Repression of basal transcription by vitamin D receptor:: evidence for interaction of unliganded vitamin D receptor with two receptor interaction domains in RIP13Δ1

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) promoter construct was observed in kidney cells in the absence of ligand 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 that strongly bound to the VDRE motif in the CYP24 promoter ablated basal repression. This VDR isoform lacked sequence in the hinge- and ligand-binding domains implicating one or both of these domains in basal repression. It is well known that thyroid hormone and retinoic acid receptors silence basal transcription of target genes in the absence of ligands and this repressor function can be mediated by the nuclear receptor corepressor IV-CoR. Two variants of N-CoR have been described, RIP13a and RIP13 Delta 1. N-CoR and the variants contain two receptor interaction domains, ID-I and ID-II, which are identical except region ID-II in RIP13 Delta 1 has an internal deletion. We have used the mammalian two hybrid system to investigate whether VDR, in the absence of ligand 1,25-(OH)(2)D-3, can interact with these domains. The data showed 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 of RIP13 Delta 1 in kidney cell lines did not alter basal expression of the CYP24 promoter construct. It is concluded that either the level of endogenous RIP13 Delta 1 in these kidney cells permits maximal repression or that repression occurs by a mechanism that is independent of RIP13 Delta 1. Alternatively, repression may be dependent on RIP13 Delta 1 but requires an additional cofactor that is limiting in these cells.