A 33 KDA PIT-1-LIKE PROTEIN BINDS TO THE DISTAL REGION OF THE HUMAN THYROTROPIN ALPHA-SUBUNIT GENE

Our previous studies demonstrated that at least two DNA regions with u pstream limits between positions -223 to -190 and positions -151 to -1 35 of the human TSH gene are important for transcriptional regulation by TRH in GH(3) 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 -19 0 bp) containing an element similar to the binding site for the pituit ary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear pro tein, in addition to the CRE-binding protein, is involved in the molec ular mechanism of TRH regulation, a gel retardation assay and Southwes tern blot analysis were performed on the distal region with GH(3) cell nuclear extracts. GH(3) extracts generated a distinct DNA-protein com plex that was effectively eliminated in the presence of excess unlabel led DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat pro lactin gene inhibited formation of the complex, but mutation of the Pi t-1 consensus sequence in the distal region did not eliminate the comp lex. In addition, Southwestern experiments showed that a 33 kDa nuclea r protein present in GH(3) cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maxi mal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also r esulted in a loss of binding affinity. Taken together, these data indi cate that the interaction of a pituitary-specific nuclear protein, ide ntical or closely related to Pit-1, with the distal region may be invo lved in the TRH stimulation of human TSH gene expression.