Opposing roles of STAT4 and Dnmt3a in Th1 gene regulation

Abstract

The STAT transcription factor STAT4 is a critical regulator of Th1 differentiation and inflammatory disease. Yet, how STAT4 regulates gene expression is still unclear. In this report, we define a STAT4-dependent sequence of events including histone H3 lysine 4 methylation, Jmjd3 association with STAT4 target loci, and a Jmjd3-dependent decrease in histone H3 lysine 27 trimethylation and DNA methyltransferase (Dnmt) 3a association with STAT4 target loci. Dnmt3a has an obligate role in repressing Th1 gene expression, and in Th1 cultures deficient in both STAT4 and Dnmt3a, there is recovery in the expression of a subset of Th1 genes that is sufficient to increase IFN-γ production. Moreover, although STAT4-deficient mice are protected from the development of experimental autoimmune encephalomyelitis, mice deficient in STAT4 and conditionally deficient in Dnmt3a in T cells develop paralysis. Th1 genes that are derepressed in the absence of Dnmt3a have greater induction after the ectopic expression of the Th1-associated transcription factors T-bet and Hlx1. Together, these data demonstrate that STAT4 and Dnmt3a play opposing roles in regulating Th1 gene expression, and that one mechanism for STAT4-dependent gene programming is in establishing a derepressed genetic state susceptible to transactivation by additional fate-determining transcription factors.

Figure 1. Gene expression, histone modification, and chromatin modifying enzyme patterns in IL-12-stimulated WT and Stat4^−/− Th1 cells

Naïve CD4⁺CD62L⁺ T cells were isolated from WT or Stat4^−/− C57BL/6 mice and cultured under Th1 polarizing conditions. On day 5, cells were harvested, stimulated with IL-12 for the indicated time points, and gene expression was examined by qRT-PCR (A, C) or used for STAT4 binding, chromatin modifying enzymes and histone modification analyses by ChIP assay using qPCR primers specific for the promoters of the indicated genes (B, D). Data are average of replicate samples ± S.D. and representative of three independent experiments with similar results. Gene names are indicated adjacent to the corresponding line (A, B) representing mRNA expression or ChIP results at the promoter.

Figure 2. IL-12-induced gene expression requires Jmjd3

(A-F) Naïve CD4⁺CD62L⁺ T cells were isolated from C57BL/6 mice and cultured under Th1 polarizing conditions. (A-D) On day 5, cells was harvested, transfected with control or Jmjd3-specific siRNA, rested overnight and stimulated with anti-CD3 for 6 h for Jmjd3 (with average of mean fluorescence intensity) and viability analyses by intracellular staining (A-B) or 24 h to measure cytokine production by ELISA (C). Transfected cells were stimulated with IL-12 for gene expression analysis by qRT-PCR (D) or chromatin modifying enzymes, histone modification and STAT4 binding by ChIP assay using primers specific for the promoters of the indicated genes (E). Data are average of three mice ± S.D. (A-C) or are average of replicated samples ± S.D. and representative of three independent experiments with similar results (D-E) *p<0.05. NS not significant

Figure 3. Dnmt3a is a negative regulator of Th1 genes

(A-G) Naïve CD4⁺CD62L⁺ T cells were isolated from WT, Dnmt3a^fl/fl CD4-Cre positive or Dnmt3a^fl/fl Stat4^−/− CD4-Cre negative (−) or positive (+) mice and cultured under Th1, Th17 or iTreg polarizing conditions. (A-B) On day 5, Th1 cells were harvested, activated with anti-CD3 or PMA and Ionomycin for 6 h before assessing cytokine production by ICS, B, Averages of percent positive cells and mean fluorescence intensity for data in (A). (C-D) Day 5 Th1 and Th17 cells were harvested and activated with anti-CD3 for 24 h before assessing cytokine production by ELISA. (E-F) Splenocytes isolated from mice with indicated genotypes or day 5 in vitro generated regulatory T (Treg) cells were assessed for natural (nTreg, CD25⁺Foxp3⁺) (E) or inducible (Foxp3⁺) (F) Treg cells by intracellular staining. Data is gated on CD4⁺ cells. (G) Day 5 Th1 cells were used to examine gene expression by qRT-PCR before (Etv5, Furin, Twist1, Il18r1, Hlx1, Runx3, Tbx21 and Jmjd3) or after (Ifng) anti-CD3 reactivation. Data are average ± S.D. of three independent experiments (A-F) or average of replicated samples ± S.D. and representative of three independent experiments with similar results (G). *p<0.05

Figure 4. Mice with double deficiency in STAT4 and Dnmt3a had partial recovery in inflammatory T cell function

(A-B) Mean clinical score of MOG peptide (35–55)-induced EAE in WT, Dnmt3a^fl/fl CD4-Cre positive (A) or in WT, Dnmt3a^fl/fl Stat4^−/− CD4-Cre negative or positive mice scored daily for 30 days (B). (C-D) Mice were sacrificed on day 14 and mononuclear cells were isolated from brain and stimulated with PMA and Ionomycin for 6 h before staining for cytokine production (C) or isolated splenocytes were stimulated with MOG peptide for 48 h and cytokine production was measured using ELISA (D). Data are average ± S.E.M. of two independent (A-B, n=6–10 mice/group/experiment) or average ± S.E.M. of 4 mice (C-D). *p<0.05 comparing Dnmt3a^fl/fl Stat4^−/− CD4-Cre negative or positive samples.

Figure 5. Gene expression, histone modification, and transcription factor binding in the absence of Dnmt3a

(A-D) Naïve CD4⁺CD62L⁺ T cells were isolated from WT, Dnmt3a^fl/fl Stat4^−/− CD4-Cre negative or positive mice and cultured under Th1polarizing conditions. On day 5, cells were harvested and stimulated with IL-12 for 4 hours before gene expression analysis by qRT-PCR (A) or histone modification analysis by ChIP assay at Ifng regulatory elements (B) or the Hlx1 promoter (C). (D)T-bet, Runx3 and Jmjd3 bound to the Ifng locus was analyzed by ChIP assay in Th1 cells. Data are average of replicated samples ± S.D. and representative of three independent experiments with similar results.

Figure 6. Ectopic Th1 transcription factor expression rescues IFNγ production

Naïve CD4⁺CD62L⁺ T cells were isolated from WT, Dnmt3a^fl/fl Stat4^−/− CD4-Cre negative (−) or positive (+) mice and cultured under Th1polarizing conditions. On day 2, cells were transduced with retrovirus expressing MIEG-EGFP (MIEG), MIEG Hlx1-EGFP (Hlx1), or MIEG T-bet-EGFP (Tbx21) (A-B), or control vector Thy1.1 or Runx3-thy1.1 (Runx3) (C-D), or both MIEG T-bet-EGFP (Tbx21) and MSCV Hlx1-Thy1.1 (Hlx1) or matching controls (E-F). After five days of differentiation, Th1 cells were activated with anti-CD3 for 6 h before measuring cytokine production by ICS. Representative plots gated on transduced cells (A, C) with average percentage of positive cells indicated in bar graphs (B, D). (E-F) After five days of differentiation, doubly transduced Th1 cells were sorted, reactivated with anti-CD3 for 24 h, and cytokine production was measured by ELISA (E) with the fold induction in cytokine production between Dnmt3a^fl/fl Stat4^−/− CD4-Cre negative and positive transduced Th1 cells compared to its control cells (F). Data are average ± S.D. of three independent experiments.*p<0.05.