RESUMO
The basic helix-loop-helix protein BETA2/NeuroD activates transcription of the secretin gene and is essential for terminal differentiation of secretin-producing enteroendocrine cells. However, in heterodimeric complexes with its partner basic helix-loop-helix proteins, BETA2 does not appear to be a strong activator of transcription by itself. Mutational analysis of a proximal enhancer in the secretin gene identified several cis-acting elements in addition to the E-box binding site for BETA2. We identified by expression cloning the zinc finger protein RREB-1, also known to exist as a longer form, Finb, as the protein binding to one of the mutationally sensitive elements. Finb/RREB-1 lacks an intrinsic activation domain and by itself did not activate secretin gene transcription. Here we show that Finb/RREB-1 can associate with BETA2 to enhance its transcription-activating function. Both DNA binding and physical interaction of Finb/RREB-1 with BETA2 are required to potentiate transcription. Thus, Finb/RREB-1 does not function as a classical activator of transcription that recruits an activation domain to a DNA-protein complex. Finb/RREB-1 may be distinguished from coactivators, which increase transcription without sequence-specific DNA binding. We suggest that Finb/RREB-1 should be considered a potentiator of transcription, representing a distinct category of transcription-regulating proteins.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Secretina/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/metabolismo , Células Cultivadas , Cricetinae , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Sequências Hélice-Alça-Hélice , Humanos , Dados de Sequência Molecular , Mutação , Sequências Reguladoras de Ácido Nucleico , Secretina/metabolismo , Homologia de Sequência de Aminoácidos , Transativadores/química , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Dedos de ZincoRESUMO
Expression of the hormone secretin in enteroendocrine cells is restricted to the nondividing villus compartment of the intestine, implying that terminal differentiation is linked to cell cycle arrest and that differentiation is repressed in actively proliferating cells. We have shown previously that the basic helix-loop-helix protein, BETA2/NeuroD, induces cell cycle withdrawal in addition to increasing secretin gene expression. A number of transcription factors important for differentiation are repressed by D cyclins. Repression by D cyclins appears to be independent of its effects on the cell cycle. We show that cyclin D1 represses BETA2/NeuroD-dependent transcription of the secretin gene. Examination of cyclin box mutants shows that repression is unrelated to Cdk4 activation. Although cyclin D1 and BETA2 associate in vivo, they do not directly interact. Cyclin D1 may be recruited to BETA2 by binding to the C-terminal domain of the p300 coactivator, downstream from the BETA2-binding site. In the small intestine, cyclin D1 expression occurs only in the actively proliferating crypts of Lieberkuhn but not in villi. Thus repression by cyclin D1 may serve to prevent secretin gene transcription from occurring in relatively immature epithelial progenitor cells.
