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Elife. 2019 Jun 17;8. pii: e46269. doi: 10.7554/eLife.46269.

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

Author information

1
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.
2
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria.
3
Gregor Mendel Institute, Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria.
4
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
5
Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
6
Medical University of Vienna, Vienna, Austria.
#
Contributed equally

Abstract

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.

KEYWORDS:

CTCF; cell biology; chromosomes; cohesin; fluorescence-correlation spectroscopy; gene expression; genome organization; human; mass spectrometry; sister chromatid cohesion

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