Nature Genetics: doi: /ng Supplementary Figure 1

Transcription

1 Supplementary Figure 1 Characterization of HPV Tg/+ ; Krt17 / mice. (a) Macroscopic images of HPV16 Tg/+ mouse ears at P40, P70 and P120. (b) Immunostaining of HPV16 Tg/+ ear tissue sections for K17 (red) and Hoechst (blue) at P20 and P40. (c) H&E staining of a wild-type P70 ear tissue section (this micrograph complements the data presented in Fig. 1b). (d) Immunostaining and quantification of vasculature in P70 ear tissue sections from wild-type (green bar), HPV16 Tg/+ (blue bar) and HPV16 Tg/+ ; Krt17 / (open bar) mice. The elements stained are PECAM (green), K14 (red) and Hoechst (blue). n = 9 biological replicates per genotype. (e) Flow cytometry analysis of total cells isolated from P70 ear tissue for four distinct genotypes. Cells double-positive for CD45 and CD4, CD207, CD11b or CD11c were analyzed. Number indicates the

2 percentage of double-positive cells per genotype. Data represent one of three biological replicates. (f) Immunoprecipitation of HPV16 E7 from P2 mouse skin keratinocytes. (g) p53 expression levels in P70 ear tissue. Actin was used as a loading control for total protein, and K14 serves as a loading control for epithelial cells. The K17 immunoblot establishes proper genotyping. PIS, preimmune sera. The blots in f and g represent one of six biological replicates. (h) TUNEL staining of P70 ear tissue, denoting apoptotic cells restricted to the suprabasal layers of epidermis, for both genotypes. Images represent one of six biological replicates. (i) Electron micrographs and aspect ratio quantification (box-and-whisker plot) of basal epithelial cells from 8-month-old ear tissue (8,400 magnification). n = 100 basal cells counted across 3 biological replicates per genotype.

3 Supplementary Figure 2 TPA- and HPV16-induced gene expression in skin tumor keratinocytes. (a) Normalized expression for target genes in intact A431 keratinocytes stimulated with TPA relative to DMSO (solid red bars) and in TPA-treated A431 cells expressing either shcontrol or shkrt17 plasmid (open bars). n = 5 biological replicates. (b) Normalized expression for gene transcripts restored upon Krt17 re-expression (n = 7 biological replicates; blue bars) relative to vector control and compared to Krt42 re-expression (n = 5 biological replicates; red bars). (c) Normalized expression for distinct regions of the Aire transcript (exon 2 or 10 or a region spanning exons 13 and 14) in TPA-treated ears (blue bars) or thymus (red bars) relative to liver, in two different genetic backgrounds (C57BL/6, left; FVB/N, right). n = 4 biological replicates. (d,e) Sense-strand RNA in situ hybridization controls in skin tissue sections (complements data reported in Fig. 2c,d). Scale bars, 200 µm. (f) Coimmunoprecipitation of K17 with RFP-Trap pulldown of mcherry-aire compared to unfused mcherry. GAPDH serves as a loading control for total protein. n = 1 of 7 biological replicates.

4

5 Supplementary Figure 3 Loss of Krt17 does not affect thymic Aire expression or function. (a) Normalized expression for Aire in P10 thymic tissue between wild-type (blue bar), HPV16 Tg/+ (red bar) and HPV16 Tg/+ ; Krt17 / (open bar) mice. *NS, no statistical significance. (b) RNA in situ hybridization for Aire in thymic tissue sections. Arrows denote regions of medullary thymus positive for hybridization. Scale bars, 200 µm. (c) H&E staining of lacrimal glands from aged (>4 month) C57BL/6 mice. Scale bars, 100 µm. (d) Histology scoring for the presence of lymphocytic infiltrate in multiple tissues from aged (>4 month) Krt17 +/+, Krt17 /, Aire +/+ and Aire / mice. *P values are from non-parametric ANOVA with Dunn s post-testing. n.s., no statistical significance. (e) Summary of histology scoring (each pie represents an individual mouse) with each tissue represented by a different color. A filled wedge indicates >10% of the tissue exhibiting inflammation.

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7 Supplementary Figure 4 Characterization of Aire and nuclear K17 punctae in tumor epithelia. (a) A431 keratinocytes transfected with mcherry-aire and immunostained in green with markers for lysosomes (LAMP-1), stress granules (eif3η) or PML bodies (PML). Scale bars, 5 µm. (b) A431 keratinocytes stably expressing shrna targeting Krt17 (shkrt17) or shcontrol plasmids transiently transfected with mcherry-aire and immunostained for HSP70 (green). Scale bars, 20 µm. (c) Singleplane confocal images of HeLa cells stably expressing GFP-K17 and either mock treated or treated with LMB. Scale bars, 5 µm. (d) Single-plane confocal images through the nucleus of HeLa cells either mock treated or treated with LMB and immunostained for K17 (green) and Hoechst (blue) using mouse (left panels) or rabbit (right panels) anti-k17 antibodies. The z plane is on top. Arrows denote intranuclear punctae immunopositive for K17. Scale bars, 20 µm. (e,f) Single-plane confocal images of immortalized keratinocytes from wild-type (e) or Krt17 / (f) mouse epidermis immunostained for K17 and K5. Scale bars, 20 µm. (g) Immunoblots for subcellular fractions of DMSO- or TPA-treated A431 cells transfected with mcherry-aire compared to unfused mcherry control. Sol, soluble (GAPDH positive); NE, nuclear envelope enriched (nesprin 3 positive); Chr, chromatin enriched (histone H3 positive). (h) Immunoblots of subcellular fractions of DMSO- or TPA-treated A431 keratinocytes probed for various type I keratins, as indicated on the left. Sol, soluble (GAPDH positive); NE, nuclear envelope enriched (nesprin 3 positive); Chr, chromatin enriched (histone H3 positive). All images in a g are representative of three biological replicates. Blots in h are representative of five biological replicates.

