Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome

Transcription

1 Epgenetcs ISSN: (Prnt) (Onlne) Journal homepage: Valdaton of a DNA methylaton mcroarray for 450,000 CpG stes n the human genome Juan Sandoval, Holger Heyn, Sebastan Moran, Jord Serra-Musach, Mguel A. Pujana, Marna Bbkova & Manel Esteller To cte ths artcle: Juan Sandoval, Holger Heyn, Sebastan Moran, Jord Serra-Musach, Mguel A. Pujana, Marna Bbkova & Manel Esteller (2011) Valdaton of a DNA methylaton mcroarray for 450,000 CpG stes n the human genome, Epgenetcs, 6:6, , DOI: /ep To lnk to ths artcle: Copyrght 2011 Landes Boscence Publshed onlne: 01 Jun Submt your artcle to ths journal Artcle vews: 4058 Vew related artcles Ctng artcles: 416 Vew ctng artcles Full Terms & Condtons of access and use can be found at

2 RESEARCH PAPER Epgenetcs 6:6, ; June 2011; 2011 Landes Boscence Valdaton of a DNA methylaton mcroarray for 450,000 CpG stes n the human genome Juan Sandoval, 1 Holger Heyn, 1 Sebastan Moran, 1 Jord Serra-Musach, 2 Mguel A. Pujana, 3 Marna Bbkova 4 and Manel Esteller 1,5,6, * 1 Cancer Epgenetcs and Bology Program (PEBC); 2 Translatonal Research Laboratory; Catalan Insttute of Oncology; Bellvtge Bomedcal Research Insttute (IDIBELL); 3 Bomarkers and Susceptblty Unt; Spansh Bomedcal Research Centre Network for Epdemology and Publc Health; Catalan Insttute of Oncology; Bellvtge Bomedcal Research Insttute (IDIBELL); L Hosptalet de Llobregat; Barcelona, Catalona, Span; 4 Illumna, Inc.; San Dego, CA USA; 5 Department of Physologcal Scences II; School of Medcne; Unversty of Barcelona; Barcelona, Catalona, Span; 6 Insttuco Catalana de Recerca Estuds Avançats (ICREA)l Barcelona; Catalona, Span Key words: DNA methylaton, epgenetcs, mcroarray, CpG stes, DNA methyltransferase, colon cancer, valdaton DNA methylaton s the most studed epgenetc mark and CpG methylaton s central to many bologcal processes and human dseases. Snce cancer has hghlghted the contrbuton to dsease of aberrant DNA methylaton patterns, such as the presence of promoter CpG sland hypermethylaton-assocated slencng of tumor suppressor genes and global DNA hypomethylaton defects, ther mportance wll surely become apparent n other pathologes. However, advances n obtanng comprehensve DNA methylomes are hampered by the hgh cost and tme-consumng aspects of the sngle nucleotde methods currently avalable for whole genome DNA methylaton analyses. Followng the success of the standard CpG methylaton mcroarrays for 1,505 CpG stes and 27,000 CpG stes, we have valdated n vvo the newly developed 450,000 (450K) cytosne mcroarray (Illumna). The 450K mcroarray ncludes CpG and CNG stes, CpG slands/shores/shelves/open sea, non-codng RNA (mcrornas and long non-codng RNAs) and stes surroundng the transcrpton start stes (-200 bp to -1,500 bp, 5'-UTRs and exons 1) for codng genes, but also for the correspondng gene bodes and 3'-UTRs, n addton to ntergenc regons derved from GWAS studes. Heren, we demonstrate that the 2011LandesBoscenc Donotdstrbut 450K DNA methylaton array can consstently and sgnfcantly detect CpG methylaton changes n the HCT-116 colorectal cancer cell lne n comparson wth normal colon mucosa or HCT-116 cells wth defectve DNA methyltransferases (DKO). The provded valdaton hghlghts the potental use of the 450K DNA methylaton mcroarray as a useful tool for ongong and newly desgned epgenome projects. Introducton The dsrupton of the DNA methylaton profle s a major hallmark of human cancer. 1,2 Among the aberrant DNA methylaton sgnatures of transformed cells there are two key events n tumorgeness: the promoter CpG sland hypermethylatonassocated nactvaton of tumor suppressor genes and the global hypomethylaton of the cancer genome, whch s probably lnked to chromosomal nstablty and the reactvaton of endoparastc sequences. 