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. 2004 Aug 24;101(34):12622-7.
doi: 10.1073/pnas.0400849101. Epub 2004 Aug 10.

Evolutionarily conserved sequence elements that positively regulate IFN-gamma expression in T cells

Maria Shnyreva  1 , William M Weaver, Mathieu Blanchette, Scott L Taylor, Martin Tompa, David R Fitzpatrick, Christopher B Wilson
Affiliations

Evolutionarily conserved sequence elements that positively regulate IFN-gamma expression in T cells

Maria Shnyreva et al. Proc Natl Acad Sci U S A. .

Abstract

Our understanding of mechanisms by which the expression of IFN-gamma is regulated is limited. Herein, we identify two evolutionarily conserved noncoding sequence elements (IFNgCNS1 and IFNg CNS2) located approximately 5 kb upstream and approximately 18 kb downstream of the initiation codon of the murine Ifng gene. When linked to the murine Ifng gene (-3.4 to +5.6 kb) and transiently transfected into EL-4 cells, these elements clearly enhanced IFN-gamma expression in response to ionomycin and phorbol 12-myristate 13-acetate and weakly enhanced expression in response to T-bet. A DNase I hypersensitive site and extragenic transcripts at IFNgCNS2 correlated positively with the capacity of primary T cell subsets to produce IFN-gamma. Transcriptionally favorable histone modifications in the Ifng promoter, intronic regions, IFNgCNS2, and, although less pronounced, IFNgCNS1 increased as naïve T cells differentiated into IFN-gamma-producing effector CD8+ and T helper (TH) 1 T cells, but not into TH2 T cells. Like IFN-gamma expression, these histone modifications were T-bet-dependent in CD4+ cells, but not CD8+ T cells. These findings define two distal regulatory elements associated with T cell subset-specific IFN-gamma expression.

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Figures

Fig. 1.
Fig. 1.
Conserved noncoding regions with enhancer activity in the Ifng locus. (A) Peaks of similarity in pairwise sequence alignments of the Ifng locus between human and mouse (Upper) or human and rat (Lower) shown as VISTA plots. Conserved sequences are shown relative to their position in the human genome on the horizontal axes below each panel. The percentage sequence identity is indicated on the vertical axes. Conserved noncoding sequences (CNS) are shown in red, coding exons are shown in blue, and 5′ and 3′ UTRs are shown in turquoise. The position in the mouse genome of the first base of IFNgCNS1, the translation start site, and IFNgCNS2 are shown at the top, where the horizontal arrow indicates the direction of transcription. (B) IFNgCNS1 and -2 exhibit enhancer activity. EL-4 cells were transiently transfected with a 9-kb murine Ifng gene alone (mIFNg) or with IFNgCNS1, IFNgCNS2, or both (see Right) and a β-actin luciferase control plasmid, and either not stimulated (unst) or stimulated with PMA (P), ionomycin (I), or PMA plus ionomycin (P+I), without or with cyclosporin A (CsA). Cells were also cotransfected with a T-bet expression vector or pcDNA. IFN-γ was assessed by ELISA and normalized to luciferase activity [relative light units (RLU)]. The graph is representative of two to five independent experiments with each sample analyzed in duplicate.
Fig. 2.
Fig. 2.
IFNgCNS2 harbors a DH site. CD4+ T cells were grown under TH1 or TH2 conditions for 2 weeks. Positions of previously described DH sites in the Ifng gene (11), with black boxes depicting exons, and the site identified in this study at the 5′ end of IFNgCNS2 are shown by downward arrows. B, BamHI sites.
Fig. 3.
Fig. 3.
T cell polarization affects histone acetylation in IFNgCNS1 and -2. (A) Naïve T cells were cultured in vitro under TH1 or TH2 conditions (for CD4+ cells) or in the presence of IL-2 only (for CD8+ cells), then harvested at different time points (0, 17, and 72 h). Chromatin was precipitated with anti-acetyl histone H3 antibody, and the amounts of precipitated DNA were determined by real-time quantitative PCR. Samples collected at 0 h are naïve unstimulated CD4+ (CD4 unstim) or CD8+ T (CD8 unstim) cells. (B) IFN-γ mRNA expression in the same cell samples as determined by real-time quantitative RT-PCR. One of two independent experiments with similar results is shown. IP, immunoprecipitated.
Fig. 4.
Fig. 4.
Histone modifications parallel extragenic transcription in the Ifng locus. Naïve T cells were polarized in vitro as described in Fig. 3. (A) Chromatin was immunoprecipitated (IP) by using anti-acetyl-histone H3 (ac-H3), antiacetyl-histone H4 (ac-H4), or anti-dimethyl-histone H3 lysine 4 (K4-H3) antibody. Each region was analyzed with two different primer sets. The horizontal axis shows the Ifng locus with positions numbered relative to the start site, and black boxes representing IFNgCNS1, the promoter (P), third intron (I), and IFNgCNS2. One of two independent experiments with similar results is shown. (B) Chromatin-bound extragenic transcripts in the IFNgCNS1 and -2 regions from CD4+ T cells cultured for 3 days in TH1 or TH2 conditions were quantified by real-time RT-PCR and normalized to β-actin transcripts. One of three independent experiments with similar results is shown.
Fig. 6.
Fig. 6.
Transcriptionally favorable histone modifications in the Ifng gene, IFNgCNS1 and -2, are T-bet-dependent in TH1 cells, but not in CD8+ T cells. T cells from T-bet–/– or control BALB/c mice were cultured for 3 days as in Fig. 3, then ChIP was performed with anti-acetyl histone H3 antibody. (Inset) IFN-γ mRNA expression is shown. One of two experiments with similar results is shown. IP, immunoprecipitated.
Fig. 5.
Fig. 5.
T-bet induces transcriptionally favorable histone modifications in IFNgCNS1 and -2. CD4+ TH2 cells were transduced with either a bicistronic T-bet-GFP retrovirus (T-bet) or a control GFP retrovirus (vector). ChIP was performed by using anti-dimethyl-H3-K4 antibody as in Fig. 4. (Inset) IFN-γ mRNA expression was assessed in parallel. One of two experiments with similar results is shown. IP, immunoprecipitated.
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