8

9 Supplementary Figure 5 ChIP and EMSA assays. (a) Immunoblot for K17 after immunoprecipitation of K17, relative to preimmune sera (PIS), from the nuclear extract used for the ChIP analysis presented in Figure 4. Input represents the sample removed before immunoprecipitation. The image represents one of seven biological replicates. (b) DNA amplicons from the K17 ChIP assay (Fig. 4) observed after equal numbers of qpcr cycles by DNA electrophoresis. The images represent one of three biological replicates. (c,d) 5 upstream sequences of the CXCL10 (c) and CCL19 (d) transcripts. The locations of the forward and reverse primer sites used for the ChIP assays are depicted with colored arrows. NF- B consensus sequences are denoted with bold text. The ATG translation start site is italicized. (e) EMSA analysis of radiolabeled Oct1 consensus oligonucleotides using nuclear extracts from A431 keratinocytes treated with TPA, relative to DMSO control. Supershift analysis was conducted with antibodies against K17, relative to preimmune sera (PIS) control. Cold competition was conducted using 50-fold excess of non-labeled oligonucleotide. The image represents one of three biological replicates. (f) Reciprocal coimmunoprecipitation demonstrating p65 enrichment with K17 immunoprecipitation relative to preimmune sera (PIS) (left panel; n = 7 biological replicates) and K17 enrichment with p65 immunoprecipitation relative to IgG control (right panel; n = 3 biological replicates).

10 R.P. Hobbs et al. Keratin 17 regulates Aire to promote skin tumorigenesis Supplementary Table 1. Summary of "Inflammatory Cytokine and Receptors" array Wildtype HPV16 tg/+ ;Krt17 -/- vs. HPV16 tg vs. HPV16 tg Cxcl Cxcl Hsp90a 1.41 Ccr Il1b Lta 1.31 Il Ccl Tnfrsf1b 1.05 Ccl Cxcl Cxcl Ccl Il1f Il13ra Il8rb Cxcl Mif Ccl Cxcl C Xcr Ccl Cxcr Ccr Ccl Il1r Ccl Ccl Il10rb Ccr Ccl Abcf Il2rg Il1a Ltb Ccr Il Gapdh Ccl Casp Il2rb Ifng Il1f Il1r Ccr Cd40lg Gusb Ccl Itgb Il17b Cxcl Spp Ccr Tnf Il Il Il5ra Hprt Crp Itgam Ccl Il10ra Il Ccr Il HPV16 tg -induced, Krt17-dependent Wildtype HPV16 tg/+ ;Krt17 -/- Cxcr Cx3cl Ccl Ccr Il Pf Ccr Tollip Cxcl Tgfb Scye Actb Il6st Il6ra Ccl Ccl Il Tnfrsf1a Ccl Ccr Ccl Bcl Il HPV16 tg -induced, Krt17-independent Wildtype Not induced by HPV16 tg

11 R.P. Hobbs et al. Keratin 17 regulates Aire to promote skin tumorigenesis Supplementary Table 2. Summary of "NfκB Signaling Pathway" array HPV16 tg/+ ;Krt17 -/- vs. HPV16 tg Il HPV16 tg -induced, Tnfrsf1a 1.09 Chuk Gja Krt17-dependent Ltbr 1.08 Tlr Nfkb Htr2b Tnf Raf Irf Relb Traf Ikbkb HPV16 tg/+ ;Krt17 -/- Rela Traf Akt Tlr vs. HPV16 tg Il1b Hsp90ab Ikbke Csf Rel Tollip Il1a Tnfrsf1b Irak Bcl Icam Ikbkg Il Zap Crebbp Ifng HPV16 tg -induced, Bcl Tgfbr Gapdh Krt17-independent Nod Ripk Casp Eif2ak Ripk Stat Nfkbia Tgfbr Tnfsf Tbk Tlr Nlrp Tnfaip Card Cd Atf Kat2b Mapk Tnfrsf10b Cflar Casp Tradd F2r Egr Lta Jun Il1r Nfkb Elk Fasl 1.49 Tlr Atf Csf C Tlr Hprt Actb Map3k Myd Cd Tlr Fadd 1.33 Tlr Fos Not induced by HPV16 tg Tlr Irak Smad Tnfsf Ccl Lpar Slc20a Gusb Not induced by HPV16 tg Not induced by HPV16 tg

12 R.P. Hobbs et al. Keratin 17 regulates Aire to promote skin tumorigenesis Supplementary Table 3. Summary of "T h 17 for Autoimmunity and Inflammation" Array HPV16 tg/+ ;Krt17 -/- vs. HPV16 tg Il12rb Hprt Isg Socs Socs Cd Ccl Cebpb 1.25 Cx3cl Il Il Gata Il Cacybp 1.16 Il Mmp Cd Mmp Cxcl Myd Cxcl Cxcl Syk 1.08 Mmp Traf Tlr Ccl Cd Nfkb Il17rb Stat S1pr HPV16 tg/+ ;Krt17 -/- Hsp90ab Cd3d vs. HPV16 tg Il17rc Nfatc Il23a Tnf Tbx Csf Il Il Il1b Il17c Il6ra Il Il23r Il17d Ccl Cd Il17rd Il Icam Cd Il12b Il7r Rorc Il17a Yy Cd3e Clec7a Il12rb Cd3g Ifng Stat5a Il Icos Actb Il Il Stat Cxcl Tirap Csf Ccl Cd8a Jak Il17f Gusb Foxp Il17re Cd40lg Jak Gapdh Stat Il Il Ccl Tgfb HPV16 tg -induced, HPV16 tg -induced, Krt17-independent Krt17-dependent Not induced by HPV16 tg Not induced by HPV16 tg