1,2 The large body of data concernng DNA methylaton landscapes n human tumors whose clncal relevance has already been recognzed 3 occurs n a scentfc context where nformaton about the altered DNA methylaton patterns n other common dsorders, 4,5 such as neurologcal 6 and cardovascular 7 dseases, s stll very scarc Part of the explanaton mght relate to the dstnct bologcal processes nvolved and the ease wth whch sample materal can be acqured n the oncology arena, but the explanaton may also concern techncal aspects. In ths regard, t s necessary to have standard and common methods for obtanng comprehensve DNA methylaton profles from dfferent types of cells and tssues wth user-frendly boanalytcal tools that allow data to be shared among scentsts wth dstnct areas of experts Lookng at DNA methylaton analyss technologes t s evdent that a wde range of optons exsts. The bsulfte genomc sequencng of multple clones s a useful approach for the detaled study of the CpG methylaton status of a gven sequence n a few samples, whlst methylaton-specfc PCR or pyrosequencng technques are of great value for studyng a few CpG stes n a greater number of samples. If we go to the global scale, avalable approaches nclude the solaton of methylated fractons of the genome by methylaton-senstve enzymes, 8-10 mmunoprecptaton wth a methyl-cpg bndng doman antbody 11,12 and methylcytosne antbody New approaches nclude representaton bsulphte sequencng (RRBS), 17 and sequencng-by-synthess (MethylC-Seq) technology. 18 Usng whole-genome bsulfte sequencng, an analyss of the DNA methylome of perpheral blood mononuclear cells from a sngle case has also been reported recently. 19 However, these technques requre a hgh level of specalzaton and are stll expensve and tme-consumng. Thus, they are a largely unavalable to a large part of the bomedcal communty who wsh to examne the relevance of extensve *Correspondence to: Manel Esteller; Emal: mesteller@dbell.cat Submtted: 04/29/11; Accepted: 04/29/11 DOI: /ep Epgenetcs Volume 6 Issue 6

3 RESEARCH PAPER RESEARCH PAPER 201 1L andesb os c enc Donotd s t r but Fgure 1. Descrpton of the 450K DNA methylaton array. (A) Functonal genomc dstrbuton (FGD) classfed n dfferent groups: promoter, body, 3'UTR and ntergenc. (B) CpG content and neghborhood context classfed n: sland, shore, shelf and other. (C) Assocated RNA transcrpton classfed n: codng, non-codng and ntergenc. (D) Chromosome locaton. DNA methylaton changes n the orgn and progresson of many human dseases. Mrrorng the extensve use of well annotated DNA mcroarrays for sngle-nucleotde polymorphsms wth massve genome coverage, DNA methylaton mcroarrays wth a user-frendly component have begun to become avalabl Among these, the 1,505 CpG (Illumna GoldenGate DNA Methylaton BeadArray),20-23 and 27,000 CpG (Illumna Infnum HumanMethylaton27 BeadChp),24-27 mcroarrays have proved to be useful for addressng varous bologcal and medcal questons n a range of ndependent studes n areas such as cancer,23,24 agng21,25-27 and tssue-specfcty.21,22 The launch of a new 450,000 CpG ste platform for DNA methylaton studes (Illumna Infnum HumanMethylaton450 BeadChp) appears to be a promsng tool n the feld. Heren, we have valdated the 450K DNA methylaton mcroarray from bologcal, functonal and techncal standponts usng colorectal cancer and DNA methylaton models. The data obtaned ndcate that ths new platform s a robust and relable approach that may advance our understandng of the contrbuton of DNA methylaton to human bology and ts dsorders. Results and Dscusson Dstrbuton and classfcaton of the 450,000 cytosne stes n the human genom The 450K DNA Methylaton array (Infnum HumanMethylaton450 BeadChp) ncludes 485,764 cytosne postons of the human genom From these cytosne stes, 482,421 postons (99.3%) are CpG dnucleotdes, whlst 3,343 stes (0.7%) correspond to CNG targets. Thus, hereafter, we