Dey A, Ozato K
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Methods. 1997 Feb;11(2):197-204. doi: 10.1006/meth.1996.0406.
Retinoic acid (RA) treatment of embryonal carcinoma (EC) cells initiates a cascade of alterations in gene regulation, leading to their differentiation into various cell types. In P19 EC cells RA treatment stimulates induction of the RAR beta gene, while it represses Oct3/4 gene expression. Here we present dimethylsulfate-based genomic footprinting analyses of these two genes. We found that the RAR beta promoter is not occupied prior to RA treatment, but following RA treatment all regulatory elements in this promoter become occupied. On the other hand, the Oct3/4 promoter is occupied at all three known elements before RA treatment, but this occupancy is coordinately lost following the treatment. Thus, factor occupancy coincides with expression of the genes. It is likely that the presence of factor binding or its absence revealed here represents a mechanism of the regulated expression of these genes in vivo. Our results demonstrate the power of genomic footprinting for studying regulatory events for transcription in vivo. In contrast, with in vitro protein-DNA binding assay, factors for both promoters are present in these cells regardless of RA treatment. It has been shown that RA receptor (RAR) and retinoid X receptor (RXR), by heterodimerization, mediate the RA action in EC cells. To elucidate the role of RAR/RXR heterodimers in the RAR beta promoter occupancy in vivo, genomic footprinting has been performed in P19 cells stably expressing dominant negative mutants of RXR. Two such mutants, lacking either the DNA binding domain or the C-terminal activation domain, inhibit RA induction of the RAR beta gene in these cells. RA-induced factor occupancy is also markedly inhibited at all elements in the RAR beta promoter in these cells. Our results show that binding of liganded RAR/RXR heterodimers to RARE is required for other factors to gain access to their respective elements in the promoter.
视黄酸（RA）处理胚胎癌细胞会引发一系列基因调控变化，使其分化为多种细胞类型。在P19胚胎癌细胞中，RA处理可刺激RARβ基因的诱导，同时抑制Oct3/4基因表达。在此，我们展示了基于硫酸二甲酯的这两个基因的基因组足迹分析。我们发现，在RA处理前，RARβ启动子未被占据，但在RA处理后，该启动子中的所有调控元件均被占据。另一方面，Oct3/4启动子在RA处理前在所有三个已知元件处被占据，但处理后这种占据情况协同丧失。因此，因子占据与基因表达一致。此处揭示的因子结合的存在或缺失可能代表了这些基因在体内调控表达的一种机制。我们的结果证明了基因组足迹法在研究体内转录调控事件方面的强大作用。相比之下，在体外蛋白质-DNA结合试验中，无论RA处理与否，这两个启动子的因子在这些细胞中均存在。已经表明，视黄酸受体（RAR）和类视黄醇X受体（RXR）通过异源二聚化介导RA在胚胎癌细胞中的作用。为了阐明RAR/RXR异源二聚体在体内RARβ启动子占据中的作用，我们在稳定表达RXR显性负突变体的P19细胞中进行了基因组足迹分析。两个这样的突变体，一个缺失DNA结合结构域，另一个缺失C端激活结构域，均可抑制这些细胞中RA诱导的RARβ基因表达。在这些细胞的RARβ启动子的所有元件处，RA诱导的因子占据也受到明显抑制。我们的结果表明，配体化的RAR/RXR异源二聚体与视黄酸反应元件（RARE）的结合是其他因子进入启动子中各自元件的必要条件。
