Methods to Study Histone Chaperone Function in Nucleosome Assembly and Chromatin Transcription

  • Parijat Senapati
  • Deepthi Sudarshan
  • Shrikanth S. Gadad
  • Jayasha Shandilya
  • Venkatesh Swaminathan
  • Tapas K. KunduEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1288)


Histone chaperones are histone interacting proteins that are involved in various stages of histone metabolism in the cell such as histone storage, transport, nucleosome assembly and disassembly. Histone assembly and disassembly are essential processes in certain DNA-templated phenomena such as replication, repair and transcription in eukaryotes. Since the first histone chaperone Nucleoplasmin was discovered in Xenopus, a plethora of histone chaperones have been identified, characterized and their functional significance elucidated in the last 35 years or so. Some of the histone chaperone containing complexes such as FACT have been described to play a significant role in nucleosome disassembly during transcription elongation. We have reported earlier that human Nucleophosmin (NPM1), a histone chaperone belonging to the Nucleoplasmin family, is a co-activator of transcription. In this chapter, we describe several methods that are used to study the histone chaperone activity of proteins and their role in transcription.

Key words

Chromatin transcription assay ACF complex p300 Nucleophosmin 



We thank R.G. Roeder and J. Kadonaga for some of the valuable reagents used in this work.


  1. 1.
    Loyola A, Almouzni G (2004) Histone chaperones, a supporting role in the limelight. Biochim Biophys Acta 1677:3–11CrossRefPubMedGoogle Scholar
  2. 2.
    Swaminathan V, Kishore A, Febitha K, Kundu T (2005) Human histone chaperone nucleophosmin enhances acetylation-dependent chromatin transcription. Mol Cell Biol 25:7534–7545CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Shandilya J, Gadad S, Swaminathan V, Kundu TK (2007) Histone chaperones in chromatin dynamics: implications in disease manifestation. Subcell Biochem 41:111–124CrossRefPubMedGoogle Scholar
  4. 4.
    Burgess RJ, Zhang Z (2013) Histone chaperones in nucleosome assembly and human disease. Nat Struct Mol Biol 20:14–22CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Okuwaki M, Tsujimoto M, Nagata K (2002) The RNA binding activity of a ribosome biogenesis factor, nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype. Mol Biol Cell 13:2016–2030CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Grisendi S, Bernardi R, Rossi M, Cheng K, Khandker L, Manova K, Pandolfi PP (2005) Role of nucleophosmin in embryonic development and tumorigenesis. Nature 437:147–153CrossRefPubMedGoogle Scholar
  7. 7.
    Tarapore P, Okuda M, Fukasawa K (2002) A mammalian in vitro centriole duplication system: evidence for involvement of CDK2/cyclin E and nucleophosmin/B23 in centrosome duplication. Cell Cycle 1:75–81CrossRefPubMedGoogle Scholar
  8. 8.
    Yung BY (2007) Oncogenic role of nucleophosmin/B23. Chang Gung Med J 30:285–293PubMedGoogle Scholar
  9. 9.
    Okuwaki M, Matsumoto K, Tsujimoto M, Nagata K (2001) Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone. FEBS Lett 506:272–276CrossRefPubMedGoogle Scholar
  10. 10.
    Shandilya J, Swaminathan V, Gadad SS, Choudhari R, Kodaganur GS, Kundu TK (2009) Acetylated NPM1 localizes in the nucleoplasm and regulates transcriptional activation of genes implicated in oral cancer manifestation. Mol Cell Biol 29:5115–5127CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Gadad SS, Shandilya J, Kishore AH, Kundu TK (2010) NPM3, a member of the nucleophosmin/nucleoplasmin family, enhances activator-dependent transcription. Biochemistry 49:1355–1357CrossRefPubMedGoogle Scholar
  12. 12.
    Kundu T, Palhan V, Wang Z, An W, Cole P, Roeder R (2000) Activator-dependent transcription from chromatin in vitro involving targeted histone acetylation by p300. Mol Cell 6:551–561CrossRefPubMedGoogle Scholar
  13. 13.
    Fyodorov DV, Kadonaga JT (2003) Chromatin assembly in vitro with purified recombinant ACF and NAP-1. Methods Enzymol 371:499–515CrossRefPubMedGoogle Scholar
  14. 14.
    Kundu T, Wang Z, Roeder R (1999) Human TFIIIC relieves chromatin-mediated repression of RNA polymerase III transcription and contains an intrinsic histone acetyltransferase activity. Mol Cell Biol 19:1605–1615PubMedCentralPubMedGoogle Scholar
  15. 15.
    Munakata T, Adachi N, Yokoyama N, Kuzuhara T, Horikoshi M (2000) A human homologue of yeast anti-silencing factor has histone chaperone activity. Genes Cells 5:221–233CrossRefPubMedGoogle Scholar
  16. 16.
    An W, Roeder RG (2004) Reconstitution and transcriptional analysis of chromatin in vitro. Methods Enzymol 377:460–474CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Parijat Senapati
    • 1
  • Deepthi Sudarshan
    • 1
  • Shrikanth S. Gadad
    • 1
  • Jayasha Shandilya
    • 1
  • Venkatesh Swaminathan
    • 1
  • Tapas K. Kundu
    • 1
    Email author
  1. 1.Transcription and Disease Laboratory, Molecular Biology and Genetics UnitJawaharlal Nehru Centre for Advanced Scientific ResearchBangaloreIndia

Personalised recommendations