Gui C Y, Dean A
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-2715, USA.
Mol Cell Biol. 2001 Feb;21(4):1155-63. doi: 10.1128/MCB.21.4.1155-1163.2001.
On stably replicating episomes, transcriptional activation of the epsilon-globin promoter by the beta-globin locus control region HS2 enhancer is correlated with an increase in nuclease sensitivity which is limited to the TATA-proximal nucleosome (N1). To elucidate what underlies this increase in nuclease sensitivity and the link between chromatin modification and gene expression, we examined the nucleoprotein composition and histone acetylation status of transcriptionally active and inactive promoters. Micrococcal nuclease digestion of active promoters in nuclei released few nucleosome-like nucleoprotein complexes containing N1 sequences in comparison to results with inactive promoters. We also observed that N1 DNA fragments from active promoters are of a subnucleosomal length. Nevertheless, chromatin immunoprecipitation experiments indicate that histones H3 and H4 are present on N1 sequences from active promoters, with H3 being dramatically hyperacetylated compared with that from inactive promoters and vector sequences. Strikingly, H3 in the adjacent upstream nucleosome (N2) does not appear to be differentially acetylated in active and inactive promoters, indicating that the nucleosome modification of the promoter that accompanies transactivation by HS2 is highly directed and specific. However, global acetylation of histones in vivo by trichostatin A did not activate transcription in the absence of HS2, suggesting that HS2 contributes additional activities necessary for transactivation. N1 sequences from active promoters also contain reduced levels of linker histone H1. The detection of a protected subnucleosomal sized N1 DNA fragment and the recovery of N1 DNA sequences in immunoprecipitations using anti-acetylated H3 and H4 antibodies argue that N1 is present, but in an altered conformation, in the active promoters.
在稳定复制的附加体上，β-珠蛋白基因座控制区HS2增强子对ε-珠蛋白启动子的转录激活与核酸酶敏感性增加相关，这种增加仅限于TATA近端核小体（N1）。为了阐明这种核酸酶敏感性增加的基础以及染色质修饰与基因表达之间的联系，我们研究了转录活性和非活性启动子的核蛋白组成和组蛋白乙酰化状态。与非活性启动子的结果相比，微球菌核酸酶对细胞核中活性启动子的消化释放出很少含有N1序列的核小体样核蛋白复合物。我们还观察到，来自活性启动子的N1 DNA片段具有亚核小体长度。然而，染色质免疫沉淀实验表明，组蛋白H3和H4存在于活性启动子的N1序列上，与非活性启动子和载体序列相比，H3的乙酰化程度显著增加。引人注目的是，相邻上游核小体（N2）中的H3在活性和非活性启动子中似乎没有差异乙酰化，这表明HS2介导的转录激活伴随的启动子核小体修饰是高度定向和特异性的。然而，在没有HS2的情况下，曲古抑菌素A在体内对组蛋白的全局乙酰化并没有激活转录，这表明HS2有助于转录激活所需的其他活性。来自活性启动子的N1序列中连接组蛋白H1的水平也降低。使用抗乙酰化H3和H4抗体在免疫沉淀中检测到受保护的亚核小体大小的N1 DNA片段以及N1 DNA序列的回收表明，N1在活性启动子中以改变的构象存在。
