Fig. 4
Ezh2 cooperates with Tcf1 to activate key TFH genes. a Venn diagram showing overlap between Ezh2 and Tcf1 binding peaks in TFH cells. b Distribution of Ezh2/Tcf1 co-occupied sites. Promoter is defined as +/–1 kb region flanking TSSs, and gene body refers to region covering +1 kb to transcription end site, with remainder as intergenic region. c De novo motif analysis of Ezh2/Tcf1 co-occupied sites. The motif logos and statistical significance are listed. d Ezh2, but not G9a, co-immunoprecipitates with Tcf1. FLAG-tagged Tcf1 was co-transfected with HA-tagged Ezh2 or G9a into 293T cells. The cell lysates were immunoprecipitated with anti-FLAG and immunoblotted with anti-HA. Data are representative of two experiments. e Ezh2 co-immunoprecipitates with Tcf1 in primary TFH cells. CXCR5+SLAMlo TFH cells were sorted from WT mice infected with LCMV-Arm and immunoprecipitated with normal rabbit IgG or anti-Tcf1 followed by immunoblotting with anti-Ezh2. ‘m’ denotes marker lane. Data are representative of two experiments. f Ezh2 is recruited by Tcf1. WT or Tcf1–/–Lef1–/– mice were infected with VacV, and CXCR5+SLAMlo TFH cells were sorted on 6 dpi and analyzed by ChIP with anti-Ezh2 antibody or control IgG. Enriched Ezh2 binding at the TSSs of Bcl6, Icos, Arf and a Cxcr5 upstream region was determined by ChIP-qPCR. Data are means ± s.d. from two experiments with each sample measured in duplicates. ns, not statistically significant; *p < 0.05; **p < 0.01 by Student’s t-test. g Diagram of dual reporter retroviral construct. Red “x” denotes Tcf1 motif mutations in mutant Bcl6 reporter. h Thy1.1 reporter expression driven by the Bcl6 promoter. In vivo primed WT or Ezh2–/– Smarta CD4+ T cells were infected with WT or mutant Bcl6 reporter retrovirus, followed by adoptive transfer and LCMV-Arm infection. On 4 dpi (equivalent to day 6 after initial priming), GFP+CD45.2+ TFH cells were detected in the recipient spleens and assessed for Thy1.1 expression. Cumulative data are means ± s.d. from experiments with ≥5 recipients analyzed for each genotype. ***p < 0.001 by Student’s t-test
