Minucci S, Zand D J, Dey A, Marks M S, Nagata T, Grippo J F, Ozato K
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Mol Cell Biol. 1994 Jan;14(1):360-72. doi: 10.1128/mcb.14.1.360-372.1994.
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.
维甲酸X受体（RXRs）与多种核激素受体形成异源二聚体，并被认为具有多效性功能。为了研究RXRs在维甲酸（RA）介导的基因调控中的作用，我们设计了一种显性负性RXRβ。这种突变受体称为DBD-，缺乏DNA结合结构域，但保留了与伴侣受体二聚化的能力，导致形成无功能的二聚体。将DBD-转染到P19小鼠胚胎癌细胞（EC）中，其中含有RA反应元件（RAREs）的报告基因通过内源性RXR-RA受体（RAR）异源二聚体的活性被RA激活。我们发现DBD-对这些细胞中的RARE报告基因活性具有显性负性作用。建立了稳定表达DBD-的P19克隆；这些克隆也不能响应RA激活RARE驱动的报告基因。此外，这些细胞在RA诱导的Hox-1.3和RARβ mRNA表达以及RA诱导的Oct3 mRNA下调方面存在缺陷。凝胶迁移率变动分析表明，RA处理对照P19细胞可诱导RARE结合活性，其中RXRβ是主要成分。然而，在表达DBD-的细胞中，RA诱导的结合活性大大降低。通过基因组足迹分析，我们表明RA处理可诱导对照P19细胞内源性RARβ基因中RARE的体内占据，但在DBD-细胞中未观察到这种占据。这些数据提供了证据，表明DBD-的显性负性作用是由于缺乏受体与靶DNA的结合。最后，我们表明在F9 EC细胞中DBD-的表达导致抑制RA诱导的分化所伴随的生长停滞。综上所述，这些结果表明RXRβ和伴侣受体在RA介导的基因调控以及EC细胞的生长和分化控制中起核心作用。
