Fig 2: Recruitment of SF-1 to the proximal region of the mouse Cx43 promoter. Chromatin immunoprecipitation (ChIP) assays for SF-1 recruitment on the Cx43 promoter were performed with TM4 Sertoli cells. Indicated Cx43 promoter regions were amplified following ChIP assays. Recruitment using a normal rabbit IgG was used as a negative control. Histone H3 as well as cJUN were used as the positive controls for protein-DNA complexes and recruitment to the proximal region of the Cx43 promoter, respectively. Amplification of a region of the second intron of Rpl30 was used as a negative control for recruitment of SF-1 to DNA (E). Results are presented as percentages of input (±SEM). Statistical analyses were performed using an ANOVA, followed by a Bonferroni multiple comparisons' test. Statistically significant differences from control (IgG) are indicated by an asterisk (*P < 0.05). The proximal region of the Cx43 promoter was investigated for SF-1 conserved regulatory elements using the CONTRAV3 platform [33] (F).

Fig 3: DP thymocytes from HDAC3-cKO mice show increased STAT5 activation in response to IL-21.(A) Surface expression of cytokine receptor chains γc, IL-4Rα, IL-7Rα, IL-15Rα, IL-2Rβ, and IL-21R on DP thymocytes from WT and HDAC3-cKO mice. Flow cytometric plots have an isotype control to illustrate background level of expression. (B) Gene expression (RNA-seq) of Il2rg, Il4ra, Il7r, Il15ra, Il2rb, and Il21r in Immature (DP) cells from OT-II and OT-II HDAC3-cKO mice. (C) Snapshot of H3K27ac ChIP-seq tracks at the Il4ra and Il21r gene loci in Immature (DP) thymocytes from OT-II and OT-II HDAC3-cKO mice. Shaded regions identify super-enhancers. (D) Protein expression of IL-21R on DP, CD4SP and CD8SP thymocytes from WT mice and DP thymocytes from HDAC3-cKO mice. (E) pSTAT5 levels in DP thymocytes from WT and HDAC3-cKO mice after 10 min ex vivo stimulation with IL-21, IL-15, IL-7, IL4, or media alone. (F) Protein expression of STAT5a and STAT5b in DP thymocytes from WT and HDAC3-cKO mice. (G) pSTAT3 and pSTAT1 levels after a 10 min in vitro IL-21 stimulation, and pSTAT6 levels after IL-4 stimulation of DP thymocytes from WT and HDAC3-cKO mice. (H) Quantitative ChIP (qChIP) of HDAC3 binding at the Il21r promoter in DP thymocytes from WT and HDAC3-cKO mice. Graph depicts fold enrichment over Rpl30 (n = 4 mice/group from four indpendent experiments). (I) DNase-seq and Hi-C arc plots at the Il4ra and Il21r gene loci in DP thymocytes and pooled DN3-to-DP thymocytes, respectively. Shaded region highlights where HDAC3 binds, as shown in Figure 5H. (DHS, DNA hypersensitivity sites). (A, E, G) Bar graph shows mean ± SEM of MFI from 4 to 5 mice from at least three independent experiments. (D, H) Plots are representative of at least three mice from three independent experiments. (F) Bar graph shows mean ± SEM of MFI from three mice from two independent experiments. (***, p < 0.001). See also Figure 5—figure supplements 1–2.

Fig 4: Arkadia degrades SKI and SnoN to regulate iTreg cell differentiation. (A) Validation of SKI and SnoN antibodies. T cells were activated by plate-bound anti-CD3ε and anti-CD28 and cytokine as indicated. Lysates from SKI- or SnoN-deficient MEFs were used as control. Multiple bands of SKI on immunoblot are due to phosphorylation (Marcelain and Hayman, 2005), and there are two isoforms of SnoN (76.4 kD and 71.1 kD). (B) Immunoblot of time course of degradation of SKI and SnoN in control and Arkadia mutant T cells differentiated under the indicated conditions. Red arrowheads indicate SKI and SnoN proteins detected by corresponding antibodies. α-Tubulin (∼50 kD) served as loading control. Data in A and B are representative of two independent experiments. (C) DKO of Ski and SnoN rescues Arkadia-deficient Treg cell differentiation in vitro. T cells electroporated with indicated gRNAs were cultured under Treg or Th17 differentiation conditions (Materials and methods). Left: Representative flow cytometry of Foxp3 expression in Treg conditions. Right: Technical replicates of one of three independent experiments with Treg and Th17 differentiation of CD4+ T cells electroporated with control (circles) or Arkadia-targeting (triangles) gRNAs plus control, Ski, SnoN, and Ski/SnoN (DKO) gRNAs. Statistical analyses were performed with unpaired t test. ***, P < 0.001. Error bars represent SD. (D) Arkadia regulates FOXP3 expression through histone modification at the Foxp3 locus. Differentiated Treg cells electroporated with indicated gRNAs were harvested for ChIP analysis using anti-acetyl-H3K27 or isotype control antibodies. Control, Ski, SnoN, and Ark represent KO of genes by sgRNAs. TKO, triple KO. CNS1, Foxp3 CNS1 region; RPL30, Rpl30 intron 2 region. Representatives of three independent experiments. Statistical analyses were performed with an unpaired t test. Error bars represent SD. **, P < 0.01. (E) DKO of Ski and SnoN rescues iTreg cell differentiation in Arkadia-deficient T cells in vivo. The experiment was performed as shown in the schematic and is described in Materials and methods. Relative ratios of iTreg frequency were calculated by dividing iTreg frequency of CD45.1/CD45.1 cells by that of CD45.2/CD45.2 cells in the recipient mice. To compare experimental groups, the relative ratio in each animal was calculated individually and combined for analysis with unpaired t test. Error bars represent SD. **, P < 0.01; ***, P < 0.001. Data shown are from one experiment with a total of 20 mice.