Abstract
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.
Highlights
Folding of mammalian genomes into structures known as Topologically Associating Domains (TADs) is thought to help regulate gene expression while aberrant misfolding has been associated with disease (Dekker and Mirny, 2016; Fudenberg and Pollard, 2019; Hansen et al, 2018a; Hnisz et al, 2017; Lupianez et al, 2015; Symmons et al, 2014)
Consistent with the key roles played by CTCF and cohesin, models of genome folding through cohesin-mediated loop extrusion, which is stopped by chromatin-bound CTCF, have been remarkably successful in reproducing the general features of genomic contact maps at the level of TADs (Fudenberg et al, 2016; Fudenberg et al, 2017; Sanborn et al, 2015)
Despite the essential roles of cohesin in sister chromatid paring and interphase genome organization, and of condensin in mitotic chromosome compaction, the stoichiometry of these SMC complexes remains a matter of debate (Nasmyth, 2011; Skibbens, 2016)
Summary
Folding of mammalian genomes into structures known as Topologically Associating Domains (TADs) is thought to help regulate gene expression while aberrant misfolding has been associated with disease (Dekker and Mirny, 2016; Fudenberg and Pollard, 2019; Hansen et al, 2018a; Hnisz et al, 2017; Lupianez et al, 2015; Symmons et al, 2014). We take the mean of C59 and C87 across the two methods, namely ~217,200 CTCF proteins per cell in mESCs, as the best and final estimate This provides additional cross-validation and suggests that FCM can be used to estimate the absolute abundance of other Halotagged proteins if the U2OS C32 Halo-CTCF cell line is used as a standard (see below; Figure 3). We previously estimated the fraction of cohesin complexes that are relatively stably associated with chromatin (~20–25 min residence time in mESC G1) and presumably topologically engaged to be ~40% in G1 (Hansen et al, 2017) If we take this as the upper bound of putatively ‘loop-extruding’ cohesin complexes, we can calculate the upper limit on the density of extruding cohesin molecules as ~5.3 per Mb assuming cohesin exists as a monomeric ring or ~2.7 per Mb if cohesin forms dimers (Figure 1G; full details on calculation in Materials and methods). This cohesin:cohesin interaction appears to be protein-mediated rather than dependent on DNA association since benzonase treatment, which leads to complete DNA degradation ring dimer oligomer wt B4 C85
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