13 R.P. Hobbs et al. Keratin 17 regulates Aire to promote skin tumorigenesis Supplementary Table 4. Summary of "Signal Transduction PathwayFinder" array HPV16 tg/+ ;Krt17 -/- vs. HPV16 tg Cdkn2a Rbp Cdh Cxcl Brca Icam Fos Hprt Vegfa Ccl Hsp90ab Pparg Selp Cdk Lta Fas Birc Hhip Il4ra Mmp Wnt Tnf Mmp Fasn Tfrc 1.20 Naip Vcam Birc Myc 1.14 Ikbkb HPV16 tg/+ ;Krt17 -/- Greb Bax vs. HPV16 tg Igfbp Nab Csf Tcf Hsf Fasl Wisp Tert Ccl Gapdh 1.10 Ei Cxcl Gadd45a 1.03 Cdkn1b Il1a Nos Ccnd Il2ra En Lef Ptgs Atf Bmp Birc Il Hoxa Cd Irf Wnt Cebpb Trp Ptch Egr Actb Hspb Sele Foxa Pmepa Hk Nfkbia Fn Tank Cdkn2b Bmp Odc Fgf Cdkn1a Igfbp Nrip Gys Mdm Bcl2l Jun Bcl Gusb Lep Cyp19a HPV16 tg -induced, HPV16 tg -induced, Krt17-independent Krt17-dependent Not induced by HPV16 tg Not induced by HPV16 tg

14 R.P. Hobbs et al. Keratin 17 regulates Aire to promote skin tumorigenesis Supplementary Table 5. Summary of "Innate and Adaptive Immune Response" array HPV16 tg/+ ;Krt17 -/- vs. HPV16 tg Ccr C8a 1.47 Mapk Pglyrp Tlr Cfp Prg Il12rb Pglyrp Tnf Pglyrp Tgfb Hc Irak Actb Ltf 1.05 Nos HPV16 tg/+ ;Krt17 -/- Ptafr 1.03 Traf Nfkb Crp vs. HPV16 tg HPV16 tg - induced, Krt17- dependent Defb Ppbp Adora2a Trem Cxcr Hmox Il1b Ncf Lalba Il Serpina1a Colec Il1f Tlr Ikbkb Clec7a Irf Tnfrsf1a Il1f Gusb Ifngr Ifnb Il1r Ifngr Tlr Cybb Camp Il Nfkbia Cd1d Il1a Cd Il1f Cd Mif Myd Il1f Mapk Stab Casp Tlr Fn Casp Gapdh Il1rapl2 N/A Il1f Serpine Il1rl2 N/A Ccl Nlrc Lyz Il1r Hprt Tlr Il1rn Nfkb Ly Irak Hsp90ab Tlr Chuk Lbp Dmbt N/A Tollip Proc 1.59 N/A Il1rap Sftpd Tlr HPV16 tg -induced, Krt17-independent Not induced by HPV16 tg Not induced by HPV16 tg

