SUPPLEMENTAL MATERIAL DOT1L-mediated H3K79me2 … · of differential mark occupancy, and heatmaps of relative gene expression generated. Genes with higher than average expression

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SUPPLEMENTAL MATERIAL

DOT1L-mediated H3K79me2 modification critically regulates gene expression

during cardiomyocyte differentiation

Paola Cattaneo1,2,6, Paolo Kunderfranco1, Carolina Greco1, Alessandro Guffanti3, Giuliano

Giuseppe Stirparo2,6, Francesca Rusconi1, Roberto Rizzi4,5, Elisa Di Pasquale1,2, Silvia

Laura Locatelli2,6, Michael V.G. Latronico1, Claudia Bearzi4,5, Roberto Papait1,2* and

Gianluigi Condorelli1,2,6*

1Humanitas Clinical and Research Center, 20089 Rozzano (MI), Italy

2Institute of Genetics and Biomedical Research, National Research Council of Italy (CNR),

UO of Milan, 20089 Rozzano (MI), Italy

3GenOmnia, via Nerviano 31/b, 20020 Lainate (MI), Italy

4Casa di Cura Multimedica Istituto di Ricovero Cura Carattere Scientifico, 20138 Milan,

Italy

5Institute of Cell Biology and Neurobiology, National Research Council of Italy (CNR),

00100 Rome, Italy;

6University of Milan, 20133 Milan, Italy

*Corresponding authors: Gianluigi Condorelli

([email protected]) and Roberto Papait

([email protected]), Humanitas Clinical and Research Center, Via

Manzoni 56, 20089 Rozzano (MI), Italy.

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Detailed Materials and Methods Purification of cardiomyocytes. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85–23, revised 1996) and approved by the local ethical committee. The study was performed on embryos (E8.5, E10.5, E12.5, and E14.5), neonatal (1 day after birth) and adult (2-month-old) mice. Mice were housed in a controlled environment on a 12h light/dark illumination schedule and fed with a chow diet. Mice were sacrificed according to the protocol of the internal ethics committee, the hearts excised, and primary embryonic, neonatal, and adult cardiac cells were isolated as described elsewhere.1, 2, 3 mES cell culture and differentiation protocol. The TBV2 mouse embryonic stem (mES) cell line was used throughout this study. Cells were cultured on a feeder layer of mitotically inactivated mouse embryonic fibroblast (MEFs) in order to keep them undifferentiated in a pluripotent state. The propagation medium was high glucose DMEM supplemented with sodium pyruvate, L-glutamine, penicillin–streptomycin, 2-mercaptoethanol, 15% ES-screened FBS (Hyclone), and 103 U/mL LIF (Millipore). mES cells were passed twice on 0.1% gelatin-coated tissue culture dishes without MEFs before starting experiments. Differentiation of mES cells into the cardiac lineage was carried out in feeder-free conditions using standard techniques.4 In differentiation medium (high glucose DMEM supplemented with sodium pyruvate, L-glutamine, penicillin–streptomycin, 2-mercaptoethanol, and 15% FBS (GIBCO) without LIF), embryoid bodies (EBs) were aggregated using the “hanging drop” method (300cells/drop): cells were cultured for 2 days (d0–d2) as hanging drops and then for 3 days (d2–d5) in suspension in low-attachment Petri dishes (Falcon).4 5-day-old EBs were plated onto 0.1% gelatin-coated tissue culture dishes in differentiation medium and harvested at different time points for mRNA analysis, ChIP, immunoblotting, and FACS. Total RNA extraction, cDNA synthesis, and qRT-PCR gene expression analysis. RNA was extracted from mES cells and cardiac cells using TRIzol (Invitrogen). RNA was reverse-transcribed to cDNA using the Super Script VILO cDNA Synthesis Kit (Invitrogen) and amplified by real-time quantitative PCR with SYBR Green PCR master mix (Applied Biosystem) and specific primers for pluripotency and cardiac markers. Primer sequences are available upon request. Each sample was analyzed in triplicate using an ABI 7900HT (Applied Biosystems). 18s or GAPDH were used as the housekeeping gene for expression normalization. Epigenetic enzyme TaqMan assay cards. RNA was extracted from mES cells and cardiac cells using TRIzol (Invitrogen). RNA was reverse transcribed to cDNA using Super Script VILO cDNA Synthesis Kit (Invitrogen). 1.2μg of cDNA of each sample was used to analyze the expression of 85 genes encoding important epigenetic enzymes. Custom microfluidic gene expression cards were drawn using TaqMan probes (Applied Biosystems). TaqMan probe information and card design is available upon request. Each sample was analyzed using an ABI 7900HT (Applied Biosystems). Data were analyzed with DataAssist software using the median normalization method.

Immunoblotting. Immunoblotting was carried out using standard protocols. The following primary antibodies were used overnight at 4°C: anti-KMT4 DOT1L (abcam ab64077), anti-

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H3 dimethyl Lys79 (abcam ab3594), anti-H3 (abcam ab1791), anti-POU domain, class 5, transcription factor 1 (Pou5F1) (abcam ab19857), anti-myosin heavy chain (MYH) (Chemicon MAB 1552), and anti-lamin B (Santa Cruz sc6216).

Viral particle production and transduction. Two distinct short hairpin (sh)RNAs against Dot1L and a non-target shRNA were cloned into the plK0.1 lentiviral vector (OpenBiosystems) and tested. Viral particles were produced in 293T cells by co-transfection with pCMV-VSVG and psPAX2 plasmids. FACS analysis. FACS was carried out using standard protocols. Cells fixed in 4% paraformaldehyde were stained with anti-troponin I (TNNI) (Millipore MAB3150) antibody. Samples acquisition was performed on FACS Canto Cell Analyzer (Becton Dickinson) and data analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Chromatin immunoprecipitation assay. Genome-wide localization of histone modifications (H3K4me3, H3K27me3, H3K79me2, and H3K9me3) for each stage was determined via chromatin immunoprecipitation followed by high-throughput sequencing with SoLiD 5500 (Life Technologies) carried out according to a standard procedure. Briefly, 5x106 cells were used for each immunoprecipitation. Cells were cross-linked for 10 min at room temperature using 1% formaldehyde. Cross-linking was quenched by adding glycine to a final concentration of 0.125M. The cells were then collected, resuspended in lysis buffer (5mM PIPES pH8, 85mM KCl, 0.5% NP40, and protease inhibitors), and incubated on ice for 15 minutes before proceeding with sonication to generate 200−400bp fragments. The efficiency of sonication was assessed with agarose gel electrophoresis. Chromatin samples were pre-cleared for 1 hour with protein-G beads and then immunoprecipitated overnight at 4°C with specific antibodies: anti-H3 dimethyl Lys79 (abcam ab3594), anti-H3 trimethyl Lys4 (Active Motif 39159), anti-H3 trimethyl Lys 27 (Millipore-Upstate 07-449), anti-H3 trimethyl Lys9 (Millipore-Upstate 07-442), anti-H3 (abcam ab1791) and rabbit IgG (Millipore-Upstate 12-370). After incubation, the immunocomplexes were bound to protein-G beads for 2 hours and subsequently washed with low-salt wash buffer (0.1% SDS, 2mM EDTA, 20mM Tris HCl pH8, 1% Triton X-100, 150mM NaCl and protease inhibitors), high-salt wash buffer (0.1% SDS, 2 mM EDTA, 20mM Tris HCl pH8, 1% Triton x-100, 500mM NaCl, and protease inhibitors), and TE buffer. Immunocomplexes were eluted in elution buffer (1% SDS, 100mM NaHCO3 and protease inhibitors) and the cross-linking was reverted overnight at 65°C. Samples were treated with proteinase K, extracted with phenol/chloroform, and precipitated with ethanol. Purified DNA was evaluated by qPCR on an ABI 7900HT with SYBR green PCR master mix (Applied Biosystem) using specific primers designed close to the promoter region and the gene transcription start site (TSS): A = -1000bp/-500bp from TSS; B = +500bp/+1000bp from TSS; C = +3500bp/+4000bp from TSS. Values obtained were normalized to the input and to the H3 content. The sequences of the primers used for ChIP-qPCR are available upon request.

