© 2011 Nature America, Inc. All rights reserved. H.Y.C. ([email protected]). University of Oslo, Oslo, Norway. Genetics, Institute for Cancer Research, Oslo University Hospital Montebello, Oslo, Radiumhospitalet, Norway. of Medicine, Baltimore, Maryland, USA. Cancer Comprehensive Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Dartmouth Medical School, Hanover, New Hampshire, USA. Hill, North Carolina, USA. of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. and Artificial Intelligence Laboratory, Institute Massachusetts of Technology, Cambridge, USA. Massachusetts, of Medicine, Stanford, California, USA. 1 transcriptional in implicated been have lncRNAs several to elements enhancer produce are ncRNAstranscribed also protein coding genes intergenic ncRNAs,(long nearby or orwithin lincRNAs), mayinterspersed be or they genes coding protein from isolation in located be in vary length from hundred several bases to infidelity tens of kb; lncRNAs may polymerase RNA of byproducts represent just or ncRNAs functional are these if However,uncertain remains it initiation RNAs (tiRNAs) and TSS-associated RNAs (TSSa-RNAs) ously named promoter-associated small RNAs (PASRs),vari transcription-been have which nucleotides, 200 to 20 from ranging ncRNAs (TSS), divergent transcription by RNA polymerase can generate small defense and regulation post-transcriptional genome in function (piRNAs), RNAs Piwi-associated and (miRNAs) microRNAs (siRNAs), RNAs interfering small including ncRNAs, regulatory Small transcribed. are (ncRNAs) RNAs coding expected Mammalian genomes are more pervasively transcribed than previously for depletion p53-dependent damage. expression regions the external Transcription Michael L Whitfield Anita Langerød Hugo M Horlings Tiffany Hung RNAs within promoterscell-cycle Extensive and coordinated transcription of noncoding Nature Ge Nature Received 19 August 2010; accepted 6 May 2011; published online 5 June 2011; Program Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA. Although evidence for function of IncRNAs as a group is lacking, lacking, is group a as IncRNAs of function for evidence Although

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6 6 :143 :127 1 4 6 9 8 6 :602 :–688 :–662 :–639 C B 1 A D :–7794 :–1860 :–64 :–79 :–53 1 1 1 1 2 2 2 2 2 2 :–172 :–6760 3 :183 :142 :15 :1926 :1417 :–276 :–238 :–223 :–196 :–767 :–225 :–139 :–982 :–311 :–554 2 :–68 C 2 B A 2 1 1 A 1 1 C :39498 :43877 :–97 6 :–36 :–6045 :–6418 :–4410 :–4044 :–48 :–5717 :–5830 :–4845 :–219 :–281 :–803 6 8 3 9 3 1 8 0 2 1 0 7 3 4 8 3 1 0 0 2 6 7 7 ). ).  - -

© 2011 Nature America, Inc. All rights reserved. be functional in the cell cycle, in self-renewal and in cancer. in self-renewal cycle, cell in the functional be ncRNAs that these suggest conditions may and cellular ent biological lation of the dynamic expression patterns of these ncRNAs and differ response or are spliced together as exons of one transcript. High corre multiple adjacent ncRNAs are either coordinatelythat suggesting regulated in locus, a sharedgenomic same the from ncRNAs the of many ‘stemnessa be ( cluster’ GFP-RAS-I and cells differentiated MYC-RAS-I and P63-knockdown with nocytes kerati ESCs, in induced strongly are that lncRNAs of composed is ( samples all across pattern expression their fide bona lncRNAs of in not mRNAs only pattern these but also expression the RAS-I E2F3- MYC-RAS-I and ESCs the with clustered four of cancers the lncRNA profiles: on their based groups different two into split carcinomas breast inva human ductal sive primary eight addition, In lncRNAs. of majority the for expression of pattern inverse an had program, expression gene mRNA ESC-like the not do express which keratinocytes, transduced for embryonic and cancer stem cells. In contrast, the E2F3-RAS-I tered with that of ESCs ( Notably, the lncRNA expression profile of MYC-RAS-I expression program and acquire properties of cancer stem cells stem cancer of properties acquire and program expression gene mRNA ESC-like an activate keratinocytes human transduced MYC that showing together, clustered to controls relative oncogenes other oncogene the with transduced were that samples keratinocyte five from patterns Expression state. undifferentiated the in role a have may ncRNAs these that gesting that of sug with ESC, clustered differentiation, keratinocyte inhibits s e l c i t r A 

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were also examined on our tiling array and calculated pairwise pairwise calculated and array tiling our on examined also were that samples 17 on arrays expression mRNA, whole-genome nearest conducted we the of expression the with correlated typically are lncRNAs gene-proximal whether To gene. determine coding nearby of including lncRNAs, Multiple and A gene Gene sets Ontology enriched are listed. Representative association. with the associated gene lncRNA set. A no black entry indicates significant set. A that blue the entry indicates gene Gene set Ontology is negatively with gene Gene the set Ontology associated gene lncRNA is positively ( gene sets 17 Processes across (rows) samples Biological Gene Ontology map Module of response. damage gene lncRNA versus sets (columns) in groups are for lncRNAs the enriched functional cell cycle and in DNA on the same locus 108 across ( samples. each between of the correlations 216 and ncRNAs transcripts nearby of Histogram Pearson transcripts. lncRNA with neighboring correlation 108 across ( samples. each between of the correlations 216 Pearson and ncRNAs the do patterns with not those of correlate the mRNAs in 3 Figure damage–related gene sets (UVA/UVB), several oncogenic signatures signatures oncogenic (UVA/UVB), gene sets several damage–related nega receptor tive, and up), negative BRCA DNA-prognosis BRCA1 overexpressed estrogen (BRCA sets gene cancer breast prognosis Cell or poor Brentani several included Cycle modules enriched addition, In Cell KEGG). Cycle example, (for sets cell-cycle–related collection) c2 (MSigDB Database Signatures Molecular from the states and clinical biological as well as pathways signaling and metabolic curated of sets with gene map module similar a constructed We cycle. cell the to related strongly still are lncRNAs these of patterns expression the gene, protein-coding neighboring their to expression in correlation to limited DNAhaving despite and Thus, response damage. splicing, RNA assembly, and biogenesis complex ribonucleoprotein bination, associated with biological processes including cell cycle, DNA recom ( terms Ontology Gene distinct with iden associated to are that lncRNAs tify biclustering performed and set gene Processes Ontology Gene Biological the versus set gene co-expression lncRNA each of 3 Fig. across the 17 samples ( that lncRNA with mRNAs correlated or that negatively are positively For each lncRNA, we a gene set defined co-expression as the group of approach a guilt-by-association using may regulate they that ways mRNA the relate with in ( transcript same the exons of are and/or other each regulate positively regulated, dinately coor be may ncRNAs neighboring positive, that was hypothesis our locus supporting same the of ncRNAs two between correlation ( 5 3 neighboring and lncRNAs of analysis (qRT-PCR) RT-PCR in function not ( expression mRNA may nearby or repress activate lncRNAs the of most mRNA, that protein-coding suggesting nearby the between and lncRNAs 216 anti-correlation the or of most correlation significant no was there Notably, wide. genome mRNA every versus lncRNA promoter cell- cycle each of patterns expression the between correlations Pearson P Fig. Supplementary Fig. 2 Fig. Supplementary ′

