Barrier insulators

多年来，我们一直在研究鸡β珠蛋白位点5端的复合绝缘子。 我们发现，除了能够阻止CTCF介导的增强子-启动子相互作用外，它还能够阻断报告基因的异染色质化。 这种特性与CTCF无关，但我们已经证明它确实依赖于另外两种蛋白质USF 1和BGP 1的结合。 我们已经广泛研究了USF 1的作用，并表明作为其绝缘子功能的一部分，它招募了各种各样的组蛋白修饰酶，这些酶用于维持附近组蛋白处于活性状态，并防止其他抑制性组蛋白修饰被引入。 我们的研究结果表明，USF 1在体内与脊椎动物Set 1复合物相互作用，该复合物在赖氨酸4处甲基化组蛋白H3，以及PRMT 1，该复合物在精氨酸3处甲基化组蛋白H4。 这些都存在于两个独立的，多组分的复合物，似乎是本地化的绝缘子元件通过特异性结合的CTCF。 这些复合物现在正在单独研究，并且正在研究PRMT 1在基因组中被USF 1招募的其他位点的作用。 我们还研究了BGP 1的性质，它结合到绝缘体中的不同位点。 我们发现小鼠ES细胞中BGP 1的小鼠同源物在DNA甲基化中起着重要作用;在其缺失的情况下，全基因组关键位点的甲基化水平被抑制。 我们已经使用了其中Vezf 1（小鼠BGP 1）被删除的ES细胞系。 与H博士合作。Stuhlmann（康奈尔医学院），我们现在已经表明，在没有Vezf 1的DNA从头甲基转移酶，Dnmt 3b，下调。 野生型表型可以通过将Vezf 1表达载体引入这些细胞中而在很大程度上恢复。 我们已经证明，Vezf 1结合在体内的Dnmt 3b基因的内含子中的一个位点。

We have been studying for some years the compound insulator at the 5 end of the chicken beta globin locus. We showed that in addition to its ability to prevent enhancer-promoter interactions, mediated by CTCF, it is also able to block the hetrochromatinization of a reporter gene. This property is independent of CTCF, but we have shown that it does depend on the binding of two other proteins, USF1 and BGP1. We have studied the role of USF1 extensively and shown that as part of its insulator function it recruits a wide variety of histone modifying enzymes, which serve to maintain nearby histones in an active state, and prevent other, repressive, histone modifications from being introduced. Our results show that USF1 interacts in vivo with the vertebrate Set1 complex, which methylates histone H3 at lysine 4, as well as PRMT1, which methylates histone H4 at arginine 3. These are present in two separate, multicomponent complexes which appear to be localized to the insulator element through specific binding of CTCF. These complexes are now being individually investigated, and the role particularly of PRMT1 is being studied at other sites in the genome where it is recruited by USF1. We have also investigated the properties of BGP1, which binds to separate sites in the insulator. We find that the mouse homolog of BGP1 in mouse ES cells plays an important role in DNA methylation; in its absence methylation levels at critical sites genome-wide are depressed. We have made use of an ES cell line in which Vezf1, the mouse BGP1, is deleted. In collaboration with Dr. H. Stuhlmann (Cornell Medical College) we have now shown that in the absence of Vezf1 the DNA de novo methyl transferase, Dnmt3b, is down regulated. Wild type phenotype can largely be restored by introducing a Vezf1 expression vector into these cells. We have shown that Vezf1 binds on vivo to a site in an intron of the Dnmt3b gene.