Supplementary Figure 1 Zranb1 gene targeting. (a) Schematic picture of Zranb1 gene targeting using an FRT-LoxP vector, showing the first 6 exons of Zranb1 gene (exons 7-9 are not shown). Targeted mice were crossed with FRT deleter (Rosa26-FLPe) mice to generate Zranb1-floxed mice, which were further crossed with different Cre mice to generate germline KO (CMV-Cre), T-cKO (Cd4-Cre), or DC-cKO (Cd11c-Cre) mice. (b) Genotyping PCR analysis of germline Zranb1 wild-type (WT), KO (KO), and heterozygous (Het) mice using P1/P2 primer pair for wild-type allele and P1/P4 primer pair for KO allele. (c,d) Genotyping PCR of T cell-conditional Zranb1 wild-type (WT, Zranb1+/+Cd4-Cre), T-cKO (Zranb1f/fCd4-Cre), and heterozygous (T-cKO Het, Zranb1+/fCd4-Cre) mice (c) or DC-conditional Zranb1 wild-type (WT, Zranb1+/+Cd11c-Cre), DC-cKO (Zranb1f/fCd11c-Cre), and heterozygous (DC-cKO Het, Zranb1+/fCd11c-Cre) mice (d) using P3/P4 primer pair to amplify WT and floxed alleles and Cre-specific primers for the Cd4-Cre and Cd11c-Cre DNA. (e) RT-PCR was performed to measure the Zranb1 mrna level in the indicated cell types.
Supplementary Figure 2 Development of cells of the immune system in mice with germline deletion of Zranb1. (a) Flow cytometry analysis of the percentage (numbers in quadrants) of CD4 + CD8 + double-positive (DP), CD4 + and CD8 + single-positive (SP) thymocytes in the thymus (Thy) and B220 + B cells (B) and CD4 + and CD8 + T cells in the spleen (Spl) of wild-type (WT) and KO mice. (b) Flow cytometry analysis of naïve (N, CD62 hi CD44 lo ) and memory (M, CD62 lo CD44 hii ) CD4 + T cells and naïve (N), central memory (CM, CD62L hi CD44 hi ), and effector memory (EM, CD62 lo CD44 hi ) CD8 + T cells in the spleen of wild-type and KO mice. (c) Flow cytometry analysis of the Foxp3 + Treg cells in the spleen and thymus of wild-type (WT) or KO mice. (d) Flow cytometry analysis of conventional DC (cdc, CD11c + CD11b hi B220 ) and plasmacytoid DC (pdc, CD11c + CD11b B220 + ) percentage in total CD11c-positive cells of Bone marrow (BM) and Spleen (Spl). Data in all panels are presented as a representative FACS plots (left) and mean ± SEM values based on multiple samples (right). Similar results were obtained in three independent experiments. *P<0.05.
Supplementary Figure 3 Trabid is dispensable in T cells for T cell activation and EAE induction. Age- and sex-matched wild-type (WT) and T-cKO mice were subjected to MOG 35-55 -induced EAE. (a) Flow cytometry analysis of the CD45 + immune cells (CD4 + and CD8 + T cells and CD11b + monocytes) infiltrated into the CNS (brain and spinal cord) (n = 3, day 14 post-immunization), presented as a representative plot (left) and summary graph (right). (b) Flow cytometry analysis of the IFN- producing T H 1 and IL-17A-producing T H 17 cells in the CNS (percent of CD4 + CD45 + cells) and draining lymph nodes (percent of CD4 + T cells) (n = 3, day 14 post-immunization). Data are presented as a representative plot (left, CNS only) and summary graph of absolute cell numbers (right). (c) ELISA analysis of the indicated cytokines in the supernatants of cultures of splenocytes isolated from WT and T-cKO EAE mice (day 14 after immunization) and restimulated in vitro with MOG peptide (20 g/ml for 48 h). (d) Flow cytometry analysis of the frequency of T H 1, T H 17, and T REG cells in wild-type and T-cKO naive CD4 + T cells, stimulated for 4 days with platebound anti-cd3 and anti-cd28 antibodies under T H 0, T H 1, T H 17, or T REG conditions. (e) Flow cytometry analysis of cell proliferation, based on CFSE dilution, of naïve T cells stimulated with plate-bound anti-cd3 (5 g/ml) and anti-cd28 (1 g/ml) for 48 h. (f) ELISA of IFN- and IL-2 using supernatants of the cell cultures described in e. Data are representative of three independent experiments. Error bars are mean ± SEM values.
Supplementary Figure 4 Effect of Trabid deficiency on the function of DCs in regulating T cell differentiation. (a,b) Flow cytometry analysis of Foxp3 + T REG cells (a) or IFN- + T H 1 and IL-17 + T H 17 cells (b) in wild-type naïve CD4 + T cells stimulated for 4 days with plate-bound anti-cd3 and anti-cd28 plus supernatant of LPS-activated wild-type (WT) or DC-cKO BMDCs (LPS-DC) in the absence ( ) or presence (+) of the indicated cytokines.
