Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.     

Positive and negative regulation of basal expression of a yeast HSP70 gene.

The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-lower level of basal expression and altered kinetics of expression after heat shock. A series of deletion and point mutations led to identification of an upstream repression sequence (URS) which overlapped HSE2. A promoter containing a mutation in the URS showed an increased level of basal expression. A URS-binding activity was detected in yeast whole-cell extracts by a gel electrophoresis DNA-binding assay. The results reported in this paper indicate that basal expression of the SSA1 promoter is determined by both positive and negative elements and imply that the positively acting yeast heat shock factor HSF is responsible, at least in part, for the basal level of expression of SSA1.