RESUMO
Glucocorticoids (GCs) are effective anti-inflammatory drugs; yet, their mechanisms of action are poorly understood. GCs bind to the glucocorticoid receptor (GR), a ligand-gated transcription factor controlling gene expression in numerous cell types. Here, we characterize GR's protein interactome and find the SETD1A (SET domain containing 1A)/COMPASS (complex of proteins associated with Set1) histone H3 lysine 4 (H3K4) methyltransferase complex highly enriched in activated mouse macrophages. We show that SETD1A/COMPASS is recruited by GR to specific cis-regulatory elements, coinciding with H3K4 methylation dynamics at subsets of sites, upon treatment with lipopolysaccharide (LPS) and GCs. By chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq, we identify subsets of GR target loci that display SETD1A occupancy, H3K4 mono-, di-, or tri-methylation patterns, and transcriptional changes. However, our data on methylation status and COMPASS recruitment suggest that SETD1A has additional transcriptional functions. Setd1a loss-of-function studies reveal that SETD1A/COMPASS is required for GR-controlled transcription of subsets of macrophage target genes. We demonstrate that the SETD1A/COMPASS complex cooperates with GR to mediate anti-inflammatory effects.
Assuntos
Elementos Facilitadores Genéticos/imunologia , Macrófagos/imunologia , Complexos Multiproteicos , RNA-Seq , Receptores de Glucocorticoides , Transcrição Gênica/imunologia , Animais , Inflamação/genética , Inflamação/imunologia , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/imunologiaRESUMO
Innate immune responses rely on rapid and precise gene regulation mediated by accessibility of regulatory regions to transcription factors (TFs). In natural killer (NK) cells and other innate lymphoid cells, competent enhancers are primed during lineage acquisition, and formation of de novo enhancers characterizes the acquisition of innate memory in activated NK cells and macrophages. Here, we investigated how primed and de novo enhancers coordinate to facilitate high-magnitude gene induction during acute activation. Epigenomic and transcriptomic analyses of regions near highly induced genes (HIGs) in NK cells both in vitro and in a model of Toxoplasma gondii infection revealed de novo chromatin accessibility and enhancer remodeling controlled by signal-regulated TFs STATs. Acute NK cell activation redeployed the lineage-determining TF T-bet to de novo enhancers, independent of DNA-sequence-specific motif recognition. Thus, acute stimulation reshapes enhancer function through the combinatorial usage and repurposing of both lineage-determining and signal-regulated TFs to ensure an effective response.
Assuntos
Elementos Facilitadores Genéticos/genética , Elementos Facilitadores Genéticos/imunologia , Células Matadoras Naturais/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Animais , Cromatina/genética , Cromatina/imunologia , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Toxoplasma/imunologia , Toxoplasmose/genética , Toxoplasmose/imunologiaRESUMO
Transposable elements (TEs) compose nearly half of mammalian genomes and provide building blocks for cis-regulatory elements. Using high-throughput sequencing, we show that 84 TE subfamilies are overrepresented, and distributed in a lineage-specific fashion in core and boundary domains of CD8+ T cell enhancers. Endogenous retroviruses are most significantly enriched in core domains with accessible chromatin, and bear recognition motifs for immune-related transcription factors. In contrast, short interspersed elements (SINEs) are preferentially overrepresented in nucleosome-containing boundaries. A substantial proportion of these SINEs harbor a high density of the enhancer-specific histone mark H3K4me1 and carry sequences that match enhancer boundary nucleotide composition. Motifs with regulatory features are better preserved within enhancer-enriched TE copies compared to their subfamily equivalents located in gene deserts. TE-rich and TE-poor enhancers associate with both shared and unique gene groups and are enriched in overlapping functions related to lymphocyte and leukocyte biology. The majority of T cell enhancers are shared with other immune lineages and are accessible in common hematopoietic progenitors. A higher proportion of immune tissue-specific enhancers are TE-rich compared to enhancers specific to other tissues, correlating with higher TE occurrence in immune gene-associated genomic regions. Our results suggest that during evolution, TEs abundant in these regions and carrying motifs potentially beneficial for enhancer architecture and immune functions were particularly frequently incorporated by evolving enhancers. Their putative selection and regulatory cooption may have accelerated the evolution of immune regulatory networks.
