Insulator function and CTCF

绝缘体功能和CTCF

• 批准号: 8349746
• 负责人: Gary Felsenfeld
• 金额: $ 26.36万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349746
• 关键词: Attention; Binding; CCCTC-binding factor; Cell Cycle Stage; Cell Nucleus; Chickens; Chromatin; DNA; DNA Sequence; Elements; Enhancers; Gene Expression; Investigation; Laboratories; Proteins; RNA; Regulation; Role; Site; Surveys; Techniques; Tertiary Protein Structure; beta Globin; cohesin; genetic regulatory protein; genome-wide; imprint; novel; prevent; protein complex; protein p68

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. We have shown that this protein is responsible for regulation of imprinted gene expression at several imprinted loci. In order to understand the mechanism of action of CTCF we have continued to extend our earlier studies showing that CTCF molecules interact with co-factors. Among the interactions now under investigation is the interaction of CTCF with the cohesin protein complex, recently shown in the laboratories of Matthias Merkenschlager and others to be essential to the recruitment of cohesin to DNA at some stages of the cell cycle. Our recent results have identified SA2 as the cohesin subunit that directly interacts with CTCF, and also identified the domain of CTCF with which it interacts. We have also used mass spec analysis to detect other co-factors that appear to be important for CTCF insulator function. Using these techniques, we have identified a novel and important co-factor of CTCF, the regulatory protein p68. We have shown that this protein, together with its associated RNA co-factor,binds to CTCF, is present genome-wide at CTCF occupies sites on chromatin, and is essential for insulator activity. We also have evidence that at least one role of p68/SRA is to help stabilize the CTCF/cohesin interaction.

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，该绝缘子既能够阻断外部增强子的影响，又能够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。我们已经表明，这种蛋白质是负责调节印迹基因的表达在几个印迹位点。为了了解CTCF的作用机制，我们继续扩展我们早期的研究，表明CTCF分子与辅因子相互作用。 目前正在研究的相互作用是CTCF与粘附素蛋白复合物的相互作用，最近在Matthias Merkenschlager和其他人的实验室中显示，在细胞周期的某些阶段，粘附素对DNA的募集至关重要。我们最近的研究结果已经确定SA 2作为直接与CTCF相互作用的粘附素亚基，并且还确定了与其相互作用的CTCF结构域。 我们还使用质谱分析来检测似乎对CTCF绝缘子功能重要的其他辅因子。使用这些技术，我们已经确定了一个新的和重要的辅因子CTCF，调节蛋白p68。 我们已经表明，这种蛋白质，连同其相关的RNA辅因子，结合到CTCF，是目前全基因组的CTCF占据染色质上的网站，是必不可少的绝缘子活动。我们也有证据表明p68/SRA的至少一个作用是帮助稳定CTCF/cohesin相互作用。