wll use the term CpG to refer to all of these, except when we refer specfcally to putatve CNG methylaton. The ncorporated CpG stes are dstrbuted among all 22 autosomal and 1 sex chromosome pars of humans. Chromosome 1 harbors the most postons (46,867, 9.6%) and chromosome Y the fewest (416, 0.08%). Accordng to ther assocated RNA transcrpts, 361,766 CpGs (74.4%) correspond to classc codng messenger RNA genes, 4,168 (0.85%) are lnked to non-codng RNAs (3,440 for mcrornas and 728 for long non-codng RNAs). For 119,830 (24.6%) stes there are no annotated transcrpts assocated wth the descrbed CpG locaton. From the CpG content and neghborhood context, 150,254 (30.9%) CpGs are n CpG Epgenetcs 693

4 201 1L andesb os c enc Donotd s t r but Fgure 2. Normal colon mucosa 1 vs. colorectal cancer cell lne HCT-116: A 450K DNA methylaton portrat. (A) Graphc showng percentage of dfferentally methylated CpG stes n Normal colon 1 respect to HCT-116. (B) Percentage of Hypermethylaton and hypomethylaton. (C) Dstrbuton of hypermethylated CpGs n HCT-116 accordng to functonal genomc dstrbuton; CpG content and neghborhood context; assocated RNA transcrpton and chromosome locaton. (D) Dstrbuton of hypomethylated CpGs n HCT-116 accordng to functonal genomc dstrbuton; CpG content and neghborhood context; assocated RNA transcrpton and chromosome locaton. slands, 112,072 (23%) n CpG shores,10 47,161 (9.7%) n CpG shelves10 and 176,277 (36.3%) are solated CpGs n the genome that we defne as Open Sea. From the functonal genome 694 dstrbuton standpont, 200,339 CpGs (41%) are located n proxmal promoters, defned as the sum of CpG stes located wthn 200 bp (62,625) or 1,500 bp (77,379) upstream of the Epgenetcs Volume 6 Issue 6

5 201 1L andesb os c enc Donotd s t r but Fgure 3. Normal colon mucosa 2 vs. colorectal cancer cell lne HCT-116: A 450K DNA methylaton portrat. (A) Graphc showng percentage of dfferentally methylated CpG stes n normal colon 2 respect to HCT-116. (B) Percentage of hypermethylaton and hypomethylaton. (C) Dstrbuton of hypermethylated CpGs n HCT-116 accordng to functonal genomc dstrbuton; CpG content and neghborhood context; assocated RNA transcrpton and chromosome locaton. (D) Dstrbuton of hypomethylated CpGs n HCT-116 accordng to functonal genomc dstrbuton; CpG content and neghborhood context; assocated RNA transcrpton and chromosome locaton. Epgenetcs 695

6 Fgure 4. Scatter plot for CpG methylaton values between normal colon mucosa 1 and 2. descrbed transcrpton start ste and n the 5'-untranslated regon (49,525) and exon 1 (10,810); moreover, 15,383 (3.16%), 150,212 (30.9%) and 119,830 (24.6%) CpG stes correspond to 3'-untranslated regons, gene body and ntergenc-open Sea sequences, respectvely. If we combne the two classfcatons descrbed above, we can determne that for the 200,339 CpG stes located n proxmal promoters, 92,374 CpGs (46.1%) are n CpG slands, 56,672 (28.3%) n CpG shores and 7,548 (3.8%) n CpG shelves, whlst 43,745 (21.8%) are n other regons of the genome wthout any enrchment of CpG content (Open Sea). Fgure 1 summarzes the genomc envronment of the 485,764 CpGs. Valdaton of the 450K DNA methylaton mcroarray n colorectal tumorgeness. We decded to test the ablty of the 450K array to detect CpG methylaton changes n a well defned model of colon cancer and DNA methylaton We dd ths n two ways. Frst, we compared the DNA methylaton profles of two human prmary normal colorectal mucosa samples wth that observed n a wdely used colorectal cancer cell lne, HCT Second, we took advantage of the fact that homologous recombnaton has been used n HCT-116 to dsrupt DNMT1 and DNMT3b n ths system. 29 Most mportantly, the HCT-116 double-knockout cells for DNMT1 and DNMT3b (DKO cells) have mnmal DNA methyltransferase actvty, a 95% reducton n 5-methylcytosne content, demethylaton of repeated sequences and loss of mprntng at the nsuln-lke growth factor II locus. 