15 R.P. Hobbs et al. Keratin 17 regulates Aire to promote skin tumorigenesis Supplementary Table 6. List of qrt-pcr target genes and primers sets MOUSE HUMAN Primer Sequence (5'-->3') Primer Sequence (5'-->3') Actb Forward GGCTGTATTCCCCTCCATCG ACTB Forward CATGTACGTTGCTATCCAGGC Actb Reverse CCAGTTGGTAACAATGCCATGT ACTB Reverse CTCCTTAATGTCACGCACGAT Aire Forward AGGTCAGCTTCAGAGAAAACCA AIRE Forward CCAGGCTCTCAACTGAAGGC Aire Reverse TCATTCCCAGCACTCAGTAGA AIRE Reverse GAATCCCGTTCCCGAGTGG Anxa2 Forward TGCTCTGAACATTGAGACAGC ANXA2 Forward GAGCGGGATGCTTTGAACATT Anxa2 Reverse AATAGGCCCAAAATCACCGTC ANXA2 Reverse TAGGCGAAGGCAATATCCTGT Axin2 Forward ATGAGTAGCGCCGTGTTAGTG AXIN2 Forward CAACACCAGGCGGAACGAA Axin2 Reverse GGGCATAGGTTTGGTGGACT AXIN2 Reverse GCCCAATAAGGAGTGTAAGGACT Ccl19 Forward CCTGGGAACATCGTGAAAGC CCL19 Forward TACATCGTGAGGAACTTCCACT Ccl19 Reverse TAGTGTGGTGAACACAACAGC CCL19 Reverse CTGGATGATGCGTTCTACCCA Ccl2 Forward TTAAAAACCTGGATCGGAACCAA CCL2 Forward CAGCCAGATGCAATCAATGCC Ccl2 Reverse GCATTAGCTTCAGATTTACGGGT CCL2 Reverse TGGAATCCTGAACCCACTTCT Ccl22 Forward AAGCCTGGCGTTGTTTTGATA CCL22 Forward ATCGCCTACAGACTGCACTC Ccl22 Reverse CCTGGGATCGGCACAGATA CCL22 Reverse GACGGTAACGGACGTAATCAC Ccl25 Forward GAGGGCGATGAGAATCTTGAC CCL25 Forward CACCACAACACGCAGACCTT Ccl25 Reverse TCCTCACGCTTGTACTGTTGG CCL25 Reverse TGCTGATGGGATTGCTAAACTT Ccl5 Forward GCTGCTTTGCCTACCTCTCC CCL5 Forward TCCAACCCAGCAGTCGTCT Ccl5 Reverse TCGAGTGACAAACACGACTGC CCL5 Reverse TTGGCGGTTCTTTCGGGTG Ccr4 Forward TGCACCAAGGAAGGTATCAAGG CCR4 Forward AGAAGGCATCAAGGCATTTGG Ccr4 Reverse GTACACGTCCGTCATGGACTT CCR4 Reverse ACACATCAGTCATGGACCTGAG Ccr7 Forward GTACGAGTCGGTGTGCTTCAA CCR7 Forward TGAGGTCACGGACGATTACAT Ccr7 Reverse GGTAGGTATCCGTCATGGTCT CCR7 Reverse GTAGGCCCACGAAACAAATGAT Cd40 Forward TTGTTGACAGCGGTCCATCTA CD40 Forward CACCTGGAACAGAGAGACACA Cd40 Reverse CCATCGTGGAGGTACTGTTTG CD40 Reverse CCTCACTCGTACAGTGCCA Cflar Forward GCTCCAGAATGGGCGAAGTAA CFLAR Forward TGCTCTTTTTGTGCCGGGAT Cflar Reverse ACGGATGTGCGGAGGTAAAAA CFLAR Reverse CGACAGACAGCTTACCTCTTTC Cxcl10 Forward CCAAGTGCTGCCGTCATTTTC CXCL10 Forward GTGGCATTCAAGGAGTACCTC Cxcl10 Reverse GGCTCGCAGGGATGATTTCAA CXCL10 Reverse TGATGGCCTTCGATTCTGGATT Cxcl11 Forward GGCTTCCTTATGTTCAAACAGGG CXCL11 Forward GACGCTGTCTTTGCATAGGC Cxcl11 Reverse GCCGTTACTCGGGTAAATTACA CXCL11 Reverse GGATTTAGGCATCGTTGTCCTTT Cxcl5 Forward TCCAGCTCGCCATTCATGC CXCL5 Forward AGCTGCGTTGCGTTTGTTTAC Cxcl5 Reverse TTGCGGCTATGACTGAGGAAG CXCL5 Reverse GCGAACACTTGCAGATTACTGA Cxcl9 Forward TCCTTTTGGGCATCATCTTCC CXCL9 Forward GTAGTGAGAAAGGGTCGCTGT Cxcl9 Reverse TTTGTAGTGGATCGTGCCTCG CXCL9 Reverse AGGGCTTGGGGCAAATTGTT Cxcr3 Forward TACCTTGAGGTTAGTGAACGTCA CXCR3 Forward CCACCTAGCTGTAGCAGACAC Cxcr3 Reverse CGCTCTCGTTTTCCCCATAATC CXCR3 Reverse AGGGCTCCTGCGTAGAAGTT Deptor Forward GATAGACGGCACCATCTCAAAA DEPTOR Forward CCTACCCAAACTGTTTTGTCGC Deptor Reverse GGCCTCCTTATGTTCAATGAGC DEPTOR Reverse CGGTCTGCTAATTTCTGCATGAG Egr1 Forward TCGGCTCCTTTCCTCACTCA EGR1 Forward GGTCAGTGGCCTAGTGAGC Egr1 Reverse CTCATAGGGTTGTTCGCTCGG EGR1 Reverse GTGCCGCTGAGTAAATGGGA Eif4e Forward AACCGGAAACCACCCCTAC EIF4E Forward TGCGGCTGATCTCCAAGTTTG Eif4e Reverse CTCTGGGTTAGCAACCTCCT EIF4E Reverse CCCACATAGGCTCAATACCATC Eif4ebp1 Forward GGGGACTACAGCACCACTC EIF4EBP1 Forward CTATGACCGGAAATTCCTGATGG Eif4ebp1 Reverse GTTCCGACACTCCATCAGAAAT EIF4EBP1 Reverse CCCGCTTATCTTCTGGGCTA Fn1 Forward TTCAAGTGTGATCCCCATGAAG FN1 Forward AGGAAGCCGAGGTTTTAACTG Fn1 Reverse CAGGTCTACGGCAGTTGTCA FN1 Reverse AGGACGCTCATAAGTGTCACC

