Fig. 5
Ezh2 promotes TFH differentiation by epigenetically repressing Cdkn2a. a Cdkn2a gene structure. The distal exon 1b and proximal exon 1a are differentially spliced with downstream exons to generate p19Arf and p16Ink4a, respectively. Targeting different exons (marked in red) achieves p19Arf- or p16Ink4a-specific deletion. b Detection of Arf and Ink4a transcripts in TFH cells. CD45.2+ WT or Ezh2–/– Smarta CD4+ T cells were adoptively transferred into congenic mice followed by infection with LCMV-Arm. On 4 dpi, CD45.2+ CXCR5+ SLAMlo TFH cells were sorted from the recipient spleens, and Arf and Ink4a transcripts from the Cdkn2a locus were detected by quantitative RT-PCR. c Detection of histone modification changes in TFH cells. WT or Ezh2–/– CD45.2+ CXCR5+ SLAMlo TFH cells were sorted on 4 dpi as in (b), and H3K27me3 and H3K27ac at the TSS of Arf and Ink4a were analyzed by ChIP-qPCR. d Detection of Ezh2 binding to the Cdkn2a locus. WT CD45.2+ TFH and TH1 cells were sorted from the recipient spleens on 5 dpi as in (b), and together with WT naive CD4+ T cells, were subjected to ChIP analysis with anti-Ezh2 antibody or control IgG, and enriched Ezh2 binding at the TSS of Arf and Ink4a was determined by ChIP-qPCR. Data in (b–d) are means ± s.d. from ≥2 experiments, with each sample measured in duplicates. e–g Effect of genetically ablating p19Arf on rectifying TFH defects due to Ezh2 deficiency. CD45.2+ Smarta CD4+ T cells from WT, Ezh2–/–, Arf–/–, or Ezh2–/–Arf–/– Smarta-Tg mice were adoptively transferred into congenic mice followed by infection with LCMV-Arm. On 4 dpi, CXCR5+SLAMlo TFH cells were detected in the recipient spleens (e), and further analyzed for caspase-3/7 activation (f) and Bcl6 expression (g). CXCR5–SLAMhi TH1 cells were analyzed for cell numbers (e) and Bcl6 expression (marked with dotted red line in g) for direct comparison with TFH cells. Data are means ± s.d. from ≥2 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 for indicated pairwise comparison by Student’s t-test, coupled with one-way ANOVA for multi-group comparisons