29 Interestngly, DKO cells also dsplay abrogaton of the methylaton-medated slencng of many tumor suppressor codng genes 29-32,34 and non-codng RNAs wth growth nhbtory functons such as mcrornas 33 and transcrbed ultraconserved regons (T-UCRs). 35 Before analyzng the CpG methylaton data, we excluded possble sources of techncal bas that could have nfluenced the results. Every b value n the Infnum HumanMethylaton450 BeadChp platform s accompaned by a detecton p-valu We found that a threshold p-value above 0.01 ndcated unrelable b values n 796 CpGs (0.16%) of the 485,764 stes analyzed. Thus, usng ths flter, 484,968 CpGs proved to be relable and were 2011LandesBoscenc used subsequently n the study. Donotdstrbut Fgure 5. Genomc envronment of the DNA methylaton dfferences observed n the proxmal promoters. (A) CpG content and neghborhood context of the CpG stes ncluded n the promoters of the Infnum HumanMethylaton 450K array. (B) CpG hypermethylaton and hypomethylaton events observed n HCT-116 cells n comparson to normal colon mucosa 1 and 2 accordng to the promoter type (sland, shore, shelf or others-open sea). 696 Epgenetcs Volume 6 Issue 6

7 2011LandesBoscenc Donotdstrbut Fgure 6. A comparson of the 450K DNA methylaton mcroarray wth the 1,505 CpGs and 27K mcroarray data n HCT-116 cells. (A) Correlaton of Infnum HumanMethylaton 27K vs. 450K array. (B) Correlaton of Infnum HumanMethylaton Golden Gate (1.5K) vs. 450K array. 450K DNA methylaton analyss of normal mucosa and colon cancer cells. The frst 450K DNA methylaton comparsons were made between the two normal colon mucosa samples (NC1 and NC2) and the colorectal cancer cell lne HCT-116. The results are summarzed for NC1/HCT-116 n Fgure 2 and for NC2/HCT-116 n Fgure 3. The data obtaned from both comparson pars are extremely smlar, beng partly assocated wth the hgh concordance of the 450K DNA methylaton pattern between the two normal colon tssues (Pearson correlaton coeffcent R 2 = and Spearman s rank correlaton coeffcent rho = ) (Fg. 4). Of the 484,968 CpGs studed, sgnfcant DNA methylaton dfferences were observed between normal colon mucosa and colorectal cancer cells at 3 6% of the stes (17,884 and 29,784 CpGs) (Fgs. 2 and 3). Most mportantly, 96 97% (17,190 and 29,112 CpGs) of the observed DNA methylaton changes corresponded to a CpG hypermethylaton event n the cancer cells, whlst hypomethylaton changes only accounted for 2 4% (672 and 694 CpGs) of the total observed n HCT-116 (Fgs. 2 and 3). The dfference between gan or loss of DNA methylaton was not only quanttatve, but also qualtatv CpG hypermethylaton events n the colorectal cancer cells mostly occurred n CpG slands (66 68%; 11,784 and 19,372 CpGs) and proxmal promoters (54 56%; 9,720 and 15,792 CpGs) (Fgs. 2 and 3). If we combne both concepts, 69 71% (6,894 and 10,843 CpGs) of the hypermethylated CpG ste events n cancer cells occurred n proxmal promoters that contaned a CpG sland (Fg. 5). On the other hand, the DNA hypomethylaton events manly occurred n the body of the genes (38 40%; 253 and 277 CpGs) and n the Open Sea areas of the genome (ncludng ntergenc regons) wth a value of 67 70% (448 and 483 CpGs) (Fgs. 2 and 3). For those few hypomethylaton events observed n the proxmal promoters of cancer cells (165 and 174 CpGs), most of them (61%) occurred n non-cpg sland-assocated promoters (Fg. 5). There was not any specal assocaton between the DNA methylaton events and the type of related transcrpt or the chromosomal locaton: both hyper- and hypomethylaton changes occurred n mrna, ncrna and non- RNA-assocated CpG stes (Fgs. 2 and 3) and all human chromosomes presented the two types of DNA methylaton events (Fgs. 2 and 3). CNG methylaton of the 3,343 stes ncluded n the 450K mcroarray was not observed