Huang Suming, Li Xingguo, Yusufzai Timur M, Qiu Yi, Felsenfeld Gary
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
Mol Cell Biol. 2007 Nov;27(22):7991-8002. doi: 10.1128/MCB.01326-07. Epub 2007 Sep 10.
The insulator element at the 5' end of the chicken beta-globin locus acts as a barrier, protecting transgenes against silencing effects of adjacent heterochromatin. We showed earlier that the transcription factor USF1 binds within the insulator and that this site is important for generating in adjacent nucleosomes histone modifications associated with active chromatin and, by inference, with barrier function. To understand the mechanism of USF1 action, we have characterized USF1-containing complexes. USF1 interacts directly with the histone H4R3-specific methyltransferase PRMT1. USF1, PRMT1, and the histone acetyltransferases (HATs) PCAF and SRC-1 form a complex with both H4R3 histone methyltransferase and HAT activities. Small interfering RNA downregulation of USF1 results in localized loss of H4R3 methylation, and other histone modifications associated with euchromatin, at the insulator. A dominant negative peptide that interferes with USF1 binding to DNA causes silencing of an insulated reporter construct, indicating abolition of barrier function. These results show that USF1 plays a direct role in maintaining the barrier, supporting a model in which the insulator works as a barrier by maintaining a local environment of active chromatin.
鸡β-珠蛋白基因座5'端的绝缘子元件起到屏障作用，保护转基因免受相邻异染色质的沉默效应影响。我们之前表明转录因子USF1结合在绝缘子内，且该位点对于在相邻核小体中产生与活性染色质相关的组蛋白修饰很重要，由此推断，与屏障功能相关。为了解USF1的作用机制，我们对含有USF1的复合物进行了表征。USF1直接与组蛋白H4R3特异性甲基转移酶PRMT1相互作用。USF1、PRMT1以及组蛋白乙酰转移酶（HATs）PCAF和SRC-1形成一个兼具H4R3组蛋白甲基转移酶和HAT活性的复合物。通过小干扰RNA下调USF1会导致绝缘子处H4R3甲基化以及其他与常染色质相关的组蛋白修饰局部缺失。一种干扰USF1与DNA结合的显性负性肽会导致绝缘报告构建体沉默，表明屏障功能丧失。这些结果表明USF1在维持屏障中起直接作用，支持了一种模型，即绝缘子通过维持活性染色质的局部环境作为屏障发挥作用。
