RESUMO
POU domain proteins contain a bipartite DNA-binding element that can confer allosteric control of coactivator recruitment. Dimerization of Oct-1 and Oct-2 on palindromic response elements results in the conformational dependent inclusion or exclusion of the transcriptional coactivator OBF-1. In this paper, we demonstrate that Oct-1 and Oct-2 can function as transcriptional repressors by recruiting and physically interacting with members of the Grg/TLE family of corepressors. In accordance with a model of DNA induced cofactor assembly, and analogous to the recruitment of the OBF-1 coactivator, the different Grg/TLE members can discriminate between both Oct-1 and Oct-2, and the monomeric or dimeric nature of the POU/DNA complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta , Fatores de Transcrição/metabolismo , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Evolução Biológica , Linhagem Celular , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Dimerização , Humanos , Fator 1 de Transcrição de Octâmero , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
BACKGROUND AND AIMS: Mutations/polymorphisms in the CARD15/NOD2 gene and in the promoter region of the TNFalpha gene are associated with susceptibility to and modulate the phenotype of Crohn's disease (CD). The molecular mechanisms for this genotype-phenotype correlation are yet to be elucidated. CARD15 is an intracellular receptor for bacterial muramyl dipeptide (MDP), and can elicit an inflammatory response via activation of the NF-kappaB pathway. MDP is also known to induce the expression of pro-inflammatory cytokines including TNFalpha, through a still poorly characterized signaling pathway. We sought to determine whether CARD15-mediated NF-kappaB activation can contribute to MDP-induced TNFalpha production and, consequently, if polymorphisms in both genes affect the control of such induction. METHODS/RESULTS: Transfection and electrophoretic mobility shift assays (EMSA) experiments in HEK293 cells demonstrated that MDP exposure stimulates TNFalpha gene transcription, as a result of CARD15-induced NF-kappaB activation and binding to TNFalpha promoter. When the CD-associated CARD15 1007fs variant was analyzed, induction of TNFalpha promoter activity was found to be defective. Different combinations of CARD15 and TNFalpha promoter polymorphisms gave rise to distinct TNFalpha transcription levels. CONCLUSIONS: CARD15 and TNFalpha promoter polymorphisms interact to exert a functional effect on MDP-induced TNFalpha production. This gene-gene interaction may contribute to interindividual variation in susceptibility to, and manifestation of, Crohn's disease.
Assuntos
Doença de Crohn/genética , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Polimorfismo Genético , Fator de Necrose Tumoral alfa/genética , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Técnicas de Cultura de Células , Doença de Crohn/fisiopatologia , Ensaio de Desvio de Mobilidade Eletroforética , Genótipo , Humanos , Rim/citologia , NF-kappa B/fisiologia , Proteína Adaptadora de Sinalização NOD2 , Fenótipo , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/biossínteseRESUMO
PU.1 and Pax5 are important regulators of immunoglobulin heavy-chain (IgH) gene expression in B lineage cells. We have previously shown that PU.1 can potentiate the transcription of an IgH HS1,2 enhancer-linked reporter gene, and that Pax5 represses the same enhancer in transient transfection assays. Here we report that PU.1, like Pax5, can recruit and physically interact with a member of the Groucho family of co-repressors, Grg4. As a consequence, PU.1 in conjunction with Pax5 represses enhancer function in a position-dependent manner when Grg4 is recruited. Interestingly, Grg4 levels decrease following B-cell activation, suggesting temporal regulation of Grg4. Moreover, the joining-chain promoter, with an activity pattern and architecture resembling HS1,2 can also be repressed by the combinatorial action of Pax5/PU.1/Grg4. These data indicate that Pax5 depends on PU.1, acting in cis, for stable recruitment of Grg co-repressors to B-cell-specific genes.
