Chromatin Structure And Function

我们继续对表达基因附近的染色质结构进行研究。鸡红系细胞中的珠蛋白基因家族是一个模型系统，在该系统中可以研究红系发育过程中家族和个体成员的调控机制。我们将注意力集中在鸡β-珠蛋白基因5‘端1.2kb的绝缘子DNA序列及其上游元件上。这种绝缘体既能阻止外部增强剂的影响，又能防止可能关闭整个区域表达的浓缩染色质的侵蚀。我们以前已经证明，增强子阻断活性与单个蛋白CTCF与增强子内的一个位点结合有关。在最近的研究中，我们已经将我们对增强子阻断活性的其他位置的搜索扩展到小鼠T细胞受体α/增量基因座。其他实验室的工作已经表明，在该基因座和下游的爸爸I基因之间存在具有增强子阻断活性的序列，可能是为了防止两个基因调控系统之间的串扰。我们发现CTCF在该区域存在一个单一的强结合位点，并且它具有预期的增强子阻断活性。虽然该区域中没有其他这样的位点，但其他序列元件具有绝缘体活性，这表明存在以前未认识到的新机制。在另一项工作中，我们证明了CTCF位与核基质组分相连，这与我们实验室早期的结果一致，该结果指出了这种绝缘体的拴系机制？S作用。 绝缘体还具有单独的能力，以防止位置效应报告基因稳定地转染到细胞系或动物中，作为防止浓缩染色质侵蚀的边界。我们发现，这种保护能力存在于1.2kb绝缘子内的250个碱基的核心元件中，并且核心中包含CTCF位点的亚区的缺失不会影响活性。然而，对应于核蛋白结合位点的另外四个亚区对于位置效应保护(边界功能)是重要的。我们现在已经证明，这些结合位点中的一个专门负责在与基因激活相关的位点上维持高水平的组蛋白乙酰化和甲基化。这个位点结合了蛋白质USF1和USF2的异源二聚体。这些结果与我们提出的一个模型是一致的，在该模型中，屏障功能与绝缘子附近的多个组蛋白修饰有关。为了了解USF蛋白是如何介导这一作用的，我们调查了USF1通过体内染色质免疫沉淀和体外共沉淀招募的辅助因子。我们确定了负责这种激活的酶？组蛋白修饰如组蛋白乙酰化和组蛋白H3赖氨酸4甲基化。此外，我们检测到与组蛋白H4上负责甲基化精氨酸3的酶PRMT1有很强的相互作用。这是另一个与转录活性染色质相关的标记。我们使用RNAi方法建立了PRMT1耗竭的稳定细胞系。不仅精氨酸残基的甲基化如预期的那样严重下调，而且H3和H4的乙酰化也受到了强烈的抑制。我们使用染色质和从这些细胞中纯化的提取物来证明这种作用是直接的，即H4Arg3的甲基化不仅是必要的，而且足以允许组蛋白乙酰化。这是一个重要的级联的新例子。其中在可以发生其他修改之前需要对Arg3进行先前的修改。它为更好地理解有助于调控基因表达的表观遗传机制指明了道路。

We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We have shown previously that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. In recent studies we have extended our search for other sites of enhancer blocking activity to the mouse T cell receptor alpha/delta locus. Work in other laboratories had shown that sequences with enhancer blocking activity existed between this locus and the downstream Dad I gene, probably to prevent cross talk between the two gene regulatory systems. We showed that a single strong binding site for CTCF is present in this region, and that it has the expected enhancer blocking activity. Although there are no other such sites in the region, other sequence elements have insulator activity, pointing to the existence of novel mechanisms not previously recognized. In other work, we showed that CTCF sites are tethered to the nuclear matrix fraction, consistent with earlier results from our laboratory that point to a tethering mechanism for this insulator?s action. The insulator also has the separate ability to protect against position effects reporter genes that are stably transfected into cell lines or animals, serving as a boundary against encroachment of condensed chromatin. We found that this protective ability is present in a ?core' element, 250 bp long, from within the 1.2 kb insulator, and that deletion of subregions within the core that contain the CTCF site do not affect activity. However four other subregions corresponding to binding sites for nuclear proteins are important for position effect protection (boundary function). We have now shown that one of these binding sites is specifically responsible for maintaining a high level of histone acetylation and methylation at sites associated with gene activation. This site binds a heterodimer of the proteins USF1and USF2. These results are consistent with a model we have proposed in which barrier function is connected with multiple histone modifications in the neighborhood of the insulator. In an effort to understand how the USF proteins mediate this action, we have investigated what co-factors are recruited by USF1, both by chromatin immunoprecipitation in vivo and by co-precipitation in vitro. We identified enzymes responsible for such ?activating? histone modifications as histone acetylation and histone H3 lysine 4 methylation. In addition, we detected a strong interaction with PRMT1, the enzyme responsible for methylating arginine 3 on histone H4. This is another mark associated with transcriptionally active chromatin. We used RNAi methods to generate stable cells lines with depleted amounts of PRMT1. Not only was methylation of the arginine residue severely down-regulated as expected, but acetylation of both H3 and H4 was also strongly inhibited. We used chromatin and extracts purified from these cells to show that the effect was direct, i.e. methylation of H4Arg3 was not only necessary but also sufficient to allow histone acetylation. This is an important new example of a ?cascade? in which prior modification of Arg3 is required before the other modifications can occur. It points the way to a better understanding of the epigenetic mechanisms that help regulate gene expression.