Histone hyperacetylated domains across the Ifng gene region in natural killer cells and T cells

Abstract

Local histone acetylation of promoters precedes transcription of many genes. Extended histone hyperacetylation at great distances from coding regions of genes also occurs during active transcription of gene families or individual genes and may reflect developmental processes that mark genes destined for cell-specific transcription, nuclear signaling processes that are required for active transcription, or both. To distinguish between these, we compared long-range histone acetylation patterns across the Ifng gene region in natural killer (NK) cells and T cells that were or were not actively transcribing the Ifng gene. In T cells, long-range histone acetylation depended on stimulation that drives both T helper (Th) 1 differentiation and active transcription, and it depended completely or partially on the presence of Stat4 or T-bet, respectively, two transcription factors that are required for Th1 lineage commitment. In contrast, in the absence of stimulation and active transcription, similar histone hyperacetylated domains were found in NK cells. Additional proximal domains were hyperacetylated after stimulation of transcription. We hypothesize that formation of extended histone hyperacetylated domains across the Ifng gene region represents a developmental mechanism that marks this gene for cell- or stimulus-specific transcription.

Fig. 1.

Stat4- and T-bet-dependent Th1 differentiation. Purified CD4⁺ T cells from C57BL/6, C57BL/6.Stat4^-/-, BALB/c, or BALB/c.T-bet^-/- mice were stimulated under neutral, Th1, or Th2 conditions in the presence or absence of TSA. After 3 d, cultures were harvested and analyzed for levels of IFN-γ by ELISA. Results are expressed as the mean level of IFN-γ in ng/ml ± SD (total of three experiments).

Fig. 2.

Comparison of DNA sequence conservation among species and long-range Q-HAc in T cells. (A) Schematic of the murine Ifng gene, including positions of known DNase hypersensitivity sites (arrows) and distal enhancer regions (rectangles) (from refs. 27-29). (B) Sites of DNA sequence conservation were obtained from the UCSC Genome Browser web site and are expressed as logarithm-of-odds (LOD) scores derived from a phylogenetic Markov model produced by the phastcons program. (C) Histone acetylation or Q-HAc was determined in chromatin that was isolated from naïve CD4⁺ T cells or T cells cultured under neutral, Th1, or Th2 conditions for 3 d in the presence or absence of TSA as indicated. Results are expressed in mean relative units ± SD.

Fig. 3.

Long-range Q-HAc depends on the transcription factors Stat4 and T-bet, which are required for Th1 lineage commitment. (A) Long-range Q-HAc is similar in Th1 cells from C57BL/6 and BALB/c mice. Chromatin was isolated from C57BL/6 and BALB/c T cells cultured under Th1 conditions for 3 d, and Q-HAc was determined as in Fig. 2C. (B) Long-range Q-HAc depends on Stat4. Chromatin was isolated from T cells from C57BL/6 and C57BL/6.Stat4^-/- mice cultured under Th1 conditions for 3 d. Q-HAc was determined as in Fig. 2C. (C) Dependence of long-range Q-HAc on T-bet. Chromatin was isolated from BALB/c and BALB/c.T-bet^-/- T cells after culture for 3 d under Th1 conditions. Q-HAc was determined as in Fig. 2C. (D) Stat4 and T-bet bind to multiple sites along the Ifng gene region in Th1 cells. ChIP assays were performed essentially as described, except that anti-Stat4 or anti-T-bet antibodies were used for immunoprecipitation.

Fig. 4.

NK cell production of IFN-γ after cytokine stimulation. (A) NK cells were harvested from tissue culture at the indicated times after isolation and stimulated with IL-12, IL-18, or IL-12 and IL-18. Cultures were harvested 24 h after stimulation, and levels of IFN-γ (ng/ml ± SD) were determined by ELISA. (B) NK cells, naïve T cells, effector Th1 cells, and effector Tc1 cells were stimulated with IL-12 and IL-18. Cultures were harvested at the indicated times. IFN-γ levels (ng/ml ± SD) were determined by ELISA.

Fig. 5.

Resting NK cells have a preexisting long-range histone acetylation pattern across the Ifng gene region. NK cells were freshly isolated from spleen or harvested after 3 wk of culture and processed for ChIP assay. Positions of the different primer pairs relative to the Ifng gene are shown. Q-HAc was determined as in Fig. 2C.

Fig. 6.

IL-12 stimulates histone acetylation at a specific site at -6kb5′ of the Ifng gene. NK cells were unstimulated or stimulated with IL-12 or IL-18 for 6 h. Samples were processed for ChIP assay and analyzed as outlined in Materials and Methods. Results of Q-HAc analysis at -6kb5′ of the Ifng gene are shown.

Fig. 7.

Stimulation of NK cells with the combination of IL-12 and IL-18 leads to selective histone acetylation of the first intron of the Ifng gene. NK cells received no stimuli or were stimulated with IL-12, IL-18, or IL-12 and IL-18 for 6 h. Samples were processed for ChIP assay and analyzed as described in the legend of Fig. 6. Results of Q-HAc analysis at positions -0.4, +0.4, and +1.1 kb from the Ifng start site are shown.