We note that JF 646-labeling is near-quantitative in live cells ( Yoon et al., 2016) moreover, a titration experiment indicated ≥90% labeling efficiency ( Figure 1-figure supplement 1A–C), although we cannot exclude slight undercounting due to incomplete labeling. We then ran a known quantity of protein side-by-side with a known number of cells labeled with the same fluorescent HaloTag ligand and quantified the total protein abundance per cell using ‘in-gel’ fluorescence ( Figure 1A Materials and methods). To establish a standard, we purified recombinant 3xFLAG-Halo-CTCF and Rad21-Halo-3xFLAG from insect cells and labeled the purified proteins with the bright dye JF 646 coupled to the covalent HaloTag ligand ( Grimm et al., 2015). We showed that these cell lines express the tagged proteins at endogenous levels by quantitative western blotting, ( Hansen et al., 2017). First, we developed an ‘in-gel’ fluorescence method based on previously validated mouse and human cell lines where either CTCF (U2OS and mouse embryonic stem cells (mESC)) or the cohesin kleisin subunit Rad21 (mESC) were endogenously and homozygously Halo-tagged ( Hansen et al., 2017). To estimate the absolute abundance (number of proteins per cell) of CTCF and cohesin, we applied a combination of three distinct methods: 1) ‘in-gel’ fluorescence, 2) Fluorescence Correlation Spectroscopy (FCS)-calibrated imaging, and 3) Flow Cytometry (FCM). Determining the number of CTCF and cohesin proteins per cell Building on our recent genomic and imaging studies of endogenously tagged CTCF and cohesin ( Hansen et al., 2017), here we (1) estimate bounds on the density of potentially loop-extruding cohesin complexes and estimate the CTCF-binding site occupancy probability in cells (2) provide biochemical evidence that at least a subset of cohesin complexes exist as dimers or oligomers and (3) develop a simple method for determining the absolute cellular abundance of any protein fused to the widely used and highly versatile HaloTag ( Los et al., 2008). Importantly, the number of CTCF and cohesin molecules, the molecular mechanism of loop extrusion and the stoichiometry of cohesin during this process remain unknown, further limiting our ability to test various models. Nevertheless, these models have been limited by a dearth of quantitative biological data to constrain the modeling. 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). Likewise, loss of the cohesin unloader WAPL strengthens TADs ( Gassler et al., 2017 Haarhuis et al., 2017 Wutz et al., 2017). Concordantly, knock-out of cohesin loading proteins NIPBL ( Schwarzer et al., 2017) and MAU2 ( Haarhuis et al., 2017) also affect TAD organization, although to different extents. CTCF and cohesin have emerged as causal regulators of TAD formation and maintenance, since acute CTCF or cohesin depletion causes global loss of most TADs ( Gassler et al., 2017 Nora et al., 2017 Rao et al., 2017 Wutz et al., 2017). 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.įolding 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 Lupiáñez et al., 2015 Symmons et al., 2014). 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. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers.

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. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved.

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