Fig. 5

Foxp1 binds to Foxp3 locus and retains permissive histone modifications. a Sorted CD4⁺ Tnv cells from Foxp3^IRES-Thy1.1 mice were activated in vitro in the presence or absence of TGF-β for 3 days. ChIP analysis was performed with either anti-Foxp1 or normal rabbit IgG (control) antibody. Data are shown as a relative enrichment of immunoprecipitated DNA corresponding to Foxp3 gene locus over Input. Gmpr serves as negative control. Data representative of at least four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test, error bars, s.e.m). b ChIP analysis with anti-Foxp1 antibody on iTreg cells differentiated for 3 days from CD4⁺CD25⁻ Tnv cells derived from Smad2^f/fSmad3^+/−CD4-Cre or littermate Smad2^f/fSmad3^+/+ mice. Data represents two independent experiments. c ChIP analysis to determine Foxp1 occupancy at Foxp3 gene locus at the indicated time points. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test, error bars, s.d.). d Sorted Tnv cells from WT and KO mice were differentiated into iTreg cells in vitro for 3 days when they were sorted, labeled with CTV, and co-transferred in RAG1^−/− hosts along with allelically marked CD4⁺ T cells derived from CD45.1⁺Foxp3^GFPKO mice. Seven days post transfer, recipient mice were sacrificed and Foxp3 expression was analyzed simultaneously with CTV dilution. Representative FACS plots within CD4⁺TCRβ⁺CD45.2⁺ gate and quantification of percentage of cells retaining Foxp3 and MFI of Foxp3 within the indicated populations are shown. Data are representative of two independent experiments. e Comparison between CpG methylation levels in the indicated regions of Foxp3 in WT- or KO-derived iTreg cells after 7 days of differentiation. Methylated CpG levels detected by bisulfite sequencing in two biological replicate samples were averaged for each residue from >200,000 reads. Percentage of methylation of individual CpG sites is shown. f Relative enrichment for the permissive H3-K4me3, H3-K9/14Ac, and non-permissive H3-K9me3 chromatin modifications throughout the Foxp3 locus in WT- or KO-derived iTreg cells after 7 days of differentiation. Relative distances (kb) from Foxp3 transcription start site (TSS) of primer probes are indicated in the x axis. Pro Promoter. Representative data from one of at least three independent experiments are shown