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CDKN1A ncRNA PANDA LAP3 CDKN1A is located approximately 5 kb upstream of of upstream kb 5 approximately located is LAP3 5 located were ncRNAs 12 those of Notably, 2 upst: gene ( qRT-PCR by on and ncRNAs array 216 tiling the the of 12 in change two fold least at showed fibroblasts lung fetal samples. cancer to compared breast epithelium breast normal ( human to ESCs D, compared fetal pancreas. day. ( (orange). in the with expression known periodic cell cycle of cell and sorting genes activated expression by cycle were phases confirmed fluorescence- in HeLa cells ( cell cycle progression synchronized during (blue) ( expression. for samples lncRNA biological independent TaqMan and probes used them to interrogate and Wehuman cancers. custom generated ESC differentiation cell cycle progression, 4 Figure doxorubicin-treated fibroblasts. In order to to order In fibroblasts. doxorubicin-treated in only one expressed of the 108 the samples, was it because lncRNAs 216 the in included by was induced but also doxorubicin was not the at ( bicin CDKN1A ). Although the the Although ). d

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are induced more rapidly and with with and rapidly more induced are Validated expression Validated of in ncRNAs expression CDKN1A c mRNA, were induced by doxoru by induced were mRNA, ) Regulated expression of in ) lncRNAs expression Regulated

a in , a b a CDKN1A ). In addition, a third lncRNA lncRNA third a addition, In ).

) and foreskin fibroblasts ) ( and fibroblasts foreskin ) Periodic expression of ) lncRNAs expression Periodic the :−4,845), and, similar to the the to similar and, :−4,845), ′ CDKN1A CDKN1A and 3 and (upst: locus, upst: locus, locus may play distinct roles PANDA

DNA-damage :−4,845, hereafter termed termed hereafter :−4,845, LAP3 Fig. Fig. 5 ′ complementary DNA complementary CDKN1A upon DNA damage. damage. DNA upon protein, p21. protein, :−800) by quantita by :−800) , qRT-PCR showed showed qRT-PCR , b locus intersects a intersects locus s e l c i t r A ). ). Notably, several ). Furthermore, Furthermore, ). CDKN1A :−1,210 and and :−1,210

response Fig. Fig.

b :−800, :−800, LAP3 ). ). Cell 2 ). ).  - - - - -

© 2011 Nature America, Inc. All rights reserved. in metastatic ductal carcinomas but not in normal breast tissue tissue breast normal in not but carcinomas ductal metastatic in of induction induce to syndrome ability the Li-Fraumeni in induce to ability the abrogated observed mutant, (p.Arg273His) damage-inducible DNA ( PANDA of expression mutant—restored (p.Val272Cys) p53 carc 9a Fig. sequencing DNA in 2–11 exon by mutation determined as inactivating harboring tumors breast primary p21. of transcript on effect on ( of induction the damage inhibited DNA before p53 of knockdown co-regulation. siRNA-mediated p53 of Indeed, possibility the supports which site, binding site CDKN1A binding p53 the of the upstream confirmed immediately analysis regulates ChIP-chip also p53 expression. whether asked we response, damage to antisense scribed 5 a is LAP3 from transcribed divergently is that transcript a 1.5-kb and revealed RNA (RACE) ends blot analysis s e l c i t r A  ( Supplementary Fig. 9b Fig. Supplementary Fig. 5 Fig. e ChIP-chip a b Because p53 is a positive regulator of of regulator positive a is p53 Because +Dox Fold siRNA/siCtrl –Dox ∆ 60 ncRNAs RNA RNA CDKN1A 0.2 0.4 0.6 0.8 1.0 1.2 1.4 i 0 noma cells by wild-type p53—but not the loss-of-function loss-of-function the not p53—but wild-type by cells noma 4 2 ′ Hours post-dox P53 PANDA pseudogene ( -capped and polyadenylated non-spliced lncRNA that is tran is that lncRNA non-spliced polyadenylated and -capped siCTRL e 100 100 ). Further, complementation of p53-null H1299 lung lung H1299 p53-null of complementation Further, ). and and 0 2 0 0 PANDA 1 8 are diametrically situated 2.5 kb inte from this situated are diametrically

siTP53 2 6 . In contrast, RNA interference of of interference RNA contrast, In . Supplementary Fig. 8 Fig. Supplementary level shows a trend of lower expression in human human in expression lower of trend a shows level 2 0 CDKN1A * –9,569 PANDA

siCDKN1A 4 expression, indicating that PANDAthat a is not linked indicating expression, –800 –9,56 –1,21 –4,84 +88 Fig. 5 5 CDKN1A 9 0 5 PANDA c without concordant concordant without Fold RNA expression nor is PANDA expression dependent on on dependent expression PANDA is nor ∆ –5,830 d 10 15 20 25 30 35 40 Fig. 5 Fig. 0 5 ). and LAP3 CDKN1A PANDA 8 0 PANDA CDKN1A –4,845 Hours post-dox CDKN1A ( . Supplementary Supplementary Fig. 7 f Fig. 5 Fig. CDKN1A ). Notably, a gain-of-function p53 p53 gain-of-function a Notably, ). CDKN1A predicted 16 RAC ( PANDA –4,845 by 70% 24 h after DNA damage damage DNA after h 24 70% by 20 E Promoter ), which is similar to its effect effect its to similar is which ), f f ). We also observed selective selective observed We also ). 24

