Fig 2: Demethylase-inactive Uty fails to support S1pr1 expression.(a) Contour plots (left) show expression of S1pr1 and Va2 on mature thymocytes from wild-type (WT) and dKO OT-II transgenic female or male mice. Dot plots (right) show the frequency of S1pr1hi cells among mature CD4 SP thymocytes. Each symbol represents one individual mouse. (b,c) Contour plots (lefts) show CD4 and CD8 expression on thymocytes (b) or splenocytes (c) from mice analysed in (a). Dot plots (right) show absolute numbers of mature Va2hi CD4 SP thymocytes (b) or CD4+ T-splenocytes (c). Each symbol represents one individual mouse. Results are representative of four mice of each gender and genotype analysed in four distinct experiments (a–c). *: P<0.05 (unpaired two-tailed t-test). Error bars indicate s.d.

Fig 3: GSK3 regulation of thymocyte egress: A model.The S1P gradient provides a driving force for the egress of mature thymocytes into the bloodstream. A Foxo1-Klf2 axis controls the expression of S1P receptor S1P1 on mature thymocytes. Constitutive expression and activity of GSK3 in WT thymocytes are required for suppressing tonic Akt signaling by phosphorylating ß-catenin, leading to its degradation. In the absence of GSK3, ß-catenin accumulates in the cytoplasm, where it promotes Akt activation by decreasing the association of Akt with its phosphatases, PHLPP2 and PP2A, resulting in Foxo1 phosphorylation and degradation, diminished Klf2 and S1P1 expression, and impaired thymocyte egress.

Fig 4: Jmjd3 and Utx are needed for S1pr1 expression.(a) Quantitative RT-PCR showing expression of Gapdh, S1pr1 and Klf2 mRNA in OT-II transgenic wild-type (WT) and dKO mature thymocytes. Results are normalized relative to expression of the 18S rRNA gene and presented as fold change relative to wild type values (set as 1). Data is from five determinations from three independently sorted sample sets, except for Gapdh (two determinations from two sample sets). (b,d) Contour plots (left) of Va2 and S1pr1 expression on gated Va2hi CD24lo CD4 SP thymocytes from wild-type (WT) and dKO (b) or Jmjd3-KO (d) OT-II mice. Dot plots (right) shows frequency of S1pr1hi cells among mature CD4 SP thymocytes; each symbol represents one individual mouse. Data comes from five (b) and four (d) distinct experiments. (c) RT-PCR analysis of S1pr1 and Klf2 mRNA expression in CD24lo CD4 SP thymocytes from wild-type (WT) and Jmjd3-KO OT-II mice, expressed as in (a). Data is from three independent sample sets. (e) Overlaid histograms show expression of surface CD69 in Va2hiCD24loCD4 SP thymocytes from wild type (dotted line) and dKO (plain line) OT-II transgenic mice. Grey shaded histograms indicate expression in DP thymocytes from non-transgenic wild-type mice. Data is representative of four mice of each genotype, analysed in four distinct experiments. (f) RT-PCR analysis of Cd69 and Nr4a1 (encoding Nur77) expression in Va2hi CD24lo CD4 SP thymocytes from indicated mice. Data is expressed as in (a) and comes from three independently sorted sample sets. (g) Bar graphs compare expression of S1pr1 and Nr4a1 in sorted mature OT-II transgenic CD4 SP thymocytes either ex vivo or after cell suspension culture. Results are presented relative to WT ex vivo thymocytes (set as 1) after normalization on 18S rRNA expression. Data is from three independently sorted sample sets. *P<0.05, ***P<0.001 and ****P<0.0001, not significant in other cases (unpaired t-test). Error bars indicate s.d.

Fig 5: Jmjd3 and Utx redundantly promote H3K27Me3 demethylation.(a) H3K27Me3 amounts (mean±s.d.) at the S1pr1 promoter were assessed by ChIP-qPCR in sorted DP thymocytes from wild-type mice carrying an endogenous TCR repertoire, or Va2hi CD24lo CD4 SP thymocytes from wild type (WT) and dKO OT-II mice. H3K27Me3 amount values are shown as percentage of input. Data is from three independent experiments. *P<0.05 (unpaired t-test). (b) IGV browser tracks show the distribution (normalized sequence reads) of H3K27Me3 ChIPseq signals at indicated loci in wild-type DP and mature (Va2hi CD24lo) OT-II transgenic CD4 SP thymocytes.