ChIP-seq analysis pipeline. ChIP-seq experiments were performed as previously described.5 Library preparation and DNA sequencing was performed at Genomnia Srl. ChIP-seq data analysis was performed using distinct bioinformatics software. For H3K4me3, H3K27me3 and H3K9me3 modifications in mES cells, bed files were downloaded from Gene Expression Omnibus (H3K4me3: GSM307618; H3K27me3: GSM307619; H3K9me3: GSM307621). Genome coordinates were converted from mm8 to mm9 mouse reference genome using Batch Coordinate Conversion (LiftOver) created by

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the UCSC Genome Bioinformatics Group. For the H3K79me2 modification in mES cells, raw sequencing reads were downloaded from Gene Expression Omnibus (GSM307150-GSM307151) and mapped to the mouse genome (version mm9) using BOWTIE (version 0.12.8).

To profile histone modifications in cardiomyocytes, sequencing reads were mapped to the mouse genome (version mm9) using BOWTIE (version 0.12.8). Uniquely mapped reads with no more than two mismatches were used for binding peak detection. To identify peaks, two peak-calling software were used: MACS (Model-based Analysis of ChIP-seq)6 and SICER (Spatial Clustering Approach for the Identification of ChIP-Enriched Regions)7 Both software detected binding peaks by comparing IP and input control. We used the following parameters: MACS: effective genome size = 1.87e+09, band width = 300, model fold = 5.30, p-value cutoff = 1.00e-05; SICER: windows size =200, gap size = 600, redundancy threshold = 1, FDR = 0.05. Occupancy analysis and differential binding affinity analysis were assessed with the R Bioconductor package DiffBind (v. 1.2.0).8 The final set of binding peaks contained those that were called by both software. Genomic annotation of the peaks identified from the ChIP-seq data were performed using the R Bioconductor package ChIPpeakAnno (v. 2.4.0).9 A summary of the genes associated with H3K79me2-, H3K4me3-, H3K27me3-, and H3K9me3-occupied regions is provided in Table S1. The complete ChIP-seq datasets are available from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nih.gov/geo/), under the accession number GSE45174.

Density profiles of chromatin marks at promoters and gene bodies were computed with HOMER v. 4.5 (Empirical Motif Optimizer).10 Functional annotation of differentially enriched modifications was performed using GREAT.11 Genome browser representations of H3K79me2-, H3K4me3-, H3K27me3-, and H3K9me3-enrichment profiles were obtained with IGV 2.1 visualization software.12 Gene expression microarray analysis pipeline. BeadChips were scanned with the Illumina iScan system. Raw data were background-subtracted and normalized using the quantile normalization method of the R Bioconductor package lumi.13 Normalized data were filtered for genes with significant expression levels compared with negative control beads. Selection for differentially expressed genes was performed on statistical significance (adjusted p-value< 0,001) according to the Illumina t-test error model (limma R Bioconductor package).14 The transcript with the highest median expression was selected to represent the expression of the gene if it was represented with several transcripts. Functional annotation of significant genes identified by microarray analysis was searched by the web-accessible program named Database for Annotation, Visualization and Integrated Discovery (DAVID) version 2009, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) (david.abcc.ncifcrf.gov).15 The complete Illumina gene expression datasets are available from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nih.gov/geo/), under the accession number GSE44829. Correlation of ChIP-seq and gene expression data. Genes were ordered by magnitude of differential mark occupancy, and heatmaps of relative gene expression generated. Genes with higher than average expression were marked in red and those with lower than average expression were marked in green (scale in standard deviations). Scatter plots were produced to see how the expression level agreed with degree of histone modification. The Pearson correlation coefficients between histone modifications and the expression level of genes were calculated using R. Network analysis considering direct and indirect

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relationship among genes was performed using Ingenuity Pathway Analysis software (v. 8.5, Ingenuity® Systems, www.ingenuity.com). Statistical analysis. Data are presented as mean ± SD. P-values were determined by two-tailed t-test. P<0.05 was considered statistically significant. Supplemental References 1. Rodgers LS, Schnurr DC, Broka D, Camenisch TD. An improved protocol for the

isolation and cultivation of embryonic mouse myocytes. Cytotechnology 2009, 59(2): 93-102.

2. Rizzi R, Di Pasquale E, Portararo P, Papait R, Cattaneo P, Latronico MV, et al. Post-

natal cardiomyocytes can generate iPS cells with an enhanced capacity toward cardiomyogenic re-differentation. Cell Death Differ 2012, 19(7): 1162-1174.

3. Catalucci D, Zhang DH, DeSantiago J, Aimond F, Barbara G, Chemin J, et al. Akt

regulates L-type Ca2+ channel activity by modulating Cavalpha1 protein stability. J Cell Biol 2009, 184(6): 923-933.

4. Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995,

7(6): 862-869.

5. Papait R, Cattaneo P, Kunderfranco P, Greco C, Carullo P, Guffanti A, et al. Genome-

wide analysis of histone marks identifying an epigenetic signature of promoters and enhancers underlying cardiac hypertrophy. Proc Natl Acad Sci U S A 2013, 110(50): 20164-20169.

6. Feng J, Liu T, Qin B, Zhang Y, Liu XS. Identifying ChIP-seq enrichment using MACS.

Nat Protoc 2012, 7(9): 1728-1740.

7. Zang C, Schones DE, Zeng C, Cui K, Zhao K, Peng W. A clustering approach for

identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 2009, 25(15): 1952-1958.

8. Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ, et al.

Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 2012, 481(7381): 389-393.

9. Zhu LJ, Gazin C, Lawson ND, Pages H, Lin SM, Lapointe DS, et al. ChIPpeakAnno: a

Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics 2010, 11: 237.

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10. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell 2010, 38(4): 576-589.

11. McLean CY, Bristor D, Hiller M, Clarke SL, Schaar BT, Lowe CB, et al. GREAT

improves functional interpretation of cis-regulatory regions. Nat Biotechnol 2010, 28(5): 495-501.

12. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al.

Integrative genomics viewer. Nat Biotechnol 2011, 29(1): 24-26.

13. Du P, Kibbe WA, Lin SM. lumi: a pipeline for processing Illumina microarray.

Bioinformatics 2008, 24(13): 1547-1548.

14. Smyth GK, Michaud J, Scott HS. Use of within-array replicate spots for assessing

differential expression in microarray experiments. Bioinformatics 2005, 21(9): 2067-2075.

15. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large

gene lists using DAVID bioinformatics resources. Nat Protoc 2009, 4(1): 44-57.