Do Normalized expression TSS ( TSS 10 15 10 14 5 2 6 ( ) x CDKN1A , antisense of the predicted predicted the of antisense , PANDA 28 but selectively preserved preserved selectively but PANDA CDKN1 Empty vector + – + – + – + – +885 CDKN1 –9,569 –800 –1,210 –2,230 Fig. 5 Fig. –1,902 CDKN1A

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b +1,420 PANDA had no no had PANDA PANDA R273H p53 (LF) TP53 and 3 7 - ,

the other hand, expression of neither neither of expression hand, other the ( also See (FL3). fibroblasts human normal CDKN1A the from transcribed lncRNAs of A cluster treatment. doxorubicin of course h time a 24 across repression or induction coordinate shows lncRNAs of also array. See tiling upst: the of upstream immediately site binding p53 the confirmed input to relative signal ChIP-chip p53 the bottom, the At point. time single one in transcripts DNA-damage–inducible all observe not did we that Note fibroblasts. lung fetal human ng/ml) (200 treated (dox) h doxorubicin 24 in or control the in intensity hybridization RNA of examples are tracks two middle the In ( 5 Figure primary human lung fibroblasts specifically retrieved endogenous endogenous retrieved specifically fibroblasts lung human primary interaction. Immunoprecipitation of NF-YA from doxorubicin-treated (refs. LSD1 or EZH2 as lncRNAs, such other bind can that complexes modification matin induced by DNA damage ( specifically retrieved NF-YA from cellular lysates of human fibroblasts 1.2-kb a not but RNA,PANDA transcribed chromatography RNA function. NF-YA NF-YA factor scription tran the for site binding the by genes target p53 other from guished apoptosis. delimits of induction CDKN1A p53-mediated with parallel in that suggest data these DNA-damage–induced to fibroblasts sitized not shown; scripts within the cells ( marker of apoptosis, revealed PARP cleavage only in ( down resulted in fivefold to sevenfold increased TUNEL-positive cells contrast, In TUNEL. by measured as apoptosis to no little had siRNA control to exposed cells FL3 in treatment bicin apoptosis not but arrest p21 of independently acts that effector P53 by affected tors of apoptosis, including ( PANDA death’ ‘cell terms Ontology ( ‘apoptosis’ and Gene 0.04) the as such apoptosis, in PANDA by twofold least at repressed were genes 193 and induced were genes 224 that showed ( knockdown the lowing siRNAs targeting custom with (FL3) fibroblasts fetal human We transduced response. selectively retains the ability to induce PANDA. induce to ability the retains selectively (p.Arg273His), p53 allele, Li-Fraumeni a gain-of-function whereas induction, restore to fails mutant loss-of-function (p.Val272Cys) p53 The of induction damage DNA restores cells H1299 student’s by determined Mean of induction Doxorubicin a d Fig. ) At the top is a map of all detected transcripts at the CDKN1A promoter. CDKN1A at the transcripts ) is a top At detected of the all map ) RNA blot of of blot ) RNA Next, we addressed whether whether addressed we Next, Core promoters of cell death genes downstream of p53 are distin are p53 of downstream genes death cell of promoters Core G1 p53-dependent triggers fibroblasts human in damage DNA CDKN1A CDKN1A

6e ± Fig. Fig. 6 s.d. are shown; * shown; are s.d.

, depletion induced several genes encoding canonical activa canonical encoding genes several induced depletion knockdown were significantly enriched for those involved involved those for enriched significantly were knockdown locus over a 24 h time course after doxorubicin treatment of treatment doxorubicin after course h time a 24 over locus f ncRNAs at the the at ncRNAs ). Immunoblot analysis of PARP, a caspase substrate and and substrate caspase PARP, a of analysis Immunoblot ). for cell cycle arrest, p53-mediated induction of of induction p53-mediated arrest, cycle cell for g Supplementary Fig. 10 Supplementary PANDA locus are induced. ( induced. are locus ). ). In contrast, six additional siRNAs targeting other tran :−4,845 closely matches the predicted transcript on the the on transcript predicted the matches closely :−4,845 PANDA CDKN1A a DVANCE ONLINE PUBLICATION ONLINE DVANCE CDKN1A PANDA PANDA S depletion ( depletion upplementary Figure 7 Figure upplementary confirms a transcript size of 1.5 kb. ( kb. 1.5 of size a transcript confirms 42,43), or p21, illustrating the specificity of the the of specificity the illustrating p21, or 42,43), P CDKN1A t P PANDA TSS after DNA damage. The RACE clone of clone RACE The damage. DNA after TSS 4 -test. ( -test. < 0.03) ( 0.03) < < 0.05 relative to siCTRL (control siRNA) siRNA) (control siCTRL to relative < 0.05 0 38 , and we reasoned that that reasoned we and , Fig. 7 Fig. 6 Fig. and then applied doxorubicin for 24 h fol doxorubicin applied and then , 3 promoter had no effect on apoptosis (data knockdown ( knockdown APAF1 9 . Consistent with this finding, doxoru finding, this with Consistent . f requires p53 but not CDKN1A. CDKN1A. not but p53 requires ) Expression of wild-type p53 in p53-null p53-null in p53 wild-type of ) Expression c locus are induced by DNA damage. damage. DNA by induced are locus a Fig. 6 Fig. ) Expression of transcripts from the the from transcripts of ) Expression a ). PANDA did not retrieve other chro ). Global gene expression analysis analysis expression gene Global ). Fig. 6 Fig. PANDA , , ). ). Thus, PANDA sen knockdown BIK d CDKN1A ), suggesting that that suggesting ), S b , , upplementary Figure 6 Figure upplementary FAS ). qRT-PCR confirmed that that confirmed qRT-PCR ). Fig. 6 Fig. . . ( 4 affects the DNA damage damage DNA the affects 1 using purified, purified, using CDKN1A b CDKN1A and ) Quantitative RT-PCR ) Quantitative LacZ

