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序列及其上游元件上。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。我们之前已经证明增强子阻断活性与单个蛋白CTCF与增强子内某个位点的结合有关。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，并利用它们从HeLa细胞提取物中分离出CTCF与其他蛋白质之间的复合物。鉴定出了几种蛋白质，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。鸡β -珠蛋白基因座的染色质免疫沉淀研究表明，核蛋白在基因座两端的两个边界元素上与CTCF共定位。此外，在携带多个5'绝缘子拷贝的细胞系中，荧光原位杂交分析显示绝缘子定位在核小体表面。定位依赖于完整的CTCF结合位点的存在。这些发现提出了一种增强子阻断活性的模型，其中核磷蛋白以ctcf依赖的方式将绝缘体系在核小体表面。这个模型与果蝇的吉普赛人绝缘体元件有有趣的相似之处，后者涉及到完全不同的蛋白质。

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.