n the normal colon mucosa samples or n HCT-116 cells. Our approach to bologcal valdaton of the robustness of the 450K mcroarray data revealed promoter CpG sland hypermethylaton of sequences prevously descrbed as densely methylated n the HCT-116 cancer cell lne usng canddate, genomc and transcrptonal approaches and further confrmed by bsulfte genomc sequencng of multple clones and other technques ,34 For example, ths was the case for the 5'-CpG slands of the tumor suppressor genes SFRP2 and SFRP5, 30 CHFR, 36 GATA-5, 37 IGFBP7, 38 PRDM2 (RIZ1), 39 PCDH8, 40 PAX6, 11 and RASGRF2. 32 DNA methylaton status was also confrmed for CpG slands encompassng the transcrpton start stes of mcrornas wth growth nhbtory functons, such as mr-124a, 33 mr-34b/c, 41,42 mr-129-2, 43 mr-152, 44 mr Further bologcal valdaton of the new platform was provded by the DNA methylaton status of mprnted and germlne-specfc genes n both normal colon mucosas. We observed that the 450K Epgenetcs 697

8 2011LandesBoscenc Donotdstrbut Fgure 7. HCT-116 vs. DKO cells: A 450K DNA methylaton portrat. (A) Total number of hypermethylated CpG stes n HCT-116 and DKO cells over a basal Beta value of (B) Graphc showng percentage of dfferentally methylated CpG stes n HCT-116 respect to DKO. (B) Percentage of hypermethylaton and hypomethylaton. (C) Dstrbuton of hypomethylated CpGs n HCT-116 accordng to functonal genomc dstrbuton; CpG content and neghborhood context; assocated RNA transcrpton and chromosome locaton. CpGs located n the dfferentally methylated regons (DMRs) of mprnted genes, such as H19, KCNQ1, SNRPN and MEST, presented 50% CpG methylaton, as expected from the parental allele-specfc methylaton. 46 In addton, 450K CpGs located n the proxmal promoter regons of male germlne-specfc genes, such as the MAGE and GAGE famles, showed 100% CpG methylaton n normal colon mucosa, as has been prevously descrbed for non-testcular tssues. 47 We were also nterested n the overall type of genes that undergo DNA methylaton changes n HCT-116 n comparson to normal colon mucosa. In order to dentfy functons assocated wth these dfferentally methylated CpGs and genes, we performed gene ontology (GO) analyss usng the DAVID program (BROAD). The CpGs dentfed as beng hypermethylated n the colon cancer cell lne were assocated wth 4,763 genes. These genes were hghly sgnfcantly enrched n cell and organ morphogeness (p = 1.2 x and p = 1.3 x 10-34, respectvely), regulaton of RNA metabolc processes and transcrptonal regulaton (p = 1.5 x ). Conversely, CpGs hypomethylated n HCT-116 cells relatve to normal colon nvolved 336 genes. These HCT-116 hypomethylated genes were sgnfcantly enrched n pathways assocated wth the regulaton of cell communcaton (p = ) and system development (p = ). Fnally, we provde addtonal techncal valdaton of the 450K DNA methylaton mcroarray data n HCT-116 cells by comparng them wth results from the well establshed and fully standardzed 1,505 CpGs (GoldenGate), and 27,000 CpG (Illumna Infnum HumanMethylaton27 BeadChp), 26,27 methylaton mcroarrays. The HumanMethylaton450 BeadChp ncludes 95% and 90% of the content contaned on the Golden Gate and HumanMethylaton27 BeadChp, respectvely. We observed that DNA methylaton data obtaned wth the new 450K mcroarray s correlated wth the methylaton levels detected at each CpG ste usng the 1,505 CpGs (Pearson correlaton coeffcent R 2 = and Spearman s rank correlaton coeffcent rho = ) and 27,000 CpG (Pearson correlaton 698 Epgenetcs Volume 6 Issue 6