apoptosis. Altogether, Altogether, apoptosis. b

itself nor nor itself ). Genes induced by induced Genes ). LRDD PANDA Nature Ge Nature mRNA fragment, fragment, mRNA . PANDA and and PANDA ( e PANDA Fig. Fig. 6 PANDA ) may affect affect may TP53 -depleted

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© 2011 Nature America, Inc. All rights reserved. are needed that not only identify ncRNAs but also provide insight insight provide also but ncRNAs identify only not that needed are and enhancers, promoters as gene well as from stand-alone produced intergenic be loci can RNAs noncoding long malian genomes is transcribed. In addition to small noncoding RNAs, Recent studies have revealed that a surprisingly large fraction of mam DISCUSSION apoptotic gene expression program ( PANDA mediatearrest,whereas to p21 NF-YA impedes of activation cally to mediate cell cycle arrest and survival. ated transcription at to chromatin. These data suggest that DNA damage activates p53-medi Thus, PANDA binding to NF-YA may evict or prevent NF-YA binding required in forpart cell death bytriggered loss of PANDA ( genes and apoptosis as measured by TUNEL, indicating that NF-YA is of NF-YA and PANDA substantially attenuated induction of apoptotic known as (also including PANDA NF-YAgenes, of target at increased substantially occupancy p53-responsivethe promoter of PANDA ( treatment. doxorubicin h of 24 after student’s by determined siCTRL to compared sample siPANDA bar, 20 Scale treatment. doxorubicin h of 28 after fibroblasts FL3 TP53 ( cells). FL3 (in treatment doxorubicin h of 28 after siRNA control to relative siPANDA with induction revealed ( cells. FL3 in treatment doxorubicin h of 24 after siRNA control to relative siPANDA * graphs. bar all in shown target specifically siRNAs Custom (FL3). fibroblasts human in doxorubicin 6 Figure Nature Ge Nature into their potential Usingfunction. biological an ultrahigh-resolution d a

Dox RNA (fold) Fold ∆ dox/ctrl Fold ∆ dox/ctrl 1.0 2.0 3.0 4.0 5.0 in FL3 cells revealed no reduction in expression with siPANDA relative to control siRNA. ( siRNA. control to relative siPANDA with expression in reduction no revealed cells FL3 in 0.5 1.0 1.5 2.0 2.5 3.0 10 12 14 16 18 2 4 6 8 0 0

PANDA Fig. 7 – – CCNB1 Ctrl Ctrl CDKN1A n Ctrl siPANDA TP53 etics A siPANDA A + b NOXA lncRNA regulates the apoptotic response to DNA damage. ( damage. DNA to response apoptotic the regulates lncRNA

). NF-YA is a nuclear transcription factor that activates B B , ,

FAS C C ADVANCE ONLINE PUBLICATION ONLINE ADVANCE CDKN1A B A ) ) ( * , , – + P Fig. 7 BBC3 < 0.05 compared to siCTRL for all panels determined by Student’s Student’s by determined panels all for siCTRL to compared < 0.05 LAP3 PANDA siPANDA e c (also known as as known (also and Merge TUNEL DAPI ). ). Moreover, concomitant knockdown * FAS – + PANDA upon DNA damage Fig. siCTRL C * 8 + ). CDKN1A thatsynergisti functions 14 PUMA , 15 b , 1 7 siCtrl . New approaches mRNA produces

) and and ) 4 0 . Depletion . Depletion t Fig. 7d siPANDA -test. ( -test. siPANDA B A PMAIP1 µ m. ( m. g , ) Protein blot of PARP cleavage in control and PANDA siRNA FL3 fibroblasts fibroblasts FL3 PANDA siRNA and control in PARP of cleavage blot ) Protein e f ) Quantification of three independent TUNEL assays. assays. TUNEL independent three of ) Quantification ). ). - - -

PANDA RNA processing Programmed celldeath Apoptosis assembly Protein-DNA complex Nucleosome assembly DNA packaging a ) siRNA knockdown of PANDA in the presence of DNA damage with with damage DNA of presence PANDA the of in knockdown ) siRNA In addition, the ncRNA product may modulate neighboring gene gene neighboring in expression modulate may product ncRNA the addition, In interference). transcriptional called (so binding with factor transcription compete or sequences regulatory over chromatin compacted open may transcription of act The ways. of variety a in activity gene oncogenic perturbation. defined with cells of those to states promoter their of similarity the authentictor p63. humanLikewise, tumorson based can be classified MYC and epithelial progenitors depleted of the differentiation regula by induced tumors ESCs, among states promoter of similarity a fied promoter states among distinct perturbations. For instance, we identi in these diverse similarities samples cell cycle unexpected highlighted amongst profiles lncRNA of comparison Second, sample. biological anyone in transcribed are these of 73 only average, on identified, we regions transcribed noncoding new 216 the Of surveyed. were condi tions few a only if missed been have may and conditions specific highly in only induced are transcripts noncoding the of many First, rapid and economical fashion is advantageous a for at in least two reasons. samples biological diverse and numerous interrogate to ability The perturbations. diverse represent that samples 108 in promoters tiling array, we interrogated the transcriptional landscape at cell-cycle Noncoding transcription through regulatory elements may affect affect may elements regulatory through transcription Noncoding with no discernable effect on the the on effect discernable no with f ( ( P c P =0.01) ) Quantitative RT-PCR of canonical apoptosis pathway genes genes pathway apoptosis RT-PCR canonical of ) Quantitative % TUNEL positive =0.03) 10 15 20 25 30 35 0 5 ( ( P P =0.0001) =0.03) –2.0 cis

