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-珠蛋白基因座的5端的1.2 kb绝缘子DNA序列以及其上游的元素上。该绝缘子能够阻止外部增强剂的影响，也可以防止凝结染色质的侵蚀，这可能会阻止整个区域的表达。我们先前已经证明，增强子阻断活性与单个蛋白CTCF与增强子内部位置的结合有关。我们已经表明，该蛋白质负责调节几个印迹基因座的印迹基因表达。为了了解CTCF的作用机理，我们继续扩展了早期的研究，表明CTCF分子与副因素相互作用。 现在正在研究的相互作用中，CTCF与粘蛋白蛋白复合物的相互作用，最近在Matthias Merkenschlager的实验室中显示，而其他人则是在细胞周期的某些阶段将粘蛋白募集到DNA至关重要的。我们最近的结果将SA2确定为直接与CTCF相互作用的粘蛋白亚基，并确定了与之相互作用的CTCF域。 我们还使用质量规格分析来检测其他对CTCF绝缘子函数重要的共同因素。使用这些技术，我们已经确定了CTCF的新颖而重要的共同因素，即调节蛋白p68。 我们已经表明，该蛋白及其相关的RNA co因子与CTCF结合，在CTCF的全基因组均存在于染色质上，并且对于绝缘剂活性至关重要。我们还有证据表明，p68/SRA的至少一种作用是帮助稳定CTCF/粘蛋白相互作用。