A NEGATIVE TYROSINE AMINOTRANSFERASE GENE ELEMENT THAT BLOCKS GLUCOCORTICOID MODULATORY ELEMENT-REGULATED MODULATION OF GLUCOCORTICOID-INDUCED GENE-EXPRESSION

Tyrosine aminotransferase (TAT) is the prototypic steroid-inducible ge ne. Recently, we have found that the modulation of TAT induction prope rties is reproduced by a novel cis-acting TAT gene element, the glucoc orticoid modulatory element (GME). This GME lies about 1 kb upstream o f the glucocorticoid response elements (GREs) of the TAT gene and bind s a heterooligomer of two recently defined proteins. We now report the existence of an additional TAT gene element between the GME and the G REs that blocks the action of the GME and thus prevents the left shift in the glucocorticoid dose-response curve caused by the GME. This neg ative element has the properties of a silencer because its activity is relatively position- and orientation-independent. The interaction app ears to be stoichiometric in that the effects of a single negative ele ment can be overcome by a second GME. This negative element also has a n intrinsic inhibitory activity in the absence of the GME. The majorit y of the negative element activity could be elicited by a 56-bp sequen ce between -3105 and -3050 bp of the TAT gene. Multiple, clustered mut ations of this sequence reduced, but did not eliminate, the negative a ctivity. Further efforts to restrict the negative element were unsucce ssful, suggesting that multiple sequences are required for full activi ty. High affinity, sequence-specific binding of a trans-acting factor( s) was observed in gel shift assays. This binding was half-maximally c ompeted by a 4.4-fold excess of nonradioactive probe and was very stab le once formed (Delta H double dagger(dissoc.) = 32 kcal/mol), suggest ing that low concentrations of a high affinity binding protein(s) exis t in nuclear extracts. Further support for this conclusion came from t he observation that cotransfection of a plasmid containing multiple co pies of the 56-bp negative element was able to relieve the negation of GME activity in a GME-56-bp-GRE reporter construct. These data direct ly support the role of a trans-acting factor(s) in binding to the 56-b p negative element and blocking GME activity. Collectively, these data suggest that glucocorticoid induction of TAT gene expression is subje ct to multiple levels of control by several new cis-acting elements an d thus is much more complex than previously appreciated.