Determining cellular CTCF and cohesin abundances to constrain 3D genome models
Determining cellular CTCF and cohesin abundances to constrain 3D genome models
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DOI：
10.7554/elife.40164
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发表时间：
2019-06-17
期刊：
影响因子：
7.7
通讯作者：
Hansen, Anders S.
中科院分区：
文献类型：
--
作者：
Cattoglio, Claudia;Pustova, Iryna;Hansen, Anders S.
实现对3D基因组结构的定量和预测性理解仍然是一个重大挑战，因为它需要对所涉及的关键蛋白质进行定量测量。在这里，我们报告CTCF和cohesin，拓扑关联结构域（TADs）在哺乳动物细胞中的两个因果调节器的定量。扩展我们先前的成像研究（汉森等人，2017），我们估计了puppelicDNA环挤出粘附素复合物的密度和CTCF结合位点占用率的界限。此外，内源性标记的亚基（Rad 21）的免疫共沉淀研究表明，存在粘着蛋白二聚体和/或寡聚体。最后，基于我们的细胞系与准确测量的蛋白质丰度，我们报告了一种方法，方便地确定任何Halo标记的蛋白质在细胞中的分子数量。我们预计，我们的结果和已建立的测量细胞蛋白质丰度的工具将促进对3D基因组组织的更定量的理解，并促进蛋白质定量，这是理解不同生物过程的关键。
Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.