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C * c d

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< 0.05 for each each for < 0.05 C C B A siPANDA s e l c i t r A Fold ∆ dox/ctrl Fold ∆ dox/ctrl 15 25 35 45 10 30 50 70 0 5 0 trans CDKN1A ± s.d. are are s.d. C Ctrl Ctrl 2 LRDD siPANDA siPANDA 2 * * BIK or even even or A A 85 kDa 116 kDa and and * B * B * C C *  - - - © 2011 Nature America, Inc. All rights reserved. cell survival by impeding the apoptotic gene expression program. program. of the ability the by tumors: exploited apparently be can expression linkage This gene apoptotic the impeding by survival cell arrest, cycle cell mediate to inhibitor cycle cell a Whereas promoters. gene target occupying NF-YA from factor transcription the sequestering by genes apoptotic of parallel. in occurs p53 by induction their that indicating damage, DNA to response other’s the on effect of depletion contrast, In show that than to antisense transcribed named we which lncRNAs, these of One damage. DNA by induced are itself, mRNA TSS. At the the of downstream kb 2 and upstream kb 10 within lncRNAs four of average an and lncRNA detected one least at have loci gene coding pathways same ( biological the in function that transcripts multiple for code that units genomic to modulate the chromatin state promoters.of cell-cycle promoterscycle may a be form ofanticipation regulatory or feedback in gene regulation in tiation. These findings suggest that cell-cycle ncRNAs may participate cancers, andhuman are in regulated and in cycle response cell to the DNA in damage expressed or ESC differendifferentially these are ncRNAs of many that validated further analysis qRT-PCR prognosis. cancer with associated been have and differentiation, cell stem and DNA cycle, pathways, cell including damage response biological cific (and presumably co-regulated) with promoter ncRNAs function inco-expressed genes The spe genome. the in elsewhere located genes with mRNA neighboring but their with are rather correlated in expression ncRNAs are norpositively neither negatively correlated in expression expression profiles across our samples. We found that most promoter els of ncRNAs and cognate mRNAs, we compared ncRNA and mRNA these different mechanisms predict distinct relationships between lev as serve a target for regulation by small regulatory RNAs in as is panel this for legend The shown. is see ( the in region downstream of promoters on sites NF-YA target known at ChIP-qPCR siPANDA. or siCTRL with nucleofected ( bottom. the at shown as NF-YA, of immunoprecipitation confirms qRT-PCR.by Immunoblot measured as retrieves specifically lysates FL3 doxorubicin-treated NF-YA from ( analysis. immunoblot by proteins retrieved the of 7 Figure s e l c i t r A  P53 mutant p.Arg273His to gain-of-function Li-Fraumeni selectively d ) Concomitant knockdown of NF-YA attenuates induction of apoptotic genes by PANDA depletion, as measured by qRT-PCR. For knockdown efficiency efficiency qRT-PCR.by knockdown For measured as PANDA by depletion, genes apoptotic of induction NF-YA of attenuates knockdown ) Concomitant PANDA Our results suggest that the human genome is organized into into organized is genome human the that suggest results Our a S upplementary Figure 11 Figure upplementary CDKN1A Input

PANDA from doxorubicin-treated FL3 cell lysates. We visualized We visualized lysates. cell FL3 doxorubicin-treated from PANDA

CDKN1A Beads . Loss-of-function and complementation experiments experiments and complementation . Loss-of-function regulates transcription factor NF-YA. ( NF-YA. factor transcription regulates LacZ induction during DNA damage is p53 dependent. DNA is dependent. during p53 damage induction PANDA CCNB1 trans promoter, five lncRNAs, similar to the PANDA FAS CDKN1A . In addition, noncoding transcription of cell- LSD1 EZH2 p21 NFYA CDKN1A Fig. Fig. , , , is a non-spliced 1.5-kb ncRNA that is is that ncRNA 1.5-kb non-spliced a is , . . ( promoter lacking the NF-YA motif. Mean Mean NF-YA motif. the lacking promoter FAS e ) Concomitant knockdown of NF-YA rescues apoptosis induced by PANDA depletion. Quantification of TUNEL staining staining TUNEL of Quantification PANDA by depletion. induced apoptosis NF-YA of rescues knockdown ) Concomitant c WB:NF-YA 8 ) ChIP of NF-YA in FL3 fibroblasts fibroblasts NF-YA FL3 of in ) ChIP , , ). ). Forty nine of protein- 56 cell-cycle NOXA and is induced with faster kinetics kinetics and faster with is induced or depletion of of depletion or b

PANDA Retrieved RNA (% input) , , 20 10 15 BBC3 0 5 d . Input GAPDH α IgG b -NF-YA ( inhibits the expression expression the inhibits ) Immunoprecipitation of ) Immunoprecipitation PUMA IgG CDKN1A PANDA a PANDA ) or a control a control ) or ) RNA chromatography chromatography ) RNA

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0 Model of coding and noncoding transcripts at the the at transcripts noncoding and coding of Model APAF1 hnRPK 2 siCTRL 4 IgG . Thus, the regulatory sequence upstream of of upstream sequence regulatory the Thus, . , that is induced by p53 and mediates p53-dependent gene siPANDA siCTRL FAS locus coordinately activates transcription of of transcription activates coordinately locus induction instead of instead induction p53 NF-YA siPANDA a *

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0 5 NF-YA CDKN1A Nature Ge Nature Cell cyclearrest 0.1 0.2 0.3 0.4 0.5 0.6 2 1 0 CDKN1A Fig. Fig. CDKN1 may provide provide may CDKN1A p21 siCTRL IgG Control t CDKN1A 8 * -test. -test. 4 3 , named named , NF-YA ). These These ). A siPANDA n locus locus etics IgG as

