Absolute quantification of cohesin, CTCF and their regulators in human cells
- Research Institute of Molecular Pathology, Austria
- European Molecular Biology Laboratory, Germany
- University of Oxford, United Kingdom
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.
Data availability
Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012712. Sequencing data have been deposited in GEO (GSE126990.
-
ChIP-seq data from Absolute quantification of cohesin, CTCF and their regulators in human cellsNCBI Gene Expression Omnibus, GSE126990.
Article and author information
Author details
Iain Finley Davidson
Jan-Michael Peters
Funding
Boehringer Ingelheim
- Johann Holzmann
- Kota Nagasaka
- Johannes Fuchs
- Gerhard Dürnberger
- Wen Tang
- Rene Ladurner
- Georg A Busslinger
- Karl Mechtler
- Iain Finley Davidson
- Jan-Michael Peters
European Molecular Biology Laboratory
- Antonio Z Politi
- Merle Hantsche-Grininger
- Nike Walther
- Birgit Koch
- Jan Ellenberg
National Institutes of Health (Common Fund 4D Nucleome Program (U01 EB021223 / U01 DA047728))
- Jan Ellenberg
Paul G. Allen Frontiers Group (Allen Distinguished Investigator Program)
- Jan Ellenberg
EMBL International PhD Programme
- Nike Walther
Austrian Research Promotion Agency (FFG-852936)
- Jan-Michael Peters
Austrian Research Promotion Agency (Laura Bassi Centre for Optimized Structural Studies grant FFG-840283)
- Jan-Michael Peters
Austrian Science Fund (Wittgenstein award Z196-B20)
- Jan-Michael Peters
Horizon 2020 Framework Programme (653706)
- Jan Ellenberg
Horizon 2020 Framework Programme (823839)
- Karl Mechtler
Austrian Science Fund (I 3686-B25 MEIOREC - ERA-CAPS)
- Karl Mechtler
Austrian Science Fund (FWF special research program SFB F34)
- Jan-Michael Peters
Austrian Research Promotion Agency (FFG-834223)
- Jan-Michael Peters
Vienna Science and Technology Fund (WWTF LS09-13)
- Jan-Michael Peters
Seventh Framework Programme (241548 (MitoSys))
- Jan Ellenberg
- Jan-Michael Peters
Horizon 2020 Framework Programme (693949)
- Jan-Michael Peters
Sixth Framework Programme (503464 (MitoCheck))
- Jan-Michael Peters
European Molecular Biology Organization (ALTF 1335-2016)
- Kota Nagasaka
Human Frontier Science Program (LT001527/2017)
- Kota Nagasaka
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Holzmann et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,227
- views
-
- 975
- downloads
-
- 94
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Absolute quantification of cohesin, CTCF and their regulators in human cells
eLife 8:e46269.
https://doi.org/10.7554/eLife.46269
Further reading
-
- Cancer Biology
- Cell Biology
Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate transporter SLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here, Fgf23 knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name: Bglap, protein name). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lower Slc34a2 and higher Bglap/Slc34a1 (protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lower SLC34A2 and a subpopulation of germ cells express phosphate transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbance in vitro using calcium or phosphate treatment induced deposition of calcium phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.
-
- Cell Biology
- Genetics and Genomics
A glaucoma polygenic risk score (PRS) can effectively identify disease risk, but some individuals with high PRS do not develop glaucoma. Factors contributing to this resilience remain unclear. Using 4,658 glaucoma cases and 113,040 controls in a cross-sectional study of the UK Biobank, we investigated whether plasma metabolites enhanced glaucoma prediction and if a metabolomic signature of resilience in high-genetic-risk individuals existed. Logistic regression models incorporating 168 NMR-based metabolites into PRS-based glaucoma assessments were developed, with multiple comparison corrections applied. While metabolites weakly predicted glaucoma (Area Under the Curve = 0.579), they offered marginal prediction improvement in PRS-only-based models (p=0.004). We identified a metabolomic signature associated with resilience in the top glaucoma PRS decile, with elevated glycolysis-related metabolites—lactate (p=8.8E-12), pyruvate (p=1.9E-10), and citrate (p=0.02)—linked to reduced glaucoma prevalence. These metabolites combined significantly modified the PRS-glaucoma relationship (Pinteraction = 0.011). Higher total resilience metabolite levels within the highest PRS quartile corresponded to lower glaucoma prevalence (Odds Ratiohighest vs. lowest total resilience metabolite quartile=0.71, 95% Confidence Interval = 0.64–0.80). As pyruvate is a foundational metabolite linking glycolysis to tricarboxylic acid cycle metabolism and ATP generation, we pursued experimental validation for this putative resilience biomarker in a human-relevant Mus musculus glaucoma model. Dietary pyruvate mitigated elevated intraocular pressure (p=0.002) and optic nerve damage (p<0.0003) in Lmx1bV265D mice. These findings highlight the protective role of pyruvate-related metabolism against glaucoma and suggest potential avenues for therapeutic intervention.
