Genomic footprinting of retinoic acid regulated promoters in embryonal carcinoma cells.
Genomic footprinting of retinoic acid regulated promoters in embryonal carcinoma cells.
复制标题
胚胎癌细胞中视黄酸调节启动子的基因组足迹。
作者：
A. Dey;K. Ozato
维甲酸(RA)处理胚胎癌细胞(EC)后，会引起一系列基因调控的改变，导致其分化为不同类型的细胞。在P19 EC细胞中，RA处理刺激RARβ基因的诱导，同时抑制Oct3/4基因的表达。在这里，我们提出了基于二甲基硫酸盐的这两个基因的基因组足迹分析。我们发现，RARβ启动子在RA治疗前没有被占据，但在RA治疗之后，该启动子中的所有调控元件都被占据。另一方面，Oct3/4启动子在RA治疗前在所有已知的元件上都被占据，但在治疗后这种占据被协调地丢失。因此，因子占有率与基因的表达是一致的。这里揭示的因子结合的存在或缺失很可能代表了这些基因在体内调节表达的一种机制。我们的结果证明了基因组足迹在研究体内转录调控事件方面的力量。相比之下，通过体外蛋白质-DNA结合分析，无论RA治疗如何，这两种启动子的因子都存在于这些细胞中。已有研究表明，维甲酸受体(RAR)和维甲酸X受体(RXR)通过异源二聚体在EC细胞中介导RA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的显性阴性突变体的P19细胞中进行了基因组足迹研究。两个这样的突变体，缺乏DNA结合结构域或C末端激活结构域，抑制了RA对这些细胞中RARβ基因的诱导。在这些细胞中，RA诱导的因子占有率在RARβ启动子的所有元件上也明显受到抑制。我们的结果表明，连接的RAR/RXR异源二聚体与RERE结合是其他因子获得其各自启动子元件所必需的。
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.