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Fig. S1. A, Cardiomyogenic differentiation of mES cells. Time-course scheme and phase-contrast microscopy images of differentiation of mES cells via the hanging-drop method. B, Relative qRT-PCRs of differentially expressed genes during differentiation of mouse embryonic stem cells towards the cardiac lineage. Pou5F1 and Nanog (pluripotency markers), Eomes, Mesp1 and Brachyury (mesodermal markers), Nkx2-5, Isl1 and Tbx5 (cardiac progenitor markers) and Myh7 and Mhy6 (cardiomyocyte markers). mRNA levels are normalized to 18s, expressed as mean±SD of experimental replicates and plotted as relative mRNA expression. Three independent experiments were performed. C, Western blot quantification and normalization of protein expression levels in mES cells at 0, 1, 2, 5 and 9 days of differentiation. The expression levels are shown as relative to d0 and normalized to that of lamin B (LAM b) for POU5F1, α/βMYH and DOT1L, and to that of unmodified H3 for H3K79me2. Results are presented as mean±SD of three biological replicates. D, Gene expression profile of CM markers confirming the developmental stage used. qRT-PCR was used to assess relative mRNA expression of markers of pluripotency (Pou5F1, Nanog) and of early (Nkx2-5, Gata4, and Myh7) and late (Myh6) stages of heart development in mouse embryonic stem cells (mES) and cardiomyocytes from E10.5, E12.5, E14.5 embryos, 1-day-old pups (P1) and 2-month-old mice (Adult). Bars represent the mean±SD of three biological replicates. E, Western blot quantification and normalization of protein expression levels in mES and CMs from E14.5 (CMe), P1 (CMp) and adult mice (CMa). The expression levels are shown relative to mES and normalized to that of lamin B (LAM b) for DOT1L, and to that of unmodified H3 for H3K79me2. Results are presented as mean±SD of three biological replicates.

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Fig. S2. Principal Component Analysis (PCA) for associations within different samples. Affinity data plotted for H3K79me2 (£), H3K4me3 (�), H3K27me3 (¯) and H3K9me3 (r) in mouse embryonic stem cells (mES, blue), cardiomyocytes from 1-day-old pups (CMp, red) and cardiomyocytes from 2-month-old mice (CMa, green). The graph displays on the x and y axes the first two principal components identified by PCA, revealing potential associations between samples and modifications.

mES vs CMp CMp vs CMa mES vs CMa

H3K

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0 5 10 15 20 25 30em bryonic lethality between im plantat ion and placentat ion

em bryonic lethality before som ite form at ioncom plete em bryonic lethality before som ite form at ion

abnorm al DNA repairincreased sensit iv ity to induced cell death

abnorm al cell cycleincreased cellular sensit iv ity to ionizing radiat ion

abnorm al induced cell deathabnorm al nucleus m orphology

abnorm al chrom osom e num berincreased cellular sensit iv ity to ult raviolet irradiat ion

increased cellular sensit iv ity to gam m a-irradiat ionabnorm al cell cycle checkpoint funct ion

abnorm al cellular replicat ive senescenceabnorm al pluripotent precursor cell m orphology/developm ent

aneuploidyabnorm al inner cell m ass proliferat ion

increased skin tum or incidenceabnorm al inner cell m ass

m am m ary adenocarcinom a

M ouse Phenot ype-log10(Binom ial p value)

Job ID: 20120824-public-Nf3oNdDisplay nam e: user-provided data

0 5 10 15 20 25 30abnorm al m yocardium layer m orphology

abnorm al m uscle fiber m orphologycardiom yopathy

abnorm al m yocardial fiber m orphologypericardial effusion

decreased cardiac m uscle cont ract ilityincreased heart weight

thin intervent ricular septumincreased heart vent ricle size

decreased vent ricle m uscle cont ract ilitycardiac fibrosis

dilated cardiom yopathyabnorm al sarcom ere m orphology

abnorm al fetal cardiom yocyte m orphologyabnorm al fat cell m orphology

m yocardial t rabeculae hypoplasiaabnorm al cell adhesion

increased heart left vent ricle sizethin m yocardium

disorganized m yocardium


Job ID: 20120824-public-2kEm cQDisplay nam e: user-provided data

0 5 10 15 20 25 30 35em bryonic lethality between im plantat ion and placentat ion

com plete em bryonic lethality before som ite form at ionem bryonic lethality before som ite form at ion

increased sensit iv ity to induced cell deathabnorm al cell cycle

abnorm al DNA repairem bryonic lethality before im plantat ion

increased cellular sensit iv ity to ionizing radiat ioncom plete em bryonic lethality before im plantat ion

increased cellular sensit iv ity to ult raviolet irradiat ionabnorm al blastocyst m orphology

increased cellular sensit iv ity to gam m a-irradiat ionabnorm al inner cell m ass

abnorm al chrom osom e num berabnorm al inner cell m ass proliferat ion

abnorm al cellular replicat ive senescenceabnorm al cell cycle checkpoint funct ion

absent som itesabnorm al sperm m idpiece m orphology

m am m ary adenocarcinom a


Job ID: 20120824-public-Lg3VWODisplay nam e: user-provided data

0.00.51.01.52.02.53.03.54.04.55.05.56.0abnorm al sarcom ere m orphology

decreased type II pneum ocyte num berabnorm al zona fasciculata m orphology

sm all adrenal glandsfusion of vertebral bodies

delayed circadian phaseabnorm al Z lines

abnorm al synchondrosisabnorm al presacral vertebrae

ectopic bone form at ionabnorm al m am m ary gland growth during lactat ion

abnorm al thym us cort icom edullary boundary m orphologyincreased growth rate

abnorm al foram en m agnum m orphologyincreased inguinal fat pad weight

decreased lum bar vertebrae num berabsent supraoccipital bone

decreased circulat ing total protein level


Job ID: 20120824-public-QusHPdDisplay nam e: user-provided data

0 2 4 6 8 10 12 14 16com plete em bryonic lethality before som ite form at ion

decreased cardiac m uscle cont ract ilitypericardial effusion

abnorm al m yocardial fiber m orphologydecreased vent ricle m uscle cont ract ility

com plete em bryonic lethality before turning of em bryoabnorm al fetal cardiom yocyte m orphology

em bryonic lethality before turning of em bryocardiom yopathy

abnorm al fat cell m orphologyincreased fat cell size

thin m yocardiumabnorm al sarcom ere m orphology

heart hem orrhagem yocardial t rabeculae hypoplasia

dilated cardiom yopathydisorganized m yocardium

trabecula carnea hypoplasiaincreased lean body m ass

hem opericardium


Job ID: 20120824-public-SChed7Display nam e: user-provided data

0.00.51.01.52.02.53.03.54.04.55.05.56.0failure to gast rulate

abnorm al m idbrain-hindbrain boundary developm entabsent cerebellum

absent spleen m arginal zoneabnorm al cell cycle checkpoint funct ion

decreased cellular sensit iv ity to gam m a-irradiat iondecreased spleen germ inal center size


