A 33 kDa Pit-1-like protein binds to the distal region of the human thyrotrophin alpha-subunit gene.
Our previous studies demonstrated that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (-151 to -135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (-223 to -190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA-protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.
Kim
D S
DS
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Republic of Korea.
Yoon
J H
JH
Ahn
S K
SK
Kim
K E
KE
Seong
R H
RH
Hong
S H
SH
Kim
K
K
Ryu
K
K
Park
S D
SD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010911:Pituitary Neoplasms / Q000473:pathology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D013972:Thyrotropin / Q000235:genetics*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D013973:Thyrotropin-Releasing Hormone; D013972:Thyrotropin; D004247:DNA