Folkers G E, van der Burg B, van der Saag P T
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CH Utrecht, The Netherlands.
J Biol Chem. 1998 Nov 27;273(48):32200-12. doi: 10.1074/jbc.273.48.32200.
Expression of retinoic acid receptor beta (RARbeta) is spatially and temporally restricted during embryonal development. Also during retinoic acid (RA)-dependent embryonal carcinoma (EC) cell differentiation, RARbeta expression is initially up-regulated, while in later phases of differentiation expression is down-regulated, by an unknown mechanism. To gain insight into the regulation of RARbeta, we studied the activity of the RARbeta2 promoter and mutants thereof in various cell lines. While the RARbeta2 promoter is activated by RA in a limited number of cell lines, synthetic RA-responsive reporters are activated in most cell types. We show that the expression levels of proteins that bind to the beta-retinoic acid response element (RAR/retinoid X receptors and orphan receptors) and also the differential expression of a number of coactivators modulate the RA response on both natural and synthetic reporters. We further show that cell type-specific activation of the RARbeta2 promoter is dependent on the promoter architecture including the spacing between retinoic acid response element and TATA-box and initiator sequence (betaINR). Mutation within these regions caused a decrease in the activity of this promoter in responsive EC cells, while an increase in activity in non-EC cell lines was observed. Cell-specific complexes were formed on the betaINR, suggesting that the betaINR contributes to cell-specific activation of the promoter. On this basis we propose that promoter context-dependent and more general RA response-determining mechanisms contribute to cell-specific RA-dependent activation of transcription.
维甲酸受体β（RARβ）的表达在胚胎发育过程中受到时空限制。同样，在依赖维甲酸（RA）的胚胎癌细胞（EC）分化过程中，RARβ的表达最初会上调，而在分化后期，其表达会通过未知机制下调。为了深入了解RARβ的调控机制，我们研究了RARβ2启动子及其突变体在各种细胞系中的活性。虽然RARβ2启动子在有限数量的细胞系中被RA激活，但合成的RA反应性报告基因在大多数细胞类型中都被激活。我们发现，与β-维甲酸反应元件结合的蛋白质（RAR/维甲酸X受体和孤儿受体）的表达水平，以及一些共激活因子的差异表达，会调节天然和合成报告基因对RA的反应。我们进一步表明，RARβ2启动子的细胞类型特异性激活取决于启动子结构，包括维甲酸反应元件与TATA盒和起始子序列（βINR）之间的间距。这些区域内的突变导致该启动子在反应性EC细胞中的活性降低，而在非EC细胞系中则观察到活性增加。在βINR上形成了细胞特异性复合物，这表明βINR有助于启动子的细胞特异性激活。在此基础上，我们提出启动子背景依赖性和更普遍的RA反应决定机制有助于转录的细胞特异性RA依赖性激活。
