Signal transducer and activator of transcription 4 is required for the transcription factor T-bet to promote T helper 1 cell-fate determination - PubMed (original) (raw)

Signal transducer and activator of transcription 4 is required for the transcription factor T-bet to promote T helper 1 cell-fate determination

Vivian T Thieu et al. Immunity. 2008.

Abstract

Transcriptional regulatory networks direct the development of specialized cell types. The transcription factors signal tranducer and activator of transcription 4 (Stat4) and T-bet are required for the interleukin-12 (IL-12)-stimulated development of T helper 1 (Th1) cells, although the hierarchy of activity by these factors has not been clearly defined. In this report, we show that these factors did not function in a linear pathway and that each factor played a unique role in programming chromatin architecture for Th1 gene expression, with subsets of genes depending on Stat4, T-bet, or both for expression in Th1 cells. T-bet was not able to transactivate expression of Stat4-dependent genes in the absence of endogenous Stat4 expression. Thus, T-bet requires Stat4 to achieve complete IL-12-dependent Th1 cell-fate determination. These data provide a basis for understanding how transiently activated and lineage-specific transcription factors cooperate in promoting cellular differentiation.

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Figures

Figure 1

Figure 1. Contribution of Stat4 and T-bet to expression of genes in Th1 cells

Wild type, Stat4-deficient (_Stat4_−/−) and T-bet-deficient (_Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions (IL−12 + anti-IL-4) for five days. RNA was isolated from cells either before (Ccr5, Il18r1, Etv5, Cxcr3) or six hours after (Ifng, Hlx1, Xcl1, Egr2, Egr3, Furin) re-stimulation of cells with anti-CD3. Quantitative PCR using TaqMan primers specific for each gene was performed and results were normalized to expression of beta2-microglobulin. Results are the average ± SD of replicate samples and are representative of four experiments with similar results.

Figure 2

Figure 2. IFNγ or IL-27 do not rescue gene expression in Stat4-deficient Th1 cells

(A) Wild type and Stat4-deficient (_Stat4_−/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) in the presence or absence of 100 ng/ml recombinant IFNγ for five days. Cells were re-stimulated with anti-CD3 for 18 hours and supernatants were analyzed for levels of IFNγ and IL-4 using ELISA. (B) Cells cultured as in (A) were analyzed for gene expression using qPCR as described in Figure 1. Results in (A) and (B) the average ± SD of replicate samples and are representative of four experiments with similar results. (C) Wild type and Stat4-deficient (_Stat4_−/−) CD4+ T cells were cultured under Th1 conditions (IL−12 + anti-IL-4) in the presence or absence of 100 ng/ml recombinant IL-27 for five days. Expression of genes was determined after activation with anti-CD3 for four hours. Results are representative of two experiments with similar results.

Figure 3

Figure 3. Stat4 and T-bet bind to the Hlx1 locus

(A) Wild type, Stat4-deficient (_Stat4_−/−) and T-bet-deficient (_Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and RNA was isolated from cells during each day of culture. Expression of Hlx1, Stat4 and Tbx21 were assessed in each of the samples using qPCR. Results are representative of two experiments. (B) Wild type, Stat4-deficient (_Stat4_−/−) and T-bet-deficient (_Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and chromatin was isolated for ChIP assay. ChIP was performed for Stat4 bound to the promoter of Hlx1, Ifng or Il18r1 in wild type and T-bet-deficient cells (left) or for T-bet bound to the same regions in wild type or Stat4-deficient cells (right). QPCR was performed using TaqMan primers specific for each promoter. Transcription factor bound to the locus is expressed as the percent of the input used for the ChIP assay. Results are the average ± SD of replicate samples and are representative of three experiments for Hlx1 and two experiments for binding to other promoters with similar results. ND, not detected. (C) Wild type, Stat4-deficient (_Stat4_−/−) and T-bet-deficient (_Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days in the presence or absence of 20 nM trichostatin A (TSA) and/or 10 µM 5-aza-deoxycytidine (5-aza). RNA was isolated for analysis of Hlx1 gene expression as described in Figure 1. Results are representative of two experiments.

Figure 4

Figure 4. Stat4- and T-bet-dependent chromatin remodeling at the Hlx1 locus

(A-E) Wild type, Stat4-deficient (_Stat4_−/−) and T-bet-deficient (_Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and chromatin was isolated for ChIP assay. ChIP was performed for acetylated-H3, -H4 and DNMT3a on days 3–5 of culture (A) or day 5 only (B), the histone acetyltransferases CBP, p300, PCAF and Gcn5 on day 5 of culture (C), acetylated H4K5 and K8 on day 5 of culture (D), or EZH2, H3K27me3, H4K20me3 and H3K4me2 on day 5 of culture (E) using qPCR primers for the Hlx1 promoter. Results are the average ± SD of replicate samples and are representative of 3–5 experiments for each modification or enzyme with similar patterns. ND, not detected.

Figure 5

Figure 5. Stat4- and T-bet-dependent chromatin remodeling at target loci

(A-F) Wild type, Stat4-deficient (_Stat4_−/−) and T-bet-deficient (_Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and chromatin was isolated for ChIP assay. ChIP assay was performed for acetylated-H3, -H4 and H3K4me2 at the Ifng promoter (−0.4 kb), and at sites +20 kb and +40 kb from the transcriptional start site (A), and at the Furin promoter (B). ChIP assay was performed for DNMT3a (C) and H4K20me3 (D) at the Ifng and Furin promoters. ChIP assay was performed for acetylated-H3, -H4 and H3K4me2 at the Xcl1 promoter (E) and for acetylated-H4 and H3K4me2 at intron 1 of Cd4 (F). Results are the average ± SD of replicate samples and are representative of 2–4 experiments for each modification or enzyme with similar patterns.

Figure 6

Figure 6. Stat4 requirement in T-bet function

(A) Wild type, Stat4-deficient (_Stat4_−/−), T-bet-deficient (_Tbx21_−/−) and Stat4-T-bet-double deficient (_Stat4-Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions (IL-12 + anti-IL-4) for five days and total cell extracts were immunoblotted for T-bet, Stat4 and GAPDH as a control. (B) Cells cultured as in (A) were assessed for the expression of Th1 genes before (Il18rap, Runx3) or after (Lta) re-stimulation with anti-CD3. (C-G) Wild type, T-bet-deficient (_Tbx21_−/−) and Stat4-T-bet-double deficient (_Stat4_−/− _Tbx21_−/−) CD4+ T cells were cultured under Th1 conditions. On day 2 of the culture period, cells were transduced with a bicistronic retrovirus expressing EGFP only (MIEG) or T-bet and EGFP (T-bet). At the end of the culture, cells were sorted for EGFP expression and stimulated for 18 hours with anti-CD3. Supernatants were analyzed for IFNγ levels using ELISA (C). RNA was isolated from each population to determine the expression levels of the indicated genes using qPCR (D). Surface expression of CXCR3 was determined using flow cytometry (E). ChIP assay was performed for acetylated-H3 or -H4 at the Hlx1 and Ifng promoters (F) or acetylated-H4 at the Xcl1 promoter (G). Results are the average ± SD of replicate samples and are representative of 2–3 experiments with similar results.

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