Skip to main content
SpringerLink
Log in
Book cover

Cohesin and Condensin pp 37–53Cite as

Biochemical and Functional Assays of Human Cohesin-Releasing Factor Wapl

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1515))

Abstract

During the cell cycle, duplicated sister chromatids become physically connected during S phase through a process called sister-chromatid cohesion. Cohesion is terminated during the metaphase-to-anaphase transition to trigger sister-chromatid segregation. The establishment and dissolution of cohesion are highly regulated by the cohesin complex and its multitude of regulators. In particular, the cohesin regulator Wapl promotes the release of cohesin from chromosomes during both interphase and mitosis. Here, we describe in vitro protein binding assays between Wapl and a cohesin subcomplex, and cellular assays in human cells that probe the functions of Wapl in cohesin release.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Nasmyth K (2001) Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu Rev Genet 35:673–745

    Article  CAS  PubMed  Google Scholar 

  2. Lee JY, Orr-Weaver TL (2001) The molecular basis of sister-chromatid cohesion. Annu Rev Cell Dev Biol 17:753–777

    Article  CAS  PubMed  Google Scholar 

  3. Schvartzman JM, Sotillo R, Benezra R (2010) Mitotic chromosomal instability and cancer: mouse modelling of the human disease. Nat Rev Cancer 10:102–115

    Article  CAS  PubMed  Google Scholar 

  4. Solomon DA, Kim T, Diaz-Martinez LA, Fair J, Elkahloun AG, Harris BT, Toretsky JA, Rosenberg SA, Shukla N, Ladanyi M, Samuels Y, James CD, Yu H, Kim JS, Waldman T (2011) Mutational inactivation of STAG2 causes aneuploidy in human cancer. Science 333:1039–1043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Remeseiro S, Cuadrado A, Losada A (2013) Cohesin in development and disease. Development 140:3715–3718

    Article  CAS  PubMed  Google Scholar 

  6. Nasmyth K (2011) Cohesin: a catenase with separate entry and exit gates? Nat Cell Biol 13:1170–1177

    Article  CAS  PubMed  Google Scholar 

  7. Nasmyth K, Haering CH (2009) Cohesin: its roles and mechanisms. Annu Rev Genet 43:525–558

    Article  CAS  PubMed  Google Scholar 

  8. Peters JM, Tedeschi A, Schmitz J (2008) The cohesin complex and its roles in chromosome biology. Genes Dev 22:3089–3114

    Article  CAS  PubMed  Google Scholar 

  9. Onn I, Heidinger-Pauli JM, Guacci V, Unal E, Koshland DE (2008) Sister chromatid cohesion: a simple concept with a complex reality. Annu Rev Cell Dev Biol 24:105–129

    Article  CAS  PubMed  Google Scholar 

  10. Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5:243–254

    Article  CAS  PubMed  Google Scholar 

  11. Tonkin ET, Wang TJ, Lisgo S, Bamshad MJ, Strachan T (2004) NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome. Nat Genet 36:636–641

    Article  CAS  PubMed  Google Scholar 

  12. Gillespie PJ, Hirano T (2004) Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts. Curr Biol 14:1598–1603

    Article  CAS  PubMed  Google Scholar 

  13. Watrin E, Schleiffer A, Tanaka K, Eisenhaber F, Nasmyth K, Peters JM (2006) Human Scc4 is required for cohesin binding to chromatin, sister-chromatid cohesion, and mitotic progression. Curr Biol 16:863–874

    Article  CAS  PubMed  Google Scholar 

  14. Bermudez VP, Farina A, Higashi TL, Du F, Tappin I, Takahashi TS, Hurwitz J (2012) In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex. Proc Natl Acad Sci U S A 109:9366–9371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Murayama Y, Uhlmann F (2014) Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505:367–371

    Article  CAS  PubMed  Google Scholar 

  16. Verni F, Gandhi R, Goldberg ML, Gatti M (2000) Genetic and molecular analysis of wings apart-like (wapl), a gene controlling heterochromatin organization in Drosophila melanogaster. Genetics 154:1693–1710

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Kueng S, Hegemann B, Peters BH, Lipp JJ, Schleiffer A, Mechtler K, Peters JM (2006) Wapl controls the dynamic association of cohesin with chromatin. Cell 127:955–967

