Barrier insulators

我们对鸡-珠蛋白位点5端的复合绝缘体进行了多年的研究。我们发现，除了能够阻止由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USF1/2和BGP1的结合。我们已经广泛研究了USF1/2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的组蛋白处于活性状态，并防止引入其他抑制性组蛋白修饰。我们的研究结果表明，USF1在体内与脊椎动物Set1复合物相互作用，Set1复合物使组蛋白H3在赖氨酸4位点甲基化，PRMT1使组蛋白H4在精氨酸3位点甲基化。它们存在于两个独立的多组分复合物中，似乎通过USF1/2的特定结合定位于绝缘体元件。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。研究已扩展到USF1/2和PRMT1在调节β -珠蛋白基因表达中的作用，并发现它们在其中发挥重要作用。

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. 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 have now extended 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.