HEPATIC NUCLEAR PROTEINS THAT BIND CIS-REGULATORY ELEMENTS IN THE PROXIMAL PROMOTER OF THE RAT CORTICOSTEROID-BINDING GLOBULIN GENE

The plasma transport protein for glucocorticoids, corticosteroid-bindi ng globulin (CBG), is produced by hepatocytes, and expression of the C BG gene (Cbg) in the liver is controlled by a variety of hormones, env ironmental stimuli, and developmental cues. The rat Cbg proximal promo ter, including 145 base pairs (bp) from the transcription start site, contains two cis-regulatory elements (designated P1 and P2), and is as transcriptionally active as a much more extended region (similar to 1 .2 kbp) of the promoter. We have now characterized the rat liver nucle ar proteins that interact with P1 and P2, Several proteins interacted specifically with P1 during an electrophoretic mobility shift assay (E MSA), and based on ultraviolet (UV) cross-linking and Southwestern blo t analyses, as well as an antibody-supershifting EMSA, one of these ha s been identified as hepatic nuclear factor (HNF)1 beta. The major ban d shift formed with P2 in an EMSA appears to comprise a protein that m igrates as a doublet of 58 and 62 KDa on sodium dodecylsulfate-polyacr ylamide gel ectrophoresis (SDS-PAGE) after UV cross-linking with an ol igonucleotide containing P2, as well as during Southwestern blot analy ses. Mutations of the CCAAT sequence within P2 also prevent binding to this protein, the physicochemical properties of which resemble the CC AAT-binding protein CP2. Functional analyses of this region of the rat Cbg proximal promoter fused to a luciferase reporter gene demonstrate d that mutations within P1, which prevent its interaction with NHF1, d o not influence adversely its transcriptional activity. Thus, although members of the HNF1 family of nuclear proteins play an essential role in the transcriptional activation of several other related genes (e.g ., thyroxin-binding globulin and alpha1-antitrypsin) in hepatocytes, H NF1 beta does not appear to be required for the basal activity of the rat Cbg promoter. In addition, deletion of P2 from the proximal promot er abolishes transcriptional activity and the CCAAT-binding protein th at interacts with P2 probably represents an important determinant of C bg expression under different physiological conditions. (C) 1997 Elsev ier Science Ireland Ltd.