THE ANALYSIS OF REGULATORY SEQUENCES REQUIRED FOR HIGH-LEVEL INDUCTION OF RAT ALPHA(2U)-GLOBULIN GENE-EXPRESSION BY DEXAMETHASONE

The alpha(2u)-globulins are the major urinary proteins in adult male r ats. They are encoded by a gene family, the expression of which is und er multihormonal control in the liver. Glucocorticoids are positive re gulatory 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 tha t sequences from -762 bp to -226 bp of clone 91 are required for the 2 4-fold level of induction that was observed. Addition of 5.5 kb of ups tream sequence beyond -762 bp did not alter the level of induction sig nificantly, whereas deletion of the DNA between -762 bp and -226 bp re duced inducibility to about 4-fold. Sequencing of this region revealed that an element, 5'-AGAACAggtTTCAAA-3', similar to the 15 bp consensu s glucocorticoid response element 5'-AGAACAnnnTGTACC-3', is situated 5 13 bp upstream of the transcription start site. We infer that this ele ment 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 s ite was not inducible. It now appears that at least three distinct cis -acting regulatory regions, all of which bind to the glucocorticoid re ceptor in vitro, may contribute to the full induction of clone 91 by d examethasone. These are: the distal upstream region identified by this study, a proximal upstream region that binds not only the receptor bu t also alpha 2uNF1, constitutively expressed nuclear protein required for induction and a region within the fourth intron that contains five tandem receptor binding sites.