Job ID: 20120824-public-BxIGdXDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9 10abnorm al DNA repair

increased sensit iv ity to induced cell deathabnorm al t rophoblast giant cells

abnorm al cellular replicat ive senescenceabnorm al chrom osom e num ber

increased cellular sensit iv ity to ionizing radiat ionabnorm al sperm m idpiece m orphology

absent prechordal plateabsent som ites

abnorm al cell cycle checkpoint funct ionabsent notochord

abnorm al ext raem bryonic ectoderm m orphologyabnorm al kerat inocyte apoptosis

abnorm al sperm m itochondrial sheath m orphologydisorganized ext raem bryonic t issue

increased kerat inocyte apoptosisabnorm al prechordal plate m orphology


Job ID: 20120902-public-2.0.2-vtONvMDisplay nam e: user-provided data

0 1 2 3 4 5 6 7abnorm al hair follicle outer root sheath m orphology

failure to gast rulateincreased infarct ion size

abnorm al sarcom ere m orphologyabsent hyoid bone

absent peripheral lym ph nodesthick vent ricular wall


Job ID: 20120902-public-2.0.2-LrrQAODisplay nam e: user-provided data

0 2 4 6 8 10 12increased sensit iv ity to induced cell death

abnorm al DNA repairabnorm al induced cell death

increased cellular sensit iv ity to ionizing radiat ionabnorm al chrom osom e num ber

increased cellular sensit iv ity to gam m a-irradiat ionabnorm al cellular replicat ive senescencedecreased t rophoblast giant cell num ber

abnorm al cell cycle checkpoint funct ionabnorm al inner cell m ass proliferat ion

abnorm al em bryo hatchinganeuploidy

intest inal adenocarcinom aearly cellular replicat ive senescence

B cell/T cell derived lym phom afailure to hatch from the zona pellucida


Job ID: 20120902-public-2.0.2-2ozqYXDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9 10 11failure to gast rulate

altered response to m yocardial infarct ionincreased infarct ion size

com plete em bryonic lethality before turning of em bryodecreased m acrophage nit ric oxide product ion

thin intervent ricular septumabnorm al sarcom ere m orphology

abnorm al hair follicle outer root sheath m orphologyabnorm al circulat ing noradrenaline level

absent cerebellumblindness

abnorm al corneal st rom a m orphologyabsent outer hair cell stereocilia

decreased circulat ing noradrenaline leveldisorganized m yocardium

abnorm al skeletal m uscle fiber type rat iothick vent ricular wall

prolonged QT intervalabnorm al cardiac st roke volum e

abnorm al QT interval


Job ID: 20120902-public-2.0.2-tSqeV2Display nam e: user-provided data

0 2 4 6 8 10 12 14 16em bryonic lethality before som ite form at ion

com plete em bryonic lethality before som ite form at ionsm all hair follicles

failure to gast rulateabnorm al fat cell m orphology

com plete em bryonic lethality before turning of em bryothin m yocardium

increased fat cell sizeabnorm al DNA repair

absent m esodermabnorm al m itochondrial physiology

altered response to m yocardial infarct ionabnorm al white fat cell size

abnorm al white fat cell m orphologyabnorm al white adipose t issue physiology

fusion of vertebral bodiesabnorm al ext raem bryonic ectoderm m orphology

abnorm al Z linesabnorm al exercise endurance

abnorm al sarcom ere m orphology


Job ID: 20120902-public-2.0.2-JJoTRaDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9 10 11 12 13abnorm al m uscle fiber m orphology

pericardial effusionincreased heart weightabnorm al heart weight

abnorm al fat cell m orphologyabnorm al neut rophil m orphology

thin intervent ricular septumsm all scala m edia

abnorm al m yocardial fiber m orphologyabnorm al circulat ing free fat ty acids level

abnorm al free fat ty acids levelincreased lean body m ass

thin vent ricular wallcardiac hypert rophy

abnorm al wound healingabnorm al cell m igrat ion

rib bifurcat ionabnorm al fetal cardiom yocyte m orphology

enlarged kidneyabnorm al long bone hypert rophic chondrocyte zone


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0 1 2 3 4 5 6 7 8 9 10 11abnorm al som at ic nervous system physiology

abnorm al m iddle ear m orphologyabnorm al m iddle ear ossicle m orphologyabnorm al basioccipital bone m orphology

abnorm al squam osal bone m orphologyincreased neuron num ber

abnorm al spinal cord dorsal horn m orphologyabnorm al cochlear hair cell physiology

abnorm al spinal cord interneuron m orphologyabsent outer hair cell stereocilia

abnorm al rhom bom ere m orphologyabnorm al neurom ere m orphology

thin ret inal ganglion layerabnorm al roof plate m orphology

abnorm al olfactory cortex m orphologyabnorm al hair cell physiology

abnorm al cerebellum anterior verm is m orphologydecreased renal tubule num ber

abnorm al tem poral bone m orphologyabsent pinna reflex


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0 1 2 3 4 5 6 7 8 9increased heart weight

pericardial effusionabnorm al circulat ing free fat ty acids level

abnorm al free fat ty acids levelthin intervent ricular septum

abnorm al ret iculocyte m orphologycardiom yopathy

abnorm al T wavedilated cardiom yopathy

ret iculocytosisaltered response to m yocardial infarct ion

abnorm al hem atopoiet ic stem cell physiologydecreased circulat ing free fat ty acid level

abnorm al cell adhesionincreased liver iron level

increased circulat ing free fat ty acid levelsm all inner m edullary pyram id

increased angiogenesisthick vent ricular wall

abnorm al fetal cardiom yocyte m orphology


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0 1 2 3 4 5 6 7failure to gast rulate

absent pituitary glandabnorm al basioccipital bone m orphology

absent cerebellumabnorm al cerebellum anterior verm is m orphology

abnorm al presphenoid bone m orphologyabnorm al auditory cortex m orphology

decreased neurot ransm it ter releaseabnorm al palat ine shelf

abnorm al m idbrain-hindbrain boundary developm entabnorm al cerebellum anterior lobe m orphology

abnorm al m axillary shelfabnorm al cerebellar hem isphere m orphology

abnorm al Mullerian duct m orphologydecreased inflam m atory response

absent uterussm all adenohypophysis

abnorm al excitatory postsynapt ic current am plitudeabsent ureter

alcohol preference


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0 1 2 3 4 5 6 7 8 9 10 11 12abnorm al neuron different iat ion

abnorm al cranial nerve m orphologyabnorm al spinal cord grey m at ter m orphology

abnorm al lim bic system m orphologyabnorm al brain vent ricle/choroid plexus m orphology

abnorm al forebrain developm entabnorm al tem poral lobe m orphology

abnorm al spinal cord dorsal horn m orphologyabnorm al brain com m issure m orphology

abnorm al hippocam pus m orphologyabnorm al spinal cord m orphology

abnorm al diencephalon m orphologyabnorm al rhom bom ere m orphology

abnorm al nervous system t ractabnorm al spinal cord interneuron m orphology

abnorm al GABAergic neuron m orphologyabsent cerebellum

abnorm al sensory ganglion m orphologyabnorm al neurom ere m orphology

abnorm al choroid plexus m orphology


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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0rib bifurcat ion

abnorm al response to vitam insabnorm al vertebral body m orphology

abnorm al splenic cell rat ioabnorm al em bryonic epiblast m orphology

absent pituitary glandabnorm al proxim al-distal axis pat terning

abnorm al cutaneous elast ic fiber m orphologyabnorm al lens epithelium m orphology