Assuntos
Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Linfócitos T/imunologia , Animais , Cromatina/genética , Cromatina/imunologia , Elementos de DNA Transponíveis/imunologia , Retrovirus Endógenos/genética , Retrovirus Endógenos/imunologia , Elementos Facilitadores Genéticos/imunologia , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Genoma Humano/imunologia , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Elementos Nucleotídeos Curtos e Dispersos/genética , Elementos Nucleotídeos Curtos e Dispersos/imunologiaRESUMO
Tcrd and Tcrg display identical developmental programs that depend on the activity of the enhancers Eδ and Eγ being "on" in pre-ß-selection thymocytes to activate transcription and V(D)J recombination of the unrearranged genes and "off" in post-ß-selection CD4+CD8+ double-positive thymocytes to inhibit transcription of the rearranged genes and avoid the expression of TCR δ- and TCR γ-chains in αß T lymphocytes. Eδ and Eγ activity depends on transcription factor binding to essential Runx and Myb sites and parallels that of Notch signaling. We performed Notch gain- and loss-of-function experiments and found that Notch signaling activates Tcrd and Tcrg transcription by favoring the recruitment of RUNX1 and MYB to the enhancers. Our results suggest that the dissociation of RUNX1 and MYB from Eδ and Eγ chromatin in double-positive thymocytes, which results in enhancer inactivation, is caused by decreased Notch signaling triggered by pre-TCR signaling, thereby deciphering the molecular mechanism of Tcrd and Tcrg silencing during ß-selection. These findings reveal a novel molecular mechanism for gene regulation via Notch signaling through the recruitment of RUNX1 and MYB to enhancer chromatin during thymocyte development.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Elementos Facilitadores Genéticos/imunologia , Proteínas Proto-Oncogênicas c-myb/imunologia , Receptores Notch/imunologia , Transdução de Sinais/imunologia , Timócitos/imunologia , Transcrição Gênica/imunologia , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Humanos , Células Jurkat , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myb/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores Notch/genética , Transdução de Sinais/genéticaRESUMO
MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.
Assuntos
Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Proteínas Nucleares/imunologia , Transativadores/imunologia , Fatores de Transcrição/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Rearranjo Gênico/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Macrófagos/citologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
Members of the NR4A subfamily of nuclear receptors have complex, overlapping roles during hematopoietic cell development and also function as tumor suppressors of hematologic malignancies. We previously identified NR4A1 and NR4A3 (NR4A1/3) as functionally redundant suppressors of acute myeloid leukemia (AML) development. However, their role in hematopoietic stem cell (HSC) homeostasis remains to be disclosed. Using a conditional Nr4a1/Nr4a3 knockout mouse (CDKO), we show that codepletion of NR4A1/3 promotes acute changes in HSC homeostasis including loss of HSC quiescence, accumulation of oxidative stress, and DNA damage while maintaining stem cell regenerative and differentiation capacity. Molecular profiling of CDKO HSCs revealed widespread upregulation of genetic programs governing cell cycle and inflammation and an aberrant activation of the interferon and NF-κB signaling pathways in the absence of stimuli. Mechanistically, we demonstrate that NR4A1/3 restrict HSC proliferation in part through activation of a C/EBPα-driven antiproliferative network by directly binding to a hematopoietic-specific Cebpa enhancer and activating Cebpa transcription. In addition, NR4A1/3 occupy the regulatory regions of NF-κB-regulated inflammatory cytokines, antagonizing the activation of NF-κB signaling. Taken together, our results reveal a novel coordinate control of HSC quiescence by NR4A1/3 through direct activation of C/EBPα and suppression of activation of NF-κB-driven proliferative inflammatory responses.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Proliferação de Células , Proteínas de Ligação a DNA/imunologia , Células-Tronco Hematopoéticas/imunologia , Proteínas do Tecido Nervoso/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Receptores de Esteroides/imunologia , Receptores dos Hormônios Tireóideos/imunologia , Transdução de Sinais/imunologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/imunologia , Células-Tronco Hematopoéticas/patologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Estresse Oxidativo/genética , Estresse Oxidativo/imunologia , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Transdução de Sinais/genética , Transcrição Gênica/imunologiaRESUMO
Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter-dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease-induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome.