Supplementary Figure 5 Trabid regulates LPS-stimulated c-rel recruitment and histone modifications at the promoters of Il12a and Il12b. (a) ChIP assays of c-rel recruitment to the promoter of the indicated genes using wild-type (WT) and DC-cKO BMDCs that were either unstimulated (0 h) or stimulated for 6 h with LPS. Data are presented as percentage of the total input DNA, as determined by QPCR assays. (b) Luciferase assays using HEK293 cells transfected with an Il12b-luciferase reporter plasmid in the presence (+) or absence ( ) of an empty vector (Vector) or expression vectors encoding c-rel, Trabid, or a catalytically inactive Trabid mutant, C443 (Mut Trabid). Luciferase activity is presented as fold based on empty vector. (c) ChIP analyses of H3K9 dimethylation (H3K9me2) and trimethylation (H3K9me3) in the Il12a promoter using wild-type or DC-cKO BMDCs that were untreated (NT) or stimulated with LPS for 6 h. The Y axis is percentage (%) of total H3. Data are presented as mean ± SEM values and representative of at least three independent experiments. Statistical analyses represent variations in technical replicates *P < 0.05; **P < 0.01.
Supplementary Figure 6 Trabid deficiency suppresses the recruitment of Pol II complex components to the Il12b promoter. ChIP analysis of the recruitment of Pol II, Pol II ps5, TFIIB, and TFIID to the indicated promoters in wild-type (WT) or DC-cKO BMDCs. The Y axis is percentage (%) of the DNA bound to the specific factors based on total input DNA. Data are presented as mean ± SEM values and representative of at least three independent experiments. Statistical analyses represent variations in technical replicates. *P < 0.05; **P < 0.01.
Supplementary Figure 7 Jmjd2d is regulated by Trabid and mediates induction of Il12 genes. (a,b) ChIP analysis of the binding of indicated Jmjd2 family members to different regions of Il12b locus in wild-type (WT) and DC-cKO BMDCs (a) or wild-type and Rel-KO BMDCs (b) that were either not treated (NT) or stimulated with LPS for 6 h. (c) Immunoblotting analysis of Jmjd2d in wild-type BMDCs transduced with pgipz lentiviral vectors encoding a non-silencing control shrna (C) or two different Kdm4d-specific shrnas. The cells were stimulated with LPS for 6 h to induce the level of Jmjd2d. (d) qrt-pcr analysis of the indicated genes in LPS-stimulated BMDCs described in c. Data are presented as fold relative to the Actb mrna level. (e) Immunoblotting analysis of HA-tagged Trabid (or Trabid C443A) and FLAG-tagged Jmjd2d in anti-flag (Jmjd2d) immunoprecipitates (top two panels) or lysates (lower panels) of HEK293 cells transfected with the indicated plasmids. (f,g) Immunoblotting analysis of Jmjd2d (f) and qrt-pcr analysis of the indicated genes in DC-cKO BMDCs reconstituted with pclxsn(gfp) (Vec) or pclxsn(gfp)- HA-Jmjd2d (Jmjd2d). Data are presented as mean ± SEM values and representative of at least three independent experiments. *P < 0.05; **P<0.01.
Supplementary Figure 8 A model of Trabid function in TLR-mediated induction of Il12b. LPS induces the expression of the Jmjd2d gene (Kdm4d) via an unknown signaling pathway. Jmjd2d mediates removal of H3K9me2 and H3K9me3 from the Il12b promoter, thereby promoting c-rel binding to the B element and induction of Il12b expression. The fate of Jmjd2d in TLR-stimulated DCs is tightly controlled by ubiquitination. An unknown E3 ubiquitin ligase mediates Jmjd2d ubiquitination and degradation, whereas Trabid deubiquitinates Jmjd2d to prevent its degradation. Trabid deficiency causes elevated ubiquitination and degradation of Jmjd2d and, thus, reduces the level of Il12b induction. Similar mechanisms may apply to the induction of Il12a and Il23a genes.
