RESUMO
Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE-Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/fisiologia , Loci Gênicos/fisiologia , Proteínas de Homeodomínio/biossíntese , Timócitos/metabolismo , Transcrição Gênica/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Antígenos CD4/genética , Antígenos CD4/metabolismo , Antígenos CD8/genética , Antígenos CD8/metabolismo , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/genética , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Camundongos , Elementos de Resposta/fisiologia , Timócitos/citologiaRESUMO
Tcrb locus V(D)J recombination is regulated by positioning at the nuclear periphery. Here, we used DamID to profile Tcrb locus interactions with the nuclear lamina at high resolution. We identified a lamina-associated domain (LAD) border composed of several CTCF-binding elements that segregates active non-LAD from inactive LAD regions of the locus. Deletion of the LAD border causes an enhancer-dependent spread of histone H3 lysine 27 acetylation from the active recombination center into recombination center-proximal LAD chromatin. This is associated with a disruption to nuclear lamina association, increased chromatin looping to the recombination center, and increased transcription and recombination of recombination center-proximal gene segments. Our results show that a LAD and LAD border are critical components of Tcrb locus gene regulation and suggest that LAD borders may generally function to constrain the activity of nearby enhancers.
