Trout GH promoter analysis reveals a modular pattern of regulation consistent with the diversification of GH gene control and function in vertebrates.
In vertebrates, growth hormone (GH) gene expression requires the pituitary-specific transcription factor Pit-1/GHF1 but is differently regulated by a variety of factors in different vertebrate species. Here, we have studied the transcriptional activity of the trout GH (tGH) promoter, which is synergistically stimulated by cAMP and glucocorticoid. Gel shift assays indicated that Pit-1 binds as a dimer to three high affinity sites in the -226/+24 tGH region, and that recombinant cAMP response element (CRE)-binding protein (CREB) binds to a CRE situated between the two distal Pit-1 sites. Deletional and mutational transfection experiments, performed in pituitary Pit-1-expressing GC cells, showed that the different Pit-1 sites play distinct roles and are obligatory elements in the mechanisms mediating cAMP and glucocorticoid responses. Remarkably, the results suggest a hierarchical modular model of regulation of the tGH promoter, according to which a critical module, triggered by Pit-1 bound to the proximal Pit-1 site, is necessary and sufficient to turn on and drive basal levels of transcription. The latter may be stimulated synergistically by two Pit-1-dependent reciprocally non-cooperative auxiliary modules, activated by cAMP and glucocorticoid, respectively. Such modularity explains, in evolutionary terms, the crucial role played by Pit-1 in transcriptional activation and the emergence of the wide variety of mechanisms regulating transcriptional levels of GH, prolactin and other Pit-1-target genes in vertebrates.
Argenton
Francesco
F
Dipartimento di Biologia, Università degli Studi di Padova, Università di Padova, Via Ugo Bassi 58/B, 35131, Padova, Italy.
Vianello
Silvia
S
Bernardini
Serena
S
Lopreiato
Raffaele
R
Colombo
Lorenzo
L
Bortolussi
Marino
M
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000378:metabolism; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; C494187:POU1F1 protein, human; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D003907:Dexamethasone; D013006:Growth Hormone; D000242:Cyclic AMP