Methods for Analyzing Histone Citrullination in Chromatin Structure and Gene Regulation

  • Pingxin Li
  • Jing Hu
  • Yanming WangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 809)


Histone posttranslational modifications play significant roles in regulating chromatin structure and gene expression. One of the histone modifications, histone citrullination, is catalyzed by an enzyme called peptidylarginine deiminase 4 (PAD4, also called PADI4), which converts both histone arginine (Arg) and mono-methyl arginine residues to citrulline. Recent studies have found that histone citrullination counteracts the effect of histone arginine methylation and functions as a repressive marker to turn off gene expression. Here, we describe assays to study histone citrullination by PAD4 in vitro and in vivo. We also describe approaches to measure histone citrullination levels at gene promoters using chromatin immunoprecipitation assay and analyze the effects of PAD4 inhibitor on cell cycle and apoptosis by flow cytometry. These methods would be useful techniques to study this unique histone modification.

Key words

Histone citrullination Histone arginine methylation Peptidylarginine deiminase 4 Nucleosome Chromatin immunoprecipitation 



The authors would like to thank the Flow Cytometry Facility at the Pennsylvania State University for its technical help. Research in the Wang laboratory is supported by NIH grant R01 CA136856.


  1. 1.
    Kornberg, R. D., and Lorch, Y. (1999) Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98 285–94.PubMedCrossRefGoogle Scholar
  2. 2.
    Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389 251–60.PubMedCrossRefGoogle Scholar
  3. 3.
    Richmond, T. J., and Davey, C. A. (2003) The structure of DNA in the nucleosome core. Nature 423 145–50.PubMedCrossRefGoogle Scholar
  4. 4.
    Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W., and Richmond, T. J. (2002) Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution. J Mol Biol 319 1097–113.PubMedCrossRefGoogle Scholar
  5. 5.
    Ramakrishnan, V. (1997) Histone H1 and chromatin higher-order structure. Crit Rev Eukaryot Gene Expr 7 215–30.PubMedGoogle Scholar
  6. 6.
    Narlikar, G. J., Fan, H. Y., and Kingston, R. E. (2002) Cooperation between complexes that regulate chromatin structure and transcription. Cell 108 475–87.PubMedCrossRefGoogle Scholar
  7. 7.
    Campos, E. I., and Reinberg, D. (2009) Histones: annotating chromatin. Annu Rev Genet 43 559–99.PubMedCrossRefGoogle Scholar
  8. 8.
    Li, B., Carey, M., and Workman, J. L. (2007) The role of chromatin during transcription. Cell 128 707–19.PubMedCrossRefGoogle Scholar
  9. 9.
    Kouzarides, T. (2007) Chromatin modifications and their function. Cell 128 693–705.PubMedCrossRefGoogle Scholar
  10. 10.
    Groth, A., Rocha, W., Verreault, A., and Almouzni, G. (2007) Chromatin challenges during DNA replication and repair. Cell 128 721–33.PubMedCrossRefGoogle Scholar
  11. 11.
    Jenuwein, T., and Allis, C. D. (2001) Translating the histone code. Science 293 1074–80.PubMedCrossRefGoogle Scholar
  12. 12.
    Kouzarides, T. (2002) Histone methylation in transcriptional control. Curr Opin Genet Dev 12 198–209.PubMedCrossRefGoogle Scholar
  13. 13.
    Kuo, M. H., and Allis, C. D. (1998) Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 20 615–26.PubMedCrossRefGoogle Scholar
  14. 14.
    Nowak, S. J., and Corces, V. G. (2004) Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet 20 214–20.PubMedCrossRefGoogle Scholar
  15. 15.
    Wang, Y., Wysocka, J., Sayegh, J., Lee, Y. H., Perlin, J. R., Leonelli, L., Sonbuchner, L. S., McDonald, C. H., Cook, R. G., Dou, Y., Roeder, R. G., Clarke, S., Stallcup, M. R., Allis, C. D., and Coonrod, S. A. (2004) Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306 279–83.PubMedCrossRefGoogle Scholar
  16. 16.
    McBride, A. E., and Silver, P. A. (2001) State of the arg: protein methylation at arginine comes of age. Cell 106 5–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Bedford, M. T., and Clarke, S. G. (2009) Protein arginine methylation in mammals: who, what, and why. Mol Cell 33 1–13.PubMedCrossRefGoogle Scholar
  18. 18.
    Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S. M., Schurter, B. T., Aswad, D. W., and Stallcup, M. R. (1999) Regulation of transcription by a protein methyltransferase. Science 284 2174–7.PubMedCrossRefGoogle Scholar
  19. 19.
