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Wapl is an essential regulator of chromatin structure and chromosome segregation

Abstract

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures1,2,3,4,5,6,7, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl8,9 stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin–DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin–DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

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Figure 1: Wapl depletion reveals arrangement of cohesin in axial chromosomal domains.
Figure 2: Wapl controls chromatin structure by regulating cohesin–DNA interactions.
Figure 3: Wapl is essential for cell cycle progression and proper chromosome segregation.
Figure 4: The prophase pathway of cohesin dissociation protects cohesin from cleavage by separase.

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Accessions

Gene Expression Omnibus

Data deposits

ChiP-seq and microarray data have been deposited in the GEO database (http://www.ncbi.nlm.nih.gov/geo) under accession code GSE41603.

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Acknowledgements

We dedicate this paper to the memory of B. Peters, who performed the first experiments on Wapl in our laboratory. We are grateful to K. Aumayr, O. F.-Capetillo, T. Hoffmann, M. E. Idarraga-Amado, S. Kueng, T. Kulcsar, M. Leeb, P. Pasierbek, D. Santamaría, G. Schmauss, A. Souabni, H. Tkadletz, K. Wendt and members of the Peters laboratory for discussions and assistance, J. Hutchins for suggesting the term vermicelli, K. Nasmyth for the separase mouse model, and M. Barbacid, T. Cremer, T. Hirano, T. Jenuwein, M. Malumbres and J. Zuber for reagents. T.N. was supported by the European Molecular Biology Organization (EMBO) and the Japanese Society for the Promotion of Science (JSPS). S.H. was supported by funds from the Agence National de la Recherche (JCJC-SVSE2-2011, ChromaTranscript project) and the European Union (FP7-PEOPLE-2011-CIG, ChromaTranscript project). D.A.C. was supported by MFPL VIPS Program (BMWF and City of Vienna). A.V. acknowledges financial support by the Vienna Science and Technology Fund (WWTF) project VRG10-11, the Research Platform Quantum Phenomena and Nanoscale Biological Systems (QuNaBioS) and by Boehringer Ingelheim. Research in the laboratory of J.-M.P. is supported by Boehringer Ingelheim, the Austrian Science Fund (FWF special research program SFB F34 ‘Chromosome Dynamics’, and Wittgenstein award Z196-B20), the Austrian Research Promotion Agency (FFG, Laura Bassi Center for Optimized Structural Studies), the Vienna Science and Technology Fund (WWTF LS09-13), and the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 241548 (MitoSys).

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Experiments were designed and data interpreted by A.T., G.W., S.H., M.J., J.E. and J.-M.P. A.T. generated Waplfl embryonic stem cells, established and maintained the mouse colony required for this study, and carried out chromosome segregation, transcriptome and ChIP-seq studies. A.T. and G.W. analysed cohesin localization by IFM, and cleavage of Scc1 in anaphase MEFs. G.W. generated MEFs expressing Scc1–LAP and Scc1–9myc, and carried out cohesin IFM experiments in telophase and G1-phase MEFs. G.W. and V.B. performed chromatin fractionation experiments. G.W. and T.N. performed cleavage assays of Scc1 with Xenopus egg extracts. A.T., G.W. and W.T analysed cell cycle progression of MEFs. S.H. performed DNA granularity analysis and Cy3-dUTP labelling of DNA. M.J. carried out bioinformatic analyses. A.Wue. performed cohesin FRAP experiments. E.S. performed FISH experiments. A.Wut. supervised the work of A.T. with embryonic stem cells, and participated in designing the Wapl targeting strategy. I.F.D. performed replication assays with Xenopus egg extracts. D.A.C. performed live imaging of cohesin vermicelli under the guidance of A.V. A.T. and J.-M.P. wrote the manuscript.

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Correspondence to Jan-Michael Peters.

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This file contains Supplementary Figures 1-18, Supplementary Methods, additional references and primer sequences for Supplementary figure 11. (PDF 2113 kb)

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Tedeschi, A., Wutz, G., Huet, S. et al. Wapl is an essential regulator of chromatin structure and chromosome segregation. Nature 501, 564–568 (2013). https://doi.org/10.1038/nature12471

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