Assuntos
Linfócitos B/imunologia , Ligante de CD40 , Edição de Genes/métodos , Regulação da Expressão Gênica/imunologia , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1 , Linfócitos T/imunologia , Reparo Gênico Alvo-Dirigido/métodos , Regiões 3' não Traduzidas/imunologia , Animais , Ligante de CD40/genética , Ligante de CD40/imunologia , Elementos Facilitadores Genéticos/imunologia , Feminino , Humanos , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/genética , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/imunologia , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/terapia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologiaRESUMO
Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain (Igh) and controlled expression of the gene encoding the transcription factor c-Myc (Myc) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage-specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.
Assuntos
Linfócitos B/imunologia , Proliferação de Células , DNA Helicases/imunologia , Elementos Facilitadores Genéticos/imunologia , Proteínas Nucleares/imunologia , Fatores de Transcrição/imunologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/imunologia , DNA Helicases/genética , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Hibridização in Situ Fluorescente , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In immune cells, as in all mammalian cells, nuclear DNA is wrapped around histones to form nucleosomes. The positioning and modifications of nucleosomes throughout the genome defines the chromatin state of the cell and has a large impact on gene regulation. Chromatin state is dynamic throughout immune cell development and activation. High-throughput open chromatin assays, such as DNase-seq, can be used to find regulatory element across the genome and, when combined with histone modifications, can specify their function. During hematopoiesis, distal regulatory elements, known as enhancers, are established by pioneer factors that alter chromatin state. Some of these enhancers are lost, some are gained, and some are maintained as a memory of the cell's developmental origin. The enhancer landscape is unique to the cell lineage-with different enhancers regulating the same promoter-and determines the mechanism of cell type-specific activation after exposure to stimuli. Histone modification and promoter architecture govern the diverse responses to stimulation. Furthermore, chromatin dynamics may explain the high plasticity of certain tissue-resident immune cell types. Future epigenomic research will depend on the development of more efficient experiments and better methods to associate enhancers with genes. The ultimate goal of mapping genome-wide chromatin state throughout the hematopoietic tree will help illuminate the mechanisms behind immune cell development and function.
Assuntos
Histonas/metabolismo , Sistema Imunitário , Imunidade Celular , Animais , Diferenciação Celular , Linhagem da Célula , Montagem e Desmontagem da Cromatina/imunologia , Elementos Facilitadores Genéticos/genética , Elementos Facilitadores Genéticos/imunologia , Epigênese Genética , Epigenômica , Ensaios de Triagem em Larga Escala , Humanos , Conformação Molecular , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologiaRESUMO
The IgH intronic enhancer region Eµ is a combination of both a 220-bp core enhancer element and two 310-350-bp flanking scaffold/matrix attachment regions named MARsEµ. In the mouse, deletion of the core-enhancer Eµ element mainly affects VDJ recombination with minor effects on class switch recombination. We carried out endogenous deletion of the full-length Eµ region (core plus MARsEµ) in the mouse genome to study VH gene repertoire and IgH expression in developing B-lineage cells. Despite a severe defect in VDJ recombination with partial blockade at the pro-B cell stage, Eµ deletion (core or full length) did not affect VH gene usage. Deletion of this regulatory region induced both a decrease of pre-B cell and newly formed B cell compartments and a strong orientation toward the marginal zone B cell subset. Because Igµ H chain expression was decreased in Eµ-deficient pre-B cells, we propose that modification of B cell homeostasis in deficient animals was caused by "weak" pre-B cell and BCR expression. Besides imbalances in B cell compartments, Ag-specific Ab responses were not impaired in animals carrying the Eµ deletion. In addition to its role in VDJ recombination, our study points out that the full-length Eµ region does not influence VH segment usage but ensures efficient Igµ-chain expression required for strong signaling through pre-B cells and newly formed BCRs and thus participates in B cell inflow and fate.