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0 2 4 6 8 10 12 14 16em bryonic lethality between im plantat ion and placentat ion

com plete em bryonic lethality before som ite form at ionem bryonic lethality before som ite form at ion

abnorm al cell cycledecreased erythrocyte cell num berabnorm al erythrocyte cell num ber

abnorm al cell adhesionincreased sensit iv ity to induced cell death

abnorm al placenta sizeincreased birth weight

absent m esodermsm all placenta

abnorm al kerat inocyte m orphologyincreased birth body size

increased cellular sensit iv ity to ult raviolet irradiat ioncardiom yopathy

increased heart weightwavy neural tube

abnorm al hem atopoiet ic stem cell physiologyabsent am nion


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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0m alocclusion

thin apical ectoderm al ridgeabnorm al vest ibular saccular m acula m orphology

abnorm al squam osal bone m orphologyabnorm al spinal cord dorsal horn m orphology

absent cerebellumabnorm al hyoid bone greater horn m orphology

abnorm al neurom ere m orphologyabnorm al rhom bom ere m orphology

absent outer hair cell stereociliaabnorm al brain ependym a m orphology

abnorm al tectum m orphologyabnorm al choroid plexus m orphology

abnorm al m idbrain roof plate m orphologylowered ear posit ion

thin ret inal ganglion layerabnorm al stom ach enteroendocrine cell m orphology

uterus hypoplasiaabnorm al corpora quadrigem ina m orphology

absent pinna reflex


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0 1 2 3 4 5 6 7 8 9increased fat cell size

pericardial effusionfetal growth retardat ion

increased heart left vent ricle sizethin m yocardium

increased cellular sensit iv ity to ult raviolet irradiat ionincreased heart weight

cardiom yopathyabnorm al fat cell m orphology

com plete em bryonic lethality before turning of em bryoincreased ovary tum or incidence

decreased white adipose t issue am ountabnorm al neural plate m orphology

abnorm al heart left vent ricle sizem yocardial t rabeculae hypoplasia

increased lean body m assabsent som ites

increased birth weightt rabecula carnea hypoplasia

dilated cardiom yopathy


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0 1 2 3 4 5 6 7abnorm al Mullerian duct m orphology

absent cerebellumabnorm al palate bone m orphology

abnorm al presphenoid bone m orphologyabnorm al palat ine shelfsm all adenohypophysis

spinal hem orrhageabnorm al m idbrain-hindbrain boundary developm ent

Wolffian duct degenerat ionabnorm al sclerotom eincreased rib num ber

m alocclusionabnorm al blood-brain barrier funct ion

absent epididym isincreased t idal volum e

abnorm al choroid plexus m orphologyabnorm al basioccipital bone m orphology

decreased lactot roph cell num berfailure of Mullerian duct regression

delayed kidney developm ent


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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5decreased white adipose t issue am ount

sm all scala m ediadecreased suscept ibility to hepat ic steatosis

tachypneaabnorm al white fat cell m orphology

abnorm al gluconeogenesisB cell derived lym phom a

abnorm al vascular perm eabilityabsent t rophoblast giant cells

abnorm al white fat cell sizeincreased cardiovascular system tum or incidence

increased param etrial fat pad weightincreased inguinal fat pad weight

absent som iteshem opericardium

heart hem orrhageincreased kidney tum or incidence

abnorm al phaeom elanin content


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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5increased infarct ion size

abnorm al cell adhesion


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GO Mouse Phenotype

S3

mES vs CMp CMp vs CMa

0 2 4 6 8 10 12 14 16 18 20 22 24 26regionalizat ion

pat tern specificat ion processcell fate com m itm ent

anterior/posterior pat tern specificat ioncent ral nervous system neuron different iat ion

em bryonic organ m orphogenesisdorsal spinal cord developm ent

stem cell different iat ionneuron fate com m itm ent

ant igen processing and presentat ion of pept ide ant igen via MHC class Ihindbrain developm ent

cell fate specificat ionm esoderm m orphogenesis

cell different iat ion in spinal cordm esoderm developm ent

ear m orphogenesiskidney m esenchym e developm entm esonephric tubule developm ent

forebrain generat ion of neuronshindbrain m orphogenesis

GO Biological Process-log10(Binom ial p value)

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0 5 10 15 20 25 30 35 40 45 50 55 60RNA processing

response to DNA dam age st im ulusDNA m etabolic process

ncRNA m etabolic processt ranslat ion

protein foldingncRNA processing

DNA repairM phase

ribosom e biogenesisM phase of m itot ic cell cycle

m itosisorganelle fission

cellular com ponent biogenesis at cellular levelm RNA m etabolic process

ribonucleoprotein com plex biogenesisRNA splicing

protein m odificat ion by sm all protein conjugat ion or rem ovalpost t ranscript ional regulat ion of gene expression

rRNA m etabolic process


Job ID: 20120824-public-Lg3VWODisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9act in filam ent organizat ioncardiac m uscle cont ract ion

m yofibril assem blyactom yosin st ructure organizat ion

regulat ion of generat ion of precursor m etabolites and energyregulat ion of lipid catabolic process

heart processRho protein signal t ransduct ion

sarcom ere organizat ionrost rocaudal neural tube pat terning

regulat ion of st riated m uscle cont ract ioncardiac m yofibril assem bly

m yoblast cell fate com m itm entm yoblast different iat ion

regulat ion of insulin receptor signaling pathwaym idbrain-hindbrain boundary developm ent

act in filam ent -based m ovem entalanine t ransport

m ucosal-associated lym phoid t issue developm entneut ral am ino acid t ransport


Job ID: 20120824-public-QusHPdDisplay nam e: user-provided data

0 5 10 15 20 25 30 35DNA m etabolic process

ncRNA m etabolic processt ranslat ion

M phaseDNA repair

ncRNA processingM phase of m itot ic cell cycle

protein foldingm itosis

organelle fissionDNA replicat ion

ribosom e biogenesistRNA m etabolic process

ribonucleoprotein com plex biogenesiscellular com ponent biogenesis at cellular level

rRNA m etabolic processrRNA processing

m acrom olecule m ethylat ionDNA recom binat ion

m ethylat ion


Job ID: 20120902-public-2.0.2-2ozqYXDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8face m orphogenesis

head m orphogenesiscent ral nervous system vasculogenesis

regulat ion of generat ion of precursor m etabolites and energyposit ive regulat ion of receptor act ivity

est rogen receptor signaling pathwayact in filam ent organizat ion

body m orphogenesisregulat ion of gene silencing

m aintenance of protein locat ion in cellface developm ent

m aintenance of locat ion in cellexplorat ion behavior

cytoskeletal anchoring at plasm a m em braneregulat ion of ARF protein signal t ransduct ion

carbohydrate t ransportestablishm ent of endothelial barrier

cerebellar cortex developm entposit ive regulat ion of glycogen biosynthet ic process

regulat ion of glycogen biosynthet ic process


Job ID: 20120902-public-2.0.2-tSqeV2Display nam e: user-provided data

0 2 4 6 8 10 12 14act in filam ent -based process

act in cytoskeleton organizat ioncellular response to pept ide horm one st im ulus

negat ive regulat ion of t ransferase act ivitynegat ive regulat ion of kinase act ivity

cellular response to insulin st im ulusact in filam ent organizat ion

regulat ion of gene expression, epigenet icregulat ion of generat ion of precursor m etabolites and energy

insulin receptor signaling pathwayregulat ion of gene expression by genet ic im print ing

