TheThe ChIPChIP AssayAssay ExpertsExperts™™

www.genpathway.com Genpathway - The ChIP Assay Experts

Genpathway: The leading provider of ChIP and DNA assays, expertise & services

Combining Genpathway’s assays with Affymetrix tiling arrays provides researchers a complete ChIP-on-Chip and Methylated DNA IP-on-Chip solution Where Can You Apply Genpathway’s Technology?

Æ Target Identification Æ Target Validation Æ Compound Efficacy Æ Mechanism of Action Æ Pharmacogenomics Æ Clinical Diagnostics

Targets Compound testing Preclinical Clinical

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s o Screening Pharm Pharm Tgx c

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R PK D Validation Tox How can Genpathway’s novel assays move your programs forward?

Æ Discover transcription factor and co-regulator binding sites, plus the genes they regulate, across the entire Æ Profile the in your cells, but at the DNA level, which gives highly reproducible results Æ Quantify changes in gene transcription and regulation at any timeframe, even within minutes Æ Discover and quantify epigenetic changes including DNA methylation and modifications Æ Identify new genes associated with drug treatment or disease mechanisms Genpathway’s ChIP Assays: FactorPath, TranscriptionPath & Methylated DNA

Genpathway’s optimized procedures give maximum sensitivity and minimal background binding. Genpathway’s analysis modes: Highly informative and reproducible

Combining assays and analysis modes provides additional information. Genpathway’s ChIP-on-Chip analysis: a high quality combination

Affymetrix‘ Tiling Arrays contain 25-mer probes separated by 10 bp spaces across the genome. How investigators are using the Genpathway-Affymetrix Total Solution

Examples of FactorPath, TranscriptionPath and Methylated DNA Therapeutic Areas assays provide new, valuable information Oncology Determine drug mechanism of action Metabolic Discover and validate drug targets disorders Toxicology Identify downstream effects of compounds Inflammation Discovery biomarkers for clinical & discovery programs Endocrinology Determine disease mechanisms for future targets Genetic disorders Identify side effects of compounds Cardiovascular Determine efficacy of compounds Diabetes Define genetic pathways for important programs Virology Validate results obtained by other assays Genpathway Total Solution for Biomarkers

Program Cancer (or other Therapeutic Areas) Customer Identify biomarkers in disease-relevant samples, e.g., need tumors, other tissues or blood Genpathway 1. Screen whole genome for MeDNA regions in sets of solution affected and unaffected tissues 2. Select MeDNA panel for custom array(s) 3. Use custom MeDNA arrays to test large numbers of samples; may develop subpanels 4. Use to classify patients, predict and correlate response to treatment, predict metastases, screen compounds, predict unwanted side effects Detect Me-DNA genome-wide by Methylated DNA IP-on-Chip

„ Methylated IP-on-Chip/Tiling for detection of methylated DNA regions in MDA-MB-231 cells.

Methylated DNA regions in promoters, Very good correlation with CpG within genes, and between genes islands (blue), but also differences

Locations of peaks representing Methylated DNA Peaks on Array F (chromosomes 8, 11, 12) Number Percent Total number on Array F 997 Extrapolated to entire genome 6979 In CpG Islands +/- 200 bp 233 23.4% In promoters (-1 to -10,000 from TSS*) 123 12.3% In genes 584 58.6% In +/- 100 bp 137 13.7% Unannotated (no gene within 10 kb) 216 22.0% *TSS = Transcription Start Site Validate peaks on the arrays as true methylated DNA regions

40 35 Untreated Large peak 35 30 Treated-AZA Small peak 30 25 Methylated DNA 25

20 tected / 1000 cells Query: Peaks on 20 Detected / 1000 Cells arrays represent 15 15

Me-DNA regions 10 10

5 5

0 Regions De Me-DNA 0 Me-DNA Regions ADCY8 ER HTR1B TPM long KLF14 FZD7 C1RL SOX2 ACTB AKNA WDR1 KLF14 TBX15 Untr12 Untr20 GATA2 HTR1B ADCY8 KCND3 C8orf61 TFAP2D ESRRA1 NMNAT2 TNFAIP8 LRSAM1 MGC45477 MGC33926 5-Azacytidine treatment: Me-DNA LOC285429 regions exhibit decreased levels