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© 2011 Nature America, Inc. All rights reserved. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturegenetics/. The authors declare no competing interests.financial T.H. and D.J.W. with input from all co-authors. M.v.d.V., A.-L.B.-D., S.S., M.L.W. and Y.X. The manuscript was prepared by H.Y.C., samples or reagents: A.K.K., Y.K., G.D.G., H.M.H., N.S., C.U., P.W., A.L., S.K.K., M.F.L. and M.K. contributed CSF analysis. The following authors contributed Yulei Wang, Yu Wang and B.K. conducted high-throughput TaqMan RT-PCRs. experiments. T.H. performed the experiments and the computational analysis. H.Y.C. and D.J.W. initiated the project. H.Y.C., D.J.W. and T.H. the designed (DoD) National Defense Science & Engineering Graduate Fellowship Foundation(NDSEG). (NSF) Graduate Research Fellowship and the DepartmentInstitute. of Defense T.H. is supported by the Stanford Graduate SocietyFellowship, (H.Y.C.). the NationalH.Y.C. Science is an Early Career Scientist ofDiabetes the HowardResearch HughesFoundation Medical (S.K.K. and H.Y.C.) and the AmericanInstitute Cancer (NCI) (R01-CA118750 to H.Y.C. and R01-CA130795 to andM.L.W.), Skin Diseases the(NIAMS) Juvenile (K08-AR054615 to D.J.W.), NIH/NationalNational CancerInstitutes of Health (NIH)/National Institute of employeesArthritis andof MusculoskeletalLife Technologies. This work was supported byreading grants of fromthe themanuscript US and P. Khavari for reagents.We Y.W., thank J. Rinn, B.K. M. andGuttman Yu and WangA. Regev forare discussions, L. Attardi for careful Note: Supplementary information is available on the been deposited with GenBank under the accession number JF803844. Expression Omnibus (GSE28631). Sequence for codes. Accessionhuman online the at http://www.nature.com/naturegenetics/. paper the of in ­version available are references associated any and Methods M URLs. and likely other ncRNAs emanating from regulatory sequences. medicine. Future studies are needed to pinpoint the functions of these of promoters, which may of be in use cancer andbiology regenerative expression provides a convenient means of tracking the chromatin state ers that may play diverse functions. At a minimum, promoter ncRNA Nature Ge Nature 12. 11. 10. COMPETING FINANCIAL INTERESTS COMPETING AUTH A ckn

ethods Our study provides an initial catalog of lncRNAs in cell-cycle promot

el, A.C. Seila, P.Kapranov, silencing. RNA in players key proteins: Argonaute M.J. Simard, & G. Hutvagner, regulation. gene in role big a with RNAs small MicroRNAs: G.J. Hannon, & L. He, Mattick, J.S. & Makunin, I.V. Small regulatory RNAs in mammals. Carninci,P. Non-coding RNAtranscription: turning onneighbours. G.A. Calin, Carninci, P. Bertone, P. Ponjavic, J., Ponting, C.P. & Lunter, G. Functionality or transcriptional noise? Evidence Core, L.J., Waterfall, J.J. & Lis, J.T. Nascent RNA sequencing reveals widespread R.J. Taft,pausing for selectionfornoncodingwithinlong RNAs. anddivergent initiation humanatpromoters. Genet. Nat. (2008). 1849–1851 transcription. pervasive Biol. Cell Mol. Rev. Nat. Genet. Rev. Nat. 14 (2008). 1023–1024 carcinomas. and leukemias (2005). 1559–1563 arrays. tiling Spec No 1, R121–R132 (2005). R121–R132 1, No Spec o O Genomica, Genomica, wl R R C e O n t al. et dgm et al. et al. NTRIBUTI

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e lrcnevd ein ecdn nRA ae lee i human in altered are ncRNAs encoding regions Ultraconserved 5 iig n mcora dt ae vial a Gene at available are data microarray and Tiling iegn tasrpin rm cie promoters. active from transcription Divergent RNA maps reveal new RNA classes and a possible function for function possible a and classes RNA new reveal maps RNA nts , 522–531 (2004). 522–531 ,

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© 2011 Nature America, Inc. All rights reserved. which has also been applied to assess new transcribed regions in mouse in regions transcribed new assess to applied been also has which method (CSF) frequencies substitution codon the using genomes lian their in alignments signatures with orthologous regions in 20 evolutionary other sequenced placental the mamma evaluated we regions, transcribed new potential. protein-coding Measuring ‘genegene, dst)’; of mRNA’;nearest symbol ‘distance from TSS’. of protein–coding cell-cycle gene, int; downstream of proteincell-cycle coding protein-coding gene, upst; exon of cell-cycle cell-cycle protein–coding of gene, exon;TSS intron of (upstream location ‘genomic follows: as annotated were Transcripts analysis. further for considered were samples the of 10% of mum samples were clustered using Galaxy array 55 all from calls Peak units. transcriptional significant considered were NimbleGen) with a window size of 50. Peaks with a peak score greater than ten each array were subjected to peak calling using the NimbleScan program (Roche calling. Peak protocol. NimbleGen (RETROscript Kit, Ambion), labeled and hybridized according to the standard transcribed reverse Ambion), Kit, (MessageAmp amplified RNA was Briefly, the In addition, including those encoding cyclins, CDKs and CDKIs ( 10 kb upstream to 2 kb downstream of each TSS from 53 other cell-cycle genes, encompasses (which region 9p21 the of kb 25 across resolution 5-bp at designed was NimbleGen) hybridization. RNA and design array Tiling ONLINE Nature Ge Nature performs a performs comparison two between statistical empirical codon models Briefly, CSF regions. and noncoding in coding known substitutions of similar frequency relative the on based codon alignment, its all within observed considering substitutions genome the in region any for score a produces CSF doxorubicin (Sigma)-treated human fetal lung fibroblasts, polyA was selected was polyA human selected lung fetal fibroblasts, (Sigma)-treated doxorubicin the FirstChoice RLM-RACE Kit (Ambion). RNA was extracted from 200 ng/ml PANDA CO2. 5% in °C at 37 (Gibco) streptomycin penicillin- 1% and (Hyclone) FBS 10% in cultured were CRL2091) (ATCC fibroblasts foreskin or AG04393) (Coriell FL3 fibroblasts lung fetal Human methods. standard using isolation RNA for processed receipt, and of washed h minced, 24 within processed were pancreata fetal Staged Washington. of University Laboratory, Research Defects Birth the from obtained were ata described as are Program Autopsy Rapid University Hopkins Johns the from tumors breast metastatic and tissues breast normal Institute. Cancer Institute Cancer Netherlands The from Netherlands tumors breast Human primary and University Hopkins Johns University, Stanford of boards review institutional from approval obtained we and tion, cells. and samples Tissue testing ( hypothesis multiple for account to used was discovery false calculation a (FDR) and rate distribution, hypergeometric the by determined was Database Signatures ( c2 Molecular (MSigDB collection) the from states clinical and biological and sets gene ( Processes Biological Ontology Gene with sets gene of ncRNA map these module a generated then we associations, functional Todetermine ncRNA. that with 0.5 a than less or had than that greater was genes that correlation Pearson protein-coding the of sets gene defined then we ncRNA, each For URLs). (see Genomica program the using visualized and clustered map was This samples. 17 different across on expression based combinations pairwise all for correlations Pearson the by computing genes protein-coding map analysis. Module conservation sequence of level for overall the controlling while explanation is a better model protein-coding the whether quantifies that ratio a likelihood and reports regions, noncoding on based other the and regions coding known of alignments from estimated P Fig. 3 Fig. < 0.05, FDR < 0.05). < FDR 0.05, < c ) and with curated gene sets of metabolic and signaling pathways pathways signaling and metabolic of sets gene curated with and ) cloning analysis. and sequence

METHODS n Robust multichip average normalized single channel data from from data channel single normalized average multichip Robust etics HOXA CDKN2A We a map generated module of the ncRNAs the versus and and Supplementary Fig. 4 Supplementary Informed consent was obtained for tissue dona tissue for obtained was consent Informed HOXD 3 0 , , . P14ARF loci were placed on the array as a control. a control. as array on the placed were loci 2 , 4 8 , , and only transcripts present in a mini To assess the coding potential of the the of potential coding the Toassess 3 ′ and and and 5 A custom tiling array (Roche (Roche array tiling custom A ) 3 ′ CDKN2B 3 RACE was using performed . . The Supplementary Supplementary Table 1 2 3 . Human fetal pancre fetal Human . P value of enrichment ), as well as from from as well as ), 30 5 5 1 2 , , , one 49 and , 1 5 4 0 ). ). - - - - , .

as an endogenous control for normalization across different samples. different across normalization for control endogenous an as used PPIA was Technologies). (Life System PCR Real-Time Fast 7900HT the replicates were run for each gene for each sample in a format 384-well plate on ufacturer’s TaqMan PreAmp Master Mix Kit Protocol (Life Technologies). Two 14-cycle PCR amplification followed by real-time PCR reaction using the man to cDNA a The Transcription resulting was Kit subjected Technologies). (Life Reverse cDNA Capacity High the using RNA total of ng 50 from generated these ncRNAs across different conditions, complementary DNAs (cDNA) were of profiling expression For gene sequences. or genomic transcripts nontarget genome-wide and assays designed between reactivity cross potential predict to matrix alignment TaqMana all using position-specific were verified assays the using regions transcribed new ‘single-exon’ 219 design mode. The and transcript specificity genome of the specificity of 60 targeting developed was assays. ncRNA custom TaqMan to (Stratagene)). Green SYBR Mix RT-PCR Step Master normalized and Assays RT-PCR for Expression Strand-specific GAPDH. Gene TaqMan using Biosystems) (Applied Mix RT-PCR Master One-Step the using performed was RNA total was DNA genomic of ng 50–250 and using RT-PCR (Ambion). DNA-free TURBO (Qiagen), using eliminated Kit Mini RNeasy the and (Invitrogen) RT-PCR. protocol. manufacturer’s standard the to according performed was RLM-RACE and (Ambion) MAG kit Poly(A)Purist the using overnight. We washed 10 10 We washed overnight. 2 and 89879), (Pierce columns spin into micro were transferred r.p.m.for 15 at min 13,000 Supernatants trifuged cen and combined were lysates cytoplasmic and Nuclear H). setting power s (30 30 s on, off, Sonicator a Diagenode for 7 using min sonicated and pestle motorized a using s 5 for mM dounced 0.5 were lysates MgCl2, Nuclear PMSF). mM mM DTT,1 4 KCl, M 0.42 glycerol, 10% 7.5, pH HEPES mM (20 as C vortex frequent with min 20 for Buffer pellet of the to added A was volume Buffer of used was equal an Next, collected. was (cytosol) supernatant the and r.p.m., 2,000 at min 3 for centrifuged were Lysates ice. on min 5 for mM (10 A Buffer 15 min on ice at of 150 r.p.m. NP-40 of to 0.25% was concentration added a final volume 2× for with DTT, PMSF) mM mM 1 0.5 KCl, mM 10 MgCl2, mM 1.5 lysed 7.5, pH HEPES After were min. 5 cells for glycine washes, M PBS 0.125 of two addition the by formaldehyde followed 1% min, with 10 for crosslinked and trypsinized h, 16 for doxorubicin immunoprecipitation. RNA antifade reagent with DAPI (Invitrogen) and imaged at 20× magnification. 1 hour at 37 °C. Slides were then washed with PBS and mounted in Prolong methanolGold at −20 °C for 10 min and incubatedslides with(Lab-Tek), the treatedTUNEL with labeling200 ng/ml doxorubicinmixture (Sigma)for for 24 h, fixedKit, TMRwith Red (Roche). Human fetal lung fibroblasts were cultured on chamber TUNEL. ( 5 Table of ON-TARGETPlus siRNAs (Dharmacon) targeting RNA interference. H-209). Biotech NF-YAanti Cruz (Santa and Biotech) LSD1 Cruz (Santa p21 anti anti AC22), Signal (Cell EZH2 anti ab6046), (ab17721), (Abcam anti-B-tubulin using 9542), Signal performed were (Cell anti-PARP blots Protein ab28). (Abcam anti-p53 and ab9050) (Abcam anti-H3K35me3 ab8580), (Abcam anti-H3K4me3 assays: cipitation Antibodies. (Agilent). Kit Labeling length full with generated were Probes protocol. manufacturer’s standard the following (Ambion) Kit and PolyA Purist Mag (Ambion). RNA blot was performed using NorthernMax RNAblot. Supplementary Table 5 Supplementary ). Validated siRNAs for mRNAs were obtained from Ambion Ambion from obtained were mRNAs for siRNAs Validated ). TUNEL assays were performed using the Total RNA was extracted from cells using the TRIzol reagent reagent TRIzol the using cells from extracted was RNA Total We obtained 5 5 We obtained The following antibodies were used for chromatin immunopre chromatin for used were antibodies following The Human fetal lung fibroblasts were transfected with 50 with nM were Human transfected lung fibroblasts fetal ). µ µ PANDA l of Protein A/G UltraLink Resin (Pierce 53132) 53132) (Pierce Resin UltraLink A/G Protein of l g of polyA RNA using an RNeasy Kit (QIAGEN) (QIAGEN) Kit RNeasy an using RNA polyA of g Ten million cells were treated with 200 ng/ml ng/ml 200 with treated were cells Tenmillion A panel of TaqMan custom ncRNA assays assays ncRNA custom TaqMan of panel A using the Prime-It RmT Random Primer Primer RmTRandom Prime-It the using µ g of antibody was added and incubated incubated and added was g antibody of PANDA was performed using the One- the using was performed insitu PANDA CellDeath Detection doi:10.1038/ng848 ( Supplementary - - -

