Nature Immunology: doi: /ni Supplementary Figure 1. Construction of lnckdm2b RFP reporter and lnckdm2b-knockout mice.

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1 Supplementary Figure 1 Construction of lnckdm2b RFP reporter and lnckdm2b-knockout mice. (a) ILC3s (Lin CD45 lo CD90 hi ) were isolated from small intestines, followed by immunofluorescence staining. LncKdm2b was detected by RNA fluorescence in situ hybridization (RNA-FISH). Green: lnckdm2b probe or scramble probe; red: CD127 or rat IgG2a- ; purple: CD45 or rat IgG2b-. Nuclei were counterstained by DAPI. Scale bar, 5 m. (b) Strategies for construction of lnckdm2b flox/flox and lnckdm2b RFP mice. (c) Total RNA from BM cells of lnckdm2b flox/flox or lnckdm2b flox/flox ;Vav-Cre mice were extracted, followed by RNA hybridization. LncKdm2b specific probe was labeled with biotin. 18S RNA served as a loading control. (d,e) Analysis of ILC1s (d) and ILC2s (e) in small intestines from lnckdm2b flox/flox or lnckdm2b flox/flox ;Vav-Cre mice by FACS. ILC1s were gated on CD3 CD45 NK1.1 NKp46 and ILC2s were gated on Lin Thy1.2 CD25 KLRG1 ST2. n=6 for each group. (f,g) Analysis of NK cells in spleen (f) and ILC1s in liver (g) from lnckdm2b flox/flox ;Vav-Cre or lnckdm2b flox/flox ;Vav-Cre mice. NK cells were gated on NKp46 NK1.1. ILC1s were gated on CD3 DX5 TCR NK1.1 CD49a. n=6 for each group. (h) Total RNAs were extracted from ILC3s derived from lnckdm2b flox/flox or lnckdm2b flox/flox Rorc-Cre mice, followed by analysis of lnckdm2b expression with RT-qPCR. (i) Numbers of NKp46 ILC3s, CD4 ILC3s and CD4 ILC3s in lnckdm2b and lnckdm2b mice were calculated and shown as means SD. n=6 per group. (j,k) LncKdm2b flox/flox and lnckdm2b flox/flox Rorc-Cre mice were infected with C. rodentium. 6 days later, bacterial counts (CFUs) in feces (j) and colon length (k) were measured. n=6 for each group. (l) LPLs were isolated from lnckdm2b and lnckdm2b mice post C. rodentium infection, and stimulated with IL-23 for 4 h. Then IL-22 ILC3s were analyzed by FACS. (m) Secreted IL-22 protein by LPLs as in l was tested by ELISA after stimulated with IL-23 for 24h. n=6 each group. *p 0.01 and **p by two-tailed Student s t test. All data are representative of three independent experiments.

2 Supplementary Figure 2 LncKdm2b interacts with Sabt1. (a) BrdU ILC3s (Lin CD45 CD127 ROR t ) were analyzed by FACS. n=6 for each group. (b) Percentages of total ILC3s (Lin CD45.2 CD127 ROR t ) in chimeras were checked by FACS. n=6 for each group. (c) Expression levels of lnckdm2b neighboring genes in lnckdm2b and lnckdm2b ILC3s were detected by RT-qPCR. Data were normalized to endogenous Actb and shown as means ± SD. (d) Identification of Satb1 as an lnckdm2b binding protein. Satb1 protein sequences were identified by LTQ Orbitrap XL. (e) Relative mrna levels of Zfp292 were examined by real-time qpcr. Fold changes were normalized to endogenous Actb. (f) Enrichment of RNA pol II on Zfp292 promoter in lnckdm2b and lnckdm2b ILC3s. Nuclei were extracted from lnckdm2b or lnckdm2b ILC3s, followed by ChIP-qPCR assays. (g) Generation of Satb1 mice. The schematic diagram of Satb1 KO construction by CRISPR/Cas9 technology (upper left). Frameshift of Satb1 gene was confirmed by DNA sequencing (lower left). Satb1 deletion was validated by immunoblotting (right panel). *p 0.01 by two-tailed Student s t test. All data are representative of three independent experiments.