Assuntos
Subpopulações de Linfócitos B/imunologia , Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica/imunologia , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Movimento Celular/genética , Movimento Celular/imunologia , Deleção de Genes , Switching de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/biossíntese , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Distribuição Aleatória , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Recombinação V(D)J/genética , Recombinação V(D)J/imunologiaRESUMO
GTP cyclohydrolase I (GCH) catalyzes the first and rate limiting step reaction for the de novo synthesis of 5,6,7,8-tetrahydrobiopterin (BH4). The expression of GCH is dramatically elevated by immune activation, while the mechanism remains to be elucidated. In this study, we investigated the transcription mechanism of the GCH gene using lipopolysaccharide (LPS) to stimulate mouse macrophage RAW264.7 cells. With luciferase assay, we found a highly conserved enhancer region spanning approximately 300 bp in intron 1 of GCH gene as a response element to LPS stimulation. The same enhancer region was also responsible for the induction of the GCH gene by IFN-γ and TNF-α in HUVECs. With electrophoresis mobility shift assay (EMSA) and site directed mutation analysis, we identified two key fragments containing C/EBP and Ets binding motifs within the enhancer. Furthermore, C/EBP-ß was involved in LPS activated GCH transcription through direct binding to the enhancer shown by supershift, chromatin immunoprecipitation, and RNA interference experiments. In conclusion, our findings uncovered a novel mechanism of GCH transcriptional regulation by immune activation.
Assuntos
Elementos Facilitadores Genéticos/imunologia , GTP Cicloidrolase/genética , Regulação Enzimológica da Expressão Gênica , Ativação Transcricional , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/efeitos dos fármacos , Elementos Facilitadores Genéticos/genética , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/genética , Macrófagos/imunologia , Camundongos , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The closely linked human IL-3 and GM-CSF genes are tightly regulated and are expressed in activated T cells and mast cells. In this study, we used transgenic mice to study the developmental regulation of this locus and to identify DNA elements required for its correct activity in vivo. Because these two genes are separated by a CTCF-dependent insulator, and the GM-CSF gene is regulated primarily by its own upstream enhancer, the main objective in this study was to identify regions of the locus required for correct IL-3 gene expression. We initially found that the previously identified proximal upstream IL-3 enhancers were insufficient to account for the in vivo activity of the IL-3 gene. However, an extended analysis of DNase I-hypersensitive sites (DHSs) spanning the entire upstream IL-3 intergenic region revealed the existence of a complex cluster of both constitutive and inducible DHSs spanning the -34- to -40-kb region. The tissue specificity of these DHSs mirrored the activity of the IL-3 gene, and included a highly inducible cyclosporin A-sensitive enhancer at -37 kb that increased IL-3 promoter activity 40-fold. Significantly, inclusion of this region enabled correct in vivo regulation of IL-3 gene expression in T cells, mast cells, and myeloid progenitor cells.
Assuntos
Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interleucina-3/biossíntese , Interleucina-3/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Desoxirribonuclease I/genética , Elementos Facilitadores Genéticos/genética , Loci Gênicos/imunologia , Humanos , Células Jurkat , Camundongos , Camundongos Transgênicos , Distribuição Tecidual/genética , Distribuição Tecidual/imunologiaRESUMO
Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription. In this article, we report that human NLRC5, which we recently showed to be involved in viral-mediated type I IFN responses, shuttles to the nucleus and activates MHC class I gene expression. Knockdown of NLRC5 in different human cell lines and primary dermal fibroblasts leads to reduced MHC class I expression, whereas introduction of NLRC5 into cell types with very low expression of MHC class I augments MHC class I expression to levels comparable to those found in lymphocytes. Expression of NLRC5 positively correlates with MHC class I expression in human tissues. Functionally, we show that both the N-terminal effector domain of NLRC5 and its C-terminal leucine-rich repeat domain are needed for activation of MHC class I expression. Moreover, nuclear shuttling and function depend on a functional Walker A motif. Finally, we identified a promoter sequence in the MHC class I promoter, the X1 box, to be involved in NLRC5-mediated MHC class I gene activation. Taken together, this suggested that NLRC5 acts in a manner similar to class II transactivator to drive MHC expression and revealed NLRC5 as an important regulator of basal MHC class I expression.
