© 2012 Nature America, Inc. All rights reserved. ture’, are over that of transcripts a set T committed of phenotype the D.M. ([email protected]) or D.M. ([email protected]) C.B. ([email protected]). Ingelheim Ridgefield, Connecticut, USA Pharmaceuticals, (S.H.). et de Biologie Moléculaire et Cellulaire, Illkirch, France. USA. Massachusetts, Illinois at Urbana, Urbana-Champaign, Illinois, USA. Department of Biomedical Engineering, and Center for BioDynamics, Boston University, Boston, USA. Massachusetts, 1 activation Foxp3 after express transiently that cells of tion effector tamoxifen and by lineage of time for are long periods stable hosts into normal T transferred cells that observation the by refuted largely been have results those active lineage from and by from obtained of results transfer T stemmed Foxp3 have a factor diseases, role in autoimmune scription Forkhead of the loss by the cells effector aggressive into T of idea for the Support cells. effector aggressive into convert they lest stable, to be phenotype suppressive for their it important makes TCRs their T for consideration important an is such chronic as bacteria challenges commensal (TCR) self a express that cells for pathway nonimmunological certain functions regulate and allergens or microbes responses to runaway prevent deviation, autoimmune control stasis, Regulatory T cells (T aspects of T of any single cofactor. A redundant genetic switch thus ‘locked in’ the T factors, and enhancing occupancy by Foxp3 at its genomic targets. Conversely, the T same outcome, acting in synergy with Foxp3 to activate expression of most of the T Enforced expression of Helios or Xbp1 elicited distinct signatures, but Eos, IRF4, Satb1, Lef1 and GATA-1 elicited exactly the network computational inference and experimental testing to assess the contribution of other factors transcription to this. (T The factor transcription Foxp3 participates dominantly in the and specification function of Foxp3 Diane Mathis AdoroStanley Wenxian Fu signature transcriptional of regulatory T cells A multiply redundant genetic switch ‘locks in’ the nature immunology nature Received 13 February; accepted 13 August; published online 9 September 2012; Division Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA. Massachusetts, reg T reg cells) but is neither strictly necessary nor sufficient to determine the T characteristic Foxp3 cell function is underwritten by a canonical ‘T canonical a by underwritten is function cell 3 , , but some also differentiate in peripheral organs in response to reg 1 - , controlled Cre recombinase system 2 cell instability, and for the proposal that T that proposal the for and instability, cell . Most T Most . - reg driven transgenes encoding Cre recombinase - 1 tracing experiments done in pulse experiments tracing cell physiology, and differentiation stability. , , Ayla Ergun 1 5 - & Christophe Benoist 5 tracing experiments that relied on continuously continuously on relied that experiments tracing , Laurie , Glimcher Laurie Department Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, USA. Massachusetts, reg reg cells) have a key role in immunological homeo­ cells differentiate in the thymus as a rescue rescue a as thymus the in differentiate cells

aDV A reg NCE ONLINE PUBLIC ONLINE NCE 1 , cells seems very stable over time over stable very seems cells 2 , 8 , , Ting Lu - reg - reactive T cell antigen receptor receptor antigen cell T reactive reg or underexpressed in T in or underexpressed cells, as the self the as cells, cells into cells hosts alymphoid 5 , , Susan Chan 4 1 9 Immune Immune Disease Institute, Program in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, . . Thus, with the excep 4 2 7 . Phenotypic stability . stability Phenotypic , Present Present addresses: Tempero Cambridge, USA Massachusetts, Pharmaceuticals, (J.A.H.), and Boehringer 3 - , chase mode with a with mode chase 8 A , , Jonathan A Hill TION reg reg - 8 - cells turned turned cells reactivity of of reactivity These These authors contributed equally to this work. should Correspondence be addressed to family tran family 8 cell signa cell

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doi:10.1038/ni.242 been demonstrated for Runx1, NFAT, Eos, phosphorylated STAT3, phosphorylated NFAT, Eos, Runx1, for demonstrated been from a variety of families, and physical or functional interactions have T promote to and Foxp3 with T full the elicit to of transduction T can also be elicited in transforming growth factor IPEX with patients some haps stability less with and numbers lower in albeit Foxp3, Foxp3 with many T T of tor’ signature X syndrome (immune polyendocrinopathy, dysregulation, IPEX enteropathy, disorder immunological the with patients in or mice mutant scurfy in absence its in develop that autoimmunity multiorgan and of T maintenance T of ators proteins with a range of cellular locations and several molecular medi CD4 lished very early the during T in expression (T their to relative 1 reg - , conv 7 Many other transcription factors have been reported to interact interact to reported been have factors transcription other Many linked) , , Sokol Haxhinasto cells derived from CD4 derived cells reg cell) counterparts of the T the of counterparts cell) locus (T locus cell phenotype, a model that would account for several reg 11 reg 6 1 cells, but a more nuanced view has since emerged. Cells Cells emerged. since has view nuanced more a but cells, 4 , Derrick , Rossi Derrick , , and plays an important part in determining the T the determining in part an important , plays and 15 cell action cell reg , 1 cell characteristics, including a transcriptionally active 0 6 reg . Foxp3 was initially considered the ‘master regula ‘master the considered initially was Foxp3 . FOXP3 reg reg cell–like cells), can differentiate in the absence of of absence the in differentiate can cells), cell–like reg reg cell signature cell cells, as shown by the lethal lymphoproliferation as lymphoproliferation shown by cells, the lethal cell signature, including key transcription cell signature was robust after inactivation 1 or its induction by TGF by induction its or 3 reg . Foxp3 is essential for the specification and and specification the for essential is Foxp3 . reg cell differentiation 1 cell signature. Here we used + 3 , T cells of scurfy mice of T scurfy cells Department Department of University Bioengineering, of 7 4 reg , , Marlys S Fassett 1 , , James J Collins 9 . A segment of the T the of segment A . cell function. These include factors factors include These function. cell reg + 11 CD4 cells , 2 0 . 2 Howard Howard Hughes Medical Institute, + 11 regulatory T cells , 1 2 . This signature is estab is signature This . + 1 6 conventional T cell cell T conventional 1 Institut Institut de Génétique , and the genes encode - - β β 1 2 (TGF ticles e l c i rt A are not sufficient sufficient not are 1 1 , . . Conversely, the , , Roi Gazit reg

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© 2012 Nature America, Inc. All rights reserved. cells cells from mutant in mice (with RAR deficiency TGF phenotypes; surface different of and T primary CD4 various following the from samples with platform ray used 129 gene Here, we perturbations). or chemical or genetic (differentiation tions a large number of related data in sets to response a range of perturba across target(s) their and regulator(s) between variation pairwise the analyze approaches, related or regression multiple on based typically genes and a of set putative regulators tory models, then infer and rank potential connections between target number of gene large a from start that methods computational by tackled be can ity complex This genes. target their and proteins regulator of networks Transcriptional regulation is governed by extensive and interconnected T of prediction Bioinformatics RESULTS switch’.‘genetic redundant T the in which perspective gain systems T of network tory regula transcriptional com the a engineer’ ‘reverse to approach used putational first we hypothesis, this test To factors. transcription these of effects cumulative the from result thus would activity tional of of the genes the T ‘condition’,tor might a Foxp3, segment with or in combination alone cofac each that is hypothesis plausible A unknown. is orchestrated T as such factors distinct on transcription dependent themselves are these and cells, T effector of T different addition, In HIF T IRF4, experiments. three of representative are Data downregulation. Down, upregulation; Up, effect). (increasing red yellow, green, to T the of portion the maximize to selected factors ( algorithm. CLR the by predicted as (red), signature Table Foxp3 of pairs 1 Figure s e l c i rt A  factors transcription various or Akt kinase the express to transfected T

