biostudies-literatureHolzmann JEuropean Molecular Biology OrganizationAustrian Science Fund FWFHuman Frontier Science ProgramHorizon 2020 Framework ProgrammePaul G. Allen Frontiers GroupVienna Science and Technology FundBoehringer IngelheimSixth Framework ProgrammeAustrian Research Promotion AgencyEuropean Molecular Biology LaboratorySeventh Framework ProgrammeNational Institutes of Healthe46269https://www.ebi.ac.uk/biostudies/studies/S-EPMC6606026biostudies-literatureUnknown8The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.eLifeAbsolute quantification of cohesin, CTCF and their regulators in human cells.PMC6606026Laura Bassi Centre for Optimized Structural Studies grant FFG-840283iNEXT 653706ALTF 1335-2016FFG-852936FWF special research program SFB F34Wittgenstein award Z196-B20EMBL International PhD Programme (EIPP)Common Fund 4D Nucleome Program (U01 DA047728)LT001527/2017FFG-834223823839693949Allen Distinguished Investigator ProgramI 3686-B25 MEIOREC - ERA-CAPSWWTF LS09-13Common Fund 4D Nucleome Program (U01 EB021223)241548 (MitoSys)I 3686-B25503464 (MitoCheck)Walther NDurnberger GBusslinger GAHolzmann JStocsits RRDavidson IFMechtler KPoliti AZKoch BLadurner REllenberg JNagasaka KFuchs JTang WPeters JMNovak BHantsche-Grininger MfalseAbsolute quantification of cohesin, CTCF and their regulators in human cells.The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.2019-01-01T00:00:00Z2019 Jun2024-11-09T01:14:27.067Z2019-07-25T07:02:45ZS-EPMC66060263120499910.7554/eLife.46269