Assuntos
Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos HLA-A/fisiologia , Antígenos HLA-B/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Transativadores/fisiologia , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , Transativadores/antagonistas & inibidores , Transativadores/deficiência , Ativação Transcricional/genéticaRESUMO
The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). We previously discovered, using the chromosome conformation-capture technique, that Ei and E3' interact with a novel DNA sequence near the 3' end of the Igκ locus, specifically in B cells. In the present investigation, we examined the function of this far downstream element. The sequence is evolutionarily conserved and exhibits a plasmacytoma cell-specific DNase I-hypersensitive site in chromatin, henceforth termed HS10 in the locus. HS10 acts as a coactivator of E3' in transient transfection assays. Although HS10(-/-) mice exhibited normal patterns of B cell development, they were tested further along with E3'(-/-) and Ed(-/-) mice for their Igκ expression levels in plasma cells, as well as for both allelic and isotype exclusion in splenic B cells. HS10(-/-) and Ed(-/-), but not E3'(-/-), mice exhibited 2.5-fold lower levels of Igκ expression in antigenically challenged plasma cells. E3'(-/-) mice, but not HS10(-/-) mice, exhibited impaired IgL isotype and allelic exclusion in splenic B cells. We have suggestive results that Ed may also weakly participate in these processes. In addition, HS10(-/-) mice no longer exhibited regional chromosome interactions with E3', and they exhibited modestly reduced somatic hypermutation in the Jκ-Cκ intronic region in germinal center B cells from Peyer's patches. We conclude that the HS10, E3', and Ed differentially regulate Igκ gene dynamics.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Genes de Imunoglobulinas/genética , Imunoglobulinas/genética , Animais , Linfócitos B/imunologia , Sequência de Bases , Southern Blotting , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Separação Celular , Elementos Facilitadores Genéticos/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Genes de Imunoglobulinas/imunologia , Imunoglobulinas/biossíntese , Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal V(H) genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3' enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show in this study that STAT5 can repress κE3' enhancer activity. We find that STAT5 binds to a site that overlaps the κE3' PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3' enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3' enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates κE3' enhancer function.
Assuntos
Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/fisiologia , Elementos Facilitadores Genéticos/imunologia , Cadeias kappa de Imunoglobulina/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transativadores/genética , Transativadores/metabolismo , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Ligação Competitiva/genética , Ligação Competitiva/imunologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Cadeias kappa de Imunoglobulina/metabolismo , Cadeias kappa de Imunoglobulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Células NIH 3T3 , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas/fisiologia , Fator de Transcrição STAT5/fisiologia , Transativadores/fisiologiaRESUMO
Class-switch recombination of Ab isotype is mediated by a recombinational DNA deletion event and must be robustly upregulated during Ag-driven differentiation of B cells. The enhancer region 3' of the Cα gene is important for the upregulation of switch recombination. Using a transgene of the entire H chain C region locus, we demonstrate in this study that it is the four 3' enhancer elements themselves (a total of 4.7 kb) that are responsible for the upregulation rather than the 24 kb of DNA in between them. Neither allelic exclusion nor transgenic µ expression is reduced by deletion of the four 3' enhancers. We also test deletions of two or three of the 3' enhancers and show that deletion of more 3' enhancers results in a progressive reduction in both switch recombination and germline transcription of all H chain genes. Nevertheless, we find evidence for special roles for some 3' enhancers; different H chain genes are affected by different 3' enhancer deletions. Thus, we find that the dramatic induction of class-switch recombination during Ag-driven differentiation is the result of an interaction among four separated regulatory elements.