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3 4 b . Such algorithms, which . arewhich Such algorithms, - β reg Foxp3 PLAGL1 - induced Foxp3 induced cell signature accounted for. Colors indicate intensity of influence, from blue (background; no effect) effect) no (background; blue from influence, of intensity indicate for. Colors accounted signature cell α c or Foxp3); and cells ) Result of mathematical optimization of the CLR scores in in scores CLR the of optimization mathematical of ) Result β -treated cultures of CD4 of cultures -treated Helios reg Eos cell function. function. cell Endod1 GATA-1 TFII-I + T cells: cells: T Lef1 + cells; cells; - TCF-1 and b ­ ­ ­ ­ ­ ­ ­ ­ . . ( reg b experiments experiments to determine whether the computational predictions had gain complementary of set a undertook We predictions computational of confirmation Loss-of-function (GATA activating repressive, on depending the target, although some to seemed be only of factors were the transcription to predicted be and both stimulatory same range as estimates obtained in transduction experiments but than lower was in the which of signature, the 10.8% covering list, T of the genes 603 of the 330 for account could that factors transcription ten identified we model, T the of fraction greatest the for account combination, in would, that factors transcription of set a identify to tion solver process with programming the Cplex ILOG mathematical an and optimiza formulated regulators predicted from those started control additive transcriptional by a panel of factors, wetranscription T the of lators ( factors scription ( expression different only modestly T some of the predicted regulators themselves had different expression in ered before with T discov not factors transcription additional some included also they such as Eos by (encoded T with associated factors other and Foxp3 included Fig. 1 tor and potential targets (regulators with the highest scores, operates by combining the relative strength of coexpression of a regula algorithm (CLR) T in signature fiers) and selected 603 target genes that compose the canonical T project (conventional transcription factors as well as chromatin modi transcription ( ) Transcription factors (blue) most connected to genes of the T the of genes to connected most (blue) factors ) Transcription Supplementary Table1 Supplementary cell signature. ( signature. cell reg + Many target genes were predicted to be influenced by several tran by Many several to be were genes influenced target predicted T cells from Foxp3-null scurfy mice (scurfy; columns ( columns (scurfy; mice scurfy Foxp3-null from T cells cells versus T versus cells c reg PLAGL1 PLAGL GATA-1 GATA-1 HDAC9 HDAC9 b Foxp Foxp Satb1 Satb Xbp1 Xbp1 Hopx Hopx cells; cells; ; results, Lef1 Lef1 Ah Ah Eo Eo R R 1 1 3 3 1 s s 1 aDV (407 and 196 genes over - Fig. control factors from an annotation of the Gene Ontology reg A Supplementary Table 2 a NCE ONLINE PUBLIC ONLINE NCE 3

) Expression profiles of genes (rows) from matched matched from (rows) genes of profiles ) Expression cell signature. In keeping with our hypothesis of of hypothesis our with keeping In signature. cell conv 1 5 - reg Fig. 1 Fig. 100 100 , a relevance network relevance a , 1 and HDAC9). and 1 a ). We used the context likelihood of relatedness relatedness of likelihood context the Weused ). cells, such cells, as and Lef1 GATA cells ( cells b Ikzf4 Up signature ). We selected as potential regulators 2,021 2,021 regulators potential as Weselected ). ), which made it difficult to infer the regu the infer to difficult it made which ), Ikzf2 20 20 reg 0 0 ) ) and Helios by (encoded cell signature ( signature cell a , , Whole signature , with combinations of transcription transcription of combinations , with Ikzf4 - and underexpressed, respectively, Gata1 A 300 300 TION and ). The top predicted regulators - 0 reconstruction method that that method reconstruction reg ).

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© 2012 Nature America, Inc. All rights reserved. (-KO) (-KO) Eos, Xbp1 or or Helios with GATA-1mutant T (Mutant mice. wild-type TGF- 2 Figure available mouse strains deficient in a subset of those predicted cofactors: valid. was targets Foxp3 of tion ( was whole T the than so more analysis, this of distribution (ref. on Foxp3 dependent strictly transcripts those to mice identify scurfy TGF of cultures induced comparing CD4 by Foxp3 of targets predicted the analyzed we First, factors. those of expression enforced for vectors of availability the and/or factors in those deficient of mice on availability the based We relevance. biological actual chose a factors of subset transcription Ahr 2610019F03Rik Lypd6b Mfsd6 Target gene Table 1 nature immunology nature ( of Data three experiments are representative Cd83 Casp7 Atp8b4 Themis Dst Dennd2d Cyfip1 Lycat Pde3b Nlk Il2ra Il1rl2 Gpr83 Entpd1 Enc1 Nrp1 Niban Mctp2 Slamf1 Rcn1 kept inthenetwork. CLR scoresofregulatorswiththemostconnections.BoldingindicatespredictedinteractionsbetweenandgenesT Stx11 Socs2 We then analyzed the transcriptomes of T a β β

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10 10 Validity of ( Foxp3 the targets. predicted Top regulators and their strongest connections based on CLR inference 1 0 1 0 0 Fig. 2 Fig. P Ranked probelist Foxp3 targets(down) Foxp3 targets(up) 4 3 2 1 = 6.9 × 10 Expression in a T reg ), which indicated that the computational predic computational the that indicated which ), Down Down Down Down Down Down Down Down Down Down Down Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up cells