    Article  CAS  PubMed  Google Scholar 

  18. Gandhi R, Gillespie PJ, Hirano T (2006) Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase. Curr Biol 16:2406–2417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Losada A, Yokochi T, Hirano T (2005) Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts. J Cell Sci 118:2133–2141

    Article  CAS  PubMed  Google Scholar 

  20. Nishiyama T, Ladurner R, Schmitz J, Kreidl E, Schleiffer A, Bhaskara V, Bando M, Shirahige K, Hyman AA, Mechtler K, Peters JM (2010) Sororin mediates sister chromatid cohesion by antagonizing Wapl. Cell 143:737–749

    Article  CAS  PubMed  Google Scholar 

  21. Hou F, Zou H (2005) Two human orthologues of Eco1/Ctf7 acetyltransferases are both required for proper sister-chromatid cohesion. Mol Biol Cell 16:3908–3918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rankin S (2005) Sororin, the cell cycle and sister chromatid cohesion. Cell Cycle 4:1039–1042

    Article  CAS  PubMed  Google Scholar 

  23. Rankin S, Ayad NG, Kirschner MW (2005) Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates. Mol Cell 18:185–200

    Article  CAS  PubMed  Google Scholar 

  24. Lafont AL, Song J, Rankin S (2010) Sororin cooperates with the acetyltransferase Eco2 to ensure DNA replication-dependent sister chromatid cohesion. Proc Natl Acad Sci U S A 107:20364–20369

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ben-Shahar Rolef T, Heeger S, Lehane C, East P, Flynn H, Skehel M, Uhlmann F (2008) Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion. Science 321:563–566

    Article  Google Scholar 

  26. Ivanov D, Schleiffer A, Eisenhaber F, Mechtler K, Haering CH, Nasmyth K (2002) Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion. Curr Biol 12:323–328

    Article  CAS  PubMed  Google Scholar 

  27. Rowland BD, Roig MB, Nishino T, Kurze A, Uluocak P, Mishra A, Beckouet F, Underwood P, Metson J, Imre R, Mechtler K, Katis VL, Nasmyth K (2009) Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity. Mol Cell 33:763–774

    Article  CAS  PubMed  Google Scholar 

  28. Zhang J, Shi X, Li Y, Kim BJ, Jia J, Huang Z, Yang T, Fu X, Jung SY, Wang Y, Zhang P, Kim ST, Pan X, Qin J (2008) Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast. Mol Cell 31:143–151

    Article  CAS  PubMed  Google Scholar 

  29. Unal E, Heidinger-Pauli JM, Kim W, Guacci V, Onn I, Gygi SP, Koshland DE (2008) A molecular determinant for the establishment of sister chromatid cohesion. Science 321:566–569

    Article  PubMed  Google Scholar 

  30. Schmitz J, Watrin E, Lenart P, Mechtler K, Peters JM (2007) Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase. Curr Biol 17:630–636

    Article  CAS  PubMed  Google Scholar 

  31. Waizenegger IC, Hauf S, Meinke A, Peters JM (2000) Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase. Cell 103:399–410

    Article  CAS  PubMed  Google Scholar 

  32. Hauf S, Roitinger E, Koch B, Dittrich CM, Mechtler K, Peters JM (2005) Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2. PLoS Biol 3, e69

    Article  PubMed  PubMed Central  Google Scholar 

  33. Dreier MR, Bekier ME 2nd, Taylor WR (2011) Regulation of sororin by Cdk1-mediated phosphorylation. J Cell Sci 124:2976–2987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nishiyama T, Sykora MM, Huis in ‘t Veld PJ, Mechtler K, Peters JM (2013) Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin. Proc Natl Acad Sci U S A 110:13404–13409

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhang N, Panigrahi AK, Mao Q, Pati D (2011) Interaction of Sororin protein with polo-like kinase 1 mediates resolution of chromosomal arm cohesion. J Biol Chem 286:41826–41837

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Huis in ‘t Veld PJ, Herzog F, Ladurner R, Davidson IF, Piric S, Kreidl E, Bhaskara V, Aebersold R, Peters JM (2014) Characterization of a DNA exit gate in the human cohesin ring. Science 346:968–972

    Article  PubMed  Google Scholar 

  37. Chan KL, Roig MB, Hu B, Beckouet F, Metson J, Nasmyth K (2012) Cohesin's DNA exit gate is distinct from its entrance gate and is regulated by acetylation. Cell 150:961–974

