RESUMEN
A growing body of literature has examined and implicated DNA methylation as a critical epigenetic modification in T helper (Th) cell differentiation. The absence of DNA methyltransferases or methyl-binding proteins derepresses many cytokine loci, allowing their ectopic expression, while methylation of specific CpG residues is sufficient to prevent expression. Here, we characterize demethylation events of the Th2 cytokine locus control region (LCR). rad50 hypersensitive site 7 (RHS7), a hypersensitive site within this LCR, becomes demethylated in a STAT6-dependent manner and only in cells stimulated under type 2 conditions. Robust demethylation appears to require signaling contributions from both IL-4 receptor, via STAT6, and CD28, but it cannot be effected by GATA3. Finally, RHS7 is demethylated independently of cell division, consistent with an "active," rather than passive, mechanism. Taken together, these findings firmly connect RHS7 demethylation and Th2 LCR activation in the type 2 differentiation program.
Asunto(s)
Metilación de ADN , Región de Control de Posición/genética , Células Th2/metabolismo , Animales , Secuencia de Bases , Factor de Transcripción GATA3/metabolismo , Interleucina-2/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Cinética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor de Transcripción STAT6/metabolismo , Transducción de SeñalRESUMEN
Gammadelta T cells have unique features and functions compared with alphabeta T cells and have been proposed to bridge the innate and adaptive immune responses. Our earlier studies demonstrated that splenic gammadelta T cells predominantly produce IFN-gamma upon activation in vitro, which is partially due to the expression of the Th1-specific transcription factor T-bet. In this study we have explored the epigenetic and transcriptional programs that underlie default IFN-gamma production by gammadelta T cells. We show that the kinetics of IFN-gamma transcription is faster in gammadelta T cells compared with CD4(+) and CD8(+) T cells and that gammadelta T cells produce significantly greater amounts of IFN-gamma in a proliferation-independent manner when compared with other T cell subsets. By analyzing the methylation pattern of intron 1 of the ifn-gamma locus, we demonstrate that this region in naive gammadelta T cells is hypomethylated relative to the same element in naive CD4(+) and CD8(+) T cells. Furthermore, naive gammadelta T cells constitutively express eomesodermin (Eomes), a transcription factor important for IFN-gamma production in CD8(+) T cells, and Eomes expression levels are enhanced upon activation. Retroviral transduction of activated gammadelta T cells from both wild-type and T-bet-deficient mice with a dominant negative form of Eomes significantly reduced IFN-gamma production, indicating a critical role for this transcription factor in mediating IFN-gamma production by gammadelta T cells in a T-bet-independent manner. Our results demonstrate that both epigenetic and transcriptional programs contribute to the early vigorous IFN-gamma production by gammadelta T cells.
Asunto(s)
Epigénesis Genética/inmunología , Interferón gamma/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Transcripción Genética/inmunología , Animales , Metilación de ADN , Interferón gamma/biosíntesis , Activación de Linfocitos , Ratones , Ratones Noqueados , Sitios de Carácter Cuantitativo/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/inmunología , Linfocitos T/metabolismoRESUMEN
To identify interleukin-10 (IL-10)-producing cells in vivo, we generated a knockin mouse where an internal ribosome entry site (IRES) green fluorescence protein (GFP) element was inserted immediately before the polyadenylation site of the IL-10 gene. GFP fluorescence in cells from these mice was found to correlate positively with IL-10 protein expression. With this model, we found that after multiple T cell receptor (TCR) stimulations, strong expression of IL-10 was produced specifically by intraepithelial lymphocytes (IEL) in the small intestine and colonic lamina propria lymphocytes (cLPL). We found that anti-CD3 treatment induces T regulatory cell 1 (Tr1)-like cells in small intestinal IEL (sIEL) and led to the accumulation of naturally occurring regulatory T (nTreg) cells in colonic LPL (cLPL). These findings highlight the intestine as a unique site for induction of IL-10-producing T cells, which play a critical role in the regulation of inflammation in the gut.
Asunto(s)
Inmunidad Mucosa , Interleucina-10/biosíntesis , Intestinos/inmunología , Subgrupos Linfocitarios/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes , Interleucina-10/inmunología , Intestinos/citología , Ratones , Ratones MutantesRESUMEN
Cytokine loci undergo changes in chromatin structure when naive CD4(+) T cells differentiate into Th1 or Th2 cells and have also been examined for regulatory sequences underlying such changes and their functional correlates. Studies have shown that distal regulatory elements control the Ifng and Th2 cytokine loci and are primary targets for tissue-specific transcription factors, serving as centers for epigenetic changes that mark heritable traits in effector cells. Reports of intra- and, remarkably, interchromosomal interactions between these regulatory elements shed light on the mechanisms by which they regulate gene expression, revealing an extraordinary new picture that conceptually extends our views on how genes are regulated from two to three dimensions. Here, we summarize these recent findings on the role of regulatory elements and their mechanisms of action, which are of broad significance for gene regulation, not only of the immune system but also of many, if not all, coregulated genes.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/inmunología , Epigénesis Genética , Elementos Reguladores de la Transcripción , Células Th2/citología , Animales , Citocinas/genética , Citocinas/inmunología , Expresión Génica/inmunología , Glicosiltransferasas/genética , Glicosiltransferasas/inmunología , Humanos , Células Th2/fisiologíaRESUMEN
A number of modified histones, including acetylated H3 and H4 and phosphorylated H2AX (gammaH2AX), are associated with V(D)J recombination and class switch recombination (CSR). In contrast, little is known concerning the chromatin modifications associated with somatic hypermutation (SHM) in vivo. Here, we report that several modifications--including histone acetylation and H3-lysine 4 methylation--fail to demarcate an actively hypermutating immunoglobulin (Ig) locus or to correlate spatially with SHM within Ig loci. Furthermore, no obvious association between SHM and gammaH2AX could be detected. Instead, we find that the phosphorylated form of histone H2B (H2B(Ser14P)) correlates tightly with SHM and CSR. Phosphorylation of H2B within Ig variable and switch regions requires AID and may be mediated by the histone kinase Mst1. These findings indicate that SHM and CSR trigger distinct DNA damage responses and identify a novel histone modification pattern for SHM consisting of H2B(Ser14P) in the absence of gammaH2AX.
Asunto(s)
Linfocitos B/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Cambio de Clase de Inmunoglobulina , Hipermutación Somática de Inmunoglobulina , Acetilación , Animales , Inmunoprecipitación de Cromatina , Islas de CpG , Daño del ADN , Metilación de ADN , Histonas/genética , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas lambda de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/metabolismo , Metilación , Ratones , Ratones Transgénicos , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
We recently identified a 3' region of the rad50 gene possessing strong enhancer activity as well as activity consistent with function as a locus control region (LCR) for the flanking Th2 cytokine genes. In this study, we identify several functional elements within this region by examining chromatin changes as well as activity in transgenic mice. We find within this region four DNase I hypersensitive clusters, three of which are highly conserved and predominantly expressed in Th2 cells. Histone acetylation of this region is elevated in Th2 cells. Further, one of the hypersensitive sites (RHS7) is rapidly demethylated in Th2, but not Th1, cells. In transgenic mice, these hypersensitive sites impart strong, Th2-specific enhancer activity as well as copy number-dependent expression of the reporter gene, recapitulating LCR function. We postulate that these sites function alone or in combination with other regulatory elements to coordinate gene expression in the Th2 cytokine locus.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Ensamble y Desensamble de Cromatina/genética , Citocinas/genética , Elementos de Facilitación Genéticos/genética , Región de Control de Posición/genética , Células Th2/metabolismo , Acetilación , Ácido Anhídrido Hidrolasas , Animales , Secuencia Conservada/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/metabolismo , Histonas/metabolismo , Humanos , Intrones/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Familia de Multigenes/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Peripheral T cell differentiation is accompanied by chromatin changes at the signature cytokine loci. Using chromatin immunoprecipitation we demonstrate that profound increases in histone acetylation occur at the IFN-gamma and IL-4 loci during Th1/Th2 differentiation. These changes in histone acetylation status are locus and lineage specific, and are maintained by the transcription factors Tbet and GATA3 in a STAT-dependent manner. Our results suggest a model of cytokine locus activation in which TCR signals initiate chromatin remodeling and locus opening in a cytokine-independent fashion. Subsequently, cytokine signaling reinforces polarization by expanding and maintaining the accessible state at the relevant cytokine locus (IL-4 or IFN-gamma). In this model, GATA3 and Tbet serve as transcriptional maintenance factors, which keep the locus accessible to the transcriptional machinery.
Asunto(s)
Histonas/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Células TH1/citología , Células TH1/metabolismo , Acetilación , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Polaridad Celular/genética , Polaridad Celular/inmunología , Células Cultivadas , Marcadores Genéticos/inmunología , Histonas/genética , Interfase/genética , Interfase/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genética , Transducción de Señal/inmunología , Células TH1/inmunología , Transcripción Genética/inmunologíaRESUMEN
The cytokine TNFalpha launches cascades of gene activation that control inflammation and apoptosis through NFkappaB and JNK/SAPK signal transduction pathways. Here we describe a function for the zinc finger transcription factor kappa recognition component (KRC) in regulating patterns of gene activation in response to proinflammatory stimuli. We demonstrate that KRC overexpression inhibits while antisense or dominant-negative KRC enhances NFkappaB-dependent transactivation and JNK phosphorylation and consequently, apoptosis and cytokine gene expression. The effect of KRC is mediated through its interaction with the adaptor protein TRAF2, which intersects both pathways. KRC is a hitherto unrecognized participant in the signal transduction pathway leading from the TNF receptor to gene activation and may play a critical role in inflammatory and apoptotic responses.