Yang Ying, Wolf Louise V, Cvekl Ales
Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY10461, USA.
J Mol Biol. 2007 Jun 15;369(4):917-26. doi: 10.1016/j.jmb.2007.04.004. Epub 2007 Apr 5.
Mouse alphaA-crystallin gene encodes the most abundant protein of the mammalian lens. Expression of alphaA-crystallin is regulated temporally and spatially during lens development with initial expression in the lens vesicle followed by strong upregulation in the differentiating primary lens fibers. Lens-specific expression of alphaA-crystallin is mediated by DNA-binding transcription factors Pax6, c-Maf and CREB bound to its promoter region. Its 5'-distal enhancer, DCR1, mediates regulation of alphaA-crystallin via FGF signaling, while its 3'-distal enhancer, DCR3, functions only in elongated primary lens fibers via other lens differentiation pathways. DCR1 and DCR3 establish outside borders of a lens-specific chromatin region marked by histone H3 K9 acetylation. Here, we identified CREB-binding protein (CBP) and p300 as major histone acetyltransferases (HATs) associated in vivo with the mouse alphaA-crystallin locus. Both HATs are expressed in embryonic lens. Expression of CBP in primary lens fiber cells coincides with alphaA-crystallin. In the chromatin of lens epithelial cells, chromatin immunoprecipitations (ChIPs) show that the alphaA-crystallin promoter is notably devoid of any significant presence of CBP and p300, though DCR1 and a few other regions show the presence of these HATs. In the chromatin obtained from newborn lens, CBP was localized specifically at the promoter region with about ten times higher abundance compared to the entire alphaA-crystallin locus. In contrast, p300 is distributed more evenly across the entire locus. Analysis of total histone H3 and H3 K9 acetylation revealed potential lower density of nucleosomes 2 kb upstream from the promoter region. Collectively, our data suggest that moderate level of alphaA-crystallin gene expression in lens epithelial cells does not require the presence of CBP and p300 in the promoter. However, the lens-specific chromatin domain contains both promoter localized CBP on the "background" of locus-spread presence of CBP and p300.
小鼠αA-晶体蛋白基因编码哺乳动物晶状体中含量最丰富的蛋白质。αA-晶体蛋白的表达在晶状体发育过程中受到时间和空间的调控，最初在晶状体泡中表达，随后在分化的初级晶状体纤维中强烈上调。αA-晶体蛋白的晶状体特异性表达由与其启动子区域结合的DNA结合转录因子Pax6、c-Maf和CREB介导。其5'-远端增强子DCR1通过FGF信号传导介导αA-晶体蛋白的调控，而其3'-远端增强子DCR3仅通过其他晶状体分化途径在伸长的初级晶状体纤维中起作用。DCR1和DCR3确定了以组蛋白H3 K9乙酰化为标志的晶状体特异性染色质区域的外部边界。在这里，我们鉴定出CREB结合蛋白（CBP）和p300是在体内与小鼠αA-晶体蛋白基因座相关的主要组蛋白乙酰转移酶（HAT）。两种HAT在胚胎晶状体中均有表达。CBP在初级晶状体纤维细胞中的表达与αA-晶体蛋白一致。在晶状体上皮细胞的染色质中，染色质免疫沉淀（ChIP）显示αA-晶体蛋白启动子明显缺乏CBP和p300的任何显著存在，尽管DCR1和其他一些区域显示有这些HAT的存在。在新生晶状体获得的染色质中，CBP特异性定位于启动子区域，其丰度比整个αA-晶体蛋白基因座高约十倍。相比之下，p300在整个基因座上分布更均匀。对总组蛋白H3和H3 K9乙酰化的分析显示，启动子区域上游2 kb处核小体的密度可能较低。总体而言，我们的数据表明，晶状体上皮细胞中αA-晶体蛋白基因的适度表达水平不需要启动子中存在CBP和p300。然而，晶状体特异性染色质结构域在基因座广泛存在CBP和p300的“背景”下，同时包含启动子定位的CBP。
