Barrier insulators

多年来，我们一直在研究鸡β珠蛋白基因5端的复合绝缘体。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，它还能够阻止报告基因的异染色质化。这一性质独立于CTCF，但我们已经证明，它确实依赖于另外两种蛋白质USF1和BGP1的结合。我们已经广泛地研究了USF1的作用，并表明作为其绝缘体功能的一部分，它招募了各种各样的组蛋白修饰酶，这些酶用于保持附近的组蛋白处于活性状态，并防止其他抑制性的组蛋白修饰被引入。我们的结果表明，USF1在体内与脊椎动物的Set1复合体和PRMT1相互作用，前者在赖氨酸4处甲基化组蛋白H3，后者在精氨酸3处甲基化组蛋白H4。它们存在于两个独立的多组分复合体中，这两个复合体似乎通过与CTCF的特异性结合而定位于绝缘体元件。这些复合体现在正在被单独研究，特别是PRMT1的作用正在基因组的其他位置进行研究，在那里它被USF1招募。 我们还研究了BGP1的性质，它结合到绝缘子中的不同位置。我们发现，小鼠ES细胞中BGP1的小鼠同源基因在DNA甲基化中起着重要作用；如果没有它，整个基因组关键部位的甲基化水平都会受到抑制。我们利用了一种ES细胞系，在该细胞系中，小鼠BGP1基因Vezf1被缺失。在与康奈尔医学院的H.Stuhlmann博士的合作中，我们现在已经证明，在缺乏Vezf1的情况下，DNA从头开始的甲基转移酶Dnmt3b下调。通过将Vezf1表达载体导入这些细胞，可以在很大程度上恢复野生型的表型。我们已经证明Vezf1在体内与Dnmt3b基因内含子的一个位点结合。

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.