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Eos-KO 2 - deficient deficient 10 b ; average ; of average duplicates). 3 T re g - ) β 10 ­ - 4 –0.54 TCF-I 1.90 9.06 5.85 2.48 2.70 1.54 1.43 2.40 7.17 2.78 0.79 0.23 3.29 3.36 1.68 2.82 6.51 2.13 2.74 2.86 1.16 1.34 0.72 3.16 2.30 11 expression profiles from purified splenic CD4 splenic purified from profiles ­expression of 1 Fig. in lymphoid organs were ( populations again normal of knockout conditional with not shown) but other known GATA and other T cells seemed normal ( expecta tions to contrary perhaps shown), not data D.R., and R.G. and and had normal T were viable and fertile with no noted abnormal autoimmune phenotype mice with fdf complete knockout of , ranked by genome-wide change , for change by ranked all in genome-wide probes mice scurfy versus 10 Helios 2 Up signature GATA-1 mutant 3 and S.A. and L.G., data not shown); and mice with knockout knockout with mice and shown); not data L.G., and S.A. and ; ; mice with deletion of the 10 reg Regulator 2 i wih T which in , cells from their wild-type littermates (WT T cells littermates from their wild-type Endod1 3.59 4.03 2.39 6.30 3.40 1.37 6.45 2.57 6.65 1.28 3.02 1.31 2.80 3.78 1.52 5.05 4.23 1.36 0.99 1.19 3.78 4.99 0.44 4.90 3.14 0.82 10 3 reg 10 4 cell numbers and phenotypes ( reg –0.09 Helios 3.06 2.36 2.79 3.49 1.59 0.46 2.83 4.97 0.99 2.68 0.52 1.42 7.05 3.97 3.34 6.18 4.39 6.02 5.58 4.45 6.24 2.84 4.29 0.74 2.49 10 el se normal seem cells Down signature 10 Xbp1-KO 2 reg Xbp1 Gata1 cellsignaturewiththehighestscores Supplementary Supplementary Fig. 1 10 –0.01 - 2.58 4.14 3.59 5.61 4.27 3.70 1.57 5.20 2.95 6.46 2.98 3.78 2.18 4.10 2.53 1.84 2.17 0.63 1.49 1.70 5.01 2.29 2.59 1.15 3.17 Ikzf4 3 1 1 target genes were affected; mice Eos (ref. (ref. reg promoter 10 cell signature (bottom) in (bottom) cell signature (which encodes Eos), which 4 3 8 3 ), in which the T the which in ), 9 W gnrtd gene generated We . GATA-1 –0.09 –0.61 –0.62 10 1.32 0.95 1.87 3.17 5.47 0.87 4.72 3.80 0.71 2.27 0.96 2.17 0.77 1.81 0.39 0.38 1.75 1.15 8.22 1.07 0.63 6.31 0.18 + Supplementary Fig. 1 reg 3 CD25 reg 7 cells deficient in cells deficient , , in which T ticles e l c i rt A ). ). 10 Helios-KO Supplementary Supplementary

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© 2012 Nature America, Inc. All rights reserved. range as that in normal normal in that general as same the range in factors transcription the other and from Foxp3 of expres sion in expression resulted ectopic manipulations These the transcripts). distinguish endogenous to us allowed (ref. Foxp3 this mouse of that to similar signature a transcriptional has (which Foxp3 human encoding cDNA containing with retrovirus together or alone factors, transcription candidate various the CD4 activated ducing gain did then We predictions computational of confirmation Gain-of-function them. of any of one elimination complete T the analysis, computational the for used types cell diverse the in naturally varied they when ture T the affected have may factors transcription these of each transcription factor ( ( whole a as in any bias of mutant T the wild their and mice these experiments. independent two of representative are Data index. signature ( (ratio). vector empty with transduced cells in expression to relative are ratios GATA-1 plus axis); Foxp3 by (vertical elicited changes with compared axes) (horizontal cofactors various plus Foxp3 by elicited signature ( group). (bottom Foxp3 encoding vector with together or group) (top alone factor transcription vector. ( empty with transduced ( axis). (EV; horizontal vector empty with transduced cells of those with compared axis), vertical (TF; Pbx1 factor transcription control the plus Foxp3 ( right). (bottom alone cofactors the or right) (top cofactors the and Foxp3 left), (top alone Foxp3 expressing cells indicate areas Outlined culture. 3 d of after sorted and reporter) (GFP) protein fluorescent green T mouse 3 Figure s e l c i rt A  We sorted cells expressing each factor,transcription alone or together c

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Gata1 Irf4 greatest amount greatest of Foxp3 alone was unable to match of the induction did have a more substantial effect on transcription. However, even the of signal around the TSS the around signal of ChIP of ( as expected TSSs, of known vicinity the in mainly localized Foxp3 that showed locations TSS to relative nals genome the of summing Overall, respectively. mogeneity, or II control for start sites transcriptional or (TSSs) for nonho sequencing polymerase RNA to of immunoprecipitation whole antibody with Immunoprecipitation (ChIP sequencing deep by anti with proteins noprecipitated GATA chose we so demanding, for in cells duced the needed numbers ChIP GATA with together or alone CD4 primary the genome (ChIP sequencing high by followed immunoprecipitation chromatin used we scenarios, those between To DNA. distinguish to bound already genes; or the cofactors might enhance the of activity Foxp3 molecules tors Foxp3 might recruit near to T locations genomic cofac the binding, cooperative through interpretations: two lowing have could fol the localization, on Foxp3 cellular or effects its global for by quantitative not accounted and was quintet of the factors with ( Pbx1 factor transcription control between interactions GATA and Foxp3 identified cells transfected from proteins of tested the other three factors. Indeed, reciprocal coimmunoprecipitationinteractions have already been demonstrated for IRF4 and with molecularly the factors of Eosthe quintet in nuclear complexes. Such ( factor quintet a with together or alone transduced conditions, as experimental it was almost exclusively nuclear whether the by unaltered was Foxp3 of distribution nuclear/cytoplasmic The activity. to tive ‘boost’ Foxp3 its quantita but transcriptional enhanced instead a provide merely not did quintet the of factors the Thus, GATA of effect cooperative T of the genes Lef1 The synergizing activity of the cofactors, which was most notable notable most was which cofactors, the of activity synergizing The interacted Foxp3 that suggested also above reported effects The Satb1

5 Xbp1 EV lo lo in in hi hi Foxp3 - t t

Seq with irrelevant control antibody have shown a paucity paucity a shown have antibody control irrelevant with Seq - wide localization wide of localization Foxp3. We prepared chromatin from GATA-1 + reg b T + + + – – – Thy-1.1 (Foxp3 vector) GFP (GATA-1vector) cell signature obtained by Foxp3 with GATA with by Foxp3 obtained signature cell conv 10 10 10 - - 1, Satb1 and Lef1 but not between Foxp3 and the the and Foxp3 between not but Lef1 and Satb1 1, 2 3 4 Seq) to assess how factors of the quintet affected affected quintet the of factors how assess to Seq) Thy-1. cells transduced to express Flag express to transduced cells d 10 2 1 Foxp3 4 0 - lo in hi 10 - , which substantiates the substantiates which , Seq statistics, statistics, Seq 1 was apparent at all amounts of Foxp3. Foxp3. of amounts all at apparent was 1 t 3 Foxp3 - - 10 cell extracts provided a cell genome extracts - 1 (the preparation of doubly trans doubly of preparation (the 1 1 as a representative factor), immu factor), representative a as 1 lo in hi 4 - Flag and assessed the bound DNA bound the assessed and Flag t Supplementary Fig. 8 Fig. Supplementary Foxp3 +GATA- Thy-1. Thy-1. Thy-1. DAPI Fig. 5 Fig. Supplementary Table 5 Table Supplementary Foxp - 1 1 1 Seq was technically very very was Seq technically lo in hi t 3 a ); other experiments experiments ); other 1 ticles e l c i rt A Fig. 4 Fig. reg