16 Foxp3 Forward ACCATTGGTTTACTCGCATGT FOXP3 Forward CTTCAAGTTCCACAACATGCG Foxp3 Reverse TCCACTCGCACAAAGCACTT FOXP3 Reverse CGTGGCGTAGGTGAAAGGG GAPDH Forward AAATGGTGAAGGTCGGTGT GAPDH Forward AAGGTGAAGGTCGGAGTCAAC GAPDH Reverse ACTCCACGACATACTCAGCAC GAPDH Reverse GGGGTCATTGATGGCAACAATA Hnrnpa2b1 Forward GTTGAGCCAAAACGTGCTGTA HNRNPA2B1 Forward TGGAGGTAGCCCCGGTTATG Hnrnpa2b1 Reverse TTTCCAGACTGCCTATCGGTA HNRNPA2B1 Reverse GGACCGTAGTTAGAAGGTTGCT Hnrnpd Forward CCCCACGACACACTGAAGC HNRNPD Forward GCGTGGGTTCTGCTTTATTACC Hnrnpd Reverse CGCCCTGTGATAGGATCTAACT HNRNPD Reverse TTGCTGATATTGTTCCTTCGACA Hnrnpk Forward ACTGATGAGATGGTTGAATTGCG HNRNPK Forward GCAGGAGGAATTATTGGGGTC Hnrnpk Reverse CTGGCATTGTAGTCTGTACGG HNRNPK Reverse TGCACTCTACAACCCTATCGG Hspa1a/1b Forward TGGTGCAGTCCGACATGAAG HSPA1A/1B Forward TTTGAGGGCATCGACTTCTACA Hspa1a/1b Reverse GCTGAGAGTCGTTGAAGTAGGC HSPA1A/1B Reverse CCAGGACCAGGTCGTGAATC Hspa5 Forward ACTTGGGGACCACCTATTCCT HSPA5 Forward CATCACGCCGTCCTATGTCG Hspa5 Reverse ATCGCCAATCAGACGCTCC HSPA5 Reverse CGTCAAAGACCGTGTTCTCG Hspa8 Forward TCTCGGCACCACCTACTCC HSPA8 Forward ATGCCAAACGTCTGATTGGAC Hspa8 Reverse CTACGCCCGATCAGACGTTT HSPA8 Reverse AGCATCATTCACCACCATAAAGG Hspa9 Forward ATGGCTGGAATGGCCTTAGC HSPA9 Forward CTTGTTTCAAGGCGGGATTATGC Hspa9 Reverse ACCCAAATCAATACCAACCACTG HSPA9 Reverse GCAGGAGTTGGTAGTACCCAAA Icam1 Forward GTGATGCTCAGGTATCCATCCA ICAM1 Forward ATGCCCAGACATCTGTGTCC Icam1 Reverse CACAGTTCTCAAAGCACAGCG ICAM1 Reverse GGGGTCTCTATGCCCAACAA Ido1 Forward GCTTTGCTCTACCACATCCAC IDO1 Forward TTGCACGTCTAGTTCTGGGAT Ido1 Reverse CAGGCGCTGTAACCTGTGT IDO1 Reverse GCAAGACCTTACGGACATCTCC Ido2 Forward AAGGCCAACCCCAAAAGGTG IDO2 Forward CCACAGACCGAATGTGAAGAC Ido2 Reverse ACCAGGATAGGCGGGAGTC IDO2 Reverse TGTTGGCAATTTCCATCCAAGG Ifng Forward ATGAACGCTACACACTGCATC IFNG Forward TCGGTAACTGACTTGAATGTCCA Ifng Reverse CCATCCTTTTGCCAGTTCCTC IFNG Reverse TCGCTTCCCTGTTTTAGCTGC Ikbkb Forward GACATCGCATCGGCTCTTAGA IKBKB Forward GTCTTTGCACATCATTCGTGGG Ikbkb Reverse AACGGTCACGGTGTACTTCTG IKBKB Reverse GTGCCGAAGCTCCAGTAGTC Il17a Forward TTTAACTCCCTTGGCGCAAAA IL17A Forward TCCCACGAAATCCAGGATGC Il17a Reverse CTTTCCCTCCGCATTGACAC IL17A Reverse TGTTCAGGTTGACCATCACAGT Il17c Forward CTCCTGCTTCTAGGCTGGTTG IL17C Forward CCACACTGCTACTCGGCTG Il17c Reverse CCACCTGGCACTTCGAGTTAG IL17C Reverse CACACGGTATCTCCAGGGTGA Il20 Forward TCTTGCCTTTGGACTGTTCTCC IL20 Forward TTTTCTGAGATACGGGGCAGT Il20 Reverse GTTTGCAGTAATCACACAGCTTC IL20 Reverse GTCTTAGCAAATGGCGCAGGA Il4 Forward GGTCTCAACCCCCAGCTAGT IL4 Forward CGGCAACTTTGTCCACGGA Il4 Reverse GCCGATGATCTCTCTCAAGTGAT IL4 Reverse TCTGTTACGGTCAACTCGGTG Il6 Forward CCAAGAGGTGAGTGCTTCCC IL6 Forward AATTCGGTACATCCTCGACGG Il6 Reverse CTGTTGTTCAGACTCTCTCCCT IL6 Reverse TTGGAAGGTTCAGGTTGTTTTCT Irf1 Forward ATGCCAATCACTCGAATGCG IRF1 Forward TTCACACAGGCCGATACAAAG Irf1 Reverse TTGTATCGGCCTGTGTGAATG IRF1 Reverse CATGGCACAGCGAAAGTTGG Krt14 Forward AGCGGCAAGAGTGAGATTTCT KRT14 Forward TGAGCCGCATTCTGAACGAG Krt14 Reverse CCTCCAGGTTATTCTCCAGGG KRT14 Reverse GATGACTGCGATCCAGAGGA Krt16 Forward GGTGGCCTCTAACAGTGATCT KRT16 Forward GACCGGCGGAGATGTGAAC Krt16 Reverse TGCATACAGTATCTGCCTTTGG KRT16 Reverse CTGCTCGTACTGGTCACGC Krt17 Forward ACCATCCGCCAGTTTACCTC KRT17 Forward GGTGGGTGGTGAGATCAATGT Krt17 Reverse CTACCCAGGCCACTAGCTGA KRT17 Reverse CGCGGTTCAGTTCCTCTGTC Mapkap1 Forward GAGACGCAGGGCTACATATACG MAPKAP1 Forward GGTGGACACCGATTTCCCC Mapkap1 Reverse GCGGAGTCGTTCTAATCTTTGA MAPKAP1 Reverse CGCTTCACTGCCTTCAGTAAGA Mlst8 Forward ATCAGTGGGCTTTCACGAGG MLST8 Forward TGTGGGCTTCCACGAAGAC Mlst8 Reverse GGTGCGTTCACCTGGAAGAT MLST8 Reverse AGTTAATGGGTGCGTTCACCT Mmp2 Forward ACCTGAACACTTTCTATGGCTG MMP2 Forward GATACCCCTTTGACGGTAAGGA Mmp2 Reverse CTTCCGCATGGTCTCGATG MMP2 Reverse CCTTCTCCCAAGGTCCATAGC