    Wang, H., Huang, Z. Q., Xia, L., Feng, Q., Erdjument-Bromage, H., Strahl, B. D., Briggs, S. D., Allis, C. D., Wong, J., Tempst, P., and Zhang, Y. (2001) Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science 293 853–7.PubMedCrossRefGoogle Scholar
  20. 20.
    Pal, S., Vishwanath, S. N., Erdjument-Bromage, H., Tempst, P., and Sif, S. (2004) Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes. Mol Cell Biol 24 9630–45.PubMedCrossRefGoogle Scholar
  21. 21.
    Nowak, S. J., Pai, C. Y., and Corces, V. G. (2003) Protein phosphatase 2A activity affects histone H3 phosphorylation and transcription in Drosophila melanogaster. Mol Cell Biol 23 6129–38.PubMedCrossRefGoogle Scholar
  22. 22.
    Bannister, A. J., Schneider, R., and Kouzarides, T. (2002) Histone methylation: dynamic or static? Cell 109 801–6.PubMedCrossRefGoogle Scholar
  23. 23.
    Chang, B., Chen, Y., Zhao, Y., and Bruick, R. K. (2007) JMJD6 is a histone arginine demethylase. Science 318 444–7.PubMedCrossRefGoogle Scholar
  24. 24.
    Webby, C. J., Wolf, A., Gromak, N., Dreger, M., Kramer, H., Kessler, B., Nielsen, M. L., Schmitz, C., Butler, D. S., Yates, J. R., 3rd, Delahunty, C. M., Hahn, P., Lengeling, A., Mann, M., Proudfoot, N. J., Schofield, C. J., and Bottger, A. (2009) Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing. Science 325 90–3.PubMedCrossRefGoogle Scholar
  25. 25.
    Cuthbert, G. L., Daujat, S., Snowden, A. W., Erdjument-Bromage, H., Hagiwara, T., Yamada, M., Schneider, R., Gregory, P. D., Tempst, P., Bannister, A. J., and Kouzarides, T. (2004) Histone deimination antagonizes arginine methylation. Cell 118 545–53.PubMedCrossRefGoogle Scholar
  26. 26.
    Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., and Pruijn, G. J. (2003) PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 25 1106–18.PubMedCrossRefGoogle Scholar
  27. 27.
    Nakashima, K., Hagiwara, T., Ishigami, A., Nagata, S., Asaga, H., Kuramoto, M., Senshu, T., and Yamada, M. (1999) Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3). J Biol Chem 274 27786–92.PubMedCrossRefGoogle Scholar
  28. 28.
    Hagiwara, T., Nakashima, K., Hirano, H., Senshu, T., and Yamada, M. (2002) Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes. Biochem Biophys Res Commun 290 979–83.PubMedCrossRefGoogle Scholar
  29. 29.
    Tarcsa, E., Marekov, L. N., Mei, G., Melino, G., Lee, S. C., and Steinert, P. M. (1996) Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin. J Biol Chem 271 30709–16.PubMedCrossRefGoogle Scholar
  30. 30.
    Thompson, P. R., and Fast, W. (2006) Histone citrullination by protein arginine deiminase: is arginine methylation a green light or a roadblock? ACS Chem Biol 1 433–41.PubMedCrossRefGoogle Scholar
  31. 31.
    Chang, X., and Han, J. (2006) Expression of peptidylarginine deiminase type 4 (PAD4) in various tumors. Mol Carcinog 45 183–96.PubMedCrossRefGoogle Scholar
  32. 32.
    Chang, X., Han, J., Pang, L., Zhao, Y., Yang, Y., and Shen, Z. (2009) Increased PADI4 expression in blood and tissues of patients with malignant tumors. BMC Cancer 9 40.PubMedCrossRefGoogle Scholar
  33. 33.
    Luo, Y., Arita, K., Bhatia, M., Knuckley, B., Lee, Y. H., Stallcup, M. R., Sato, M., and Thompson, P. R. (2006) Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization. Biochemistry 45 11727–36.PubMedCrossRefGoogle Scholar
  34. 34.
    Li, P., Yao, H., Zhang, Z., Li, M., Luo, Y., Thompson, P. R., Gilmour, D. S., and Wang, Y. (2008) Regulation of p53 target gene expression by peptidylarginine deiminase 4. Mol Cell Biol 28 4745–58.PubMedCrossRefGoogle Scholar
  35. 35.
    Li, P., Wang, D., Yao, H., Doret, P., Hao, G., Shen, Q., Qiu, H., Zhang, X., Wang, Y., and Chen, G. Coordination of PAD4 and HDAC2 in the regulation of p53-target gene expression. Oncogene 29 3153–62.Google Scholar
  36. 36.
    Wang, Y., Li, M., Stadler, S., Correll, S., Li, P., Wang, D., Hayama, R., Leonelli, L., Han, H., Grigoryev, S. A., Allis, C. D., and Coonrod, S. A. (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184 205–13.PubMedCrossRefGoogle Scholar
  37. 37.
    Fang, J., Wang, H., and Zhang, Y. (2004) Purification of histone methyltransferases from HeLa cells. Methods Enzymol 377 213–26.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Center for Eukaryotic Gene RegulationUniversity ParkUSA
  2. 2.Department of Biochemistry and Molecular BiologyPennsylvania State UniversityUniversity ParkUSA
  3. 3.Genetics Graduate ProgramPennsylvania State UniversityUniversity ParkUSA
  4. 4.Center for Eukaryotic Gene Regulation, and Genetics Graduate ProgramPennsylvania State UniversityUniversity ParkUSA

Personalised recommendations