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. 2015 Sep 14;6:8266. doi: 10.1038/ncomms9266

Figure 5. Foxp3 binds to the Il9 locus and inhibits IL-9 expression by recruiting histone deacetylases.

Figure 5

(a) Graph depicting Il9 locus and putative Foxp3 binding sites (red) at the Il9 promoter and CNS regions. (b) ChIP analysis of Foxp3 binding to the Il9 gene locus in CD4+ T cells transduced with retrovirus expressing GFP alone (Ctrl) or GFP-Flag-Foxp3 (Foxp3), then cultured under iTreg-polarizing conditions in the presence of DTA-1. GFP+ cells were sorted and fixed for ChIP using anti-Flag antibody. Data represent mean values±s.d. (n=3). (c,d) Flow cytometry plots showing IL-9-producing cells in CD4+ T cells transduced with retrovirus expressing GFP alone or Foxp3-GFP, and cultured under iTreg-polarizing conditions for 3 days in the presence of DTA-1. Numbers indicate the percentage of cells in the quadrants (c). Graph depicts the percentage of IL-9+ cells in GFP+ population (d). Data represent mean values±s.d. (n=6). (e) Co-immunoprecipitation of flag-foxp3 in CD4+ T cells transduced with retrovirus expressing flag-foxp3, and cultured under iTreg-polarizing conditions for 2 days. Anti-flag and control IgG immunoprecipitates (IP) were subjected to immunoblot analysis (IB) with anti-HDAC1, anti-HDAC2, anti-Sirt1 and anti-Sirt7 antibodies, and representative plots of 3 experiments are shown. (f) ChIP analysis of H3Ac and H4Ac modifications at Il9 promoter and CNS regions in GFP+ cells sorted from naive CD4+ T cells transduced and activated as in (b) for 2 days. Data represent mean values±s.d. (n=3). (g,h) Flow cytometry analysis of IL-9-producing cells in naive CD4+ T cells transduced and activated as in (c) for 3 days in the presence or absence of HDAC inhibitor Trichostatin A (TSA, 2 nM) and Sirt1 inhibitor EX-527 (EX, 0.5 μM). Numbers in the quadrants indicate the percentage of positive cells (g). Graph depicts the percentage of IL-9+ cells in GFP+ population. Data represent mean values±s.d. (n=9) (h). (i) ChIP analysis of H3Ac and H4Ac modifications at Il9 promoter and CNS regions in Foxp3−GFP+ cells sorted from naive CD4+ T cells transduced with retrovirus expressing Foxp3-GFP and cultured as in (g) for 2 days. Data represent mean values±s.d. (n=3). P values were determined by Student's t-test (*P<0.05).