Stat5 binds the Foxp3 gene. (A) Schematic of the mouse Foxp3 gene. Vertical lines depict potential Stat binding sites in the first intron and the putative promoter (I, II, III). Site IV does not contain a Stat-binding site and was used as a control. (B) Stat-binding sites in the mouse Foxp3 gene are underlined and aligned with sequences from other species. Site I is located between 006010267 and 006010275 in the mouse genome (http://genome.ucsc.edu/cgi-bin/hgGateway?hgsid = 83 436 504&clade = vertebrate&org = Mouse&Db = mm7). Site II is located between 006012112 and 006012120. Intronic sites designated III are located between 006017209 and 006017217, 006017406 and 006017414, and 006017523 and 006017531. All sequences are from the sense strand; note that the previous Stat-binding site sequences identified by Zorn et al³⁶  were from the antisense strand, although the same sites were interrogated in our analysis. (C) Sorted thymic CD25⁺CD4 SP (▪) and CD25⁻CD4 SP (⊡) T cells were treated with IL-2 for 1 hour. Proteins and DNA were cross-linked with formaldehyde, cells were lysed, and DNA was sheared. Chromatin immunoprecipitation was performed using either normal rabbit serum or anti-Stat5 antibody. Quantification of immunoprecipitated DNA fragments was performed by real-time PCR using primers and probes for sites I, II, III, and the irrelevant site IV. Values were normalized to corresponding input control and are expressed as fold enrichment relative to normal rabbit serum for each experiment. Means ± SE are shown.