Assuntos
Switching de Imunoglobulina/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Recombinação Genética/imunologia , Deleção de Sequência/imunologia , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos/genética , Elementos Facilitadores Genéticos/imunologia , Éxons/genética , Feminino , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/genéticaRESUMO
The aim of this study was to determine the genetic regulation of macrophage migration inhibitory factor (MIF). DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients. Two cis-elements within the first intron were found to be responsible for the enhancer activity. Mutation of the consensus Sp1 GC box on each cis-element abrogated enhancer activity and EMSA indicated Sp1 binding to one of the cis-elements contained in the intron. SiRNA knock-down of Sp1 alone or Sp1 and Sp3 together was incomplete and did not alter the enhancer activity. Mithramycin inhibited expression of MIF in CEMC7A cells. This effect was specific to the intronic enhancer and was not seen on the MIF promoter. These results identify a novel, cell type-specific enhancer of MIF. The enhancer appears to be driven by Sp1 or related Sp family members and is highly sensitive to inhibition via mithramycin.
Assuntos
Elementos Facilitadores Genéticos/efeitos dos fármacos , Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica , Oxirredutases Intramoleculares/genética , Íntrons/genética , Íntrons/imunologia , Fatores Inibidores da Migração de Macrófagos/genética , Plicamicina/farmacologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Fator de Transcrição Sp1/imunologia , Fator de Transcrição Sp3/imunologiaRESUMO
In the macrophage, toll-like receptors (TLRs) are key sensors that trigger signaling cascades to activate inflammatory programs via the NF-κB gene network. However, the genomic network targeted by TLR/NF-κB activation and the molecular basis by which it is restrained to terminate activation and re-establish quiescence is poorly understood. Here, using chromatin immunoprecipitation sequencing (ChIP-seq), we define the NF-κB cistrome, which is comprised of 31,070 cis-acting binding sites responsive to lipopolysaccharide (LPS)-induced signaling. In addition, we demonstrate that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome, and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6-deficient macrophages are acutely hypersensitive to LPS and, using comparative ChIP-seq analyses, we found that the Bcl-6 and NF-κB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6-binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Macrófagos/imunologia , NF-kappa B/genética , Animais , Sítios de Ligação , Células Cultivadas , Epigênese Genética , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6 , Receptor 4 Toll-Like/genéticaRESUMO
CD8(+) T lymphocytes mediate the immune response to viruses, intracellular bacteria, protozoan parasites, and tumors. We provide evidence that the transcription factor Bcl11b/Ctip2 controls hallmark features of CD8(+) T cell immunity, specifically antigen (Ag)-dependent clonal expansion and cytolytic activity. The reduced clonal expansion in the absence of Bcl11b was caused by altered proliferation during the expansion phase, with survival remaining unaffected. Two genes with critical roles in TCR signaling were deregulated in Bcl11b-deficient CD8(+) T cells, CD8 coreceptor and Plcgamma1, both of which may contribute to the impaired responsiveness. Bcl11b was found to bind the E8I, E8IV, and E8V, but not E8II or E8III, enhancers. Thus, Bcl11b is one of the transcription factors implicated in the maintenance of optimal CD8 coreceptor expression in peripheral CD8(+) T cells through association with specific enhancers. Short-lived Klrg1(hi)CD127(lo) effector CD8(+) T cells were formed during the course of infection in the absence of Bcl11b, albeit in smaller numbers, and their Ag-specific cytolytic activity on a per-cell basis was altered, which was associated with reduced granzyme B and perforin.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Ativação Linfocitária/imunologia , Proteínas Repressoras/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transferência Adotiva , Animais , Apresentação de Antígeno/imunologia , Apoptose/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/genética , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Proliferação de Células , Células Clonais/citologia , Células Clonais/imunologia , Células Clonais/metabolismo , Elementos Facilitadores Genéticos/genética , Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica/fisiologia , Granzimas/genética , Granzimas/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Orthomyxoviridae/imunologia , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Fosfolipase C gama/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ligação Proteica/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Proteínas Supressoras de Tumor/genética , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1(-/-) fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages.