Supplementary Material for. Inflammation induced repression of Foxp3-bound chromatin in regulatory T cells
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1 Supplementary Material for Inflammation inue repression of -oun hromatin in regulatory T ells Aaron Arvey 1,2, Joris van er Veeken 1,2, Roert M. Samstein 1,2, Yongqiang Feng 1,2, John A. Stamatoyannopoulos 4, Alexaner Y. Ruensky 1.2,3 1 Howar Hughes Meial Institute, 2 Immunology Program, 3 Luwig Center, Memorial Sloan-Kettering Caner Center, New York, NY 165; 4 Department of Genome Sienes, University of Washington, 175 NE Paifi Street, Seattle, WA 98195, USA Corresponing author, ruenska@msk.org Nature Immunology: oi:1.138/ni
2 Diphtheria Toxin -DT +DT DTR Mie Express Diphtheria Toxin Reeptor on Treg Cells # + Cells Depletion Reovery Day Day 1 Day 11 DT Treatment an Cell Isolation Total ell ounts ( 1 6 ) Total ell ounts ( 1 6 ) CD4 + + Sp. Sp. LN LN -DT +DT CD4 + CD44 + CD62L - - -DT +DT CD KLRG Ki CXCR3 e Total ell ounts ( 1 6 ) CD4 + CD44 - CD62L + - Sp. LN -DT +DT GITR All Present own in (vs ) up in (vs ) DTR : vs r = r =.57 r = B16 Melanoma: vs Supplementary Figure 1: Charaterization of in vivo ativate Treg ells. (a) Shemati of generation of in vivo ativate Treg an ells. The time ourse of DT aministration to DTR mie an susequent reoun of Treg ells are shown. () DT-treate mie isplay splenomegaly an lymphaenopathy (representative of n > 5 experiments, eah with n > 3 DT treate an n > 3 untreate DTR mie). () DT-treatment results in inrease numers of + Treg an ells on ay 11. () Cell surfae effetor moleules an proliferation markers are inrease in ells (representative of n = 3 experiments, eah with n > 3 mie). (e) Ativate Treg ells isolate from iphtheria toxin treate DTR mie are representative of ativate Treg ells in other inflammatory settings. Intra-tumoral Treg an ells were flow ytometry sorte from B16 melanoma tumors estalishe in GFP mie. Gene expression atasets were ompare for tumor infiltrating Treg vs. ells (x-axis) an vs. ells isolate from DT-treate DTR mie (y- axis). Plots show all alle as present (left), those that are expresse at a lower level in ompare to ells (enter), an those that are expresse at a higher level in ells ompare to ells (right). Pearson orrelation values are shown. Tumor Treg an array analysis was performe using two iologial repliates. Nature Immunology: oi:1.138/ni.2868
3 ChIP RPM ChIP RPM ChIP RPM (Reps 3,4) ChIP RPM (Reps 1,2) ChIP RPM (Rep 2) ChIP RPM (Rep 1) Density vs Reps Reps Fol Change ChIP RPM Supplementary Figure 2: ins similar loi in an ells. (a) Quantitative assessment of ChIP in resting an ativate Treg ells. Peaks were alle in oth resting an ativate ells an RPM was ompute in eah onition on the union of peaks. Data was quantile normalize prior to averaging aross n = 4 an n = 2 repliates. Raw ata were also analyze y negative inomial moeling as esrie in main text an methos. () Same as A, exept x- an y-axes show repliate-to-repliate variation for (left) an (right) ChIP-seq RPM ata. This analysis emonstrates that the variation oserve in A oes not surpass reprouiility variation. () Analysis of the istriution of RPM-fol hanges shows that the -vs- omparison oes not surpass that oserve in reprouing the ata in either onition. Nature Immunology: oi:1.138/ni.2868
