DOMINANT-NEGATIVE RETINOID-X RECEPTOR-BETA INHIBITS RETINOIC ACID-RESPONSIVE GENE-REGULATION IN EMBRYONAL CARCINOMA-CELLS

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormo ne receptors and are thought to exert pleiotropic functions. To addres s the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXRbeta. This mutated receptor, termed D BD-, lacked the DNA binding domain but retained the ability to dimeriz e with partner receptors, resulting in formation of nonfunctional dime rs. DBD- was transfected into P19 murine embryonal carcinoma (EC) cell s, in which reporters containing the RA-responsive elements (RAREs) we re activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activit y on the RARE reporter activity in these cells. P19 clones stably expr essing DBD- were established; these clones also failed to activate RAR E-driven reporters in response to RA. Further, these cells were defect ive in RA-induced mRNA expression of Hox-1.3 and RARbeta, as well as i n RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays d emonstrated that RA treatment of control P19 cells induces RARE-bindin g activity, of which RXRbeta is a major component. However, the RA-ind uced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occup ancy of the RARE in the endogenous RARbeta gene in control P19 cells b ut that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is cause d 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 a rrest that accompanies RA-induced differentiation. Taken together, the se results demonstrate that RXRbeta and partner receptors play a centr al role in RA-mediated gene regulation and in the control of growth an d differentiation in EC cells.