Hou Chunhui, Zhao Hui, Tanimoto Keiji, Dean Ann
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20398-403. doi: 10.1073/pnas.0808506106. Epub 2008 Dec 12.
The mechanism underlying enhancer-blocking by insulators is unclear. We explored the activity of human beta-globin HS5, the orthologue of the CTCF-dependent chicken HS4 insulator. An extra copy of HS5 placed between the beta-globin locus control region (LCR) and downstream genes on a transgene fulfills the classic predictions for an enhancer-blocker. Ectopic HS5 does not perturb the LCR but blocks gene activation by interfering with RNA pol II, activator and coactivator recruitment, and epigenetic modification at the downstream beta-globin gene. Underlying these effects, ectopic HS5 disrupts chromatin loop formation between beta-globin and the LCR, and instead forms a new loop with endogenous HS5 that topologically isolates the LCR. Both enhancer-blocking and insulator-loop formation depend on an intact CTCF site in ectopic HS5 and are sensitive to knock-down of the CTCF protein by siRNA. Thus, intrinsic looping activity of CTCF sites can nullify LCR function.
绝缘子介导增强子阻断的机制尚不清楚。我们研究了人β-珠蛋白HS5的活性，它是依赖CTCF的鸡HS4绝缘子的直系同源物。转基因上位于β-珠蛋白基因座控制区（LCR）和下游基因之间的额外一份HS5拷贝符合增强子阻断剂的经典预测。异位的HS5不会干扰LCR，但通过干扰RNA聚合酶II、激活因子和共激活因子的募集以及下游β-珠蛋白基因的表观遗传修饰来阻断基因激活。在这些效应的背后，异位的HS5破坏了β-珠蛋白与LCR之间的染色质环形成，而是与内源性HS5形成了一个新的环，在拓扑结构上隔离了LCR。增强子阻断和绝缘子环形成都依赖于异位HS5中完整的CTCF位点，并且对通过siRNA敲低CTCF蛋白敏感。因此，CTCF位点的内在环化活性可以使LCR功能无效。
