A PROP1-binding factor, AES cloned by yeast two-hybrid assay represses PROP1-induced Pit-1 gene expression

• Published in: Molecular and cellular endocrinology 2013-08, Vol.376 (1-2), p.93-98
• Main Authors: Sugiyama, Yuka, Ikeshita, Nobuko, Shibahara, Hiromi, Yamamoto, Daisuke, Kawagishi, Mayuko, Iguchi, Genzo, Iida, Keiji, Takahashi, Yutaka, Kaji, Hidesuke, Chihara, Kazuo, Okimura, Yasuhiko
• Format: Article
• Language: English
• Subjects: AES; Assaying; Binding Sites; Brain; Brain - metabolism; cDNA libraries; Cell Line, Tumor; CPHD; Gene expression; Gene Expression Regulation; Gene Library; Genes, Reporter; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Human; Humans; Hypopituitarism - genetics; Hypopituitarism - metabolism; Hypopituitarism - pathology; Leucine; Mutation; Mutations; Pit-1; Pituitary Gland - cytology; Pituitary Gland - metabolism; precipitin tests; PROP1; Protein Binding; Protein Interaction Domains and Motifs; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; reporter genes; Repressor Proteins - genetics; Repressor Proteins - metabolism; TLE1; Transcription Factor Pit-1 - genetics; Transcription Factor Pit-1 - metabolism; transcriptional activation; Two-hybrid assay; Two-Hybrid System Techniques; Yeast
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/j.mce.2013.05.022

•Nine Prop1-interacting protein candidates were cloned using a yeast two-hybrid assay.•Amino-terminal enhancer of split (AES) was the most abundant of the candidates.•AES suppressed the Prop1-induced Pit-1 gene expression.•AES was associated with Prop1 in an immunoprecipitation assay. 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.