17 Mmp3 Forward ACATGGAGACTTTGTCCCTTTTG MMP3 Forward CTGGACTCCGACACTCTGGA Mmp3 Reverse TTGGCTGAGTGGTAGAGTCCC MMP3 Reverse CAGGAAAGGTTCTGAAGTGACC Mmp9 Forward CTGGACAGCCAGACACTAAAG MMP9 Forward TGTACCGCTATGGTTACACTCG Mmp9 Reverse CTCGCGGCAAGTCTTCAGAG MMP9 Reverse GGCAGGGACAGTTGCTTCT Mpz Forward CGGACAGGGAAATCTATGGTGC MPZ Forward CATCGTGGTTTACACCGACAG Mpz Reverse GCGCCAGGTAAAAGAGATGTCA MPZ Reverse TGGAAGATCGAAATGGCATCTCT Mpzl1 Forward TGGATGGTTGACCACGGTCT MPZL1 Forward ACGCCAAAAGAAATCTTCGTGG Mpzl1 Reverse CCCAGGTGACTCGGTCTTT MPZL1 Reverse TCAACCCGCCAGTCGTACTA Mpzl2 Forward GATGCGCTAACTGTGACGTG MPZL2 Forward TTAATGGGACAGATGCTCGGT Mpzl2 Reverse GGTCTTTGAACCGTCCGCT MPZL2 Reverse AAGACACCCGGTCCTTAAACC Mpzl3 Forward CCTTGGAGATAAGTGCCGATG MPZL3 Forward ATGCCCATGTCCGAGGTTATG Mpzl3 Reverse CGGTATGTCCAGTCTATGGTCA MPZL3 Reverse GGAGGGCGATATGTCCAGTCT Mtor Forward CAGTTCGCCAGTGGACTGAAG MTOR Forward ACTCGCTTCTATGACCAACTGA Mtor Reverse GCTGGTCATAGAAGCGAGTAGAC MTOR Reverse CACCTTCCACTCCTATGAGGC Myd88 Forward TCATGTTCTCCATACCCTTGGT MYD88 Forward GGCTGCTCTCAACATGCGA Myd88 Reverse AAACTGCGAGTGGGGTCAG MYD88 Reverse CTGTGTCCGCACGTTCAAGA Nfkbia Forward TGAAGGACGAGGAGTACGAGC NFKBIA Forward CTCCGAGACTTTCGAGGAAATAC Nfkbia Reverse TTCGTGGATGATTGCCAAGTG NFKBIA Reverse GCCATTGTAGTTGGTAGCCTTCA Prkca Forward GTTTACCCGGCCAACGACT PRKCA Forward AAGGGACCCGACACTGATGA Prkca Reverse GGGCGATGAATTTGTGGTCTT PRKCA Reverse AAGGTGGGGCTTCCGTAAGT Ptgs2 Forward TGCACTATGGTTACAAAAGCTGG PTGS2 Forward CCAGTATAAGTGCGATTGTACCC Ptgs2 Reverse TCAGGAAGCTCCTTATTTCCCTT PTGS2 Reverse TCAAAAATTCCGGTGTTGAGCA Rela Forward AGGCTTCTGGGCCTTATGTG RELA Forward GTGGGGACTACGACCTGAATG Rela Reverse TGCTTCTCTCGCCAGGAATAC RELA Reverse GGGGCACGATTGTCAAAGATG Rictor Forward GCTGCGCTATCTCATCCAAGA RICTOR Forward TCCAAAGACTCGACAGTATGTGC Rictor Reverse CTTTCTGACTAAGCGAAGGGC RICTOR Reverse GGCTAGAAATCGTGCTTCTCTG Rn18s Forward CCTGTGCCTTCCTTGGA RPS18 Forward GCGGCGGAAAATAGCCTTTG Rn18s Reverse CATTCGAACGTCTGCCCTATC RPS18 Reverse GATCACACGTTCCACCTCATC Rps6kb1 Forward GGGGCTATGGAAAGGTTTTTCA RPS6KB1 Forward AGAACTTCTGGCTCGAAAGGT Rps6kb1 Reverse CGTGTCCTTAGCATTCCTCACT RPS6KB1 Reverse CGACAGGTGTCTGACGTGTAA Rptor Forward TGTGAGAAAATCGAAGGCTCC RPTOR Forward AATGCTGCAATCGCCTCTTCT Rptor Reverse TTTGCACCGATGGTTTCCAGA RPTOR Reverse GCCAAAGGTAGGTTCCAGTCTG Runx1 Forward GCAGGCAACGATGAAAACTACT RUNX1 Forward CTGCCCATCGCTTTCAAGGT Runx1 Reverse GCAACTTGTGGCGGATTTGTA RUNX1 Reverse GCCGAGTAGTTTTCATCATTGCC S100a10 Forward TGGAAACCATGATGCTTACGTT S100A10 Forward GGCTACTTAACAAAGGAGGACC S100a10 Reverse GAAGCCCACTTTGCCATCTC S100A10 Reverse GAGGCCCGCAATTAGGGAAA S100a4 Forward TGAGCAACTTGGACAGCAACA S100A4 Forward GATGAGCAACTTGGACAGCAA S100a4 Reverse TTCCGGGGTTCCTTATCTGGG S100A4 Reverse CTGGGCTGCTTATCTGGGAAG S100a5 Forward ATTCAGGGAGAGAGGGTAGCA S100A5 Forward GGAGAGAGGGTAGCAAACTGA S100a5 Reverse CTCTGCAAGACTCAGCTCTGT S100A5 Reverse TCCCCAAGACACAGCTCTTTC S100a7a Forward AGGAGTTGAAAGCTCTGCTCT S100A7 Forward ACGTGATGACAAGATTGACAAGC S100a7a Reverse GCTCTGTGATGTAGTATGGCTG S100A7 Reverse GCGAGGTAATTTGTGCCCTTT S100a8 Forward AAATCACCATGCCCTCTACAAG S100A8 Forward ATGCCGTCTACAGGGATGAC S100a8 Reverse CCCACTTTTATCACCATCGCAA S100A8 Reverse ACACTCGGTCTCTAGCAATTTCT S100a9 Forward ATACTCTAGGAAGGAAGGACACC S100A9 Forward GGTCATAGAACACATCATGGAGG S100a9 Reverse TCCATGATGTCATTTATGAGGGC S100A9 Reverse GGCCTGGCTTATGGTGGTG Sdc1 Forward CTTTGTCACGGCAGACACCTT SDC1 Forward CTGCCGCAAATTGTGGCTAC Sdc1 Reverse GACAGAGGTAAAAGCAGTCTCG SDC1 Reverse TGAGCCGGAGAAGTTGTCAGA Sfn Forward GTGTGTGCGACACCGTACT SFN Forward TGACGACAAGAAGCGCATCAT Sfn Reverse CTCGGCTAGGTAGCGGTAG SFN Reverse GTAGTGGAAGACGGAAAAGTTCA Socs1 Forward CTGCGGCTTCTATTGGGGAC SOCS1 Forward TTTTCGCCCTTAGCGTGAAGA Socs1 Reverse AAAAGGCAGTCGAAGGTCTCG SOCS1 Reverse GAGGCAGTCGAAGCTCTCG

