Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression
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.
Indexing termsDNA methylation T-lymphocytes Gene expression regulation
methyl-CpG-binding protein 2
- 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