Multiple binding sites for nuclear factors in the 5'-upstream region of two alpha 2u-globulin genes: implications for hormone-regulated and tissue-specific control.

In order to understand the tissue- and hormone-specific control of alpha 2u-globulin synthesis we isolated the 5'-upstream putative regulatory region of two alpha 2u-globulin genes: RAP 01 and RAO 01. Both clones seem to be expressed in rat liver. DNAseI footprinting analysis after incubation with rat liver nuclear extracts was used to identify regions of potential interest. Particular attention was paid to protected regions located in the neighbourhood of domains which, according to our previous studies, interact specifically with androgen- and glucocorticoid-receptor complexes. Fifteen DNAseI footprints could be mapped in clone RAO 01 (bp -758 up to the cap site). Nineteen footprints were observed in the corresponding region of RAP 01. Differences in the footprinting patterns were mainly observed in the more distal regions. Our data confirm the presence in both clones of two binding sites for the liver enriched factor pseudo-NF1 and one site for C/EBP previously observed in other alpha 2u-globulin genes. In addition we have been able to demonstrate, in RAP 01 only, a binding site for transferrin-liver factor 1. No differences in footprinting patterns could be demonstrated using liver nuclear extracts derived from animals with a high hepatic expression of alpha 2u-globulins (normal male rats) and animals with low to absent expression (prepubertal rats, female rats, rats with the testicular feminization syndrome, diabetic rats and hypophysectomized animals). Transfection experiments indicate that a fragment of RAP 01 (bp -643 up to -617) is able to act as a glucocorticoid and as an androgen response element. Larger fragments of RAP 01 and fragments of RAO 01 are ineffective. It is concluded that the expression of individual alpha 2u-globulin genes is probably the result of combinatorial interactions of several trans-acting factors with appropriate cis-acting elements. Moreover, important sites for tissue-specific and hormone-regulated expression may be situated outside the regions investigated.