No abstract

The 2u -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 2uNF1, a constitutively expressed nuclear protein required for induction and a region within the fourth intron that contains five tandem receptor binding sites.

Expression of the rat 2u -globulin gene family is regulated in the adult male liver by a number of hormones, including growth hormone, thyroid hormone and several steroids. Upon injection into ovariectomized females, estrogens first induce 2u -globulin expression and then suppress this gene after several days of hormone administration. To study this phenomenon, we developed a mouse L-cell line that expressed the human estrogen receptor. High levels of rat 2u -globulin transcript were induced in stable transfectants of this line carrying a cloned 2u -globulin gene, following exposure to 17 -estradiol. Since this induction was inhibited by cycloheximide, the response to estrogen, as to other steroids, appears to be secondary.Using genes with variously deleted 5 -upstream regions, sequences responsible for this induction were located between 730 bp and 223 bp relative to the start of transcription. Examination of the DNA in this region revealed that an estrogen receptor element was located at 590 bp in an area that is highly conserved in most known 2u -globulin genes. Administration of both dexamethasone and estrogen produced a synergistic effect in this system. The induction of 2u -globulin RNA by estrogen in L-cells may re-capitulate the initial response to estrogen in vivo, and therefore represents a good model system to seek the identity of the other factors required to effect full induction.