18 Stat1 Forward GCTGCCTATGATGTCTCGTTT STAT1 Forward GCAGGTTCACCAGCTTTATGA Stat1 Reverse TGCTTTTCCGTATGTTGTGCT STAT1 Reverse TGAAGATTACGCTTGCTTTTCCT Stat2 Forward TTTGGCTACCTGGATTGAAGAC STAT2 Forward CCAGCTTTACTCGCACAGC Stat2 Reverse GGCTGAATTTTCGCAAGTTATGC STAT2 Reverse AGCCTTGGAATCATCACTCCC Stat3 Forward CACCTTGGATTGAGAGTCAAGAC STAT3 Forward CAGCAGCTTGACACACGGTA Stat3 Reverse AGGAATCGGCTATATTGCTGGT STAT3 Reverse AAACACCAAAGTGGCATGTGA Stat4 Forward GCACTCAGTAAGATGACGCAG STAT4 Forward TGTTGGCCCAATGGATTGAAA Stat4 Reverse CCAGTAGGGTAAAGCAGTTCTG STAT4 Reverse GGAAACACGACCTAACTGTTCAT Stat5a Forward CAGCCGTGGGATGCTATTGA STAT5A Forward GCAGAGTCCGTGACAGAGG Stat5a Reverse GGGACAGCGGTCATACGTG STAT5A Reverse CCACAGGTAGGGACAGAGTCT Stat5b Forward TGTCCCTGAAACGAATCAAGAG STAT5B Forward GAACACCCGCAATGATTACAGT Stat5b Reverse CTGAACTGTGAGTCAAACAGGAT STAT5B Reverse ACGGTCTGACCTCTTAATTCGT Stat6 Forward TGGAGAGCATCTATCAGAGGGA STAT6 Forward GTTCCGCCACTTGCCAATG Stat6 Reverse GCGGAACTCTTCTATAACAGCTT STAT6 Reverse TGGATCTCCCCTACTCGGTG Tcf4 Forward GATGGGACTCCCTATGACCAC TCF4 Forward AAAAATGGACCAACTTCTTTGGC Tcf4 Reverse GAAAGGGTTCCTGGATTGCCC TCF4 Reverse GGAATTGACAAAAGGTGGAGAGA Thbs1 Forward CCTGCCAGGGAAGCAACAA THBS1 Forward AGACTCCGCATCGCAAAGG Thbs1 Reverse ACAGTCTATGTAGAGTTGAGCCC THBS1 Reverse TCACCACGTTGTTGTCAAGGG Thbs2 Forward GGGGACACTTTGGACCTCAAC THBS2 Forward GACACGCTGGATCTCACCTAC Thbs2 Reverse GCAGCCCACATACAGGCTA THBS2 Reverse GAAGCTGTCTATGAGGTCGCA Tnfrsf11a Forward CAGATGTCTTTTCGTCCACAGA TNFRSF11A Forward TCCTCCACGGACAAATGCAG Tnfrsf11a Reverse AGACTGGGCAGGTAAGCCT TNFRSF11A Reverse CAAACCGCATCGGATTTCTCT Tnfsf10 Forward ATGGTGATTTGCATAGTGCTCC TNFSF10 Forward TGCGTGCTGATCGTGATCTTC Tnfsf10 Reverse GCAAGCAGGGTCTGTTCAAGA TNFSF10 Reverse GCTCGTTGGTAAAGTACACGTA Traf2 Forward AGAGAGTAGTTCGGCCTTTCC TRAF2 Forward TCCCTGGAGTTGCTACAGC Traf2 Reverse GTGCATCCATCATTGGGACAG TRAF2 Reverse AGGCGGAGCACAGGTACTT Trim28 Forward CCCTGTCTACATTCGGCCTG TRIM28 Forward TTTCATGCGTGATAGTGGCAG Trim28 Reverse ACGATGTCTTTGGAGTAGCACT TRIM28 Reverse GCCTCTACACAGGTCTCACAC Wnt1 Forward CGACTGATCCGACAGAACCC WNT1 Forward GGTGGGGTATTGTGAACGTAG Wnt1 Reverse CCATTTGCACTCTCGCACA WNT1 Reverse CGTATCAGACGCCGCTGTTT Wnt10a Forward CATGCTCGAATGAGACTCCAC WNT10A Forward AAGCCTGGAGACTCGCAAC Wnt10a Reverse CCCTACTGTGCGGAACTCAG WNT10A Reverse CGATGGCGTAGGCAAAAGC Wnt10b Forward ATCGCCGTTCACGAGTGTC WNT10B Forward GTGAGCGAGACCCCACTATG Wnt10b Reverse GGAAACCGCGCTTGAGGAT WNT10B Reverse CACTCTGTAACCTTGCACTCATC Wnt11 Forward CAGCCTTCGTGTATGCCCTG WNT11 Forward GGAGTCGGCCTTCGTGTATG Wnt11 Reverse CCGTAGCTGAGGTTGTCCG WNT11 Reverse GCTGAGGTTGTCCGCACAT Wnt2 Forward CCTCCGAAGTAGTCGGGAATC WNT2 Forward GCCTTTGTTTATGCCATCTCCT Wnt2 Reverse GCAGGACTTTAATTCTCCTTGGC WNT2 Reverse CTTGGCGCTTCCCATCTTCTT Wnt2b Forward GACACGTCCTGGTGGTACATA WNT2 Reverse GGGTGGTACAGTTCCAGCG Wnt2b Reverse GTCTGGGTAGCGTTGACACA WNT2B Forward GTTACCCAGACATCATGCGTT Wnt3 Forward TGGCTACCCAATTTGGTGGTC WNT3 Forward GGAGAGGGACCTGGTCTACTA Wnt3 Reverse CTTCACACCTTCTGCTACGCT WNT3 Reverse CTTGTGCCAAAGGAACCCGT Wnt4 Forward TGCGAGGTAAAGACGTGCTG WNT4 Forward CTCCACACTCGACTCCTTGC Wnt4 Reverse CTTGAACTGTGCATTCCGAGG WNT4 Reverse CCGAAGAGATGGCGTACACG Wnt5a Forward GGACCACATGCAGTACATTGG WNT5A Forward ATTCTTGGTGGTCGCTAGGTA Wnt5a Reverse CGTCTCTCGGCTGCCTATTT WNT5A Reverse CGCCTTCTCCGATGTACTGC Wnt7b Forward CTTCACCTATGCCATCACGG WNT7B Forward GAAGCAGGGCTACTACAACCA Wnt7b Reverse TGGTTGTAGTAGCCTTGCTTCT WNT7B Reverse CGGCCTCATTGTTATGCAGGT

