Figure 4.
Figure 4. The RB family controls the expression of Foxn1. (A) Expression of Foxn1 in CD45– MHCII+ TECS from control (Ctrl) and mutant (Mut) mice relative to Eva1 expression (n = 4; P = 0.03). (B) Immunoblot analysis of Foxn1 levels in control and mutant TECs from 12 pooled control and 5 pooled mutant thymi. α-Tubulin is included as a loading control (n = 2). (C) Immunofluorescence staining of Foxn1 and CD205 individually and merged (right) in control (top) and mutant (bottom) thymi (one of two shown). Bars, 100 µm. (D) Schematic representation of the Foxn1 promoter. Region 1a is active in the skin and thymus, whereas region 1b is only active in the skin. (E) Sequence alignment on the UCSC genome browser (Fujita et al., 2011) of the promoter region 1a encompassing two E2F sites shows conservation across multiple mammalian species (Kent, 2002). Gray highlighting indicates conserved sequences, and black highlighting indicates putative E2F binding sites. (F) Transfection into 210R TECs of an expression plasmid coding for E2F3 together with a luciferase reporter plasmid for either the wild-type region 1a of the Foxn1 promoter or a region 1a where site 3 has been mutated (n = 3; P = 0.03). (G) Transfection into 210R cells of E2F3 and RB expression and the Foxn1 luciferase reporter plasmid (n = 3; P = 0.02). (H) ChIP analysis of E2F3 and E2F4 to sites 1 and 2–3 in TEC100 cells. To adjust for inter-experiment variability of scale of percent input, experiments were normalized to nonspecific antibodies (IgG and anti-p16INK4a). This ratio is represented here. In addition, binding to a nonspecific region of the fifth chromosome was included as a negative control (n = 3; for site 1 P = 0.02, for site 2–3 P = 0.04). (I) Fold change in percent input as assessed by ChIP analysis of E2F3 and E2F4 binding to the Foxn1 promoter in TEC100 cells after transient overexpression of RB (n = 3 compared with untransfected controls; p-values: E2F3 Foxn1 site 1 = 0.06 and site 2–3 = 0.04; E2F4 Foxn1 site 1 = 0.01 and site 2–3 = 0.02). Asterisks indicate P < 0.05; ns indicates no significant difference. All error bars indicate standard error.

The RB family controls the expression of Foxn1. (A) Expression of Foxn1 in CD45– MHCII+ TECS from control (Ctrl) and mutant (Mut) mice relative to Eva1 expression (n = 4; P = 0.03). (B) Immunoblot analysis of Foxn1 levels in control and mutant TECs from 12 pooled control and 5 pooled mutant thymi. α-Tubulin is included as a loading control (n = 2). (C) Immunofluorescence staining of Foxn1 and CD205 individually and merged (right) in control (top) and mutant (bottom) thymi (one of two shown). Bars, 100 µm. (D) Schematic representation of the Foxn1 promoter. Region 1a is active in the skin and thymus, whereas region 1b is only active in the skin. (E) Sequence alignment on the UCSC genome browser (Fujita et al., 2011) of the promoter region 1a encompassing two E2F sites shows conservation across multiple mammalian species (Kent, 2002). Gray highlighting indicates conserved sequences, and black highlighting indicates putative E2F binding sites. (F) Transfection into 210R TECs of an expression plasmid coding for E2F3 together with a luciferase reporter plasmid for either the wild-type region 1a of the Foxn1 promoter or a region 1a where site 3 has been mutated (n = 3; P = 0.03). (G) Transfection into 210R cells of E2F3 and RB expression and the Foxn1 luciferase reporter plasmid (n = 3; P = 0.02). (H) ChIP analysis of E2F3 and E2F4 to sites 1 and 2–3 in TEC100 cells. To adjust for inter-experiment variability of scale of percent input, experiments were normalized to nonspecific antibodies (IgG and anti-p16INK4a). This ratio is represented here. In addition, binding to a nonspecific region of the fifth chromosome was included as a negative control (n = 3; for site 1 P = 0.02, for site 2–3 P = 0.04). (I) Fold change in percent input as assessed by ChIP analysis of E2F3 and E2F4 binding to the Foxn1 promoter in TEC100 cells after transient overexpression of RB (n = 3 compared with untransfected controls; p-values: E2F3 Foxn1 site 1 = 0.06 and site 2–3 = 0.04; E2F4 Foxn1 site 1 = 0.01 and site 2–3 = 0.02). Asterisks indicate P < 0.05; ns indicates no significant difference. All error bars indicate standard error.

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