Skip to main content

Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression

Abstract

Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoidspecific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5′ flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail.

Abbreviations

PHA:

phytohemagglutinin

5-azaC:

5-azacytidine

dC:

deoxycytosine

SAM:

Sadenosylmethionine

MeCP2:

methyl-CpG-binding protein 2

References

  1. Bird AP, Wolffe AP. Methylation-induced repression--belts, braces, and chromatin. Cell 1999; 99:451–454.

    Article  PubMed  CAS  Google Scholar 

  2. Attwood JT, Yung RL, Richardson BC. DNA methylation and the regulation of gene transcription. Cell Mol Life Sci 2002; 59:241–257.

    Article  PubMed  CAS  Google Scholar 

  3. Richardson BC. Role of DNA methylation in the regulation of cell function: autoimmunity, aging and cancer. J Nutr 2002; 132(8 Suppl):2401S-2405S.

    PubMed  CAS  Google Scholar 

  4. Nguyen CT, Gonzales FA, Jones PA. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation. Nucleic Acids Res 2001; 29:4598–4606.

    Article  PubMed  CAS  Google Scholar 

  5. Fraga MF, Esteller M. DNA methylation: a profile of methods and applications. Biotechniques 2002; 33(3):632, 634, 636–649.

    PubMed  CAS  Google Scholar 

  6. Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res 1994; 22:2990–2997.

    Article  PubMed  CAS  Google Scholar 

  7. Lu Q, Wu A, Ray D, Deng C, Attwood J, Hanash S, Pipkin M, Lichtenheld M, Richardson B. DNA methylation and chromatin structure regulate T cell perforin gene expression. J Immunol 2003; 170:5124–5132.

    PubMed  CAS  Google Scholar 

  8. Oelke K, Lu Q, Richardson D, Wu A, Deng C, Hanash S, Richardson B. Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors. Arthritis Rheum 2004; 50:1850–1860.

    Article  PubMed  CAS  Google Scholar 

  9. Cornacchia E, Golbus J, Maybaum J, Strahler J, Hanash S, Richardson B. Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity. J Immunol 1988;140:2197–2200.

    PubMed  CAS  Google Scholar 

  10. Lu Q, Kaplan M, Ray D, Zacharek S, Gutsch D, Richardson B. Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus. Arthritis Rheum 2002; 46:1282–1291.

    Article  PubMed  CAS  Google Scholar 

  11. Lu Q, Ray D, Gutsch D, Richardson B. Effect of DNA methylation and chromatin structure on ITGAL expression. Blood 2002; 99:4503–4508.

    Article  PubMed  CAS  Google Scholar 

  12. Cornwell RD, Gollahon KA, Hickstein DD. Description of the leukocyte function-associated antigen 1 (LFA-1 or CD11a) promoter. Proc Natl Acad Sci USA 1993; 90:4221–4225.

    Article  PubMed  CAS  Google Scholar 

  13. Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 1997;13:335–340.

    Article  PubMed  CAS  Google Scholar 

  14. Lichtenheld MG, Podack ER. Structure of the human perforin gene. A simple gene organization with interesting potential regulatory sequences. J Immunol 1989; 143:4267–4274.

    PubMed  CAS  Google Scholar 

  15. Zhang J, Scordi I, Smyth MJ, Lichtenheld MG. Interleukin 2 receptor signaling regulates the perforin gene through signal transducer and activator of transcription (Stat)5 activation of two enhancers. J Exp Med 1999; 190:1297–1308.

    Article  PubMed  CAS  Google Scholar 

  16. Jones PA. Gene activation by 5-azacytidine. In: Razin A, Cedar H, Riggs A, editors. DNA Methylation. Biochemistry and Biological Significance. New York: Springer-Verlag; 1984. p 165–187.

    Google Scholar 

  17. Lu Q, Richardson B. DNaseI hypersensitivity analysis of chromatin structure. In: T T, editor. Methods in Molecular Biology. Volume 287. Totowa NJ: Humana Press; 2004. p 77–86.

    Google Scholar 

  18. Macnab JC, Adams RL, Rinaldi A, Orr A, Clark L. Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus. Mol Cell Biol 1988; 8:1443–1448.

    PubMed  CAS  Google Scholar 

  19. Javed A, Zaidi SK, Gutierrez SE, Lengner CJ, Harrington KS, Hovhannisyan H, Cho BC, Pratap J, Pockwinse SM, Montecino M et al. Chromatin immunoprecipitation. Methods Mol Biol 2004; 285:41–44.

    PubMed  CAS  Google Scholar 

  20. Bird A. The essentials of DNA methylation. Cell 1992; 70:5–8.

    Article  PubMed  CAS  Google Scholar 

  21. Cihak A. Biological effects of 5-azacytidine in eukaryotes. Oncology 1974; 30: 405–422.

    Article  PubMed  CAS  Google Scholar 

  22. Nyce J, Liu L, Jones PA. Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells. Nucleic Acids Res 1986; 14:4353–4367.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Published: September 16, 2004.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, Q., Richardson, B. Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression. Biol. Proced. Online 6, 189–203 (2004). https://doi.org/10.1251/bpo89

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo89

Indexing terms