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作为一个模型系统，可以研究在红系发育过程中该家族的集群和个体成员的调控机制。我们将重点放在鸡β -珠蛋白位点5'端的1.2 kb绝缘子DNA序列上。这种绝缘体既能阻断外部增强子的影响，又能防止可能关闭整个区域表达的浓缩染色质的侵入。我们之前已经将增强子阻断活性缩小到1.2 kb序列内的一个小DNA片段，并表明该活性与普遍存在的脊椎动物核蛋白CTCF的结合有关。我们发现CTCF结合位点在小鼠和人的Igf2/H19位点的印迹中也起着重要作用。目前我们不仅在鸡的-珠蛋白位点上发现了类似的位点，而且在小鼠和人类的-珠蛋白位点上也发现了类似的位点。在人类基因座中，下游位点能够阻断可能破坏珠蛋白基因表达程序的内源性增强子。我们一直在探索小鼠和人类的基因座，以寻找额外的调控元件。该绝缘体还具有保护被稳定地转染到细胞系或动物中的报告基因免受位置效应的能力。我们现在已经证明，这种保护能力存在于一个“核心”元件，250 bp长，从1.2 kb绝缘体。核心内的亚区缺失表明CTCF位点不参与该活性，但与尚未确定的核蛋白结合位点相对应的其他四个亚区对位置效应保护很重要。这些结果意味着现在有可能分离出绝缘子活性的两个方面——增强子阻断和位置效应保护——并单独研究它们。我们也一直在研究红系发育过程中染色质结构变化和珠蛋白位点及其邻域修饰的模式。我们之前已经发现，一个红系特异性叶酸受体（FR）基因位于鸡β -珠蛋白位点的上游，被16千碱基的浓缩非表达染色质隔开。利用高分辨率免疫沉淀方法，我们已经表明，在细胞的不同发育阶段，这些基因的组蛋白乙酰化水平发生重大变化，但在整个发育过程中，浓缩染色质结构域和某些调节元件分别保持低和高乙酰化水平。我们现在已经检查了组蛋白H3甲基化在同一区域的分布。在发育过程中，赖氨酸4位点的甲基化与乙酰化模式之间存在显著的相关性，而这两种修饰与组蛋白H3位点赖氨酸9位点的甲基化之间存在显著的反相关性。这些结果为高级真核生物中大区域组蛋白甲基化与基因表达之间的关系提供了第一个强有力的支持。我们还发现-球蛋白绝缘子元件的邻域特征是组蛋白H3和H4在所有位点高度乙酰化，H3在赖氨酸4位点高度甲基化，在赖氨酸9位点几乎未甲基化。这些观察结果使我们建立了绝缘体防止位置效应的模型，这与最近关于凝聚染色质结构域的传播的结果是一致的。

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. 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 had earlier narrowed down enhancer blocking activity to a small DNA fragment within the 1.2 kb sequence, and shown that this activity involves binding of the ubiquitous vertebrate nuclear protein CTCF. We showed that the CTCF binding site also plays an important role in imprinting at the Igf2/H19 locus in mouse and humans. Now we have found that similar sites flank not only the chicken beta-globin locus, but also the beta-globin loci of mouse and human. In the human locus the downstream site is in a position to block endogenous enhancers that might otherwise disrupt the program of globin gene expression. We have been exploring the mouse and human loci to search for additonal regulatory elements. The insulator also has the ability to protect against position effects reporter genes that are stably transfected into cell lines or animals. We have now shown that this protective ability is present in a 'core' element, 250 bp long, from within the 1.2 kb insulator. Deletion of subregions within the core shows that the CTCF site is not involved in this activity, but four other subregions corresponding to binding sites for as yet unidentified nuclear proteins are important for position effect protection. These results mean that it is now possible to separate two aspects of insulator activity - enhancer blocking and position effect protection-and study them in isolation. We have also been studying the pattern of chromatin structural changes and modifications over the globin locus and its neighborhood during erythroid development. We had found earlier that an erythroid specific folate receptor (FR) gene lies upstream of the chicken beta-globin locus, separated from it by 16 kilobases of condensed non-expressed chromatin. Making use of high resolution immunoprecipitation methods, we had shown that there are major changes in histone acetylation levels over these genes in cells corresponding to various developmental stages, but that the condensed chromatin domain and certain regulatory elements maintain their low and high acetylation levels (respectively) throughout development. We have now examined the distribution of histone H3 methylation over the same region. There is a striking correlation between methylation at lysine 4 and patterns of acetylation during development, and a remarkable anti-correlation of those two modifications with methylation of histone H3 at lysine 9. These results provide the first strong support in higher eukaryotes for a relationship between histone methylation over large regions and gene expression. We also found that the neighborhood of the beta- globin insulator element is characterized by histones H3 and H4 highly acetylated at all sites, H3 highly methylated at lysine 4, and nearly unmethylated at lysine 9. These observations have led us to a model for the way in which the insulator may protect against position effects, which is consistent with recent results on the propagation of condensed chromatin domains.