19 R.P. Hobbs et al. Keratin 17 regulates Aire to promote skin tumorigenesis Supplementary Table 7. List of primer sets used for ChIP and mutagenesis Primer set Forward Sequence (5'-->3') Reverse sequence (5'-->3') hcxcl11 1 TCTTTAGGAACTCATTTACAGGT CCTTTCACCCACCCTTCAT 2 TGTATAGATATGTGAAGGATGAAGGG GATAAGAATGTGAAAGCACTGTGAA 3 GCTGGTTACCATCTGGGTTT GGCACAGGAATTTCCCTCTT 4 GGAAAGGTGCATGACTRCAAAG CAGCTTTGCTGCTCTTCTTG hcxcl10 1 GAATGGATTGCAACCTTTGTT ATTTCCCTCTGCTCCTCTTT 2 AAAGAGGAGCAGAGGGAAAT CTGATTGGATAAGGGCATTACAG 3 AGTCAACTGTAATGCCCTTATCC TGGAGGTTCCTCTGCTGTA hmmp9 1 GGAAGGCATTTACTCCAGGTTA CAAGGATACAGTGAGCCATGAT 2 GCTGGAGTGCAGTGACATAA CTAAAGCCAAGACCCAAGATT 3 TTACAGGAATGAGCCACCATAC TGTCCCTAAATTGATGTGAGGATTA 4 CCTCACATCAATTTAGGGACAAAG GCTGAATCTTCAGGGCAGTA 5 TCTAGAGGCTGCTACTGTCC CTCCCTGACAGCCTTCTTTG 6 AGGGAGAGGAAGCTGAGT CCTGAGACTTCTTTCTGAAACTAATG 7 GGAGGATATCTGACCTGGGA GTGGGAACTGTATGAAAGGGA 8 CTGACCACTGGAGGCTTTC CAGTCTCCACTGCCAAGTC 9 GGTTTCCTGCGGGTCTG TGTGTGTGTGTGTGTGTGT 10 GTGGTGTAAGCCCTTTCTCA TGAGGGCAGAGGTGTCT hccl19 1 AGGGCAATCCCTCTCTCT CCCTTTCCTTCTGCTTTCCT 2 AGAGCAGGAGCCAAGGA GCCAGAGCTTCACTTTCTCTT 3 TCACAGAATGGGACATGAAGG GAAATGCAAGGTGTGAGTGATG 4 CATCACTCACACCTTGCATTTC AACAGAGGCAGGCCAAC hgapdh 1 TGAGCAGTCCGGTGTCACTA GGACTTTGGGAACGACTGAGATG 2 AACTTTCCCGCCTCTCA CAGGACACTAGGGAGTCAAG 3 TCCTTGACTCCCTAGTGTCCTG TAGTAGCCGGGCCCTACTTT 4 ATCGGGCCAATCTCAGT TCTTGAGGCCTGAGCTA K17 Lys399Ala mutagenesis ACTCAGTACGCGAAAGAACC CAGGTGGGCATCCTCTC