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Vezf1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的Vezf1基因被缺失。在与H.Stuhlmann博士(康奈尔医学院)的合作中，我们早些时候证明了在缺乏Vezf1的情况下，DNA从头甲基转移酶Dnmt3b下调，反映了编码活性Dnmt3b的RNA剪接变异体丰度的下降。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置结合，这表明Vezf1可能干扰RNA聚合酶II的伸长率，允许不同的选择性剪接途径。 现在我们将我们的研究扩展到全基因组调查，试图了解Vezf1是如何抑制Dnmt3b的表达的。在这些研究中，我们询问了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，正如上述用小鼠ES细胞进行的实验所表明的那样。我们发现，在HeLaS3细胞中，Vezf1结合与延长的Ser2-P RNA聚合酶(Pol)11的峰值在全基因组范围内有很强的相关性，反映了Vezf1依赖的延伸放缓。在WTMES细胞中，延长形式的RNA PolII聚集在Dnmt3b基因内Vezf1结合位点附近和其他几个Vezf1位点，而在Vezf1(-/-)MES细胞中，这种积累在这些位点上显著减少。 我们现在假设，Vezf1减缓RNA PolII通过的能力也与其作为鸡β珠蛋白基因座屏障蛋白的作用有关。在另一项工作中，我们已经证明了上述16kb的异染色区是由低水平的转录和DICER依赖的机制维持的。Vezf1可以阻止聚合酶通过16kb区域的前进，阻止异染色质延伸到屏障绝缘体之外。

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 also investigated the properties of BGP1, which binds to separate sites in the insulator. We found that Vezf1, 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 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 earlier that in the absence of Vezf1 the DNA de novo methyl transferase, Dnmt3b, is down regulated, reflecting a decrease in the abundance of the RNA splice variant coding for active Dnmt3b. 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, and this suggested that Vezf1 might interfere with RNA polymerase II elongation rates, allowing for different alternative splicing pathways. Now we have extended our investigation to a genome-wide survey, in an attempt to understand how Vezf1 suppresses Dnmt3b expression. In these studies, we asked 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. We find that in HeLaS3 cells there is a strong genome-wide correlation between Vezf1 binding and peaks of elongating Ser2-P RNA polymerase (Pol) ll, reflecting Vezf1-dependent slowing of elongation. In WT mES cells, the elongating form of RNA pol II accumulates near Vezf1 binding sites within the dnmt3b gene and at several other Vezf1 sites, and this accumulation is significantly reduced at these sites in Vezf1(-/-) mES cells. We now hypothesize that the ability of Vezf1 to slow the passage of RNA pol II is also related to its role as a barrier protein at the chicken beta globin locus. In other work we had shown that the 16 kb heterochromatic region described above is maintained by low levels of transcription and a dicer-dependent mechanism. Vezf1 could block the advance of the polymerase past the 16 kb region, preventing the extension of heterochromatin beyond the barrier insulator.