DNA Methylation and Chromatin Structure Regulate T Cell Perforin Gene Expression

Abstract

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4+ and CD8+ T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4+ and CD8+ cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at −1 kb and the distal promoter at −0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5′ flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.

Tissue-resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

“…1a), and regulates the expression of perforin [23]. This enhancer region is CpG‐rich, with 10 identified CpG sites (chr10: 72, 363, 525–72, 363, 863) (Fig.…”

Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors

“…Messenger RNA (mRNA) was isolated from untreated or 2-deoxy-5-azaCtreated T cells, and analyzed using Affymetrix U95A oligonucleotide arrays, as previously described (9).…”

“…CD70 transcripts were quantitated by realtime RT-PCR using a LightCycler (Roche, Indianapolis, IN) and previously published protocols (9). The following primers were used: forward, 5Ј-TGCTTTGGTCCCATTGGTCG-3Ј and reverse, 5Ј-TCCTGCTGAGGTCCTGTGTGATTC-3Ј.…”

“…The following primers were used: forward, 5Ј-TGCTTTGGTCCCATTGGTCG-3Ј and reverse, 5Ј-TCCTGCTGAGGTCCTGTGTGATTC-3Ј. The transcripts were measured relative to ␤-actin as described previously (9).…”

Perforin level in CD4+ T cells from patients with systemic lupus erythematosus

“…An increased number of cytotoxic CD4 + T cells has been observed in patients with multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and acute coronary syndromes [20][21][22][23]. Lu et al (2003) demonstrated that DNA methylation and chromatin structure regulate PRF1 expression in T cells [40]. Moreover, Kaplan et al (2004) showed that DNA methylation inhibitors increased PRF1 expression in CD4 + T cells.…”