CHROMATIN STRUCTURE AND FUNCTION

我们继续研究了表达基因附近的染色质结构。 鸡红系细胞中的珠蛋白基因家族作为一个模型系统，其中有可能研究红系发育过程中与家族的簇和个体成员的调节相关的机制。 我们将注意力集中在鸡β-珠蛋白基因座5'端的1.2kb绝缘子DNA序列上，当它位于增强子-启动子之间时，它能够阻断增强子-启动子相互作用。 我们先前已经将这种活性缩小到1.2 kb序列内的一个小DNA片段。 我们还纯化了负责这种活性的蛋白质，并将其鉴定为CTCF，这是一种在脊椎动物中普遍存在的已知因子，对多种基因的表达具有刺激和抑制作用，并且我们表明CTCF位点存在于其他脊椎动物基因位点中，包括鸡β珠蛋白位点的3'端。我们还研究了小鼠和人类Igf 2（胰岛素样生长因子2）/H19位点附近的潜在绝缘子活性。 这是一个印记基因座：在母系传播的等位基因中，Igf 2是沉默的，H19是表达的。 在父系传递的等位基因中，情况正好相反。 两个基因之间的区域仅在父系等位基因上甲基化，并且已经表明绝缘子在该区域内。 我们现在已经表明，该区域包含CTCF的多个结合位点（小鼠中4个，人中7个）。 它们作为非常强的增强子阻断元件，阻止下游增强子激活母体等位基因中的Igf 2。 删除这些位置就取消了绝缘。 我们还表明，甲基化的网站阻止CTCF结合。 因此，父亲等位基因上Igf 2的选择性激活可以通过绝缘子的失活来解释，这允许下游增强子打开该基因。 我们的研究结果还表明，绝缘子活动可以调制在发展过程中的网站甲基化。 这极大地拓宽了绝缘体元件的潜在作用范围。 在其他研究中，我们已经检查了在发育过程中珠蛋白基因座及其附近的染色质结构变化的模式。 我们先前已经表明，红细胞特异性叶酸受体（FR）基因位于鸡β-珠蛋白基因座的上游，由16个浓缩的非表达染色质的内切酶与其分离。 利用高分辨率的免疫沉淀方法，我们已经表明，有重大的变化，组蛋白乙酰化水平超过这些基因在细胞中对应于不同的发育阶段，但浓缩的染色质结构域和某些调控元件保持其低和高乙酰化水平（分别）在整个发展。

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, which is capable of blocking enhancer-promoter interaction when it lies between them. We had earlier narrowed down this activity to a small DNA fragment within the 1.2 kb sequence. We also purified the protein responsible for this activity and identified it as CTCF, which is a known factor ubiquitous in vertebrates, with both stimulatory and inhibitory effects on expression of a variety of genes, and we showed that CTCF sites are present in other vertebrate gene loci, including the 3' end of the chicken beta globin locus. We were also led to investigate a potential insulator activity in the neighborhood of the Igf2 (Insulin-like growth factor 2)/H19 locus in mouse and human. This is an imprinted locus: In the maternally transmitted allele Igf2 is silent and H19 is expressed. The reverse is true in the paternally transmitted allele. A region between the two genes is methylated on the paternal allele only, and it had been suggested that the insulator was within this region. We have now shown that this region contains multiple binding sites for CTCF (four in mouse, seven in human). They serve as very strong enhancer blocking elements, preventing a downstream enhancer from activating Igf2 in the maternal allele. Deletion of these sites abolishes insulation. We also have shown that methylation of the sites prevents CTCF binding. Thus the selective activation of Igf2 on the paternal allele is explained by inactivation of the insulator, which allows the downstream enhancer to turn on this gene. Our results also show that insulator activity can be modulated during development by site methylation. This greatly widens the potential range of action of insulator elements. In other studies we have examined the pattern of chromatin structural changes over the globin locus and its neighborhood during development. We had shown 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 have 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.