Dey A, Minucci S, Ozato K
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Mol Cell Biol. 1994 Dec;14(12):8191-201. doi: 10.1128/mcb.14.12.8191-8201.1994.
Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.
维甲酸（RA）可激活胚胎癌细胞（EC）中RA受体β2（RARβ2）基因的转录。这种激活涉及RAR/类视黄醇X受体（RAR/RXR）异二聚体与RA反应元件（βRARE）的结合。采用基于硫酸二甲酯的基因组足迹法检测P19 EC细胞中该启动子的占据情况。在RA处理前，未在βRARE处检测到足迹，但在RA处理的第一小时内检测到了一个足迹。同时，启动子中的其他元件，即环磷酸腺苷反应元件和十四烷酰佛波醇乙酸酯样反应元件也出现了足迹。这些元件处的足迹由RA诱导，无需新的蛋白质合成，并且在RA处理的整个过程中都存在，但在撤去RA后迅速消失。在起始位点观察到的延迟保护在撤去RA后也消失了。RA诱导的足迹并非由于与这些元件结合的因子的诱导，因为体外试验表明，这些因子在RA处理前就存在于P19细胞提取物中。值得注意的是，在表达显性负性RXRβ的P19细胞中，在这些元件中的任何一个处均未观察到RA诱导的足迹，在这种细胞中，RXR异二聚体无法与βRARE结合。结果表明，配体化的异二聚体受体与βRARE的结合是使其他元件能够接触到这些因子的初始事件。相应地，报告基因分析表明，βRARE中的突变而非其他元件中的突变可消除RA对启动子的激活。RARβ2启动子可能以分级有序的方式开放，由配体化异二聚体的占据发出信号。