© 2011 Nature America, Inc. All rights reserved. for 15 min. The nuclear pellet was resuspended in 1 ml RIP buffer (150 mM mM (150 buffer RIP ml 1 in 2,500 resuspended at was pellet nuclear The centrifugation min. 15 for by pelleted were Nuclei min. 20 for ice on X-100) Triton 4% MgCl2; mM 20 7.5; pH Tris-HCl mM 40 sucrose; M (1.28 buffer 200 in incubated were h) (16 cells 2 and (Ambion) cocktail A/T1 RNase of 20 min for before conjugation to °C) RNAsebeads. digestion was with performed 5 (22–25 temperature room to shifted and (Ambion) buffer structure RNA °C (90 for was RNAfolded 2 with for ice reaction. min, 2 supplied min), RNA( of pmols 6 tions: raphy RNA RNAse chromatography. mediated Kit Mini (QIAGEN). digest DNAse RNEasy column on the the with (QIAGEN) using extracted was RNA of and ml 1 sample, the added We to °C. 45 TRIzol at h 1 for 5 incubated plus and PMSF) (Ambion) mM K 1 and Proteinase DTT mM 0.5 deoxycholate, sodium 0.5% 40, NP- 1% SDS, 0.1% EDTA, mM 1 NaCl, mM 150 7.4, pH Tris mM (50 RIPA 200 in resuspended were Beads PMSF). mM 1 and DTT mM 0.5 deoxycholate, sodium 0.5% NP-40, 1% SDS, 0.1% EDTA, mM 1 NaCl, M 1 times with RIP wash buffer and two times with 1 M RIPA (50 mM Tris pH 7.4, four washed were Samples °C. 4 at h 1 for reaction immunoprecipitation the glycerol, 10% 7.9, pH TrisHcl MgCl mM mM 5 KCl, mM (50 100 buffer wash RIP with times three doi:10.1038/ng848 Cellular lysates were prepared as follows: 10 million doxorubicin treated treated doxorubicin million 10 follows: as prepared were lysates Cellular 4 1 was performed as previously described with the following modifica following the with described previously as performed was PANDA 2 , 10 mM B-me and 0.1% NP-40) and added it to to it added and NP-40) 0.1% and B-me mM 10 , or a 1.2-kb fragment of of fragment 1.2-kb a or µ l PBS, 600 600 l PBS, µ RNAse mediated RNA RNAse mediated chromatog l of RNase V1 (Ambion). V1 RNase of l µ l H20 and 200 and 200 l H20 LacZ ) were used per per used were ) µ l nuclear lysis lysis l nuclear µ l 150 mM mM 150 l µ µ g - - l l

53. 52. 51. 50. 49. 48. CCAAT motif ( consensus NF-YA the surround to designed were BAX and Morachis from obtained were sites binding YA described ously (ChIP). immunoprecipitation Chromatin at 18,000 by centrifugation pelleted were debris nuclei were sheared using a motorized douncer for 5 s. Nuclear membrane and inhibitor (Roche Complete Protease Inhibitor Cocktail Tablets)). Resuspended KCl, 25 mM Tris pH 0.5 mM 7.4, DTT, 1 NP40, mM 0.5% PMSF and protease Supplementary Table 5 Supplementary

in JL, ode C, ldtn, .. Bon PO & hn, .. Anatomic H.Y. Chang, & P.O. Brown, H.B., Gladstone, C., Bondre, J.L., Rinn, M.J. Vijver, de van Kosiol, C., Holmes, I. & Goldman, N. An empirical codon model for protein sequence scalability and Performance M. Kellis, & M.D. M.F.,Rasmussen, Lin, A.N., Deoras, Lin, M.F. perform to galaxy Using A. Nekrutenko, & D. Blankenberg, I., Schenck, Taylor,J., demarcation by positional variation in fibroblast gene expression programs. expression gene fibroblast in variation Genet. PLoS positional by demarcation cancer. breast evolution. 12 in identification gene genomes. comparative for metrics discriminative of genomes. fly 12 using (2007). 10.5 Unit ag-cl itrcie aa analyses. data interactive large-scale et al. PLOS Comput. Biol. Comput. PLOS Mol. Biol. Evol. Biol. Mol.

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