NMNAT2; 321 bp 7000 Untreated 6000 Treated-AZA

5000 Bisulfite 4000 sequencing: ADCY8; 291 bp Peaks on arrays 3000 2000

represent Me- Relative RNA Levels DNA regions 1000 GAPDH; 314 bp 0 ADCY8 ER HTR1B TPM1 long KLF14 5-Azacytidine treatment: for corresponding genes increase Genpathway: The Only Whole-Genome Methylated DNA Total Solution

No discrimination against any  High Sensitivity sequences (vs. enzyme digestion)

Whole-genome tiling arrays (vs. CpG  Comprehensive island or promoter analysis only)

Whole-genome tiling arrays (vs.  High Throughput bisulfite-sequencing)

Robust and Consistent results (vs. variability of  Reproducible bisulfite PCR)

Genpathway’s optimized protocols  High Specificity result in low background binding Genpathway Total Solution for Targets Based on Pathways

Programs Metabolic Diseases (or other Therapeutic Areas) Customer need Identify and validate targets based on nuclear hormone pathways (or other factors or pathways) Genpathway 1. Identify nuclear hormone receptors and co- solution regulators important to program (by Customer) 2. Carry out whole genome FactorPath ChIP-on-Chip for each factor in cells or tissues 3. Identify factor binding sites (and their regulated genes) across the genome 4. Compare binding results for factors and co- regulators with genes and cell systems 5. Use subset of binding sites and genes as biomarkers for testing compounds Identify factor binding sites across the genome

„ FactorPath ChIP-on-Chip/Tiling to Affymetrix GeneChip whole-genome tiling arrays.

Examples of array peaks on chromosome 19 Distance Distance to gene to gene Interval start Interval end Gene start end Location 1132744 1133240 KIAA0963 -7981 -74599 Upstream 2486540 2486829 GNG7 110449 -24611 In gene 14815918 14816464 OR7A10 -2775 -3704 Upstream 15460283 15460958 PGLYRP2 -9646 -20495 Upstream 15947884 15948282 LOC126537 22555 3333 Downstream 16434947 16436450 CALR3 31553 14438 Downstream 34854025 34854662 PLEKHF1 6222 -4179 In gene 35865774 35865953 None* 36463389 36464254 ZNF537 -2239 -6561 Upstream 37545573 37546208 ZNF507 111726 -20256 In gene *No known gene within 20 kb

Excerpt of list of binding sites SRC-3 binding sites in MCF7 cells provided by Genpathway’s software Hybridization to duplicate arrays shows high reproducibility. Validate peaks on arrays as true factor binding sites

„ FactorPath ChIP-on-Chip/Tiling for detection of SRC-3 binding sites in MCF7 cells.

45 40 E2-treated MCF7 35 30 FactorPath Query: 25 peaks on arrays Normalized 20 15 represent SRC-3 10 binding sites 5 0 HES1 SAE1 Untr5 MLPH SCOC Untr20 GRLF1 RASEF DAPK2 HNRPL GREB1 MAML3 THADA1 c21orf63 c21orf93 SLC12A8 FLJ21128 FLJ16641 TMPRSS3 ADAMTS9 DSCAM (1) LOC389169 Negative regions Large peaks Medium peaks

Number of SRC-3 array peaks Peak locations relative to genes Tiling Array Type No. of peaks Peaks on Array B Number Percent B (chromosomes 2, 9, 19) 371 Upstream (-5 kb) 16 4.3% C (chromosomes 3, 21, 22, X) 307 Upstream (-5 to -20 kb) 49 13.2% In gene 174 46.9% D (chromosomes 4, 15, 18, 20) 474 Downstream (+20 kb) 26 7.0% Extrapolated to whole human genome 2688 No gene within 20 kb 106 28.6% TOTAL 371 100.0%

A minority of SRC-3 sites are upstream of genes Genpathway Total Solution for Toxicology or Discovery

Program Toxicology Customer need Identify mechanisms of toxicity/action for compounds Genpathway 1. Screen whole genome for differentially solution transcribed genes in cells or tissues treated with prototype compounds 2. Identify genes differentially transcribed in treated vs. untreated cells or tissues 3. Screen additional compounds and identify useful biomarkers 4. Develop custom panels of genes for screening large numbers of compounds in variety of cells and tissues, including clinical samples Discover transcribed genes across the genome using TranscriptionPath

„ TranscriptionPath ChIP-on-Chip/Tiling in estradiol-treated vs. untreated MCF7 cells.

