cause of indefinite allograft survival in the absence of miR-142 maps specifically to the T cell compartment. Of therapeutic relevance, temporal deletion of miR-142 in adult mice prior to transplantation of a fully MHC mismatched skin allograft resulted in prolonged allograft survival. Mechanistically, miR-142 directly targets Tgfbr1 for repression in regulatory T cells (TREG ). This leads to increased TREG sensitivity to transforming growth factor - beta and promotes transplant tolerance via an augmented peripheral TREG response in the absence of miR-142. These data identify manipulation of miR-142 as a promising approach for the induction of tolerance in human transplantation.
Assuntos
Rejeição de Enxerto , MicroRNAs , Aloenxertos , Animais , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Linfócitos T Reguladores , Tolerância ao Transplante , Transplante HomólogoRESUMO
In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.
Assuntos
Granzimas/imunologia , Neoplasias/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Humanos , Interferon gama/imunologia , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/citologia , Microambiente Tumoral/imunologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes.
Assuntos
Complexo Repressor Polycomb 2/metabolismo , Precursores de RNA/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Quadruplex G , Histonas/metabolismo , Humanos , Camundongos , Nucleossomos/metabolismo , Ligação Proteica , Precursores de RNA/química , Ativação TranscricionalRESUMO
Tregs play a fundamental role in immune tolerance via control of self-reactive effector T cells (Teffs). This function is dependent on maintenance of a high intracellular cAMP concentration. A number of microRNAs are implicated in the maintenance of Tregs. In this study, we demonstrate that peripheral immune tolerance is critically dependent on posttranscriptional repression of the cAMP-hydrolyzing enzyme phosphodiesterase-3b (Pde3b) by microRNA-142-5p (miR-142-5p). In this manner, miR-142-5p acts as an immunometabolic regulator of intracellular cAMP, controlling Treg suppressive function. Mir142 was associated with a super enhancer bound by the Treg lineage-determining transcription factor forkhead box P3 (FOXP3), and Treg-specific deletion of miR-142 in mice (TregΔ142) resulted in spontaneous, lethal, multisystem autoimmunity, despite preserved numbers of phenotypically normal Tregs. Pharmacological inhibition and genetic ablation of PDE3B prevented autoimmune disease and reversed the impaired suppressive function of Tregs in TregΔ142 animals. These findings reveal a critical molecular switch, specifying Treg function through the modulation of a highly conserved, cell-intrinsic metabolic pathway. Modulation of this pathway has direct relevance to the pathogenesis and treatment of autoimmunity and cancer.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Tolerância Imunológica , MicroRNAs/imunologia , Sistemas do Segundo Mensageiro/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , AMP Cíclico/genética , AMP Cíclico/imunologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Regulação Enzimológica da Expressão Gênica/genética , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Sistemas do Segundo Mensageiro/genética , Linfócitos T Reguladores/patologiaRESUMO
Transcription and chromatin function are regulated by proteins that bind to DNA, nucleosomes or RNA polymerase II, with specific non-coding RNAs (ncRNAs) functioning to modulate their recruitment or activity. Unlike ncRNAs, nascent pre-mRNA was considered to be primarily a passive player in these processes. In this Opinion article, we describe recently identified interactions between nascent pre-mRNAs and regulatory proteins, highlight commonalities between the functions of nascent pre-mRNA and nascent ncRNA, and propose that both types of RNA have an active role in transcription and chromatin regulation.