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gligoris TG, Scheinost JC, Burmann F, Petela N, Chan KL, Uluocak P, Beckouet F, Gruber S, Nasmyth K, Lowe J (2014) Closing the cohesin ring: structure and function of its Smc3-kleisin interface. Science 346:963–967

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kitajima TS, Sakuno T, Ishiguro K, Iemura S, Natsume T, Kawashima SA, Watanabe Y (2006) Shugoshin collaborates with protein phosphatase 2A to protect cohesin. Nature 441:46–52

    Article  CAS  PubMed  Google Scholar 

  40. Tang Z, Shu H, Qi W, Mahmood NA, Mumby MC, Yu H (2006) PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation. Dev Cell 10:575–585

    Article  CAS  PubMed  Google Scholar 

  41. McGuinness BE, Hirota T, Kudo NR, Peters JM, Nasmyth K (2005) Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells. PLoS Biol 3, e86

    Article  PubMed  PubMed Central  Google Scholar 

  42. Watanabe Y, Kitajima TS (2005) Shugoshin protects cohesin complexes at centromeres. Philos Trans R Soc Lond B Biol Sci 360:515–521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Xu Z, Cetin B, Anger M, Cho US, Helmhart W, Nasmyth K, Xu W (2009) Structure and function of the PP2A-shugoshin interaction. Mol Cell 35:426–441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Liu H, Rankin S, Yu H (2013) Phosphorylation-enabled binding of SGO1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis. Nat Cell Biol 15:40–49

    Article  CAS  PubMed  Google Scholar 

  45. Hara K, Zheng G, Qu Q, Liu H, Ouyang Z, Chen Z, Tomchick DR, Yu H (2014) Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion. Nat Struct Mol Biol 21:864–870

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Liu H, Jia L, Yu H (2013) Phospho-H2A and cohesin specify distinct tension-regulated Sgo1 pools at kinetochores and inner centromeres. Curr Biol 23:1927–1933

    Article  CAS  PubMed  Google Scholar 

  47. Hirano T (2015) Chromosome dynamics during mitosis. Cold Spring Harb Perspect Biol 7:pii:a015792. doi:10.1101/cshperspect.a015792

    Article  Google Scholar 

  48. Hauf S, Waizenegger IC, Peters JM (2001) Cohesin cleavage by separase required for anaphase and cytokinesis in human cells. Science 293:1320–1323

    Article  CAS  PubMed  Google Scholar 

  49. Jia L, Kim S, Yu H (2013) Tracking spindle checkpoint signals from kinetochores to APC/C. Trends Biochem Sci 38:302–311

    Article  CAS  PubMed  Google Scholar 

  50. Foley EA, Kapoor TM (2013) Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nat Rev Mol Cell Biol 14:25–37

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ouyang Z, Zheng G, Song J, Borek DM, Otwinowski Z, Brautigam CA, Tomchick DR, Rankin S, Yu H (2013) Structure of the human cohesin inhibitor Wapl. Proc Natl Acad Sci U S A 110:11355–11360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgement

We thank Drs. Kodai Hara and Qianhui Qu for their input in the purification of SA2 and Wapl proteins. We thank all Yu lab members for helpful discussions and critical reading of the manuscript. H.Y. is an investigator with the Howard Hughes Medical Institute. Work in the Yu lab is supported by grants from the Welch Foundation (I-1441 to H.Y.), the Clayton Foundation, and Cancer Prevention and Research Institute of Texas (RP110465-P3 and RP120717-P2 to H.Y.).

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, 75390, USA

    Ge Zheng, Zhuqing Ouyang & Hongtao Yu

Authors
  1. Ge Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhuqing Ouyang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hongtao Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hongtao Yu .

Editor information

Editors and Affiliations

  1. Department of Biological Chemistry, School of Medicine University of California— Irvine, Irvine, California, USA

    Kyoko Yokomori

  2. Laboratory of Genome Structure and Function Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences The University of Tokyo, Tokyo, Japan

    Katsuhiko Shirahige

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Zheng, G., Ouyang, Z., Yu, H. (2017). Biochemical and Functional Assays of Human Cohesin-Releasing Factor Wapl. In: Yokomori, K., Shirahige, K. (eds) Cohesin and Condensin. Methods in Molecular Biology, vol 1515. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6545-8_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6545-8_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6543-4

  • Online ISBN: 978-1-4939-6545-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation