The analysis of regulatory sequences required for high level induction of rat alpha 2u-globulin gene expression by dexamethasone

The a2u-globulins are the major urinary proteins in adult male rats. They are encoded by a gene family, the expression of which is under multihormonal control in the liver. Glucocorticoids are positive regulatory hormones and we have analyzed the contribution of 5*-upstream sequences to the induction by dexamethasone of two cloned members of the family transfected into mouse L-cells. The results demonstrate that sequences from "762 bp to "226 bp of clone 91 are required for the 24-fold level of induction that was observed. Addition of 5·5 kb of upstream sequence beyond "762 bp did not alter the level of induction significantly, whereas deletion of the DNA between "762 bp and "226 bp reduced inducibility to about 4-fold. Sequencing of this region revealed that an element, 5*-AGAACAggtTTCAAA-3*, similar to the 15 bp consensus glucocorticoid response element 5*-AGAACAnnnTGTACC-3*, is situated 513 bp upstream of the transcription start site. We infer that this element or its left half site is necessary for the dexamethasone-induced expression of clone 91 from the observation that a second gene, clone 2, that contained a base substitution at position 5 in the left half site was not inducible. It now appears that at least three distinct cis-acting regulatory regions, all of which bind to the glucocorticoid receptor in vitro, may contribute to the full induction of clone 91 by dexamethasone. These are: the distal upstream region identified by this study, a proximal upstream region that binds not only the receptor but also a2uNF1, a constitutively expressed nuclear protein required for induction and a region within the fourth intron that contains five tandem receptor binding sites. Journal of Molecular Endocrinology (1998) 20, 129–139