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© 2012 Nature America, Inc. All rights reserved. of of However, the retroviral vectors used contained only the coding regions and Satb1, which are repressed in T Lef1 for paradoxical particularly was This outcome? transcriptional ture, DNA and activity, functional specificity sequence elicit the same how could five distinct transcription factors, of widely different struc ( above presented results The modeling computational loops: feedback by ‘lock’ Signature sites. target its to bound of Foxp3 independently activity the Foxp3 and bilize enhance of sta recruitment to functionally but the seemed locations genomic Foxp3 to different spread not did quintet the of factors the Thus, genome quantified we when or binding site in the first intron Foxp3 of the the representative gene for shown as cells, transduced singly the in as locations same the at Foxp3 by occupancy enhanced quantitatively was there Instead, Foxp3. of binding significant of sites additional observe not binding. Foxp3 highest the with over not were Foxp3 by repressed scripts ChIP for noted often is as expression, in change transcriptional significant a to correspond not did binding This was not absolutely true, however, and manyset; sites of data strong Foxp3 entire the for 4.7% with compared transduction, >1.6 of expression in change a had ‘reads’,12.2% >75 the of in height peak Foxp3 binding Foxp3 a by with genes 57 activated the (of data genes microarray those Foxp3 of for peaks enriched highest was the binding with genes of group the expected, be of As Foxp3 might binding. peaks the range of significant showing genes for cofactors, and Foxp3 by expressing affected retrovirus of was transduction expression whose genes of distribution the puted com we data, these confirm Tofurther communication). personal from data with similar comparison by sites these of many on signals binding firmed ChIP of Foxp3 5,000 than more of tion robust detec here. The statistically data allowed observed the signals experiments. multiple of representative are Data analysis; ( the along Foxp3 height). peak that with genes all among vector, GATA-1 empty versus plus Foxp3 express to cells of transduction after (blue) <0.5-fold or (red) >2-fold of expression in change a with genes of proportion (the Foxp3 by regulation and a gene) of kb 10 within ‘pileup’ tag sequence ( genes. closest the of TSSs ( CD4 in distribution 5 Figure s e l c i rt A  endogenous of regulation the in involved be to shown been have a d

a ) Cumulative distribution of Foxp3 (in 25–base pair bins) in a 20-kilobase (kb) window relative to the the to relative window (kb) a 20-kilobase in bins) pair 25–base (in Foxp3 of distribution ) Cumulative ) Comparison of the read number for significant Foxp3-binding peaks (model-based analysis of ChIP-Seq ChIP-Seq of analysis (model-based peaks Foxp3-binding significant for number read the of ) Comparison In cells doubly transduced to express Foxp3 and GATA and Foxp3 express to transduced doubly cells In Satb1 Reads 600 0 –10

- or P Genome-wide analysis of Foxp3. Mapping of Foxp3 by ChIP-Seq to assess its genome-wide genome-wide its assess to ChIP-Seq by Foxp3 of Mapping Foxp3. of analysis Genome-wide Seq analysis; analysis; Seq < 10 Lef1 Foxp3 −7 0 Icos and lacked the 3 the lacked and ); representative Foxp3-bound genes confirmed by an independent data source are noted. noted. are source data independent an by confirmed genes Foxp3-bound representative ); + Distance toTSS(kb) T ex vivo ex genomic locus. IP, immunoprecipitation; PolII, RNA polymerase II; −, no precipitation. precipitation. no −, II; polymerase RNA PolII, IP, locus. immunoprecipitation; genomic conv +10 P cells transduced to express Flag-tagged Foxp3 with or without GATA-1. without or with Foxp3 Flag-tagged express to transduced cells < 10 < b –10 T ) Relationship between Foxp3 binding (peak height is the maximum maximum the is height (peak binding Foxp3 between ) Relationship reg Foxp3 +GATA-1 Fig. 3 Fig. −7 cells (R. Samstein and A. Rudensky, Rudensky, A. and Samstein (R. cells - ; ; binding sites (model sites binding - ′ wide binding in parallel ( parallel in binding wide Supplementary Table Supplementary and 5 and reg 0 d ) raised the following question: question: following the raised ) cells ′ untranslated regions, which which regions, untranslated - Seq data. In addition, tran In addition, data. Seq 2 - +10 6 represented among those those among represented ( Supplementary Fig. 7 Supplementary b Genes (%) 10 15 0 5 - based analysis analysis based Icos

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Satb1 expression Satb1 cell cell state and were ‘locked in‘ when Foxp3 was T overexpressed together the to transitioned genes signature all but alone, expressed was the T and ( perturbations and program the outcome of the experimental bi the reproduced successfully model minimal this fixed, was set parameter reasonable a After model. the in engaged kinetics the biochemical described up Hill with functions paired equations Differential occur. to likely were which cofactors, network calculations and omitted cross F*. to F from conversion the influenced tantly impor but most genes of signature set a larger controlled then logic, ‘and’ with operate or ‘or’could themselves which subnetworks, these U as (such F* regulate to able others (U some of mere effectors factors, subnetworks smaller controlled themselves genes signature those by encoded molecules ( F regulator main the (U upregulated either were genes whose F), T the of factors stabilize regulatory downstream or of set a and potentiate that assays, coimmunoprecipitation by post induction, modification (such as acetylation) or quantitative complex formation, as mean suggested can F* to F from transition (the F* conformation active its with (F), Foxp3 regulator ( oped a mathematical model to simulate the dynamics of such a system We function. actually devel could regulators repressed incorporated self a such whether determine to simulation by double also but feedback positive by only not achieved be in’ could ‘locking of that T is ‘pushed’ beyond cofactors some ability to autoassemble and ‘lock in’ once the expression of Foxp3 and the has it that such negative, and positive both loops, feedback tory along with the factors regulatory it includes, is organized with regula ( factors transcription of expression endogenous on effect the by suggested was interpretation plausible following The period. culture the during Satb1 and Lef1 exogenous Fig. Fig. To make the model computationally manageable, we bypassed sub computational used we proposal, that of plausibility the To assess χ

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© 2012 Nature America, Inc. All rights reserved. Our work here arrived at a conceptual framework different from from different framework conceptual a at arrived here work Our DISCUSSION T of aspects several with and outcomes experimental most with compatible tion at above. Thus, arrived the simulation a of model T T unstable the with consistent scenario ( stimulus differentiating the of withdrawal T T the to partial reverted only which features, showed cell cells the however, Foxp3, inactive With This activation was then stable after removal of the inductive stimulus. external inducing conditions were modeled to activate both F and U3. in activation of the whole network ( Fig. U as (such cofactors its of one and Foxp3 of expression ­transient T of differentiation the that postulation the by model the in resolved be could discrepancy The T contain do protein Foxp3 T some with inactivated cells with mice of thymuses as results, experimental the from somewhat deviated finding last That T but the results, tion of any of the cofactors ( D example, T in repressed those including cofactors, the of any with ( elimination. ( overexpression. factor of the window time indicates shading green (AU); units arbitrary in presented (key), factors transcription of expression indicate lines experiments: overexpression and D of T the factors regulatory downstream of a set and activation) post-transcriptional or transcriptional represent can transition this (where F* conformation 6 Figure nature immunology nature inference network computational use to was intent The origin. its reg 1 a and D and c cell signature and a transcriptionally active active transcriptionally a and signature cell

6 reg Gene expression d cell signature whose genes are upregulated (U upregulated are genes whose signature cell , top). In the simulation, transient activation of F and U (AU) F*

F 120 2 Mathematical modeling of a ‘self-locking’ network. ( network. a ‘self-locking’ of modeling Mathematical 40 80 ) positively activate the transition from F to F*, directly or through the subnetworks they control. ( control. they subnetworks the through or directly F*, F to from transition the activate ) positively 0 1 reg ). The model showed no effect of the single of single the no showed ). effect The model d Mathematical model 2 0 ) Activation of the T the of ) Activation cell physiology. cell Wild typ reg reg 4 0 cell signature ‘shut off ’ with extinction of Foxp3. of Foxp3. extinction ’ with ‘shut off signature cell U cell features, including partial activation of the the of activation partial including features, cell 1 U e

0 U aDV 2 2 0 Fig. Fig. 6 D D D U U U U reg 1 2 3 4 3 1 2 A In silico U 1 -KO cells triggers, directly or indirectly, indirectly, or directly triggers, cells 3 NCE ONLINE PUBLIC ONLINE NCE reg 4 0 c cell program; blue shading (bottom) indicates the time window of the inducing conditions. inducing the of window time the indicates (bottom) shading blue program; cell ), ), consistent with the experimental Fig. 6 U c knockout Tim ) Simulated knockout experiments (presented as in in as (presented experiments knockout ) Simulated 4 0 0 conv e d reg D , bottom left), but only if the cell state some time after after time some state cell cell–like cells mentioned mentioned cells cell–like 1 D Fig. 6 Fig. 1 -KO Subnetwork architectures 20 S1 S2 D S3 S4 S 1 1 1 1 2 1 d reg –U 40 , bottom right), a right), bottom , Foxp3 A S2 S2 S3 S3 S4 T T T T S4 12 D S 5 S 2 S 5 S 2 TION cell differentia cell ype ype ype ype 5 S 3 4 3 ) or downregulated (D downregulated ) or 2 0 Foxp3-KO AND in in silico 1 4 3 2 S1 a 27 24 S4 S4 reg 37 34 S 4 44 locus OR ) Mathematical model consisting of the main regulator Foxp3 (F), with its active active its with (F), Foxp3 regulator main the of consisting model ) Mathematical 7 6

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autoimmunity phen by T expressed TCRs bi the to self the over time, must persist memory Much as neural and switch a of operation the inherent to are indirect, or direct either suppression, with combined cells stem embryonic in pluripotency of potentiation memory tors have been demonstrated in diverse phenomena such as long fac transcription of action reciprocal the on based switches genetic bacteriophage the of state lysogenic stable the to explain shown First signal. initiating of the termination changes inheritable phen and the in stable describes switch genetic A switch. T the of control Foxp3. of exception the with factors, the of any of one removal different to Accordingly, triggers. the T response in state same the reaches and autoassembles of degree high and such positive that negative feedback, the signature T the which in perspective different very a at arrived we Instead, synergy. of degree some with probably by each of affected gene modules the cofactors, discrete would define by loss confirmation experimental with together act Foxp3 to determine that the canonical T factors transcription of panel the predict to Gene expression (AU) 120 120 40 80 40 80 0 0 1 Although it does so with much more complex determinism, the the determinism, complex more much with so does it Although F* F –D T reg Triggering conditions o 3 0 2 0 Foxp3 + type to be stable over the course of an infection and to prevent of an infection course over the to stable be type cellprogramtrigger ). Subsets of molecules encoded by the signature genes (U genes signature the by encoded molecules of Subsets ). None 20 4 0 o b U typic state of a genetic system that are conserved after after conserved are that system genetic a of state typic ); pink shading corresponds to the time frame after factor factor after frame time the to corresponds shading pink ); 1 9 . . For T Insilico 40 reg 0 D U U U U D D 3 4 3 2 1 1 2 cell signature acts much like a classic genetic genetic classic a like much acts signature cell 2 0 2 0 Foxp3 + reg reg transductionandoverexpression 4 cells makes it important for their suppressive suppressive for it makes their important cells 2 U cells, modifications of cells, modifications the methylation status , cell transformation cell , 1 4 0 4 0 U reg b 4 ) ) T cell signature to proved the impervious

In silico In Gene expression ime 0 0 (AU) 0 0 120 180 60 Foxp3 + 0 - reg or gain reg Wild-type D simulation of transduction transduction of simulation cell signature. We thatexpected λ 2 0 20 20 1 cell signature involves a very very a involves signature cell driven by the cI repressor cI the by driven D 4 1 4 - . Positive feedback loops loops feedback Positive . 40 40 of 4 0 Foxp - stable states achieved. achieved. states stable 4 - 3 function experiments experiments function 2 0 2 0 or the maintenance maintenance the or Foxp3 + 3 Tim Foxp3 0 ticles e l c i rt A e 4 0 4 0 Deficien 2 0 D 3 - reactivity of reactivity 0 0 t Foxp3 4 0 1 –U - term term 3 D D D U U U U F , * 3 2 1 4 3 2 1 0 4 1  ­ , © 2012 Nature America, Inc. All rights reserved. S. S. Smale (University of Los Angeles)California, for mouse cDNA encoding Helios; We thank R. Samstein and A. Rudensky for the unpublished ChIP Note: Supplementary information is available in the G codes. Accession the pape the of in version available are references associated any and Methods M in’ to ‘lock able T cofactor the produced by molecules gut microbes may each induce one or another tiation to the different thymic and extra physiological pathways to arrive different at the several same state.allows This also may but be relevantmice, knockout with obtained data redundancy not only ensures additional stability, as exemplified bySuch complete. the is list the that think to reason no is there and it, had here tested factors transcription seven the of five but potential), that have not did Xbp1 and (Helios universal not was switch the flip to ability This quintet. the of factors the of actions the by exemplified switch. the activate and Foxp3 with synergy in act one settings induced in of some experimental the to cofactors needed such as transduction, retroviral supplementation with IL for activation cell of and culture of conditions precise The Foxp3. of T of those to similar amounts in expression Foxp3 after even Foxp3 for transduction expression after groups different by obtained results divergent somewhat the for accounts probably model the addition, cell under the influence of IL of influence the under cell Foxp3 with two a consistent is simulation computational the by modeled nario NF factors the activating by transcription example, (for duality this achieve might fate cell receptor 2 interleukin and TCR (IL along Signaling cells. T effector of induction transient the as such expression gene ‘noisy’ from case this in activation, erroneous from long the of risk the diminish ‘two to advantages distinct are There in’ T ‘lock to the sufficient were cofactors any of the T the of establishment the for necessary were inputs two Second, loops. and pathways distinct eral a synergistic manner, and the factors of the quintet had to activate sev T fluctuating with even flora. microbial persist should that cells are which bacteria, T for or periphery, the in form processed same the in encountered be not may which antigens, T self of induction thymic the for important T the of to establishment led the that milieu cytokine and the TCR ligand the bacteriophage by it: lysogeny set that conditions the a that outlives state ensure also of the s e l c i rt A  A