3 Supplementary Figure 3 Sabt1 interacts with the NURF complex. (a) Identification of Bptf and Snf2l as Satb1 binding proteins. Bptf and Snf2l protein sequences were identified by LTQ Orbitrap XL. (b) Generation of Zfp292 mice. The schematic diagram of Satb1 KO construction by CRISPR/Cas9 technology (upper left). Frameshift of Zfp292 gene was confirmed by DNA sequencing (lower left). Zfp292 deletion was verified by immunoblotting (right panel). (c) LPLs from Zfp292 and Zfp292 mice were isolated and stimulated with IL-23 for 4 h. Numbers of IL-22 ILC3s were examined by FACS. *p 0.01 by two-tailed Student s t test. All data are representative of three independent experiments.

4 Supplementary Figure 4 Deletion of Sabt1 or Bptf leads to reduced numbers of ILC3s. (a,b) Percentages (a) and numbers (b) of ILC3s in small intestines derived from Satb1 and Satb1 mice. Percentages of indicated cells were calculated and shown at right panel (a). n=6 per group. (c,d) Percentages (c) and numbers (d) of ILC3s in small intestines derived from Bptf flox/flox and Bptf flox/flox Rorc-Cre mice. Percentages of indicated cells were calculated and shown at right panel (c). n=6 per group. (e) Bptf flox/flox and Bptf flox/flox Rorc-Cre mice were infected with C. rodentium (5x10 9 CFUs per mouse). 6 days later, bacterial counts (CFUs) in feces were measured. n=6 for each group. (f) Bptf flox/flox Rorc-Cre and Bptf flox/flox Rorc-Cre mice were infection with C. rodentium (5x10 9 CFUs per mouse) for 6 days, followed by H&E staining for colons. Scale bar, 50 m. (g) 1x10 6 CD45.1 BM cells with 1x10 6 CD45.2 LPLs from WT or TKO mice infected with Zfp292 expressing retroviral were transplanted into lethally irradiated CD45.1 recipients. Percentages of ILC3s derived from CD45.2 donors were analyzed by FACS. n=6 for each group. (h) Numbers of ILC3s as in g were analyzed by FACS. n=6 for each group. (i) Percentages of Ki67 ILC3s in Zfp292, lnckdm2b overexpression in Zfp292 Zfp292 LPLs were examined by FACS. n=6 for each group. *p 0.01 and **p<0.001 by two-tailed Student s t test. All data are representative of at least three independent experiments.

5 Supplementary Table 1 sgrna sequences for gene editing by CRISPR/Cas9 technology Name grna sequences lnckdm2b (up) 5 - TATCACCTGATGCAGACCTGAGG -3 lnckdm2b (down) 5 - GGAATCAGCTGACCTGG CTGAGG -3 Satb1 5 - AACAATGTGAGTGATCCG AAGGG-3 Zfp TTTGCTGGAGGTATACACAGTGG -3 Bptf (up) 5 - CAAGTATAATGCCTAAAGCCCGG -3 Bptf (down) 5 - AGTGGGAACAAACCTGCCTGAGG -3 lnckdm2b RFP reporter 5 - CTTAGTCTCTGAGTTAGATGTGG -3

