RESUMO
The CD4 versus CD8 lineage specification of thymocytes is linked to coreceptor expression. The transcription factor MAZR has been identified as an important regulator of Cd8 expression. Here we show that variegated CD8 expression by loss of Cd8 enhancers was reverted in MAZR-deficient mice, which confirms that MAZR negatively regulates the Cd8 loci during the transition to the double-positive (DP) stage. Moreover, loss of MAZR led to partial redirection of major histocompatibility complex (MHC) class I-restricted thymocytes into CD4(+) helper-like T cells, which correlated with derepression of Th-POK, a central transcription factor for helper-lineage development. MAZR bound the silencer of the gene encoding Th-POK, which indicated direct regulation of this locus by MAZR. Thus, MAZR is part of the transcription factor network that regulates the CD8 lineage differentiation of DP thymocytes.
Assuntos
Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Animais , Transplante de Medula Óssea , Antígenos CD4/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/genética , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Transdiferenciação Celular/genética , Transdiferenciação Celular/imunologia , Células Cultivadas , Redes Reguladoras de Genes , Antígenos H-2/genética , Antígenos H-2/metabolismo , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Quimera por Radiação , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Elementos Silenciadores Transcricionais/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Ativação Transcricional/imunologiaRESUMO
CD8 coreceptor expression is dynamically regulated during thymocyte development and is tightly controlled by the activity of at least 5 different cis-regulatory elements. Despite the detailed characterization of the Cd8 loci, the regulation of the complex expression pattern of CD8 cannot be fully explained by the activity of the known Cd8 enhancers. In this study, we revisited the Cd8ab gene complex with bioinformatics and transgenic reporter gene expression approaches to search for additional Cd8 cis-regulatory elements. This led to the identification of an ECR (ECR-4), which in transgenic reporter gene expression assays, directed expression preferentially in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like CD8(+) T cells. ECR-4, designated as Cd8 enhancer E8VI, was bound by Runx/CBFß complexes and Bcl11b, indicating that E8VI is part of the cis-regulatory network that recruits transcription factors to the Cd8ab gene complex in CD8(+) T cells. Transgenic reporter expression was maintained in LCMV-specific CD8(+) T cells upon infection, although short-term, in vitro activation led to a down-regulation of E8VI activity. Finally, E8VI directed transgene expression also in CD8αα(+) DCs but not in CD8αα-expressing IELs. Taken together, we have identified a novel Cd8 enhancer that directs expression in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like and antigen-specific effector/memory CD8(+) T cells and in CD8αα(+) DCs, and thus, our data provide further insight into the cis-regulatory networks that control CD8 expression.
Assuntos
Antígenos CD8/genética , Linfócitos T CD8-Positivos/metabolismo , Sequência Conservada , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Antígenos CD8/biossíntese , Mapeamento Cromossômico , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Cães , Elementos Facilitadores Genéticos , Genes Reporter , Humanos , Receptores de Hialuronatos/análise , Memória Imunológica , Selectina L/análise , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Ratos , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Subpopulações de Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Human articular chondrocytes (HACs) were isolated from cartilage samples of normal joints and cultivated in monolayer for 56 days. Collagen type I, II, IX, aggrecan, and versican expression was quantified by real-time polymerase chain reaction (PCR). The expression of the genes was highly time-dependent and changed over the culture time. Collagen type I was not present at the beginning of the culture and increased 100-fold during the culture time. Collagen type II and IX expression was found during the entire culture period and decreased more than 100-fold. Aggrecan expression was downregulated 100-fold whereas versican expression increased 10 times. Indices of cell differentiation, defined as ratios of collagen type II to I (CII/I), collagen type IX to I (CIX/I), and aggrecan to versican (Agg/Ver), were significantly higher at the beginning of the culture and decreased over the culture period. The CII/I and CIX/I ratios formed three phases, with a plateau at the beginning, followed by a transition phase and a steady state at the end of the culture time, whereas the Agg/Ver ratio declined constantly. The accurate quantitative assessment of extracellular matrix gene expression in samples of chondrocytes taken from monolayer culture can be used to monitor chondrocyte metabolism as well as changes in the cell differentiation status.