Cloning and characterization of gonadotropin-inducible ovarian transcription factors (GIOT1 and -2) that are novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family.
Gonadotropins are essential for ovarian follicular development and differentiation. To identify genes that are rapidly induced by gonadotropin in the immature rat ovary, ovarian genes were screened by a subtraction cloning procedure. cDNA clones encoding novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family GIOT1 and -2 (gonadotropin-inducible transcription factor 1 and 2), were identified. Two isoforms of GIOT2 (GIOT2 alpha and 2 beta), which are probably produced by alternative splicing, also exist. Nucleotide sequence analysis revealed that GIOT1, but not GIOT2, contains the krüppel-associated box-A domain at the NH(2) terminus. RNA analyses revealed that these mRNAs were rapidly and temporarily induced by gonadotropins in the rat testis as well as in the ovary. In situ hybridization study revealed that expression of GIOT1 was induced in theca interna cells in the ovary and Leydig cells in the testis. Interestingly, the gene expression of GIOT1 is restricted to the pituitary, adrenal, testis, and ovary, while GIOT2 gene is expressed ubiquitously. A functional analysis of GIOT1 and -2 by a GAL4-based mammalian one-hybrid system revealed that GIOT1, but not GIOT2, is a transcriptional repressor and that the krüppel-associated box-A domain of GIOT1 is responsible for the transcriptional repressor activity. A GAL4-based yeast two-hybrid system was also used to identify proteins that interact with the rat GIOT1. We cloned genes encoding rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta, both of which are transcription-regulatory proteins. Interaction of these proteins with GIOT1 was directly demonstrated by GST pull-down assay. Our data strongly suggest that GIOT1 may function as a novel transcriptional repressor by working with rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta proteins and may play a significant role at the transcription level in the folliculogenesis.
Mizutani
T
T
Department of Biochemistry, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan.
Yamada
K
K
Yazawa
T
T
Okada
T
T
Minegishi
T
T
Miyamoto
K
K
D016475:3T3 Cells; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004268:DNA-Binding Proteins; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression / Q000187:drug effects; D006064:Gonadotropins, Equine / Q000494:pharmacology; D006107:Granulosa Cells / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D017403:In Situ Hybridization; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014018:Tissue Distribution; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D014162:Transfection; D016335:Zinc Fingers
D006063:Chorionic Gonadotropin; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C486721:Giot1 protein, rat; D006064:Gonadotropins, Equine; D020662:Guanine Nucleotide Exchange Factors; C491077:Mdfi protein, mouse; D008565:Membrane Proteins; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins; C105592:RIC1 protein, S cerevisiae; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C447180:Zfp347 protein, rat; C093517:transcriptional intermediary factor 1; D005640:Follicle Stimulating Hormone; D004262:DNA Restriction Enzymes