JAK-STAT cascade involved in growth horm one signaling pathwaynegat ive regulat ion of protein kinase act ivity

regulat ion of m yotube different iat iongenet ic im print ing

gene silencingregulat ion of st riated m uscle cont ract ion

pept idyl-lysine m odificat ionheart t rabecular m orphogenesis

heart t rabecula form at ion


Job ID: 20120902-public-2.0.2-LrndOHDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9 10 11 12 13cerebellar cortex developm ent

floor plate developm entfloor plate m orphogenesis

regulat ion of cerebellar granule cell precursor proliferat ionm etencephalon developm ent

posit ive regulat ion of cerebellar granule cell precursor proliferat ioncerebellar cortex m orphogenesis

vent ral m idline developm entcerebellum developm ent

cerebellum m orphogenesisnegat ive regulat ion of epiderm is developm ent

hindbrain m orphogenesiscerebellar Purkinje cell-granule cell precursor cell signaling involved in regulat ion of granule cell precursor cell proliferat ion

epithelial cell developm entrenal vesicle m orphogenesis

m esoderm m orphogenesism etanephric renal vesicle m orphogenesis

head m orphogenesishead developm ent

body m orphogenesis


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0 2 4 6 8 10 12 14 16m acrom olecule catabolic process

cellular m acrom olecule catabolic processprotein catabolic process

act in filam ent -based processact in cytoskeleton organizat ion

cellular protein catabolic processproteolysis involved in cellular protein catabolic process

m odificat ion-dependent protein catabolic processpost t ranscript ional regulat ion of gene expression

pept idyl-lysine m odificat ionregulat ion of gene expression, epigenet ic

nuclear-t ranscribed m RNA catabolic process, nonsense-m ediated decayregulat ion of t ranslat ion

erythrocyte hom eostasiserythrocyte different iat ion

hom eostasis of num ber of cellsprotein acylat ion

nuclear-t ranscribed m RNA catabolic processinternal pept idyl-lysine acetylat ion

pept idyl-lysine acetylat ion


Job ID: 20120902-public-2.0.2-oU7a0jDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9head m orphogenesisbody m orphogenesisface m orphogenesis

form at ion of prim ary germ layeroviduct developm ent

ant igen processing and presentat ion of pept ide ant igen via MHC class Im etanephric nephron developm ent

m esoderm m orphogenesisnephron developm ent

dorsal/vent ral pat tern form at ionkidney m esenchym e developm ent

cell different iat ion in spinal cordbranching involved in ureteric bud m orphogenesis

head developm entm esoderm developm ent

cell fate specificat ionneuron fate com m itm ent

est rogen receptor signaling pathwayforelim b m orphogenesis

regulat ion of kidney developm ent


Job ID: 20120902-public-2.0.2-jhJj5YDisplay nam e: user-provided data

mES vs CMa

H3K

9me3

H

3K27

me3

H3K

4me3

H

3K79

me2

0.00.51.01.52.02.53.03.54.04.55.05.56.0regulat ion of cholesterol t ransport

m idbrain-hindbrain boundary developm entt rophoblast giant cell different iat ionrost rocaudal neural tube pat terning

t ranscript ion from RNA polym erase I prom oternucleot ide-excision repair

regulat ion of lipid t ransportposit ive regulat ion of lipid t ransport

negat ive regulat ion of m yeloid cell different iat ionestablishm ent of endothelial barrier

notochord developm ent


Job ID: 20120824-public-BxIGdXDisplay nam e: user-provided data

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0sterol biosynthet ic process

cholesterol biosynthet ic processglial cell m igrat ion


Job ID: 20120902-public-2.0.2-m xydErDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9 10pept idyl-lysine m odificat ion

protein acetylat ionpept idyl-lysine acetylat ion

internal pept idyl-lysine acetylat ionregulat ion of gene expression, epigenet ic

protein acylat ioninternal protein am ino acid acetylat ion

histone acetylat ionact in filam ent organizat ionhistone lysine m ethylat ion

m etencephalon developm entcellular response to insulin st im ulus

protein m ethylat ionregulat ion of generat ion of precursor m etabolites and energy

cerebellum developm entprotein tet ram erizat ionhistone H3 acetylat ion

regulat ion of horm one biosynthet ic processposit ive regulat ion of horm one biosynthet ic process

cerebellar cortex developm ent


Job ID: 20120902-public-2.0.2-JJoTRaDisplay nam e: user-provided data

0 2 4 6 8 10 12 14 16 18pat tern specificat ion process

regionalizat ioncell fate com m itm entneuron different iat ion

anterior/posterior pat tern specificat ionposit ive regulat ion of t ranscript ion from RNA polym erase II prom oter

cell m orphogenesis involved in different iat ionregulat ion of cell developm ent

em bryonic m orphogenesisneuron fate com m itm ent

forebrain developm entcent ral nervous system neuron different iat ion

stem cell different iat ionregulat ion of nervous system developm ent

negat ive regulat ion of developm ental processem bryonic organ developm ent

cell m orphogenesis involved in neuron different iat ionregulat ion of neurogenesis

signal t ransduct ion involved in regulat ion of gene expressionnegat ive regulat ion of cell different iat ion


Job ID: 20120902-public-2.0.2-wVAuCADisplay nam e: user-provided data

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0m etencephalon developm ent

bone rem odelingform at ion of prim ary germ layer

cerebellum developm entt issue rem odeling

fat ty acid elongat ion, m onounsaturated fat ty acidcerebellar cortex developm ent

coronary artery m orphogenesisdorsal/vent ral axis specificat ion

stem cell divisioncardiac vascular sm ooth m uscle cell different iat ion

non-canonical Wnt receptor signaling pathwaycell-cell junct ion organizat ion

endoderm form at ionskin developm ent

posit ive regulat ion of BMP signaling pathwaycerebellum m orphogenesis

regulat ion of cardioblast different iat ionposit ive regulat ion of canonical Wnt receptor signaling pathway

som at ic stem cell division


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0 1 2 3 4 5 6 7 8 9covalent chrom at in m odificat ion

regulat ion of t ranslat ioncell-subst rate adhesion

regulat ion of gene expression, epigenet icpept idyl-lysine m odificat ion

Golgi organizat ionprotein m odificat ion by sm all protein rem oval

genet ic im print ingendosom e t ransport

gene silencingprotein acetylat ion

protein deubiquit inat ionhistone acetylat ion

internal pept idyl-lysine acetylat ionpept idyl-lysine acetylat ion

protein acylat ionregulat ion of protein im port into nucleus, t ranslocat ion

internal protein am ino acid acetylat ionfloor plate m orphogenesis

cellular senescence


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0 1 2 3 4 5 6 7m acrom olecule m ethylat ion

body m orphogenesishead m orphogenesis

endoderm developm entface m orphogenesis

regulat ion of gene expression, epigenet ichistone lysine m ethylat ion

protein m ethylat ionpept idyl-lysine m odificat ion

histone m ethylat ionpept idyl-lysine acetylat ion

internal pept idyl-lysine acetylat ion


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0 5 10 15 20 25 30 35protein m odificat ion by sm all protein conjugat ion or rem oval

int racellular t ransportcytoskeleton organizat ion

protein ubiquit inat ionprotein m odificat ion by sm all protein conjugat ion

m RNA m etabolic processm acrom olecule catabolic process

post t ranscript ional regulat ion of gene expressionprotein catabolic process

cellular m acrom olecule catabolic processRNA splicing

int racellular protein t ransportact in cytoskeleton organizat ion

m RNA processingact in filam ent -based process

cellular protein catabolic processregulat ion of t ranslat ion

proteolysis involved in cellular protein catabolic processhistone m odificat ion

covalent chrom at in m odificat ion


Job ID: 20120824-public-SChed7Display nam e: user-provided data

0 2 4 6 8 10 12 14 16 18ncRNA m etabolic process

t ranslat ionncRNA processing

pept idyl-lysine m odificat ionribosom e biogenesis

regulat ion of t ranslat ionDNA replicat ion

protein acetylat ioncellular com ponent biogenesis at cellular level

ribonucleoprotein com plex biogenesism acrom olecule m ethylat ion

protein acylat ionpept idyl-lysine acetylat ion

rRNA m etabolic processinternal pept idyl-lysine acetylat ion

histone acetylat ioninternal protein am ino acid acetylat ion

rRNA processingregulat ion of gene expression, epigenet ic

t ranslat ional init iat ion


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0 5 10 15 20 25 30act in filam ent -based process

cytoskeleton organizat ionact in cytoskeleton organizat ion

act in filam ent organizat ionm uscle cell developm ent

m yofibril assem blyst riated m uscle cell developm entcardiac m uscle cell developm ent

cardiac cell developm entactom yosin st ructure organizat ion

cardiac m yofibril assem blycardiac m uscle cont ract ion

cell junct ion organizat ionsarcom ere organizat ion

heart processregulat ion of m uscle cont ract ion

regulat ion of generat ion of precursor m etabolites and energycell junct ion assem bly

regulat ion of m uscle system processresponse to react ive oxygen species


Job ID: 20120824-public-2kEm cQDisplay nam e: user-provided data 0 5 10 15 20 25 30 35 40 45 50 55