9 201 1L andesb os c enc Donotd s t r but Fgure 8. A comparson of the 450K DNA methylaton data wth the expresson mcroarray nformaton n HCT-116 and DKO cells. (A) Gene Set Enrchment Analyss (GSEA) comparng upregulated genes (>2-fold) n HCT116-DKO wth ranked methylaton dfferences of both cell lnes. Methylaton values >0 dsplay hypo- and values <0 represent hypermethylaton n HCT116-DKO. (B) Methylaton level of CpGs assocated to upregulated genes represented by b values. (C) Methylaton level of CpGs wthn the gene body assocated to upregulated genes. (D) Methylaton level of CpGs wthn the promoter regon assocated wth upregulated genes. (E) Methylaton level of CpG sland related promoter probes assocated wth upregulated genes. (F) Methylaton level of low densty promoters assocated wth upregulated. TSS, transcrpton start ste; CGI, CpG sland. coeffcent R 2 = and Spearman s rank correlaton coeffcent rho = ) mcroarrays (Fg. 6). These results hghlght the relablty of ths novel DNA methylaton platform. 450K DNA methylaton analyss of HCT-116 and DKO cells. The n vvo valdaton of the 450K DNA methylaton mcroarray was confrmed usng the aforementoned sogenc cell lne wth the double defect n DNMT1 and DNMT3B (DKO cell lne).29 HCT-116 cells had 301,067 CpGs methylated over a basal b value of 0.66, whle DKO cells showed a 51% reducton (146,834 CpGs retaned the descrbed methylaton values) (Fg. 7). When we analyzed HCT-116 n comparson wth DKO for each CpG poston, takng a 0.33-fold change n b values as the cut-off, we observed that 110,611 (37%) CpGs stes were hypomethylated n DKO cells (Fg. 7). These demethylated CpG stes were wdely dstrbuted throughout the genome, affectng gene bodes (33.3%, 36,870 CpGs), promoters (33.2%, 36,718 CpG) and ntergenc regons (30.2%, 33,368 CpGs) (Fg. 7). Wth respect to the CpG content and neghborhood context, the over-representaton of hypomethylated CpG stes n the shore regons (25.7%, 28,464 CpGs) (Fg. 7) s partcularly noteworthy. As an addtonal nternal control, the 450K mcroarray also detected the loss of promoter CpG sland hypermethylaton of the prevously reported hypermethylated tumor suppressor codng genes n DKO cells29-32,34 and non-codng RNAs.33,35 Interestngly, there were 191,000 (74.3%) CpG stes that had a methylaton loss below the threshold of the 0.33-fold change n b values. Several explanatons for ths observaton can be proposed, such as the suggested hypomorphc nature of the dsrupted DNMT1 locus n DKO cells48,49 or the role of other DNA methyltransferases (such as DNMT3a) n mantanng the DNA methylaton status of these CpG stes.50 Epgenetcs 699

10 Further functonal valdaton of the 450K DNA methylaton data was obtaned by confrontng CpG methylaton status and gene expresson n HCT-116 and DKO cells. Usng the GeneChp Human Gene 1.0ST Array platform (Affymetrx, Santa Clara, USA) and fve bologcal replcates per sample, we dentfed 823 upregulated and 289 downregulated genes (>2-fold) n double-knockout cells n comparson wth the wld-type HCT- 116 cell ln Regardng ther DNA methylaton profle, genes wth ncreased expresson n DNMT1/DNMT3B-defectve cells were hghly enrched n hypermethylated CpGs n HCT-116 and hypomethylated CpGs n DKO, determned by gene set enrchment analyss of ranked methylaton levels (Fg. 8). Ths observaton was even more strkng when probes located wthn the transcrpton start ste or wthn the gene body were analyzed separately: promoter-assocated postons, partcularly wthn CpG slands, revealed a large methylaton dfference related to expresson (Fg. 8). Fnally, the 450K DNA methylaton data mght also be llustrated by cluster representatons, as has been done extensvely wth the 1,505 CpG and 27K CpG DNA methylaton mcroarrays. Although specfc bonformatc tools for the analyses of the 450K platform contnue to be developed, we can already present a partal herarchcal clusterng and a heatmap of the 450K DNA methylaton data that enable normal colon mucosa, HCT-16 and DKO cells to be classfed nto dstnct arms n the analyss (Fg. 9). In addton to the nternal consstency showng that 2011LandesBoscenc Donotdstrbut the two normal colon mucosa samples (NC1 and NC2) cluster together, t s worth hghlghtng that the loss of DNA methylaton n HCT-116 upon genetc dsrupton of DNMT1 and DNMT3 (DKO cells) shfts ths sample to a more normal colon DNA methylaton profle (Fg. 9). Ths fndng emphaszes the role of DNMTs n cellular transformaton and the mportant aberrant DNA methylaton changes that cancer cells undergo, such as occurs n the HCT-116 sample, whch s solated n a separate clusterng arm (Fg. 9). Materals and Methods Fgure 9. Clusterng analyss usng the most sgnfcantly dfferent 2,000 CpG stes from the 450K DNA methylaton mcroarray n normal colon mucosa (NC1 and NC2), colorectal cancer cell lne HCT-116 and ts derved cell lne DKO. Human prmary samples and cancer cell lnes. HCT-116 and DKO cells were a generous gft from Dr. Bert Vogelsten (Johns Hopkns Kmmel Comprehensve Cancer Center, Baltmore, MD). HCT-116 colon cancer cells and double DNMT1/DNMT3b (DKO) cells were grown as prevously descrbed n reference 33. DNA qualty check. (A) Orgnal DNA quantfcaton. 100 μl of a 1/2,500 Orgnal DNA dluton were quantfed by addng 100 μl of a 1/200 dluton of Quant-T TM PcoGreen dsdna Reagent (Invtrogen # P7589), usng a seral dluton of λ DNA (1,000, 500, 250, 100, 50, 5, 2.5 ng/ml) as a standard. Fluorescence sgnal was measured wth GloMax Mult+ fluormeter (Promega Corp.,) and concentratons were from a lnear regresson of the standard ponts. (B) Normalzaton. Orgnal DNAs were normalzed to a concentraton of 50 ng/μl by dlutng DNAs wth TE ph 8.0 buffer 1x. (C) Normalzed DNA quantfcaton. Normalzed samples were requantfed to ensure the normalzaton was performed properly. (C) Electrophoress. Electrophoress usng a 1.3% agarose 1/100,000 SyberSafe gel 700 Epgenetcs Volume 6 Issue 6

11 was performed to check for hgh molecular weght DNA fragments. 50 ng of DNA were loaded onto the gel usng GeneRuler 1 kb as a ladder (Fermentas Cat. No. SM0314). The gel was run for 60 mn at 120 ma. Samples showng bands or smears smaller than 10,000 bp were dscarded. (D) DNA purty. 1 ul of normalzed DNA was employed to measure the 260/280 and 260/230 ratos usng a Nanodrop Samples showng RNA contamnaton (260/280 rato >2) were dscarded, and no samples wth a 260/230 rato less than 1 were accepted. Bsulfte converson. Bsulfte converson of 600 ng of each sample was performed accordng to the manufacturer s recommendatons for the Illumna Infnum Assay (EZ DNA methylaton kt. Zymo Research. Cat. No. D5004). The ncubaton profle was 16 cycles at 95 C for 30 s, 50 C for 60 mn and a fnal holdng step at 4 C. The effectveness of the bsulphte converson was checked for three controls that were converted smultaneously wth the samples. Enzymatcally methylated Jurkat genomc DNA (New England Bolabs Cat. No. N4002S), genomc Jurkat DNA (New England Bolabs Cat. No. N4001S), and whole genome amplfed genomc DNA were used as total methylated, ntermedate-methylated and non-methylated controls. After bsulfte treatment, methylaton-specfc PCR 51 was performed on the control samples usng a set of prmers to amplfy a 301 bp regon of the 28s rbosomal DNA. (5'-AAA ATT CTT TTC AAC TTT CCC T-3' and 5'-GAG TGA ATA GGG AAG AGT TTA G-3'). PCR condtons were: 2 mm MgCl 2 at 95 C GenomeStudo normalzes data usng dfferent nternal controls that are present on the HumanMethylaton 450 BeadChp. It also normalzes data dependng on nternal background proves. CpG methylaton dfferences were consdered sgnfcant above a cut-off of a 0.6-fold change n the β value, unless specfcally ndcated otherws DNA methylaton clusterng. We used the GenomeStudo (2010.3) Methylaton module (1.8.5) software to generate heatmaps of clustered CpGs. Unsupervsed herarchcal clusterng of the 2,000 most varable B values (standard devaton >0.37) wth a detecton p value > 0.01 was performed, calculatng Eucldan dstances. Conclusons In the near future t wll be possble to obtan a greater number of complete human DNA methylomes at a reasonable cost usng the new emergng sequencng technologes. However, ths s stll an extremely challengng process and currently there are only a very few whole methylomes avalable n whch every CpG has been counted. 