4 Os Ratio Enrihment vs Upregulate Downregulate Peak Rank Os Ratio Enrihment vs GFPKO Upregulate Downregulate Peak Rank Cumulative fration of Present P < expression hange vs GFPKO P < expression hange vs Down OR=1.76; P < 1-9 OR=1.23; P <.3 OR=2.5; P < 1-19 OR=.9; P <.15 Up vs vs GFPKO OR=1.3; P <.6 OR=1.1; P <.48 vs Down OR=2.78; P < 1-17 OR=1.23; P <.15 + vs Δ OR=2; P < 1-11 OR=2.3; P < 1-14 Up vs Distal Upstream Distal Downstream First Intron Exon Promoter vs p < 1-7 **** p < 1-5 *** ** Cumulative fration of ** Fol Change Fol Change Fol Change Fol Change Fol Change ** * ** p < 1-3 * p <.5 vs GFPKO Cumulative fration of **** **** Fol Change Fol Change Fol Change Fol Change Fol Change * *** Supplementary Figure 3: ats preominantly as a transriptional repressor. (a) Peak-rank enrihment of ining for ifferentially expresse. Down- an up-regulate gene sets (lue an re, respetively) are shown at various q-value utoffs. The y-axis shows the os ratio of enrihment of the hypergeometri test to estimate the extent of overlap etween ifferentially expresse an target. () Cumulative istriution of target gene expression hanges in vs. GFPKO an vs ells. () Overlap of ifferentially expresse with oun. Experimental onitions are esrie in the main text. Os-ratio (OR) enrihment value an hypergeometri p-values are shown. () -ining at istal upstream an first-intron sites is assoiate with inrease repression of -target. Cumulative istriution (y-axis) of Log2 expression fol hange (x-axis) for vs ells (top row) an vs GFPKO ells (ottom row). Nature Immunology: oi:1.138/ni.2868
5 ifferential DNase RPM vs ifferential DNase RPM vs DHS -oun DHS DHS average hange -oun DHS average hange average DNase RPM an average DNase RPM an Numer of ifferential DNase loi unoun -oun OR=.88 p <.78 Up OR=14.6 p < Down fol hange in DNase-seq RPM vs 2 2 oun DNase-site fol hange in DNase-seq RPM vs Cumulative fration of Present DNase Up DNase Down Present GFPKO DNase Up GFPKO DNase Down GFPKO Present DNase Up ATE DNase Down expression fol hange (legen shows ompare ell types) Supplementary Figure 4: oun enhaners are assoiate with erease DNase-aessiility. (a) assoiation with hanges in DNase-aessiility is uniform aross average lous aessiility. Lines show running average of fol hange (y-axis) relative to total aessiility DNase-seq RPM (x-axis). () DHSs with erease aessiility are enrihe for ining. This plot represents the same ataset shown in Fig. 4 exept for the inlusion of DHSs with q <.5 instea of q <.1 in Fig. 4. () DHSs that are -oun (shown in re) have erease hromatin aessiility in vs. ells (y-axis) that are not represse in vs. ells (x-axis). () near loi with inrease DNase aessiility are frequently upregulate in a -inepenent manner. In ontrast, many Treg-speifi ereases in hromatin aessiility are uniquely assoiate with -epenent ereases in neary gene expression. Nature Immunology: oi:1.138/ni.2868
6 fol hange RPKM vs ifferential on y-axis fol hange RPKM vs Frequeny in speif >4 # peaks in -oun loi Frequeny in all >4 # peaks in -oun loi Cumulative fration of P < 1-6 P < 1-8 an All present target gene expression fol hange versus ells Cumulative fration of P < 1-7 P < 1-6 an All present target gene expression fol hange versus ells fol hange RPKM vs Pe ifferential on y-axis 1.5 fol hange RPKM vs e DNase < I18Rik Pe3 > < Cala < Cyp2r1 121,52, 121,576, 121,632, 121,688, 121,744, 121,8, Position on Chromosome 7 Supplementary Figure 5: (a) Ativation signals ause hanges in in oth (y-axis) an ell populations (x-axis) relative to their resting ounterparts as well as -speifi tri-methylation at a sizale numer of loi. () Histograms emonstrate that a larger fration of speifi loks (left) have multiple ining sites ompare to all loks (right) (p <.2, KS-test). () Derease expression of target with ell speifi inreases in. Gene expression hanges (x-axis) in ells ompare to (left) an (right) ells show that -oun with (re) have signifiantly lower expression than those that are alle as present (lak) or are only oun y without inreases in (lue). P values shown are estimate y KS-tests. () The lansape of ells (y-axis) is largely estalishe in a -inepenent fashion in ells (x-axis). (e) The Pe3 lous ontains a -oun enhaner that is erease in aessiility an enrihe for. Nature Immunology: oi:1.138/ni.2868