RNA processingncRNA m etabolic process

ncRNA processingribosom e biogenesis

t ranslat ionrRNA m etabolic process

cellular com ponent biogenesis at cellular levelrRNA processing

ribonucleoprotein com plex biogenesisresponse to DNA dam age st im ulus

protein foldingM phase

cell cycle phaseDNA repair

M phase of m itot ic cell cycleorganelle fission

m itosisRNA splicing

covalent chrom at in m odificat ionchrom osom e segregat ion


Job ID: 20120824-public-Nf3oNdDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9regulat ion of ret inoic acid receptor signaling pathway

head m orphogenesisface m orphogenesisfibroblast m igrat ionm yofibril assem bly

body m orphogenesisactom yosin st ructure organizat ion

em bryonic arm m orphogenesissarcom ere organizat ion

regulat ion of branching involved in lung m orphogenesiscardiac m yofibril assem bly

regulat ion of cardioblast different iat ionnegat ive regulat ion of cytokine secret ion involved in im m une response

ant igen processing and presentat ion via MHC class Ib


Job ID: 20120902-public-2.0.2-LrrQAODisplay nam e: user-provided data

0 2 4 6 8 10 12 14act in filam ent -based process

act in cytoskeleton organizat ionm uscle cell developm ent

act in filam ent organizat ionregulat ion of lipid m etabolic process

t rabecula form at ioncell junct ion organizat ion

regulat ion of histone m odificat ionresponse to react ive oxygen species

negat ive regulat ion of kinase act ivityst riated m uscle cell developm ent

GTP catabolic processnegat ive regulat ion of t ransferase act ivity

t rabecula m orphogenesisregulat ion of t ransform ing growth factor beta receptor signaling pathway

heart t rabecula form at iont ranscript ion from RNA polym erase II prom oter

axis specificat ioncell junct ion assem bly

regulat ion of pept ide secret ion


Job ID: 20120902-public-2.0.2-m jNbznDisplay nam e: user-provided data

0 1 2 3 4 5 6 7 8 9post t ranscript ional regulat ion of gene expression

pept idyl-lysine m odificat ioninduct ion of program m ed cell death

induct ion of apoptosiscovalent chrom at in m odificat ion

histone m odificat ionprotein acylat ion

protein acetylat ionpept idyl-lysine acetylat ion

internal pept idyl-lysine acetylat ionm acrom olecule m ethylat ion

internal protein am ino acid acetylat iont ranscript ion init iat ion, DNA-dependent

regulat ion of gene expression, epigenet icregulat ion of horm one biosynthet ic process

regulat ion of t ranslat ionposit ive regulat ion of steroid horm one biosynthet ic process

DNA dam age response, signal t ransduct ion result ing in induct ion of apoptosishistone acetylat ion

leukocyte act ivat ion involved in im m une response


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0 2 4 6 8 10 12 14regionalizat ion

anterior/posterior pat tern specificat ionaxis specificat ion

neuron fate com m itm entstem cell different iat ionforelim b m orphogenesis

dorsal spinal cord developm entm esoderm m orphogenesis

m esonephric tubule developm entm esoderm developm ent

negat ive regulat ion of m uscle cell different iat ioncell different iat ion in spinal cord

posit ive regulat ion of BMP signaling pathwaykidney rudim ent form at ion

em bryonic forelim b m orphogenesisdichotom ous subdivision of term inal units involved in ureteric bud branching

rost rocaudal neural tube pat terningm esonephric tubule form at ion

em bryonic pat tern specificat ionm etanephric nephron developm ent


Job ID: 20120902-public-2.0.2-ZK1u42Display nam e: user-provided data

GO Biological Process

Fig. S3. Functional and molecular characterization of histone methylation marks in cardiomyocytes during differentiation. Functional annotation of differentially enriched modifications was performed using GREAT. The top 5 over-represented categories belonging to Gene Ontology (GO) Biological Process and GO Mouse Phenotype are shown. The x axis (in logarithmic scale) gives the binomial raw (uncorrelated) P-values. Results from three different comparative analyses are shown for each mark (H3K79me2, H3K4me3, H3K27me3 and H3K9me3): mES vs. CMp (left), mES vs. CMa (middle), and CMp vs. CMa (right). Blue histograms represent mES cells, red histograms represent CMp, and green histograms represent CMa.

PROM Pou5F1

A B C PROM

Nanog

A1 B C A2 PROM

Gata4

A B C PROM

Nkx2-5

A B PROM

Myh7

A B C PROM

Myh6

A B C PROM

TnnI3

A B C

ChIP enrichm

ent rela/

ve to

inpu

t Ch

IP enrichm

ent rela/

ve to

inpu

t normalize

d to H3

0.0

1.0

2.0

3.0

4.0

5.0 IgG

mES CMp CMa

0

5

10

15

20

25 H3

0.0

1.0

2.0

3.0

4.0

H3K79me2

0.0

0.5

1.0

1.5

2.0

2.5

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S4 A

B

Fig. S4. Histone methylation enrichment and mRNA expression at key cardiac differentiation genes. A, Relative mRNA expression was assessed through qRT-PCR in mouse embryonic stem cells (mES), post-natal cardiomyocytes (CMp) and adult cardiomyocytes (CMa). mRNA levels for Nanog, Pou5F1, Gata4, Nkx2-5, Myh7, Myh6, and TnnI3 are normalized to 18s and expressed as the mean±SD of three independent experiments. **, P<0.01 vs mES; ##, P<0.01 vs CMp. B, ChIP assay of H3K79me2, H3K4me3, H3K27me3 and H3K9me3 binding to Nanog, Pou5F1, Gata4, Nkx2.5, Myh7, Myh6 and TnnI3. Levels were determined by qPCR and are expressed as fold change to the input and relative to H3. Three different regions were analysed for each gene locus: −1000bp/−500bp from TSS (_A), +500bp/+1000bp from TSS (_B), and +3500bp/+4000bp from TSS (_C). Data are the result of three independent experiments. The enrichment levels for IgG (negative control) and the unmodified H3 relative to input are given as controls.

S5

Fig. S5. Gene expression profile of cardiomyocytes at different stages of differentiation. Illumina mRNA gene expression datasets are presented for three biological replicates of mouse embryonic stem cells (mES), post-natal cardiomyocytes (CMp) and adult cardiomyocytes (CMa). Data were subjected to two-way hierarchical clustering (centred correlation distance, centroid linkage); the heatmap was created using the limma R Bioconductor package. Samples with higher than average expression are represented in red; samples with lower than average expression are represented in green.

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S6

Fig S6. Network analysis of direct relationships among genes acquiring H3K4me3 and indirect relationships among genes acquiring H3K27me3 with transcriptional potential. The analyses were performed using Ingenuity Pathway Analysis software (v. 8.5, Ingenuity® Systems, www.ingenuity.com). Networks are displayed graphically as nodes and edges. Distinct shapes indicate distinct gene functions. Results from three different comparative analyses are shown: mES vs. CMp (top), mES vs. CMa (middle) and CMp vs. CMa (bottom). Green indicates gene downregulation and red indicates gene upregulation in CMp (mES vs. CMp), CMa (mES vs. CMa) and CMa (CMp vs. CMa), with a more intense shade indicating a greater change.

