title: Identification and analysis of prophet of Pit-1-binding sites in human Pit-1 gene.
abstract: 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.
authors.affiliation: Department of Basic Allied Medicine, Kobe University School of Medicine, Kobe, Japan.
authors.forename: Nobuko
authors.initials: N
authors.lastname: Ikeshita
authors.forename: Mayuko
authors.initials: M
authors.lastname: Kawagishi
authors.forename: Hiromi
authors.initials: H
authors.lastname: Shibahara
authors.forename: Keizo
authors.initials: K
authors.lastname: Toda
authors.forename: Tomoe
authors.initials: T
authors.lastname: Yamashita
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamamoto
authors.forename: Yuka
authors.initials: Y
authors.lastname: Sugiyama
authors.forename: Genzo
authors.initials: G
authors.lastname: Iguchi
authors.forename: Keiji
authors.initials: K
authors.lastname: Iida
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Hidesuke
authors.initials: H
authors.lastname: Kaji
authors.forename: Kazuo
authors.initials: K
authors.lastname: Chihara
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Okimura
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017930:Genes, Reporter; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D050817:Transcription Factor Pit-1 / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D018398:Homeodomain Proteins; C102134:Prophet of Pit-1 protein; D050817:Transcription Factor Pit-1