reg reg ck S ethods Finally, there was multiple redundancy in the T the in redundancy multiple was there Finally, Unlike the minimalist simplicity Unlike of simplicity the the minimalist bacteriophage E - cell switch was complex. in very First, factors participated several cells, other studies have reported much more functional activity activity functional more much reported have studies other cells, R–TT ptwy ta pooe omtet o h T the to commitment promote that pathways 2R)–STAT5 no 7 - 5 − Foxp3 step process of T of process step 4 9 CD25 wledgme . Lymphopenic conditions or chronic exposure to antigen or antigen to exposure chronic or conditions Lymphopenic . 6 reg , , G cells need not be maintained. This remanence could be be could remanence This maintained. be not need cells 11 locus also contribute to contribute stability this also locus S , hi 15 E intermediate cell that secondarily converts to a Foxp3 7 , λ 1 8 6 is self is 5 . Although we observed only very limited effects, effects, limited very only observed we Although . GEO: microarray and ChIP and microarray GEO: n 2 r ts , , . G S - reg perpetuating once established; for T for established; once perpetuating E 1 cell differentiation, which goes through a through goes which differentiation, cell - 3 κ 3 ad op, epciey. h sce The respectively). Foxp3, and B 0 reg - 6 reg - term consequences that would result result would that consequences term and and 2 or other trophic cytokines trophic other or 2 cell transcriptional network. transcriptional cell - reg cell state. Neither Foxp3 alone nor nor alone Foxp3 Neither state. cell thymic thymic contexts of T Foxp3 cells induced by gut commensal commensal gut by induced cells G S - E key’ control systems, which which systems, control key’ after the activation of CD4 activation the after 4 online version of the pape 0 reg 2 7 cell differentiation by by differentiation cell 8 . - Seq data, data, Seq 4 5 . Genetic switches switches . Genetic reg reg reg cell switch, as as switch, cell - cell signature. signature. cell seq seq data; cell differen λ - 2, 2, may have switch, the G S reg 46 ex vivo ex E online online

r , . cells, cells, 7 4 7 4 . In . 6 reg 0 + + ­ ­ ­ ,

25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 2. 1. reprints/index.html. R Published online at The authors declare no competing interests.financial M.S.F.,R.G., J.J.C., D.M. and C.B. analyzed data and wrote manuscript. L.G., S.C., P.K. and D.R. provided mice and advice; and W.F., A.E., T.L., J.A.H., W.F., J.A.H., S.H., M.S.F. and did R.G. experiments; A.E. and T.L. did computation; W.F., A.E., T.L., M.S.F., J.A.H., S.A., J.J.C., D.M. and C.B. experiments; designed (7 Cancer Research Foundation (S.H.), the American Diabetes Association 3R24AI072073 C.B., D.M. and J.C.; training grant T32 DK7260 for support of M.S.F.; and US National Institutes of Health (AI051530 to C.B. and D.M.; AI072073 to generated by the Immunological Genome Project consortium. Supported by the J. LaVecchio and G. forBuruzala This sorting. work frombenefited public data for genomic data analysis; H. Chen and Q. Cai for support; experimental and cDNA; P. for Rahl advice on ChIP and the Center M. Calderwood for Cancer Systems forBiology expression 6. 5. 4. 3. C AUTH eprints and permissions information is available online at http://www.nature.com/ at online available is information permissions and eprints O

- 07

MPETI hu L. Zhou, Y. Zheng, a, F. Pan, Y. Wu, M. Ono, N. Sugimoto, K. Otsubo, W. Lin, M.A. Gavin, J.D. Fontenot, by development cell T regulatory of Control S. Sakaguchi, & T. Nomura, S., Hori, men. and mice of FOXP3: S.F. Ziegler, L.W.Collison, D.A., Vignali, Workman,& work. cells T regulatory How C.J. Foxp3 C. Benoist, & D. Mathis, J.A., Hill, M., Feuerer, J.A. Hill, N. Komatsu, Y.P.Rubtsov, X. Zhou, M. Murai, Feuerer, M. Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M. Regulatory T cells and immune sj, M. Tsuji, J.H. Duarte, foxp3 adaptive and Natural J.J. Lafaille, & M.A. Lafaille, de Curotto of Mechanisms Cells: T. Regulatory, & A.Y. Rudensky, L.F., Lu, S.Z., Josefowicz, antagonizing ROR antagonizing T control to IRF4 cells. T regulatorycellsthataffectT metabolic parameters. tolerance. NFAT. Runx1. Immunol. CD25 (2011). polyendocrinopathy, enteropathy, X-linked syndrome. (CD4 Immunol. Nat. Nature foxp3. factor transcription Foxp3. factor transcription the (2006). Immunol. subphenotypes. (2009). specification, differentiation, signature. transcriptional cell T regulatory the of USA Sci. Acad. T-cellplasticity.retaining population minor uncommitted an and lineage (2010). 1667–1671 cells T memory pathogenic of colitis. with mice in function suppressive and Immunol. Nat. Foxp3 factor transcription the patches. Peyer’s gut in cells T conditions. lymphopenic in cells helper labor? of division a or same the of more cells: T function. and differentiation - BETA O + R R C + CD25 Cell

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Instability of the transcription factor Foxp3 leads to the generation the to leads Foxp3 factor transcription the of Instability

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ucil M.A. Burchill, Lio, C.W. & Hsieh, C.S. A two-step process for thymic regulatory T cell development. hepatic of Regulation L.H. Glimcher, & D.E. Cohen, E.F., Scapa, A.H., Lee, Huehn, J., Polansky, J.K. & Hamann, A. Epigenetic control of FOXP3 expression: FOXP3 of control state. Epigenetic A. Hamann, & cell J.K. Polansky, J., Huehn, stem embryonic the of Control R.A. Young, NF- involving switch epigenetic An K. Struhl, & H.A. Hirsch, D., Iliopoulos, Pittenger,Kandel,genetic&A C. E.switch long-term for memory. A.D. Johnson, M. Giraud, Q. Cai, C. Yu, development of the regulatory T cell repertoire. cell T regulatory the of development Immunity lineage? T-cell regulatory stable (2009). a to key the (2011). transformation. cell to 139 inflammation links IL6 and MicroRNA, Let-7 Lin28, 321 switch. (2012). cells. epithelial thymic in expression function. XBP1. factor transcription the by lipogenesis (2002). 1387–1395 lineage eosinophil the of loss selective to leads promoter , 91–96(1998)., , 693–706 (2009). 693–706 , et al. et Nature t al. et