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Fig S7. mRNA levels of Nestin, Gata6, Sox17, Isl1, Gata4, Nkx2-5, Mef2C, Myl4 and Myl7 in undifferentiated (day 0) mouse embryonic stem cells and after days 1, 2, 3, 4, 5, 7 and 9 of differentiation in control (sh-Ctrl, grey line) and Dot1L-knockdown (sh-Dot1L, black line) cells. Levels are normalized to 18s or GAPDH, expressed as the mean±SD of experimental replicates and plotted as relative mRNA expression. Three independent experiments were performed.

7

SUPPLEMENTAL FIGURE LEGENDS

Fig. S1. A, Cardiomyogenic differentiation of mES cells. Time-course scheme and phase-contrast microscopy images of differentiation of mES cells via the hanging-drop method. B, Relative qRT-PCRs of differentially expressed genes during differentiation of mouse embryonic stem cells towards the cardiac lineage. Pou5F1 and Nanog (pluripotency markers), Eomes, Mesp1 and Brachyury (mesodermal markers), Nkx2-5, Isl1, and Tbx5 (cardiac progenitor markers), and Myh7 and Mhy6 (cardiomyocyte markers). mRNA levels

are normalized to 18s, expressed as meanSD of experimental replicates, and plotted as relative mRNA expression. Three independent experiments were performed. C, Western blot quantification and normalization of protein expression levels in mES cells at 0, 1, 2, 5, and 9 days of differentiation. The expression levels are shown as relative to day 0 and

normalized to that of lamin B (LAM b) for POU5F1, /MYH, and DOT1L, and to that of

unmodified H3 for H3K79me2. Results are presented as meanSD of three biological replicates. D, Gene expression profile of CM markers confirming the developmental stage used. qRT-PCR was used to assess relative mRNA expression of markers of pluripotency (Pou5F1, Nanog) and of early (Nkx2-5, Gata4, and Myh7) and late (Myh6) stages of heart development in mouse embryonic stem cells (mES) and cardiomyocytes from E10.5, E12.5, and E14.5 embryos, 1-day-old pups (P1), and 2-month-old mice (Adult). Bars

represent the meanSD of three biological replicates. E, Western blot quantification and normalization of protein expression levels in mES and CMs from E14.5 (CMe), P1 (CMp), and adult (CMa) mice. The expression levels are shown relative to mES and normalized to that of lamin B (LAM b) for DOT1L, and to that of unmodified H3 for H3K79me2. Results

are presented as meanSD of three biological replicates.

Fig. S2. Principal Component Analysis (PCA) for associations within different samples. Affinity data plotted for H3K79me2 (), H3K4me3 (), H3K27me3 (), and H3K9me3 () in mouse embryonic stem cells (mES, blue), cardiomyocytes from 1-day-old pups (CMp, red), and cardiomyocytes from 2-month-old mice (CMa, green). The graph displays on the x and y axes the first two principal components identified by PCA, revealing potential associations between samples and modifications.

Fig. S3. Functional and molecular characterization of histone methylation marks in cardiomyocytes during differentiation. Functional annotation of differentially enriched modifications was performed using GREAT. The top 5 over-represented categories belonging to Gene Ontology (GO) Biological Process and GO Mouse Phenotype are shown. The x axis (in logarithmic scale) gives the binomial raw (uncorrelated) P-values. Results from three different comparative analyses are shown for each mark (H3K79me2, H3K4me3, H3K27me3, and H3K9me3): mES vs. CMp (left), mES vs. CMa (middle), and CMp vs. CMa (right). Blue histograms represent mES cells, red histograms represent CMp, and green histograms represent CMa.

Fig. S4. Histone methylation enrichment and mRNA expression at key cardiac differentiation genes. A, Relative mRNA expression was assessed through qRT-PCR in mouse embryonic stem cells (mES), post-natal cardiomyocytes (CMp), and adult cardiomyocytes (CMa). mRNA levels for Nanog, Pou5F1, Gata4, Nkx2-5, Myh7, Myh6,

and TnnI3 are normalized to 18s and expressed as the meanSD of three independent experiments. **, P <0.01 vs mES; ##, P <0.01 vs CMp. B, ChIP assay of H3K79me2, H3K4me3, H3K27me3, and H3K9me3 binding to Nanog, Pou5F1, Gata4, Nkx2.5, Myh7,

8

Myh6, and TnnI3. Levels were determined by qPCR and are expressed as fold change to the input and relative to H3. Three different regions were analyzed for each gene locus: −1000bp/−500bp from TSS (_A), +500bp/+1000bp from TSS (_B), and +3500bp/+4000bp from TSS (_C). Data are the result of three independent experiments. The enrichment levels for IgG (negative control) and the unmodified H3 relative to input are given as controls.

Fig. S5. Gene expression profile of cardiomyocytes at different stages of differentiation. Illumina mRNA gene expression datasets are presented for three biological replicates of mouse embryonic stem cells (mES), post-natal cardiomyocytes (CMp), and adult cardiomyocytes (CMa). Data were subjected to two-way hierarchical clustering (centered correlation distance, centroid linkage); the heatmap was created using the limma R Bioconductor package. Samples with higher than average expression are represented in red; samples with lower than average expression are represented in green.

Fig S6. Network analysis of direct relationships among genes acquiring H3K4me3 and indirect relationships among genes acquiring H3K27me3 with transcriptional potential. The analyses were performed using Ingenuity Pathway Analysis software (v. 8.5, Ingenuity® Systems, www.ingenuity.com). Networks are displayed graphically as nodes and edges. Distinct shapes indicate distinct gene functions. Results from three different comparative analyses are shown: mES vs. CMp (top), mES vs. CMa (middle), and CMp vs. CMa (bottom). Green indicates gene downregulation and red indicates gene upregulation in CMp (mES vs. CMp), CMa (mES vs. CMa), and CMa (CMp vs. CMa), with a more intense shade indicating a greater change. Fig S7. mRNA levels of Nestin, Gata6, Sox17, Isl1, Gata4, Nkx2-5, Mef2C, Myl4, and Myl7 in undifferentiated (day 0) mouse embryonic stem cells and after days 1, 2, 3, 4, 5, 7 and 9 of differentiation in control (sh-Ctrl, grey line) and Dot1L-knockdown (sh-Dot1L, black line)

cells. Levels are normalized to 18s or Gapdh, expressed as the meanSD of experimental replicates, and plotted as relative mRNA expression. Three independent experiments were performed.

SUPPLEMENTARY TABLE LEGENDS Table 1. Differential histone modification sites. Differential histone modification sites were found comparing mES vs. CMp, mES vs. CMa, and CMp vs. CMa. mES, mouse embryonic stem cells; CMp, cardiomyocytes from 1-day-old mouse pups; CMa, cardiomyocytes from 2-month-old adult mice.

Table 2. Significantly modulated genes. Illumina microarray was used to assess differential gene expression between mES, CMp and CMa. mES, mouse embryonic stem cells; CMp, cardiomyocytes from 1-day-old mouse pups; CMa, cardiomyocytes from 2-month-old adult mice.

Documents

SUPPLEMENTAL MATERIAL DOT1L-mediated H3K79me2 … · of differential mark occupancy, and heatmaps of relative gene expression generated. Genes with higher than average expression