Upper green line: Untreated MCF7. Middle green line: Treated MCF7. Lower green line: P-values between untreated & treated. Red arrow: Upregulated gene. Blue arrow: Downregulated gene. Orange arrow: Gene in unannotated region.

A powerful assay to discover novel genes in unannotated regions and quantify transcription genome-wide. Evaluate the transcriptome at the DNA level

Characteristics of regions undergoing transcription TranscriptionPath ChIP-on-Chip/ Vehicle-treated Estradiol-treated Tiling identified approx. 10,000 cells cells Differential transcribed genes in MCF7 cells. Number of regions 1360 1301 136 Approx. 10% were novel genes. On chromosome 1 890 863 76 On chromosome 6 470 438 60 Approx. 10% were differentially Annotated genes 1239 1160 98 transcribed. Unannotated regions 121 141 38

14

12 Vehicle TranscriptionPath Query 10 Estradiol validated both transcription 8 and differential transcription. 6 Chr1-85 and chr1-196 represent novel genes. 4 2

Normalized Transcription 0 ) IB 2 1 4 5 B K 1 A 2 2 K I G -85 P 5 A K 2L1 1 2L4 126 P 05 P HEY2 S P R (0.5x Untr12APON 1 D5 B C NR J C25 KCN P RBM24TGFB2chr FC L L T chr1-196T TRERFGP KIAA0478F S ACT Ensure ChIP/DNA IP quality with validated and chromatin

„ FactorPath Query of ChIP DNA demonstrating specific selection of desired /DNA complexes, and differential binding or transcription if possible.

150 60 Control IgG 50 Control IgG 120 Vehicle Estradiol 40 SRC3 Ab 90 30

20 60

10 30 Normalized Binding

0 Normalized Transcription TFF1 GREB1 Negative TFF1 GREB1 Negative 0 region region ACTB Untr20 GREB1 ACTB Untr20 GREB1

Post-ChIP Post-amplification Post-ChIP Post-amplification

FactorPath assay for TranscriptionPath assay SRC-3 binding sites for transcribed genes

Validation prior to array hybridization, including testing as needed, ensures high quality array results. Genpathway’s ChIP and DNA IP Assay Services

ChIP Query Assays FactorPath Query TranscriptionPath Query Methylated DNA Query ChIP Discovery Assays FactorPath Discovery ChIP Expanded Discovery FactorPath Expanded Discovery TranscriptionPath Expanded Discovery ChIP-on-Chip Assays FactorPath ChIP-on-Chip/Tiling FactorPath ChIP-on-Chip/Promoter TranscriptionPath ChIP-on-Chip/Tiling Methylated DNA IP-on-Chip/Tiling Methylated DNA IP-on-Chip/Promoter Genpathway + Affymetrix - The Total Solution

„ Genpathway - The ChIP Assay Experts „ Affymetrix’ Tiling Arrays + Genpathway’s Assays & Services provide customers with an immediate total ChIP solution „ Optimized procedures for ChIP/IP give superior results - Validation of Customer’s chromatin and Ab’s fully qualified „ ChiP Analysis Software (ChAS) provides customers with easily understandable and compiled results „ Superior Technical and Customer Support regarding samples, genomic regions, antibodies, and more „ Quality Control at various steps in the assay - we take the variability out of the assay „ Affymetrix + Genpathway is the only whole-genome ChIP-on- Chip and Methylated DNA IP-on-Chip Assay Total Solution TheThe ChIPChIP AssayAssay ExpertsExperts™™

www.genpathway.com