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© 2012 Nature America, Inc. All rights reserved. 1 d before transfection with those plasmids, together with the packaging packaging the with together plasmids, those with transfection before d 1 Plat Smale. S. by provided was Helios MSCV encoding cDNA vector expression GATA retroviral the Thy factors, transcription transduction. Retroviral set. tool and AMPL language the modeling for IBM) tool; (ILOG package optimization CPLEX9.0 the with mixed a is This genes. most the T the influenced together factors that transcription the identify to was objective whose problem mization computations. CLR of T network CLR the false a at filtered were rate ­discovery of 0.005 (computed scores with to the correction) Bonferroni CLR corrected of background distribution The scores. background MI the above significantly were scores combined whose those are interactions probable most transcription the correction, the background After this with interest. of gene the with associated associated factors genes transcription all all or factor of scores MI of distribution to pair background the factor–gene MI the of compares a rithm transcription factor–T transcription pairwise all for 603 and regulators transcriptional T 2,021 of network background relevance a a structed applying by network relevance strategies the on builds algorithm CLR The regulator targets. a of potential and coexpression of strength relative the combining by operates in T respectively, underexpressed, 196 and overexpressed (407 genes 603 of total a in resulted This arrays. M430v2 Affymetrix with sets, data four all in cells genes those with consistent 1.5 T in expression the of ratio consensusT peripheral brought had resultsbeen independent experiments from together. several The analysis. for used were in algorithm platform analysis genomic the GenePattern average multiarray robust the by preprocessed were data raw cultures and acid–treated retinoic T cultured locations; anatomical various contexts: experimental several in tions gene lished prediction. CLR regulatory network 02954). (protocol Committee Use and Care Animal Institutional by approved the procedures under School at Medical Harvard Medicine and Comparative Resources for Animal Center abnormality. All mice cared for in accordance with the ethical guidelines of the RT quantitative by Eos Homozygous PCR. confirmed was 1–4 exons of deletion probes, external 5 with analysis blot Southern by confirmed was locus genomic the of Ikzf4 in Eos deficient ( Helios for 6 experiments weeks after of the Mice injection in final deficient poly(I:C). 2 with Gen) mice were injected intraperitoneally three times with 250 Mx1 flanked of knockout conditional with Mice Laboratory. The Jackson from were exon) hematopoietic first of the GATA upstream pairs base 21 site Gata1 Mice. METHODS ONLINE nature immunology nature reg In the second phase, starting from the CLR scores, we formulated an opti an formulated we scores, CLR the from starting phase, second In the algorithm CLR the used we connections, regulatory Topredict matureT of signature transcriptional the of definition robust a For cell signature genes signature cell reg - ) on the C57BL/6 background have been described been have background C57BL/6 the on ) were crossed with mice that have germline expression of Cre. Targetingof Cre. expression germline have that mice with crossed were 1.1/GFP C57BL/6 mice were from the Jackson Laboratory. Mice with a mutated with Mice Laboratory. Jackson the from were mice C57BL/6 promoter on the BALB/c background BALB/c on the promoter cells) - (Ikzf2) 1, Eos, IRF4, Lef1, Satb1 or Xbp1 was from human ORFeome. Mouse Mouse ORFeome. human from was or Xbp1 Satb1 Lef1, IRF4, Eos, 1, Xbp1 - - 1 expression data sets obtained with purified CD4 purified with obtained sets data expression day intervals for the induction of for the induction day intervals 1 alleles deleted by Cre expressed from the poly(I:C) have described been . 1 1 was used throughout. The cDNA encoding human Foxp3, Foxp3, human encoding cDNA The throughout. used was Ikzf4 ), mice with insertion of ), mice with insertion reg reg - 1 cells. MATLAB software (MathWorks) was used for used was (MathWorks) software MATLAB cells. deficient mice are viable and fertile with no apparent with and fertile are viable mice deficient 1 cell signature had been defined by of signature cell the calculation defined had been . After computing mutual information (MI) values values (MI) information mutual computing . After reg For enforced expression of Foxp3 and other other and Foxp3 of expression enforced For cells to that in T in that to cells - reg fold overexpression or underexpression in T - integer optimization problem that we solved solved we that problem optimization integer cell signature the most and accounted for for accounted and most the signature cell 11 3 9 , 12 . . For the generation of mice constitutively For we this analysis, compiled 129 pub , 4 reg 8 . The Affymetrix M430v2 micr M430v2 . The Affymetrix reg - ex vivo ex cell signature gene pairs, the algo the pairs, gene signature cell orcin tp Frt e recon we First step. correction cells; TGF cells; 4 9 3 , , and values expression averaged 7 conv Cre (with a deletion in the double in the a deletion (with lox T P sites flanking P exons sites 1–4 flanking of cells, with retention of only retentionof with cells, expression; mice were mice used expression; conv β cells or T or cells - induced Foxp3 induced µ - 3 E cells were plated plated were cells E 8 g g poly(I:C) (invivo ; 5 ; Xbp1 + - - to 6 to T cell popula cell T inducible gene reg (with (with cells from from cells - 3 week

generate generate 5 reg , which which , ′ - and 3 and + o IRES cells, cells, cells; cells; array array lox - old old reg P - - - ­ ­ ­ ­ ­ ­ - - ′

anti with staining for intracellular permeabilized and fixed were cells sorted cells, protein. Foxp3 of analysis and profiling microarray for low) and mediate Thy of intensity the of basis the on fractions into in experiments the For respectively. on Thy as CD4 cytometry by flow were sorted cells 72 h. Infected were for Cells cultured then an 32 supernatants. additional °C) with retroviral for were 24 cultured h and were then spin penicillin U/ml 100 (RPMI IL human recombinant anti purity of >95%. Cells were then activated with beads coated with anti CD4 then anti (130 beads washed, were cells After BioLegend). from all (PK136; anti phycoerythrin with CD4 ciation and red blood cells were lysed in 0.8% ammonium chloride lysis buffer. and spleens lymph of nodes 6 (Invitrogen). instructions manufacturer’s pCL construct the values presented ( atarriveexpression to wereaveraged theirnormalized valuesprobes,andand (more than 5 mismatches among 25 nucle Information. Features with substantial dissimilarity between mouse and human with the basic local alignment search tool of the retrieved,National and Centerthe sequencefor similaritiesBiotechnology between mouse features and the human(25 all were of analyzedsequences nucleotide First, origin. human of factors, were which transcription ectopic the from factors transcription mouse genous of expression the endo­ us to allowed distinguish characteristic gene. the This of length full the across (‘features’) spread oligonucleotides 27 approximately whole offers Array by array on the represented is genes ST 28,853 of the each as coverage, transcript 1.0 Gene Mouse Affymetrix The data. microarray feature we used counterparts, endogenous of their T ite done for downregulated (Down) genes of the T I ( genes to the expression in control cells transduced for all upregulated (Up) signature then we calculated the median value of the change in expression ( lated the of fraction signature genes upregulated under various conditions ( genes of the T module. Multiplot GenePattern on visualized and probes notated unan remove to prefiltration Toolsafter Power Affymetrix in implemented Expression average algorithm the with robust multiarray at Data were normalized Analysis. done were and pipeline Project Genome Immunological the followed steps was These cRNA (Affymetrix). arrays resulting ST 1.0 Gene The Mouse to hybridized (Qiagen). kit mini RNeasy the with purification and Sciences) Life (Enzo kit labeling transcription RNA yield high BioArray the with labeling biotin by followed (Ambion), kit aRNA with MessageAmp rounds the two for conducted instruc was manufacturer’s amplification RNA the (Invitrogen). to tions according prepared was RNA and reagent TRIzol in lysed were populations cell sorted transduction, retroviral after or profiling. Gene-expression protein. Foxp3 of localization the of analysis for Zeiss) 200M; (Axiovert microscopy confocal by or expression protein Foxp3 of analysis BD) II; for (LSR quantitative by cytometry flow and were analyzed Up For confirmation and quantification For of and quantification Foxp3 confirmation in expression the transduced of CD4 transduction For the To distinguish the expression of transduced transcription factors from that factors transcription To of transduced expression the distinguish We a developed T = - - - - residue oligonucleotides) for one particular gene (such as as (such gene particular one for oligonucleotides) residue reg Foxp3 (FJK CD28 CD28 (Invitrogen) at a ratio of one bead per cell, with addition of 20 U/ml + CD19 (6D5), anti (6D5), CD19 F T cells were negatively purified by magnetic selection (with labeling labeling (with selection magnetic by purified negatively were cells T cell signature index was calculated as follows: follows: as calculated was index signature cell * - - M 1.1 1.1 and GFP that report expression of Foxp3 and of the other cofactors, M 1640 medium supplemented with 10% FCS, 2 mM mM 2 FCS, 10% with supplemented medium 1640 ), and the T the and ), + *2. As expected, *2. As expected, - T 048 conv reg - - Eco through the use of Lipofectamine 2000 according to the the to according 2000 Lipofectamine of use the through Eco - cell signature in cells ( the transduced 801; Miltenyi Biotec) were added to the cell suspension and and suspension cell the to added were Biotec) Miltenyi 801; cells were purified with MACS LD columns (Miltenyi) to a to (Miltenyi) columns LD MACS with purified were cells 16s; eBioscience) according to the manufacturer’s instructions, reg - - conjugated anti conjugated Fig. 4 streptomycin and 50 50 and streptomycin reg cell signature index to estimate the expression global of - - CD8 cell Up signature index was established as follows: follows: as established was index signature Up cell 2 (Proleukin; Chiron) in complete culture medium medium culture complete in Chiron) (Proleukin; 2 I a Up and For of analysis gene in expression mice knockout α - = was 1 for calculation control Similar samples. to 8 (53 + T cells, cell suspensions were prepared from from prepared were suspensions cell T cells, Supplementary Table 4 - - week 6.7), anti 6.7), Figure 4b Figure - CD11b (M1/70), anti (M1/70), CD11b o - tides) were considered mouse old C57BL/6 old mice by C57BL/6 disso physical - infected (for 2 h infected at r.p.m.2,000 and µ - M 2 M reg CD25 (PC61) and anti and (PC61) CD25 , c cell signature, and a compos , infected T cells were sorted sorted were cells T infected , - - 1.1 expression (high, inter (high, expression 1.1 - mercaptoethanol). T cells cells T mercaptoethanol). level analysis of the 1.0 ST 1.0 of the analysis level Fig. Fig. 3 I = [ = ). + doi:10.1038/ni.2420 I cells further gated gated further cells Up f ). ). First, we calcu - + CD11c (N418), (N418), CD11c - phycoerythrin phycoerythrin I Down - l fold) relative Foxp3 - glutamine, glutamine, - ]/2. CD3 CD3 and - - specific NK1.1 NK1.1 were ) F ); ); - ­ ­ ­ ­ ­ ­

