RESUMEN
PROP1 mutation causes combined pituitary hormone deficiency (CPHD). Several mutations are located in a transactivation domain (TAD) of Prop1, and the loss of TAD binding to cofactors is likely the cause of CPHD. PROP1 cofactors have not yet been identified. In the present study, we aimed to identify the PROP1-interacting proteins from the human brain cDNA library. Using a yeast two-hybrid assay, we cloned nine candidate proteins that may bind to PROP1. Of those nine candidates, amino-terminal enhancer of split (AES) was the most abundant, and we analyzed the AES function. AES dose-dependently decreased the PROP1-induced Pit-1 reporter gene expression. An immunoprecipitation assay revealed the relationship between AES and PROP1. In a mammalian two-hybrid assay, a leucine zipper-like motif of the AES Q domain was identified as a region that interacted with TAD. These results indicated that AES was a corepressor of PROP1.
Asunto(s)
Proteínas de Homeodominio/genética , Hipopituitarismo/genética , Mutación , Hipófisis/metabolismo , Proteínas Represoras/genética , Factor de Transcripción Pit-1/genética , Sitios de Unión , Encéfalo/metabolismo , Línea Celular Tumoral , Proteínas Co-Represoras , Regulación de la Expresión Génica , Biblioteca de Genes , Genes Reporteros , Proteínas de Homeodominio/metabolismo , Humanos , Hipopituitarismo/metabolismo , Hipopituitarismo/patología , Hipófisis/citología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción Pit-1/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Prophet of Pit-1 (Prop1) is a transcription factor that regulates Pit-1 gene expression. Because Pit-1 regulates the differentiation of pituitary cells and the expressions of GH, prolactin and TSHbeta genes, Prop1 mutation results in combined pituitary hormone deficiency in humans. However, Prop1-binding sites in human Pit-1 gene and the mechanism leading to combined pituitary hormone deficiency have remained unclear. In this study, we identified and analyzed Prop1-binding elements of the human Pit-1 gene. Prop1 stimulated the expression of the reporter plasmid containing Pit-1 gene from translation start site to -1340 dose dependently in GH3 cells. The activation by Prop1 was observed in GH3 and TtT/GF cells but not COS7, HeLa, JEG3, and HuH7 cells. Deletion analysis of Pit-1 gene showed that the Prop1-responsive elements were present within the -257-bp region. Within the -257-bp region, there are four elements similar to consensus sequence of paired-like transcription factors. Because Prop1 is a member of paired-like transcription factors, we assessed the elements. EMSA and transient transfection assay using the mutation of the elements revealed that the element from -63 to -53 (the proximal Prop1 binding element) was essential to Prop1-binding and Prop1-induced activation of Pit-1 reporter plasmid. A region at -8kb of human Pit-1 gene is similar to the distal region containing Prop1-binding elements in mouse Pit-1 gene. We showed the region functioned as an enhancer. Furthermore, chromatin immunoprecipitation assay showed that the proximal element could bind Prop1 in vivo cultured cells. Taken together, these findings indicated the novel functioning binding elements of Prop1 in human Pit-1 gene.