7 Cw27 Eif4e3 Lrp11 Lef1 Ifng T12 Mapk11 Dep1 Cana1f Ptprs Denn5a Rp7 Spry4 Klf9 Sox17 Sos2 S4 Tasp1 Arhgap2 Itsn1Cp A3 C33Nfil3 Pou2f3 Gng7 Asl2 Ppfip2 Cyfip1 Ar1 Zfp821 Mmp9 Tgm2 Lmo2 C83 Myo1e F9315N5Rik Exo6 Camk2 Lam1 Nav2 Zfp827 Nhlr2 Cyth3 Pln Rnf216 Eps8l3 Nr4a3 Gar1 Iglon5 Prke C32 Marh1 Cfp1 Ppfip1 Myo3 C248 Sr Erg Ra23 Pik3ap1 Atp6va4 Rnf19 Lif Fam89a Pe4 Rnf43 Pk1 Maf Aa13 Lmtk2 T Dlre1a Fnip2 Folr2 F2r Zt32Grik4 Dusp14 St6galna6 a DNase GFPKO a GFPKO DNase GFPKO a GFPKO fol hange K27me3 RPKM vs GFPKO Pe3 oun y fol hange K27me3 RPKM GFPKO vs 5.4 < Parp8 Skp1a > < Tf7 117,6, 117,649, 117,698, 117,747, 117,796, 117,845, Position on Chromosome 13 52,6, 52,8, 52,1, 52,12, 52,14, Position on Chromosome 11 e Cumulative fration of loi fol hange in K27me3 RPM vs other All marke loi (vs ) All marke loi (vs GFPKO\ ) -speifi (vs ) -speifi (vs GFPKO ) -oun -speifi (vs ) -oun -speifi (vs GFPKO ) All -oun s (vs ) All -oun s (vs GFPKO ) Cumulative fration of loi fol hange in K27me3 RPM vs other All marke loi (vs ) All marke loi (vs a GFPKO ) All marke loi (vs ) -speifi (vs ) -speifi (vs a GFPKO ) -speifi (vs ) -oun -speifi (vs ) -oun -speifi (vs a GFPKO ) -oun -speifi (vs ) All -oun s (vs ) All -oun s (vs a GFPKO ) All -oun s (vs ) fol hange K27me3 RPKM vs fol hange K27me3 RPKM GFPKO vs fol hange K27me3 RPKM vs fol hange K27me3 RPKM GFPKO vs LAMC _at TBC1D _at NFIL _at ADAMTS _at PLP _a_at CD _at PPP1R3F _at CORO2A _at SDC _at DENND5A _at GFPKO SOCS _s_at SYP _at SYS _at TASP _at TMEM _at Supplementary Figure 6: (a) Traks showing epenene of on at the Parp8 (left) an Tf7 (right) gene loi. Represse hromatin is present in ells, ut not in any other population, with the most notale eing ativate GFPKO ells isolate from inflammatory onitions an expressing reporter null allele. () at the Pe3 lous in ells is epenent on. (, ) ell-speifi is epenent. Quantifiation of hanges in vs. other ell type () or vs. other ell type () at -oun loi an genome wie. The inrease in K27me3 is epenent on (as shown in the omparison to a GFPKO ) an only in inflammatory onitions. (e) Treg lineage speifi ereases in marks are not epenent on an an e foun in GFPKO ells (left), ut not in ells (enter) an these are upregulate in Treg an Treg preursor GFPKO ells (right). Nature Immunology: oi:1.138/ni.2868
8 Input RPKM.3 Rep2 Rep1 Rep2.6 a Rep H3k27me3 RPKM GFPKO H3k27me3 RPKM 5 2 x C x C f7 Rep1 T Rep2 Ba h 2 Rep1 t P e3 Rep2 Rep1 Rep2 1 8 Rep1 15 A Rep2 Rep1 Fol Enrihment relative to input Rep2 2 Supplementary Figure 7: (a) Distriution of peaks is highly reprouile etween repliates. () Seletion of peaks. Two representative ChIP-seq experiments (x-axes) are shown relative to input ChIP-seq (y-axis). ChIP peaks with fewer reas (e.g. those with RPKM <.75) are more likely to e ause y high input signal rather than genuine ining of moifie histones. A Poisson test was use to test for statistial enrihment of reas in peaks with p < 1 7 set as a utoff. () ChIP-qPCR valiation of ommon an ifferential peaks. Experiment one in tripliate. Nature Immunology: oi:1.138/ni.2868
b PolyA RNA-seq c RNA-seq read distribution FPKM
a Repliate 2 (FPKM) Poly+ RN-seq 14 1-2 R2 =.998 1-4 -4-2 1 1 14 Repliate 1 (FPKM) Repliate 2 (FPKM) Poly RN-seq 14 1-2 R2 =.996 1-4 1-4 1-2 14 Repliate 1 (FPKM) RN-seq rea istriution Intron 12% 14% Intergeni
% of Nestin-EGFP (+) cells
8 7 6 3 Nestin CD % of Nestin-EGFP (+) ells 8 6 Nestin-EGFP Marge Nestin-EGFP Marge Expression levels of Expression levels of Tumorigeniity y stemness markers ifferentiation markers limiting ilution assay
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HA1 (%) Insulin seretion (SI) e 7. p=....... 7 p=.7 1 7 a-ell granules -ell granules BMI (kg/m ) -ells / (a- + -ells) p=. 1 7 1. p=.1..... 7 Age: -ell ontent: % Age: -ell ontent: % Age: -ell ontent: %
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