REPRESSION OF BASAL TRANSCRIPTION BY VITAMIN-D-RECEPTOR - EVIDENCE FOR INTERACTION OF UNLIGANDED VITAMIN-D-RECEPTOR WITH 2 RECEPTOR INTERACTION DOMAINS IN RIP13-DELTA-1

Repression of basal transcription of a 1,25-dihydroxyvitamin D-3 (1,25 -(OH)(2)D-3) responsive 25-hydroxyvitamin D-3-24-hydroxylase (CYP24) p romoter construct was observed in kidney cells in the absence of ligan d and this repression was dependent on a functional vitamin D response element (VDRE). Basal repression was also seen with a construct where a consensus DR-3-type VDRE was fused to the thymidine kinase promoter . Expression of a dominant negative vitamin D receptor (VDR) isoform t hat strongly bound to the VDRE motif in the CYP24 promoter ablated bas al repression. This VDR isoform lacked sequence in the hinge- and liga nd-binding domains implicating one or both of these domains in basal r epression. It is well known that thyroid hormone and retinoic acid rec eptors silence basal transcription of target genes in the absence of l igands and this repressor function can be mediated by the nuclear rece ptor corepressor IV-CoR. Two variants of N-CoR have been described, RI P13a and RIP13 Delta 1. N-CoR and the variants contain two receptor in teraction domains, ID-I and ID-II, which are identical except region I D-II in RIP13 Delta 1 has an internal deletion. We have used the mamma lian two hybrid system to investigate whether VDR, in the absence of l igand 1,25-(OH)(2)D-3, can interact with these domains. The data showe d that unliganded VDR does not interact with either ID-I or ID-II from RIP13a and RIP13 Delta 1, but does interact strongly with a composite domain of ID-I and ID-II from RIP13 Delta 1 (but not from RIP13a) and this strong interaction is abrogated in the presence of ligand. This finding implicates RIP13 Delta 1 in VDR-dependent basal repression of the promoter constructs under investigation. However, overexpression o f RIP13 Delta 1 in kidney cell lines did not alter basal expression of the CYP24 promoter construct. It is concluded that either the level o f endogenous RIP13 Delta 1 in these kidney cells permits maximal repre ssion or that repression occurs by a mechanism that is independent of RIP13 Delta 1. Alternatively, repression may be dependent on RIP13 Del ta 1 but requires an additional cofactor that is limiting in these cel ls.