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序列及其上游元件上，该绝缘子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。我们以前已经表明，增强子阻断活性与单一蛋白质CTCF与增强子内的位点结合有关。为了了解其作用机制，我们与Yoshihiro Nakatani实验室合作构建了表位标记的CTCF分子，并使用它们从HeLa细胞提取物中分离CTCF与其他蛋白质之间的复合物。鉴定了几种蛋白质，其中最突出的是核磷蛋白，一种倾向于集中在核仁中的蛋白质。对鸡β-珠蛋白基因座的染色质免疫沉淀研究表明，核磷蛋白与CTCF共定位于基因座两端的两个边界元素。此外，在携带多个拷贝的5'绝缘子的细胞系中，荧光原位杂交分析显示绝缘子位于核小体表面。定位依赖于一个完整的CTCF结合位点的存在。这些发现表明了一个增强子阻断活性的模型，其中核磷蛋白以CTCF依赖的方式将绝缘子拴在核小体表面。这个模型与果蝇中的吉普赛绝缘子元件有着有趣的相似之处，其中涉及非常不同的蛋白质。 绝缘子还具有保护稳定转染到细胞系或动物中的位置效应报告基因的单独能力，作为防止浓缩染色质侵入的边界。我们发现这种保护能力存在于一个？2kb绝缘子内的250 bp长的“核心”元件，并且核心内含有CTCF位点的亚区的缺失不影响活性。然而，其他四个亚区对应的结合位点的核蛋白是重要的位置效应保护（边界功能）。我们现在已经表明，这些结合位点之一是专门负责维持高水平的组蛋白乙酰化和甲基化的网站与基因激活。该位点结合蛋白USF 1和USF 2的异源二聚体，这反过来又招募各种组蛋白修饰酶到该位点，包括已知的乙酰化酶和甲基化酶。这些结果与我们提出的模型是一致的，在该模型中，屏障功能与绝缘体附近的多个组蛋白修饰有关。我们还发现，核心区的其他位点负责抑制附近基因座的DNA甲基化。珠蛋白绝缘子似乎在体内充当防止侵犯浓缩染色质上游区域的屏障。 我们还与Hannah Gould的实验室合作，研究染色质结构与人类免疫球蛋白重链基因座中的类别转换重组之间的关系。早期的观点认为，同种型的选择是由单个生殖系基因的染色质选择性开放决定的。我们通过对单个B细胞的分析表明，单个细胞含有来自一个以上生殖系基因的转录本，这意味着在给定细胞的开放染色质结构中可以存在一个以上的基因。 我们还与Marisa Bartolomei的实验室合作研究小鼠中的印迹Igf 2/H19位点。我们早期的工作表明，印迹依赖于基因座内印迹控制区内CTCF结合序列的父系等位基因的甲基化。新的数据是在CTCF位点突变的小鼠中获得的，这些突变阻止了甲基化，但不能阻止CTCF结合。突变的母系遗传使Igf 2和H19的表达模式保持完整，而父系遗传允许绝缘和随后的Igf 2对该等位基因的抑制以及通常在野生型细胞中观察到的H19的抑制的缓解。结果提供了关于压印标记的生成的进一步信息。

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 order to understand its mechanism of action, we constructed in collaboration with the laboratory of Yoshihiro Nakatani epitope-tagged CTCF molecules and used them to isolate complexes between CTCF and other proteins from HeLa cell extracts. Several proteins were identified, the most prominent of which was nucleophosmin, a protein that tends to concentrate in the nucleolus. Chromatin immunoprecipitation studies across the chicken beta-globin locus showed that nucleophosmin co-localizes with CTCF at the two boundary elements at either end of the locus. Furthermore, in cell lines carrying multiple copies of the 5' insulator, fluorescence in situ hybridization analysis revealed that the insulators were localized on the nucleosome surface. Localization depended on the presence of an intact CTCF binding site. These findings suggest a model for enhancer blocking activity in which nucleophosmin tethers the insulator to the nucleosome surface in a CTCF-dependent manner. This model has interesting parallels to one proposed for the gypsy insulator element in Drosophila, where quite different proteins are involved. 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, which in turn recruit a variety of histone modifying enzymes to the site, including known acetylases and methylases. 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. We have also shown that other sites in the core region are responsible for inhibiting DNA methylation at a nearby locus. The globin insulator appears to serve in vivo as a barrier against encroachment of an upstream region of condensed chromatin. We have also collaborated with the laboratory of Hannah Gould to examine the relationship between chromatin structure and class switch recombination in the human immunoglobulin heavy chain locus. Earlier opinion has held that the choice of isotype was governed by the selective opening of chromatin at a single germ line gene. We showed by analysis of single B-cells that single cells contain transcripts from more than one germ line gene, implying that more than one gene can exist in an open chromatin structure in a given cell. We have also collaborated with the laboratory of Marisa Bartolomei in a study of the imprinted Igf2/H19 locus in mouse. Our earlier work had shown that imprinting depends on the methylation in the paternal allele of CTCF binding sequences within the imprinting control region within the locus. The new data were obtained in mice which had mutations in the CTCF sites that prevented methlyation but not CTCF binding. Maternal inheritance of the mutation left Igf2 and H19 patterns of expression intact, while paternal inheritance allowed insulation and consequent Igf2 suppression on that allele and relief of repression of H19, normally observed in wild type cells. The results provide further information on the generation of the imprinting marks.