Skip to main content
Book cover

SUMO pp 205–216Cite as

Isolation of In Vivo SUMOylated Chromatin-Bound Proteins

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1475))

Abstract

SUMO posttranslational modification directs gene transcription and epigenetic programming to support normal cell function. The dynamic nature of SUMO-modification makes it difficult to identify endogenous protein substrates. Isolation of chromatin-bound SUMO targets is exceptionally challenging, as conventional immunoprecipitation assays are inefficient at concentrating this protein population. This chapter describes a protocol that effectively precipitates chromatin-associated fractions of SUMOylated heterochromatin protein 1α in cultured cells. Techniques to enrich endogenous SUMO substrates at the chromatin are also demonstrated and discussed. This approach could be adapted to evaluate chromatin-bound SUMO targets in additional in vivo systems.

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
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   169.99
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. Bawa-Khalfe T, Yeh ET (2010) The in vivo functions of desumoylating enzymes. Subcell Biochem 54:170–183. doi:10.1007/978-1-4419-6676-6_14

    Article  CAS  PubMed  Google Scholar 

  2. Bawa-Khalfe T, Yeh ET (2010) SUMO losing balance: SUMO proteases disrupt SUMO homeostasis to facilitate cancer development and progression. Genes Cancer 1(7):748–752. doi:10.1177/1947601910382555

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cubenas-Potts C, Matunis MJ (2013) SUMO: a multifaceted modifier of chromatin structure and function. Dev Cell 24(1):1–12. doi:10.1016/j.devcel.2012.11.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zilio N, Williamson CT, Eustermann S, Shah R, West SC, Neuhaus D, Ulrich HD (2013) DNA-dependent SUMO modification of PARP-1. DNA Repair 12(9):761–773. doi:10.1016/j.dnarep.2013.07.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Psakhye I, Jentsch S (2012) Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair. Cell 151(4):807–820. doi:10.1016/j.cell.2012.10.021

    Article  CAS  PubMed  Google Scholar 

  6. Shiio Y, Eisenman RN (2003) Histone sumoylation is associated with transcriptional repression. Proc Natl Acad Sci U S A 100(23):13225–13230. doi:10.1073/pnas.1735528100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cheng J, Bawa T, Lee P, Gong L, Yeh ET (2006) Role of desumoylation in the development of prostate cancer. Neoplasia 8(8):667–676. doi:10.1593/neo.06445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kim JM, Kee Y, Gurtan A, D’Andrea AD (2008) Cell cycle-dependent chromatin loading of the Fanconi anemia core complex by FANCM/FAAP24. Blood 111(10):5215–5222. doi:10.1182/blood-2007-09-113092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hall JR, Kow E, Nevis KR, Lu CK, Luce KS, Zhong Q, Cook JG (2007) Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage. Mol Biol Cell 18(9):3340–3350. doi:10.1091/mbc.E07-02-0173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schaefer M, Steringer JP, Lyko F (2008) The Drosophila cytosine-5 methyltransferase Dnmt2 is associated with the nuclear matrix and can access DNA during mitosis. PLoS One 3(1):e1414. doi:10.1371/journal.pone.0001414

    Article  PubMed  PubMed Central  Google Scholar 

  11. Bawa-Khalfe T, Lu LS, Zuo Y, Huang C, Dere R, Lin FM, Yeh ET (2012) Differential expression of SUMO-specific protease 7 variants regulates epithelial-mesenchymal transition. Proc Natl Acad Sci U S A 109(43):17466–17471. doi:10.1073/pnas.1209378109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tatham MH, Geoffroy MC, Shen L, Plechanovova A, Hattersley N, Jaffray EG, Palvimo JJ, Hay RT (2008) RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat Cell Biol 10(5):538–546. doi:10.1038/ncb1716

    Article  CAS  PubMed  Google Scholar 

  13. Desterro JM, Rodriguez MS, Hay RT (1998) SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell 2(2):233–239

    Article  CAS  PubMed  Google Scholar 

  14. Azuma Y, Arnaoutov A, Anan T, Dasso M (2005) PIASy mediates SUMO-2 conjugation of Topoisomerase-II on mitotic chromosomes. EMBO J 24(12):2172–2182. doi:10.1038/sj.emboj.7600700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dou H, Huang C, Singh M, Carpenter PB, Yeh ET (2010) Regulation of DNA repair through deSUMOylation and SUMOylation of replication protein A complex. Mol Cell 39(3):333–345. doi:10.1016/j.molcel.2010.07.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Garvin AJ, Densham RM, Blair-Reid SA, Pratt KM, Stone HR, Weekes D, Lawrence KJ, Morris JR (2013) The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair. EMBO Rep 14(11):975–983. doi:10.1038/embor.2013.141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kang X, Qi Y, Zuo Y, Wang Q, Zou Y, Schwartz RJ, Cheng J, Yeh ET (2010) SUMO-specific protease 2 is essential for suppression of polycomb group protein-mediated gene silencing during embryonic development. Mol Cell 38(2):191–201. doi:10.1016/j.molcel.2010.03.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Maison C, Romeo K, Bailly D, Dubarry M, Quivy JP, Almouzni G (2012) The SUMO protease SENP7 is a critical component to ensure HP1 enrichment at pericentric heterochromatin. Nat Struct Mol Biol 19(4):458–460. doi:10.1038/nsmb.2244, nsmb.2244 [pii]

    Article  CAS  PubMed  Google Scholar 

  19. Shin JA, Choi ES, Kim HS, Ho JC, Watts FZ, Park SD, Jang YK (2005) SUMO modification is involved in the maintenance of heterochromatin stability in fission yeast. Mol Cell 19(6):817–828. doi:10.1016/j.molcel.2005.08.021

    Article  CAS  PubMed  Google Scholar 

  20. Zhu J, Zhu S, Guzzo CM, Ellis NA, Sung KS, Choi CY, Matunis MJ (2008) Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification. J Biol Chem 283(43):29405–29415. doi:10.1074/jbc.M803632200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Maison C, Bailly D, Roche D, Montes de Oca R, Probst AV, Vassias I, Dingli F, Lombard B, Loew D, Quivy JP, Almouzni G (2011) SUMOylation promotes de novo targeting of HP1alpha to pericentric heterochromatin. Nat Genet 43(3):220–227. doi:10.1038/ng.765

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tasneem Bawa-Khalfe .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Bawa-Khalfe, T. (2016). Isolation of In Vivo SUMOylated Chromatin-Bound Proteins. In: Rodriguez, M. (eds) SUMO. Methods in Molecular Biology, vol 1475. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6358-4_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6358-4_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6356-0

  • Online ISBN: 978-1-4939-6358-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics