Absolute quantification of cohesin, CTCF and their regulators in human cells

• Published in: eLife Vol. 8
• Main Authors: Holzmann, Johann, Politi, Antonio Z, Nagasaka, Kota, Hantsche-Grininger, Merle, Walther, Nike, Koch, Birgit, Fuchs, Johannes, Dürnberger, Gerhard, Tang, Wen, Ladurner, Rene, Stocsits, Roman R, Busslinger, Georg A, Novák, Béla, Mechtler, Karl, Davidson, Iain Finley, Ellenberg, Jan, Peters, Jan-Michael
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 17.06.2019; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Binding proteins; Binding sites; Biochemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; CCCTC-Binding Factor - genetics; CCCTC-Binding Factor - metabolism; Cell Biology; Cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Line; Cells (Biology); Chromatids - genetics; Chromatin; Chromatin - genetics; Chromatin - metabolism; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Chromosome Segregation - genetics; Chromosomes; Chromosomes and Gene Expression; Cohesin; Cohesins; CTCF; Deoxyribonucleic acid; DNA; Fluorescence; Fluorescence recovery after photobleaching; Fluorescence Recovery After Photobleaching - methods; fluorescence-correlation spectroscopy; G1 Phase - genetics; Gene Dosage; Gene Expression; Genes; genome organization; Genome, Human - genetics; Genomes; Genomics; HeLa Cells; Humans; Immunoprecipitation; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Photobleaching; Physiological aspects; Proteins; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Recombination; Scientific imaging; sister chromatid cohesion; Spectroscopy; Stem cells; United States; United Kingdom > UK; Vienna Austria; cell biology; cohesin; sister chromatid cohesion; fluorescence-correlation spectroscopy; genome organization; chromosomes; mass spectrometry; CTCF; human; gene expression
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.46269

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.