© 2012 Nature America, Inc. All rights reserved. protein G–conjugated Dynal beads (Invitrogen). Immunoprecipitated DNA Immunoprecipitated (Invitrogen). beads Dynal G–conjugated protein anti Cruz), incubated were then 10 Misonix), with s; 60 of intervals at s 30 of on cycles sonicated (eight were ice lysates Cell (11%). formaldehyde with crosslinked were assay. in this as done was were described used above, ChIP ChIP-Seq. anti and Abcam) (FJK 1 h at room temperature for with antibodies immunoblot analysis (anti Tween 0.02% and milk 5% in h (2 ing bind nonspecific of blockade After membranes. PVDF to electrotransferred Bound proteinsrotation. were eluted constant by boiling, were with separated by SDS Cruz))), Santa (sc628; IgG rabbit and BD) (553454; IgG2a mouse eBioscience), (eBR2a; G IgG2a (rat immunoglobulin or control anti Abcam), (Ab28839; tion (anti immunoprecipita for antibodies to coupled beads Sepharose G Protein with post and mM, 5 of was by stopped digestion Chromatin of the addition EDTA to a concentration Roche). S7; (nuclease MNase and ‘cocktail’) inhibitor protease complete free EDTA and KCl mM 20 NaCl, mM 300 HEPES, mM (20 buffer lysis nuclear with treated subsequently were pellets Nuclear (Roche). inhibitors protease and 0.05% NP KCl mM 10 MgCl2, mM 1.5 HEPES, mM (10 solution hypotonic with ice on GATA Lef1, (Satb1, factors transcription another plus Flag for vectors with transfected doubly were cells (HEK293) Immunoprecipitation and analysis. immunoblot doi:10.1038/ni.2420 - 16s; eBioscience), anti µ - g antibody (antibody to total RNA polymerase II (sc II polymerase RNA total to (antibody antibody g Flag (M2; Sigma), anti Flag (M2; Sigma), Mouse primary CD4 primary Mouse - Flag (M2; Sigma) Flag or (M2; Sigma) anti - 40–like 40–like IgePal Ca - Satb1 (611182; BD)). (611182; Satb1 - nuclear supernatants were incubated overnight at 4 °C °C 4 at overnight incubated were supernatants nuclear - Lef1 (Ab124271; Abcam), anti Abcam), (Ab124271; Lef1 - GATA - + - 630) 630) supplemented with EDTA Foxp3 Foxp3 (FJK T cells, transduced and sorted as described described as sorted and transduced cells, T - 1 (Ab28839; Abcam), anti - GATA - 20 in 1x PBS), blots were probed for for probed were blots PBS), 1x in 20 - 1 (ab28839; 1 Abcam) prebound to (ab28839; - 16s; eBioscience), anti 16s; eBioscience), Human kidney embryonic - 1 or Pbx1), were lysed lysed were Pbx1), or 1 - Satb1 (611182; BD) BD) (611182; Satb1 5 0 - . Cells (~1 × (~1 10 . Cells - Lef1 Lef1 (Ab124271; PAGE and were - - tagged Foxp3 Foxp3 tagged free free complete - 899; Santa Santa 899; - GATA - Foxp3 - 7 1 - ­ ­ )

48. Kolmogorov analysis. Statistical the in modeling. Mathematical libraries. sequencing of construction the during amplification in variability for compensation appropriate allows amount background genome the from calculated factor normalization a that total number of reads) might be to expected vary between parallel samples and the hence (and signal true when even constant be should noise experimental from 60 kb from to the stemmed that assumption the 650 kb). This correction Foxp3 any of devoid regions in noise by of from were values values the a integrated rescaled the constant calculated ing in samples transduced to express either Foxp3 alone or Foxp3 plus GATA densities in regions peak of the fordifferent samples Foxp3(particularly bind tag local of the comparison For accurate sample. the in of reads number total (1.4.0rc2). The number of reads in each tag ‘pileup’ were first normalized to model the with bind of ‘called’ Peaks were ing genome. mouse the of mm9) (UCSC 36 Build Information Biotechnology for Center National to 0.12.7) (version software Bowtie with sequencing Illumina for Kit Prep was 50. 49.

il J.A. Hill, Rahl, P.B.Rahl, M. Reich, (2010). Cells. CD4+CD44hi from inhibition

purified and used for library construction with a ChIP with construction for library used and purified Supplementary Note Supplementary - t al. et et al. et et al. et Smirnov test or the the or test Smirnov c-Myc regulates transcriptional pause release. pause transcriptional regulates c-Myc GenePattern 2.0. GenePattern eioc cd nacs op idcin niety y relieving by indirectly induction Foxp3 enhances acid Retinoic Statistical significance was determined with the the with determined was significance Statistical Information on mathematical modeling is Information on modeling available mathematical . - based analysis of ChIP of analysis based χ 5 Nat. Genet. Nat. 2 0 Immunity test. . Sequences were aligned to the genome genome the to aligned were Sequences . - binding peaks (seven regions ranging ranging regions (seven peaks binding

29

38 , 758–770 (2008). 758–770 , , 500–501 (2006). 500–501 , nature immunology nature - Seq analysis software software analysis Seq - Cell Seq DNA Sample Seq

141 , 432–445 , - 1), ­ ­