ABSTRACT
The transcription factor Pit-1/GHF-1 plays an important role in regulating the prolactin (Prl) and growth hormone (GH) genes in mammals. In this study, the role that Pit-1 plays in regulating the prolactin and growth hormone genes in avian species was examined by cotransfection assays and immunofluorescence staining of pituitary sections. In cotransfection assays, turkey Pit-1 activated the turkey Prl, turkey GH, and rat Prl promoters 3.8-, 3.7-, and 12.5-fold, respectively. This activation was comparable to rat Pit-1 activation of these same promoters. A point mutation in the turkey Pit-1 cDNA, which changed leu-219 to ser-219, resulted in a 2-, 2-, and 10-fold reduction in the activation of the turkey Prl, turkey GH, and rat Prl promoters, respectively. Unexpectedly, coexpression of tPit-1 (leu-219) and tPit-1(ser-219) activated turkey Prl and rat Prl promoters 9.4- and 35.9-fold, respectively, but had no effect on the turkey GH promoter. Dual-label immunofluorescence analysis of turkey pituitary sections revealed that Pit-1 was not detectable in prolactin-staining cells but was detectable in GH-staining cells. Taken together, these data indicate that in the domestic turkey, Pit-1 can activate the turkey Prl promoter in vitro, but does not appear to play a role in regulating Prl gene expression in vivo. Pit-1, however, still likely plays a role in regulating GH gene expression.
Subject(s)
DNA-Binding Proteins/pharmacology , Growth Hormone/genetics , Pituitary Gland/chemistry , Prolactin/genetics , Promoter Regions, Genetic , Transcription Factors/pharmacology , Turkeys/genetics , 5' Flanking Region , Animals , Base Sequence , Cell Line , Cell Nucleus/chemistry , Cloning, Molecular , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique , Gene Expression Regulation/drug effects , Genetic Vectors , Growth Hormone/analysis , HeLa Cells , Humans , Mice , Molecular Sequence Data , Pituitary Gland/metabolism , Pituitary Gland/ultrastructure , Prolactin/analysis , Rats , Transcription Factor Pit-1 , Transcription Factors/analysis , Transcription Factors/genetics , TransfectionABSTRACT
The gene encoding turkey Pit-1/GHF-1 (tPit-1) spans approximately 12 kilobases (kb) and consists of 7 exons. One exon, which is located between exons 2 and 3, is designated exon 2a and codes for 38 amino acids not found in mammalian Pit-1. Because all tPit-1 variants contain exon 2a, they are denoted with an asterisk (*) to distinguish them from comparable mammalian Pit-1s. Three tPit-1 variants are generated by alternative splicing and transcription initiation. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1beta* relative to tPit-1*. A transcript unique to the turkey has been identified by RT-PCR and RNase mapping. This transcript, designated tPit-1W*, arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. In turkey pituitary, the mRNA for the tPit-1* variant is the most abundant, the tPit-1W* variant is intermediate, and the tPit-1beta* variant is the least abundant.