18,19 However, f researchers n the pure genome sequencng area are encounterng dffcultes n obtanng a massve number of completed human genomes n a cost-effectve and user-frendly manner, those of us n the epgenetc feld are even further behnd by several years. However, the development of a standardzed comprehensve DNA methylaton array that 2011LandesBoscenc Donotdstrbut for 1 mn, followed by 30 cycles at 95 C for 30 s, 58 C for 30 s and 72 C for 45 s, wth a fnal step at 72 C for 10 mn. Results were checked by loadng 5 μl of the PCR control onto a 1.3% agarose gel usng 1:100,000 dluton of SyberSafe and electrophoresng at 80 ma for 30 mn. DNA methylaton assay. 4 μl of bsulfte-converted DNA were used for hybrdzaton on Infnum HumanMethylaton 450 BeadChp, followng the Illumna Infnum HD Methylaton protocol. Ths conssted of a whole genome amplfcaton step followed by enzymatc end-pont fragmentaton, precptaton and resuspenson. The resuspended samples were hybrdzed on HumanMethylaton 450 BeadChps at 48 C for 16 h. Then unhybrdzed and non-specfcally hybrdzed DNA were washed away, followed by a sngle nucleotde extenson usng the hybrdzed bsulfte-treated DNA as a templat The nucleotdes ncorporated were labeled wth botn (ddctp and ddgtp) and 2,4-dntrophenol (DNP) (ddatp and ddttp). After the snglebase extenson, repeated rounds of stanng were performed wth a combnaton of antbodes that dfferentated DNP and botn by fxng them dfferent fluorophores. Fnally the BeadChp was washed and protected n order to scan t. Scannng beadchps. The Illumna HScan SQ scanner s a two-color laser (532 nm/660 nm) fluorescent scanner wth a μm spatal resoluton capable of exctng the fluorophores generated durng the stanng step of the protocol. The ntenstes of the mages were extracted usng GenomeStudo (2010.3) Methylaton module (1.8.5) softwar The methylaton score for each CpG was represented as a b value accordng to the fluorescent ntensty rato. B values may take any value between 0 (nonmethylated) and 1 (completely methylated). can be shared by dfferent laboratores and agences, employng a common methodology and bonformatc package would be an mportant step n the rght drecton. Heren we have demonstrated that a newly avalable DNA methylaton mcroarray (HumanMethylaton450 BeadChp) mght be an approprate tool for ths purpos The 450K analyses of a colorectal tumorgeness model that allows the study of normal and transformed cells n a varety of DNA methylaton settngs show that the data obtaned are confrmed by the results of prevously publshed studes n the area and provde bologcal, functonal and techncal valdaton of the new mcroarray. In the comng years, the applcaton of the 450K DNA methylaton platform n a userfrendly 12-sample per array format that requres only 500 ng of bsulfte-converted DNA per sample wll provde extensve nformaton about the DNA methylaton landscape of human tumors. Most mportantly, t mght also be decsve n establshng that DNA methylaton defects have a central role n other common human dseases and n fndng answers to many currently unresolved bologcal questons. Acknowledgments Ths work was supported by the Fondo de Investgacones Santaras Grant PI , Consolder Grant MEC09-05, Dr. Josef Stener Cancer Research Foundaton Award, Llly Foundaton Bomedcal Research Award, European Research Councl Advanced Grant EPINORC, the Health Department of the Catalan Government (Generaltat de Catalunya) and Fundacó Cellex. J.S. s a Juan de la Cerva Researcher. M.E. s an Insttuco Catalana de Recerca Estuds Avançats (ICREA) Research Professor. Epgenetcs 701

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