Figure 1. Identification and deletion of CNS0.

A. Placental sequence conservation and ATAC-seq tracks of indicated populations from Foxp3^GFP mice. Double negative thymocytes (DN): CD4⁻CD8⁻; Double positive thymocytes (DP): CD4⁺CD8⁺; CD4 Single Positive thymocytes (SP): CD4⁺CD8⁻TCRβ⁺Foxp3-GFP⁻ from thymus; and naïve CD4 T cells (Tn): CD4⁺TCRβ⁺CD62L⁺CD44⁻Foxp3-GFP⁻; resting Treg cells (rTreg): CD4⁺TCRβ⁺CD62L⁺CD44^loFoxp3-GFP⁺; activated Treg cells (aTreg): CD4⁺TCRβ⁺CD62L⁻CD44^hiFoxp3-GFP⁺ from SLO. CNS2 and CNS0 are indicated by dashed lines.

B. Conservation and positions of STAT5 motifs within CNS0. Multiple sequence alignment of the motifs with other placental mammals are shown. “.” indicates conserved base, “-” indicates deletion, and “*” indicates missing data.

C. Tracks for STAT5 ChIP-seq showing the region surrounding the Foxp3 locus for ex vivo Treg cells (nTreg) and in vitro induced Treg cells (iTreg) stimulated with IL-2. Indicated peaks: CNS2, CNS0, the Foxp3 promoter, the Ppp1r3f promoter (STAT5RE2), and a non-conserved region (STAT5RE1). RE, response element. Data represent 3 biological replicates.

D-F. Schematic of ChIP-qPCR experiment (D) and representative plots showing phosphorylation of STAT5 upon IL-2 stimulation of CD4⁺ Tn cells after 0, 12, and 24 hours of activation (E). Normalized ChIP-qPCR signals by input DNA for indicated regions after IL-2 stimulation at different time points after activation (F). Icos and Gm5069 refer to gene promoter regions. See STAR Methods for details. Plot shows means and SD, n = 3 per group. Data represent 2 independent experiments.

G. Strategy for generating CNS0^FL and CNS0^Δ animals. CNS0 was defined as chr X: 7,570,674 – 7,571,274 (mm10). See STAR Methods for details.

H-I. Representative plots (H) and summarized data (I) of the frequencies of Foxp3-GFP⁺ (Foxp3⁺) and CD25⁺Foxp3-GFP⁻ (CD25⁺) among CD4⁺CD8⁻TCRβ⁺ cells from thymuses of 5- to 18-week-old CNS0^FL or CNS0^KO male littermates. Each point indicates 1 mouse and data are pooled from 3 independent experiments.

Unpaired t-tests with correction for multiple comparisons using the Holm-Sidak method. p <0.01 **, <0.0001 ****. </P/>See also Figure S1.