Detection of Histone H3 Phosphorylation in Cultured Cells and Tissue Sections by Immunostaining

  • Jaya Padmanabhan
Part of the Methods in Molecular Biology book series (MIMB, volume 523)


Growth factor stimulation results in phosphorylation of histone H3 at ser 10 and this correlated with expression of immediate early genes suggesting that this phosphorylation is associated with transcriptional activation. Although Western immunoblot analysis allows the detection of protein modifications in histones, in order to determine the localization of histones during different phases of cell cycle or during treatment of cells with different drugs we have to use immunohistochemistry. The protocol described here allows the detection of phosphorylated histones in tissue-cultured cells and tissue sections by fluorescent or bright-field immunostaining analysis. Here we used a serine 10 specific P-histone H3 antibody to determine the localization of this phosphoprotein in an asynchronously growing H4 glioma cell line and brain sections. It has been shown that long-term potentiation (LTP) is associated with gene transcription, and histone acetylation plays a major role in LTP formation (Wood et al., Learn Mem 13:241–244, 2006; Wood et al., Hippocampus 15:610–621, 2005; Alarcon et al., Neuron 42:947–959, 2004; Korzus et al., Neuron 42:961–972, 2004). Stimulus-induced phosphorylation of histone H3 at serine 10 has also been implicated in hippocampal neurons and striatal neurons (Li et al., J Neurochem 90:1117–1131, 2004; Crosio et al., J Cell Sci 116:4905–4914, 2003). Co-staining with a cell-specific antibody will allow us to determine the type of cells that show activation of histone phosphorylation in the brain.

Key words

Histone phosphorylation immunofluorescence immunohistochemistry 


  1. 1.
    Hans, F., and Dimitrov, S. (2001). Histone H3 phosphorylation and cell division. Oncogene 20:3021–3027.PubMedCrossRefGoogle Scholar
  2. 2.
    Peterson, C.L., and Laniel, M.A. (2004). Histones and histone modifications. Curr Biol 14:R546–551.PubMedCrossRefGoogle Scholar
  3. 3.
    Johansen, K.M., and Johansen, J. (2006). Regulation of chromatin structure by histone H3S10 phosphorylation. Chromosome Res 14:393–404.PubMedCrossRefGoogle Scholar
  4. 4.
    Koshland, D., and Strunnikov, A. (1996). Mitotic chromosome condensation. Annu Rev Cell Dev Biol 12:305–333.PubMedCrossRefGoogle Scholar
  5. 5.
    Bradbury, E.M. (1992). Reversible histone modifications and the chromosome cell cycle. Bioessays 14:9–16.PubMedCrossRefGoogle Scholar
  6. 6.
    Wei, Y., Mizzen, C.A., Cook, R.G., Gorovsky, M.A., and Allis, C.D. (1998). Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena. Proc Natl Acad Sci U S A 95:7480–7484.PubMedCrossRefGoogle Scholar
  7. 7.
    Wei, Y., Yu, L., Bowen, J., Gorovsky, M.A., and Allis, C.D. (1999). Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 97:99–109.PubMedCrossRefGoogle Scholar
  8. 8.
    Gurley, L.R., D'Anna, J.A., Barham, S.S., Deaven, L.L., and Tobey, R.A. (1978). Histone phosphorylation and chromatin structure during mitosis in Chinese hamster cells. Eur J Biochem 84:1–15.PubMedCrossRefGoogle Scholar
  9. 9.
    Mahadevan, L.C., Willis, A.C., and Barratt, M.J. (1991). Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors. Cell 65:775–783.PubMedCrossRefGoogle Scholar
  10. 10.
    Thomson, S., Mahadevan, L.C., and Clayton, A.L. (1999). MAP kinase-mediated signalling to nucleosomes and immediate-early gene induction. Semin Cell Dev Biol 10:205–214.PubMedCrossRefGoogle Scholar
  11. 11.
    Van Hooser, A., Goodrich, D.W., Allis, C.D., Brinkley, B.R., and Mancini, M.A. (1998). Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation. J Cell Sci 111 (Pt 23):3497–3506.PubMedGoogle Scholar
  12. 12.
    Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli, T., Brinkley, B.R., Bazett-Jones, D.P., and Allis, C.D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106:348–360.PubMedCrossRefGoogle Scholar
  13. 13.
    Nowak, S.J., and Corces, V.G. (2004). Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet 20:214–220.PubMedCrossRefGoogle Scholar
  14. 14.
    Prigent, C., and Dimitrov, S. (2003). Phosphorylation of serine 10 in histone H3, what for? J Cell Sci 116:3677–3685.PubMedCrossRefGoogle Scholar
  15. 15.
    Allis, C.D., and Gorovsky, M.A. (1981). Histone phosphorylation in macro- and micronuclei of Tetrahymena thermophila. Biochemistry 20:3828–3833.PubMedCrossRefGoogle Scholar
  16. 16.
    Hendzel, M.J., and Bazett-Jones, D.P. (1997). Fixation-dependent organization of core histones following DNA fluorescent in situ hybridization. Chromosoma 106:114–123.PubMedCrossRefGoogle Scholar
  17. 17.
    Crosio, C., Fimia, G.M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C.D., and Sassone-Corsi, P. (2002). Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol Cell Biol 22:874–885.PubMedCrossRefGoogle Scholar
  18. 18.
    Wood, M.A., Hawk, J.D., and Abel, T. (2006). Combinatorial chromatin modifications and memory storage: a code for memory? Learn Mem 13:241–244.PubMedCrossRefGoogle Scholar
  19. 19.
    Wood, M.A., Kaplan, M.P., Brensinger, C.M., Guo, W., and Abel, T. (2005). Ubiquitin C-terminal hydrolase L3 (Uchl3) is involved in working memory. Hippocampus 15:610–621.PubMedCrossRefGoogle Scholar
  20. 20.
    Alarcon, J.M., Malleret, G., Touzani, K., Vronskaya, S., Ishii, S., Kandel, E.R., and Barco, A. (2004). Chromatin acetylation, memory, and LTP are impaired in CBP+/– mice: a model for the cognitive deficit in Rubinstein-Taybi syndrome and its amelioration. Neuron 42:947–959.PubMedCrossRefGoogle Scholar
  21. 21.
    Korzus, E., Rosenfeld, M.G., and Mayford, M. (2004). CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron 42:961–972.PubMedCrossRefGoogle Scholar
  22. 22.
    Li, J., Guo, Y., Schroeder, F.A., Youngs, R.M., Schmidt, T.W., Ferris, C., Konradi, C., and Akbarian, S. (2004). Dopamine D2-like antagonists induce chromatin remodeling in striatal neurons through cyclic AMP-protein kinase A and NMDA receptor signaling. J Neurochem 90:1117–1131.PubMedCrossRefGoogle Scholar
  23. 23.
    Crosio, C., Heitz, E., Allis, C.D., Borrelli, E., and Sassone-Corsi, P. (2003). Chromatin remodeling and neuronal response: multiple signaling pathways induce specific histone H3 modifications and early gene expression in hippocampal neurons. J Cell Sci 116:4905–4914.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jaya Padmanabhan
    • 1
  1. 1.Johnnie B. Byrd, Sr. Alzheimer’s Center and Research InstituteTampaUSA

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