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USF1/2和BGP1的结合。我们已经广泛地研究了USF1/2的作用，并表明作为其绝缘功能的一部分，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，并防止其他抑制性组蛋白修饰的引入。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。这些存在于两个独立的多组分复合体中，它们似乎通过与USF1/2的特异性结合而定位于绝缘体元件。最近的研究集中在PRMT1的作用上。我们已经证明，这种酶对H4R3的甲基化是组蛋白乙酰化和基因激活所必需的。研究已经扩展到USF1/2和PRMT1在调控β-珠蛋白基因表达中的作用，并发现它们在其中发挥着重要作用。 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。我们已经证明，这是绝缘体上BGP1结合丧失的相当特异的，并涉及与沉默相关的DNA甲基化水平的变化。我们设计了实验来测试Vezf1与APRT报告基因附近的位点结合是否会影响甲基化模式，发现在没有Vezf1位点的情况下，该区域的DNA会发生广泛的甲基化；Vezf1的结合抑制了甲基化。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，我们发现在缺乏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。 我们正在扩大我们的研究，试图了解Vezf1是如何抑制Dnmt3b的表达的。在这些研究中，我们正在询问Vezf1是否可能以一种改变基因特定剪接变体的产生的方式干扰转录延长，正如上面描述的小鼠ES细胞实验所表明的那样。

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, USF1/2 and BGP1. We have studied the role of USF1/2 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 USF1/2. Recent studies have focused on the role of PRMT1. We have shown that methylation of H4R3 by this enzyme is necessary for histone acetylation and gene activation. Studies have been extended to the roles of USF1/2 and PRMT1 in regulation of beta-globin gene expression, where they are found play an important role. 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 shown that this is quite specific for loss of BGP1 binding at the insulator, and involves changes in the levels of DNA methylation associated with silencing. We designed experiments to test whether Vezf1 binding to sites near an APRT reporter could affect methylation patterns, and found that in the absence of Vezf1 sites the region's DNA became methylated extensively; binding of Vezf1 inhibited methylation. 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 showed 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 in vivo to a site in an intron of the Dnmt3b gene. We are extending our investigation in an attempt to understand how Vezf1 suppresses Dnmt3b expression. In these studies, we are asking whether Vezf1 may interfere with transcription elongation in such a way as to alter the production of specific splice variants of a gene, as suggested by the experiments described above with mouse ES cells.