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Characterisation of tissue-specific trans-acting factor binding to a proximal element in the rat growth hormone gene
Abstract:
Using an exonuclease III protection assay, tissue-specific binding of rat pituitary tumour cell (GH3 cell) nuclear factors to a proximal region (-68 to -138) of the rat growth hormone gene promoter has been detected. The binding is particularly strong between the borders -68 to -102. The binding is eliminated in the presence of excess unlabelled rat growth hormone gene promoter sequences but also by proximal (-423 to +38) or distal (-1960 to -1260) rat prolactin gene promoter sequences and simian virus 40 enhancer/promoter sequences. Extracts of rat pituitaries showed identical binding characteristics. Methylation interference analysis indicated that the contact points between the pituitary-specific factor and the proximal rat growth hormone gene promoter-binding element (-65 to -95) are over a conserved sequence which occurs twice in the rat growth hormone gene promoter and at least eight times in the rat prolactin gene 5'-flanking sequences. This sequence has previously been proposed to constitute the binding site for the somatotroph/lactotroph tissue-specific transcription factor. Gel-retardation and exonuclease III competition analysis showed that three of the rat prolactin gene promoter elements (-46 to -71, -156 to -180 and -174 to -204) share the ability to bind the pituitary-specific factor. The binding to the most proximal rat prolactin gene promoter element (-46 to -71) was clearly more avid than to the rat growth hormone gene promoter (-65 to -95) proximal element. However, both these elements displayed the formation of two gel-retarded complexes while the more distal rat prolactin gene binding elements (-156 to -180 and -174 to -204) formed only the smaller of the two complexes. Finally, we demonstrated by co-transfection competition analysis that plasmids containing the most proximal rat prolactin gene promoter binding element completely inhibited transcription from the rat growth hormone gene promoter while rat growth hormone gene promoter sequences only partially inhibited transcription from the rat prolactin gene promoter. This suggests that the higher affinity for factor binding displayed by the proximal rat prolactin gene promoter binding site in vitro is reflected in factor binding activity in vivo.