Supplementary Table 1 List of antibodies used in biochemical assays. Antibody Product number Vender RelB C- 19 Santa Cruz Biotechnology Lamin B C- 20 Santa Cruz Biotechnology TRAF2 C- 20 Santa Cruz Biotechnology TRAF3 H- 122 Santa Cruz Biotechnology ERK K- 23 Santa Cruz Biotechnology JNK1 C- 17 Santa Cruz Biotechnology p38 H- 147 Santa Cruz Biotechnology IKKα H744 Santa Cruz Biotechnology Ubiquitin P4D1 Santa Cruz Biotechnology p105/p50 C- 19 Santa Cruz Biotechnology cebp- β C- 19 Santa Cruz Biotechnology STAT3 C- 20 Santa Cruz Biotechnology c- Rel C Santa Cruz Biotechnology TFIIB (SI- 1)X Santa Cruz Biotechnology TFIID (SI- 1)X Santa Cruz Biotechnology Pol II (N- 20)X Santa Cruz Biotechnology HSP60 H- 1 Santa Cruz Biotechnology Rabbit IgG sc- 2027 Santa Cruz Biotechnology Phospho- ERK (Y204) E- 4 Santa Cruz Biotechnology Phospho- IκBα (S32) 14D4 Cell Signaling Technology Phospho- JNK (T180/T185) 81E11 Cell Signaling Technology Phospho- p38 (T180/T182) 9211 Cell Signaling Technology Phospho- p105 (S933) 18E6 Cell Signaling Technology Phospho- IKKα/β (S176/S180) 16A6 Cell Signaling Technology Actin C- 4 Sigma- Aldrich HRP- anti- HA HA- 7 Sigma- Aldrich HRP- anti- FLAG M2 Sigma- Aldrich Ubiquityl- H2B 05-1312 Millipore H3K4me3 07-473 Millipore H3K27me3 07-449 Millipore H2B ab1790 Abcam H3K9me2 ab1220 Abcam H3K9me3 ab8898 Abcam JMJD2A ab105953 Abcam JMJD2B ab27531 Abcam JMJD2C ab85454 Abcam JMJD2D ab93694 Abcam Pol II Phospho- Ser 5 ab5131 Abcam p100/p52 TB4 NCI Preclinical Repository
Supplementary Table 2 Gene-specific primers used in qrt-pcr experiments Gene Forward primer Reverse primer Il1b AAGCCTCGTGCTGTCGGACC TGAGGCCCAAGGCCACAGGT Il6 CACAGAGGATACCACTCCCAACA TCCACGATTTCCCAGAGAACA Tnf CATCTTCTCAAAATTCGAGTGACAA CCAGCTGCTCCTCCACTTG Il12a ACTAGAGAGACTTCTTCCACAACAAGAG GCACAGGGTCATCATCAAAGAC Il12b GGAGACACCAGCAAAACGAT TCCAGATTCAGACTCCAGGG Il23a GCCAAGAAGAC CATTCCCGA TCAGTGCTACAATCTTCTTCAGAGGACA Il10 CCAGAGCCACATGCTCCTAGA GGTCCTTTGTTTGAAAGAAAGTCTTC Nos2 GTGGTGACAAGCACATTTGG AAGGCCAAACACAGCATACC Kdm4a GGAGCCTTGCTGAGCATCAC TCACTGAAGGAGCCGTCGTC Kdm4b GGCTTTAACTGCGCTGAGTC GTGTGGTCCAGCACTGTGAG Kdm4c CACGGAGGACATGGATCTCT CGAAGGGAATGCCATACTTC Kdm4d Zranb1 GTCTTGGTCGTCGTCCTTGT GGCTATTCCTCAATGCCTGT AATCCCCCTTCAGAAGCTGT TGTCTCCTCCCGATGACTT
Supplementary Table 3 QPCR primers used in ChIP assays. Gene Forward primer Reverse primer Il1b pro (κb site) GTTCCGCACATCCTGACTTA CAATTGTGCAGATGGTGTCAA Il6 pro (κb site) TTTCCAATCAGCCCCACC CAGAATGAGCTACAGACATCCC Il12a pro (κb site) ACGCACTTGTCCTTGAGATG CTGACCTTGGGAGACACATTT Il12b pro (κb site) CATTTCCTCTTAACCTGGGATTTC CTGCTCCTGGTGCTTATATACT Il12b pro - 981-780 AGGCCAGGGACAGGAAT AAGAGGTGTGCAATCCTGTAAG Il12b pro - 792-584 TGCACACCTCTTTGCAATTT GAGGGCGTCTCTTATGCTTAT Il12b pro - 600-395 CATAAGAGACGCCCTCAAA GTTACAGGCCCAAAGAATAAAG Il12b pro - 406-193 GGGCCTGTAACACCTACTTATTT ACGTCGAAATCCCAGGTTAAG Il12b pro - 220-16 CATTTCCTCTTAACCTGGGATTTC CTGCTCCTGGTGCTTATATACT Il1b TATA GAATTCCCATCAAGCTTCTCC AGACAGAGAGAGAGAGAGACTTAC Il6 TATA CCCACCCTCCAACAAAGATT ACTCCTCTCTCACAGTCTCAATA Il12a TATA CTGGCGTCTACACTGCTG TTTGGAAATGGGAATCAACACAG Il12b TATA CTGCTCCTGGTGCTTATATACT CTGCTCCTGGTGCTTATATACT Il23a TATA AGGCACTAGGAAAGAGGATCTA GTTCATACCTGGAGGAGTTGG