6 Supplementary Table 2 Primer oligonucleotides for qpcr and ChIP-qPCR assays in this study Genes Forward Reverse lnckdm2b 5 - TGGAAACCACAGAGAACTCC ATCACACCCAACACCATACG-3 Actb 5 - TGACGGGGTCACCCACACTGTGCCCATCTA CTAGAAGCATTTGCGGTGGACGATGGAGGG-3 Hmbs 5 - ATGAGGGTGATTCGAGTGGG TTGTCTCCCGTGGTGGACATA-3 Gapdh 5 - GCAGTTAAGTTCAGGAGCTTCAGG GAAGCACGGTTGTATGTGCAAGTG-3 U1 5 - CTTACCCACGATTCTCCATCTGT CCGTTGTCTTGTCAATCAGGC-3 Rorc 5 -ACCTCCACTGCCAGCTGTGTGCTGTC-3 5 -TCATTTCTGCACTTCTGCATGTAGACTGTCCC-3 Rora 5 - CGCAGCGATGAAAGCTCAAAT ACGAGGACAGGAGTAGGTGG-3 Notch1 5 - TGCGTCAACGTCCGATCC CATTCAGGCAGGTCCCACTT-3 Kit 5 - CTGGGATCTGCTCTGCGTC GGTGTCGCCAGCTTCAACTA-3 Zfp TGAAGACAGCACACCCTGAC AGCTGCTCACTTGTGATGGC-3 Ctnnbl1 5 - CCTGGCCTTGTTGGAGAACT TGGCGGGAAGCTTACACATA-3 Il23r 5 - CACTGCCGACCAAGGAATCT GCATGAGGTTCCGAAAAGCC-3 Ncor1 5 - GCATTTGATGCAGCCCAACA GTGTTCTCTTTTTAACTCCGCCA-3 Ncr1 5 -AGCCAGAGGATCAACACTGAA ACACCAGATGTTCACCGAGT-3 Satb1 5 - TATTTTTGCCCTTCCCCCGC-3 5 -CTTTGCAGCCGGGATTCTTG-3 Satb2 5 - GCCGTGGGAGGTTTGATGAT TCTTTCCGCACCAAGACGAA-3 Ahr 5 -CGCGGGCACCATGAGCAG-3 5 -CTGTAACAAGAACTCTCC-3 Notch2 5 - TATCGCCCAGACATTCTCGC ACACACTGACGGGGATCAAC-3 Anapc5 5 - TTGACCGCCTGATTCTCACTG AGCTGTTTTCTTGGAATCTCTCC -3 Rnf AAGGCGGGTGCTACTTCTATG CACAGCAGACATGCTTCTTTCTA-3 Kdm2b 5 - GATGCTGAGCGGTATCATCCG GAGACAGCGATCCATGAGCAG-3 Tmem120b 5 - AGGCACCTGAAGGACCTGAA GGCCTCCATGTCAAAGAAGAC-3 Rhof 5 - AAGATAGTGATCGTAGGTGACGG GTACTTCTCAAACACCGACGG-3 Hpd 5 - CGCCTGTGTCACATCGCTT CTCATTCACTAGAAAGACAGCGT-3 Morn3 5 - ATGGCCGTTTCTTCCACCTG CCACCTTAGGTATGGGGAACT-3 Il TTGTGCGATCTCTGATGGCT CCAGCATAAAGGTGCGGTTG-3 Zfp292 pro#1 5 - AGATGTCTCTCTTTATTTTCAA TGGGCAAGTGACTTAGTGACAAA-3 Zfp292 pro#2 5 - CTGGAGAACTGTTAGTTACAG TGCTGTGAAGAGACACCAGGACCA-3 Zfp292 pro#3 5 - AACTCACAGACAGCTGTGAAG GATTAGGAGCTGGAGAGATGGGC-3 Zfp292 pro#4 5 - TCTTTCTCTCCACACCTGCTG TAAATGCAAGGACTGTACAATAA-3 Zfp292 pro#5 5 - TCCTTAGCCTTACAGTGTGCTCC GCTTTTAAATCATTACATAAG-3 Zfp292 pro#6 5 - GAGACCCAGCATTACAATGTA ACAACCAGCCTGGAGGTTGGC-3 Zfp292 pro#7 5 - GTGCCTTTTGCCTGGTTTATGCAG TAGTCGGAAAATCAATGTGCT-3 Zfp292 pro#8 5 - CGACTACCTCTTATTCTACC TCCTCCTCCAGCCTATGCCAGAT-3 Zfp292 pro#9 5 - TTCGACCTACTTTCCCAAACTA ATTCAAACAGACCGAAATGAT-3 Zfp292 pro# TTCCAGGAGGCGTTCTCGGGT AGGCCTCTGCTGCTGCGGCGGC-3