Ns Ns Ns 1.4 1.4 1.8 1.2 1.2 1.6 1.4 1.0 1.0 1.2 0.8 0.8 1.0 0.6 0.6 0.8

A DNA looping

CTCF CTCF CTCF Chr.10:117,578,000-117,945,000 (mm9) -70 +1 +66

Mdm1 Il22 Iltifb Ifng

conservation rat human

B BALB/c C57BL/6 2.3% 1.2% IL-22 FSC

C ns ns ns 1.4 1.4 1.8 1.2 1.2 1.6 1.4 1.0 1.0 1.2 0.8 0.8 1.0 0.6 0.6 0.8

Il22 (RU) 0.6

0.4 Il17a (RU) 0.4 Runx1 (RU) 0.4 0.2 0.2 0.2 0 0 0

BALB/c BALB/c BALB/c C57BL/6 C57BL/6 C57BL/6

Supplemental Figure 1. Comparison of IL-22 expression in two mouse strains, BALB/c and C57BL/6. (A) Mouse genomic locus on chromosome 10 containing Mdm1, Il22, and Ifng genes. The nearest neighbors of Il22 are Ifng and a housekeeping gene Mdm1. The binding sites for the CCCTC-binding factor (CTCF) in the Ifng locus are indicated. In some strains of mice, including C57BL/6, this locus contains an inverted and non-expressed duplication of Il22, known as Iltifb, flanked by six tandem repeats of a highly conserved repetitive element (hatched bar). Arrows indicate the direction of gene transcription. Shown at the bottom are comparisons of the corresponding genomic regions of several species using Conservation and PhastCons tracks in the UCSC genome browser. (B) Intracellular staining for IL-22 in naive CD4+ T cells cultured under Th22 conditions for 5 d, and then restimulated with PMA and ionomycin for 5 h prior to staining. Numbers in plots indicate percent IL-22+ cells (FSC, forward scatter). (C) Real-time qRT-PCR analysis of the transcript expression, presented relative to Hprt expression (mean of n = 3 ± SD) (RU, relative units). Mann-Whitney U test where ns, not significant. A 1.2 1.2 1.6 1.2 1.0 1.0 1.4 1.0 1.2 0.8 0.8 1.0 0.8 0.6 0.6 0.8 0.6 0.4 0.4 0.6 0.4

Il4 (RU) 0.4 Il22 (RU) 0.2 Ifng (RU) 0.2 0.2 0.2 Il17a (RU) 0 0 0 0

Th1 Th2 Th1 Th2 Th1 Th2 Th1 Th2 Th17Th22 Th17Th22 Th17Th22 Th17Th22

1.4 1.2 1.4 1.2 1.2 1.0 1.2 1.0 1.0 0.8 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4

Il23r (RU) 0.2 0.2 0.2 0.2 Rora (RU) Gata3 (RU) 0 Tbx21 (RU) 0 0 0

Th1 Th2 Th1 Th2 Th1 Th2 Th1 Th2 Th17Th22 Th17Th22 Th17Th22 Th17Th22

1.2 1.4 1.2 1.2 1.0 1.2 1.0 1.0 0.8 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 Ahr (RU) Maf (RU)

0.2 0.2 Batf (RU) 0.2 Rorc (RU) 0.2 0 0 0 0

Th1 Th2 Th1 Th2 Th1 Th2 Th1 Th2 Th17Th22 Th17Th22 Th17Th22 Th17Th22

1.4 1.4 1.4 1.2 1.2 1.2 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4

0.2 0.2 Cbfb (RU) 0.2 Runx1 (RU) 0 Runx3 (RU) 0 0

Th1 Th2 Th1 Th2 Th1 Th2 Th17Th22 Th17Th22 Th17Th22

B 1.2 1.0 0.8 0.6 0.4

Il22 (RU) 0.2 0

naiveTh22 MEF Liver

Supplemental Figure 2. Real-time qRT-PCR analysis of Th cell-related gene expression. (A) Gene expression of naive CD4+ T cells differentiated for 5 d under Th1, Th2, Th17, or Th22 conditions was determined. (B) Expression of Il22 mRNA was measured in naive CD4+ T, Th22, and MEF cells, as well as hepatocytes. The results are presented relative to Hprt expression (RU, relative units). Data are representative of three independent experiments (mean of n = 3 ± SD). CNS-32B minP Luc * *

50 *

P/I * * *

Relative luciferase activity 10

0 t t t t γ γ γ γ pcDNA3 Ets-1 Fosl2 ROR Runx1 pcDNA3 BATF Fosl2 ROR Runx1 pcDNA3 BATF Ets-1 ROR Runx1 pcDNA3 BATF Ets-1 Fosl2 Runx1 pcDNA3 BATF Ets-1 Fosl2 ROR pcDNA3 BATF Ets-1 Fosl2 RORγt Runx1 (-)

Supplemental Figure 3. Contribution of Runx1 and RORγt for Il22 enhancer activity. The luciferase reporter construct containing minP linked to CNS-32B was cotransfected with an empty vector (pcDNA3) or in combination with an expression construct encoding BATF, Ets-1, Fosl2, RORγt, or Runx1. Cotransfected expression vectors are indicated under the graph. Transient transfections were performed in EL4 cells stimulated with PMA and ionomycin (P/I), and luciferase activities were normalized with a Renilla control and presented relative to the empty vector control sample (mean of n = 3 ± SD). *P < 0.05, Mann-Whitney U test.