ACTIVATION OF THE MURINE THYROTROPIN BETA-SUBUNIT PROMOTER BY GH4 RAT PITUITARY CELL-FREE-EXTRACTS

Expression of the TSH-beta subunit gene is restricted to the thyrotroph cells of the anterior pituitary. Previously we identified several AT-rich DNA elements within the murine (m) TSH-beta 5'-flanking region, denoted as D1 (-253 to -227), P4 (-142 to -131), P3 (-126 to -112), P2 (-106 to -98), and P1 (-76 to -68) which bind thyrotroph-specific factor(s). These sites are related to, but distinct from GHF-1 and LSF-1-binding sites, which restrict GH and PRL gene expression to pituitary somatotrophs and lactotrophs, respectively. To determine whether different pituitary cell types contain related factors capable of activating the mTSH-beta promoter, cell-free transcription studies were performed using extracts from GH4 rat pituitary somatomommotroph cells. Although the endogenous mTSH-beta gene is not expressed in GH4 cells, in vitro transcription of the mTSH-beta promoter, normalized to the Rous sarcoma virus internal control, revealed faithful transcription initiation from the authentic mTSH-beta-CAP sites in GH4 but not in HeLa cell extracts. Cell-free transcription analysis of mTSH-beta 5'-deletion mutants revealed consistent promoter activity with deletion to position -46 but complete loss of activity when deleted to position -9. To better define the specific factors in pituitary somatomammotrophs which interact with and activate the mTSH-beta promoter, DNase I protection and gel-shift studies were performed using extracts from GC rat pituitary somatomammotroph cells and DNA affinity-purified lactotroph-specific transcription factor, LSF-1, required for rat PRL promotor activity, and purified from GC cells. These cells contain a factor(s) which binds to thyrotroph-specific elements of the mTSH-beta promoter. These studies also show that LSF-1 binds the D1 and proximal thyrotroph-specific elements of the mTSH-beta activation of the mTSH-beta promoter in HeLa nonpituitary cell extracts in vitro. Conversely, nuclear factors present in TtT-97 murine thyrotrophs bind the proximal lactotroph-specific elements on the rPRL promoter. This in vitro transcription assay provides a means to biochemically dissect the trans-activation of the mTSH-beta promoter and to determine the functional overlap of distinct pituitary cell-specific factors in regulating GH, PRL, and TSH-beta gene expression (Molecular Endocrinology 4: 1887-1896, 1990)