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. 1998 Jul 6;188(1):103-17.
doi: 10.1084/jem.188.1.103.

Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells

D R Fitzpatrick  1 , K M Shirley, L E McDonald, H Bielefeldt-Ohmann, G F Kay, A Kelso
Affiliations

Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells

D R Fitzpatrick et al. J Exp Med. .

Abstract

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-gamma and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-gamma and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-gamma promoter was widespread across >300 basepairs in clones expressing high levels of IFN-gamma mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-gamma mRNA exhibited symmetrical methylation of four to six of the IFN-gamma promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-gamma gene methylation and mRNA expression in families of clones derived from resting CD44(low)CD8+ T cells or from previously activated CD44(high)CD8+ T cells. The patterns of IFN-gamma gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44(high) cells, but variable in clones derived from CD44(low) cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.

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Figures

Figure 1
Figure 1
Homology of CpG sites in the IFN-γ (A) and IL-3 (B) genes in different species. The mouse sequence is numbered relative to the transcription start site (||===>). Marmoset (MARM), human, rat, and/or rhesus monkey (RHES) sequences are also shown. The mouse sequence is the reference for the alignments, determined using the FASTA program, with identical residues (.) and gaps (–) indicated. CpG sites (upper case and bold, and asterisked for the mouse sites), TATA motifs (++++), and ATG start codons (###) are also indicated. Potential transcription factor–binding sites defined for the human sequence are underscored and annotated with the sequence element and/or transcription factor acronyms.
Figure 2
Figure 2
5-azacytidine treatment increases IFN-γ and IL-3 secretion by primary mouse CD8+ T lymphocytes. CD8+ T cells from normal C57BL/6 mouse LNs were activated in low density in vitro cultures using solid-phase mAb against CD3ε, CD8, and LFA-1 in the presence of IL-2 and varying concentrations of 5-azacytidine for the indicated periods of time. Cumulative secreted cytokine levels were quantitated by ELISA (A, IFN-γ) or reporter cell bioassay (B, IL-3). The results shown are representative of three independent experiments.
Figure 2
Figure 2
5-azacytidine treatment increases IFN-γ and IL-3 secretion by primary mouse CD8+ T lymphocytes. CD8+ T cells from normal C57BL/6 mouse LNs were activated in low density in vitro cultures using solid-phase mAb against CD3ε, CD8, and LFA-1 in the presence of IL-2 and varying concentrations of 5-azacytidine for the indicated periods of time. Cumulative secreted cytokine levels were quantitated by ELISA (A, IFN-γ) or reporter cell bioassay (B, IL-3). The results shown are representative of three independent experiments.
Figure 3
Figure 3
Primary bisulfite genomic DNA sequencing data. Nuclear DNA from clonal cultures of primary mouse CD8+ T cells was purified, bisulfite modified, amplified, and sequenced directly using dye terminator chemistry and automated fluorescent sequence analysis. Sequence results for the noncoding strands of the IFN-γ promoter between bases −27 and −60 in four different clones are shown in the left panel. Sequence results for the noncoding strands of the IL-3 promoter between bases −40 and −72 in three different clones are shown in the right panel. Methylated cytosines are displayed by retained blue cytosine peaks while nonmethylated cytosines are converted by bisulfite modification and PCR to red thymidine peaks. These peaks were scored for each clone and are shown below the chromatograms by the following symbols: ▪, methylated; □, demethylated. The presence of clear coincident cytosine and thymidine peaks, e.g., at position −52 in the third IL-3 sequence, was scored: ▵, partially methylated.
Figure 4
Figure 4
Differential methylation of the IFN-γ and IL-3 genes in CD8+ T cell clones and independence of clone size. CpG sites in the two promoters are depicted schematically and numbered as described in Fig. 1. Asterisks above the −53 site in the IFN-γ promoter indicate the only CpG site hitherto characterized in T cells. Methylation of the coding (upper line of symbols) and noncoding (lower line of symbols) strands for the IFN-γ and IL-3 promoters are shown for the control cell line FDCP1 and 16 CD8+ T cell clones ranging in size from 35 to >256 cells. Methylation patterns were scored as described in Fig. 3.
Figure 5
Figure 5
Quantitation of IFN-γ and IL-3 mRNA levels in a panel of CD8+ clones by competitive PCR after 4–5 d of stimulation. Levels of mRNA were determined by QCPCR and corrected for CD3ε mRNA levels as described in Materials and Methods. A shows relative IFN-γ mRNA levels in relation to clone size. B shows relative IL-3 mRNA levels in relation to clone size in the same set of clones. C shows the relationship between IFN-γ and IL-3 expression levels for each clone in the panel. The results for 53 clones are shown.
Figure 6
Figure 6
Regional IFN-γ promoter methylation patterns are related to IFN-γ mRNA expression levels in CD8+ T cells. Clone sizes and methylation patterns are shown as described in the legend to Fig. 4. Levels of mRNA are also shown as described in the legend to Fig. 5. The results depicted are representative of a panel of 40 clones for which complete DNA and mRNA data were obtained. Clones with undetectable mRNA levels are ranked by methylation pattern, whereas mRNA-positive clones are ranked by mRNA level.
Figure 7
Figure 7
Site-specific IL-3 promoter methylation patterns are related to IL-3 mRNA expression levels in CD8+ T cells. Clone sizes, methylation patterns, and mRNA levels for the panel of clones are shown as described in the legend to Fig. 6.
Figure 8
Figure 8
Heritability of IFN-γ promoter methylation patterns and IFN-γ mRNA expression levels in families of CD8+ T cells. CD8+ LN T cells from normal mice were separated by FACS® into CD44low and CD44high subpopulations, and seeded by automated deposition as single cells into stimulation cultures for 4 d as described in Materials and Methods. Parental CD8+ T cell clones were then subcloned by micromanipulation, with harvesting of progeny subclones after 2–3 d of further growth. Families of clones derived from CD44low cells are shown in A, whereas those derived from CD44high cells are shown in B. Each family has a separate letter code with the parent clone (0) and sibling subclones (–6) numbered. The parent clone for family C did not survive micromanipulation. Methylation patterns and mRNA levels for the families of clones are shown as described in the legends to Figs. 4 and 5.
Figure 8
Figure 8
Heritability of IFN-γ promoter methylation patterns and IFN-γ mRNA expression levels in families of CD8+ T cells. CD8+ LN T cells from normal mice were separated by FACS® into CD44low and CD44high subpopulations, and seeded by automated deposition as single cells into stimulation cultures for 4 d as described in Materials and Methods. Parental CD8+ T cell clones were then subcloned by micromanipulation, with harvesting of progeny subclones after 2–3 d of further growth. Families of clones derived from CD44low cells are shown in A, whereas those derived from CD44high cells are shown in B. Each family has a separate letter code with the parent clone (0) and sibling subclones (–6) numbered. The parent clone for family C did not survive micromanipulation. Methylation patterns and mRNA levels for the families of clones are shown as described in the legends to Figs. 4 and 5.
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