1. March 6, 2016 EpiQ Chromatin Analysis Kit A New Tool for Epigenetic Research Gábor Kohut PhD Field Application Specialist Central and Eastern Europe

2. Epigenetics – what is it? Source: embryology.med.unsw.edu.au Source: hgu.mrc.ac.uk Inherited phenotype or gene expression changes due to mechanisms other than DNA sequence changes

3. Only 1%-6% of all cytosines in the genome are methylated DNA methylation is associated with a closed chromatin structure and gene inactivation Aberrant DNA methylation occurs in most cancers DNA Methylation

4. Santos-Rosa H, Caldas C. Eur J Cancer;41:2381-402 (Nov, 2005). Histone Modifications Acetylation of lysine (K) Methylation of lysine and arginine (R) mono, di and tri-methylation Phosphorylation of serine (S) Ubiquitination of lysine

5. Common Methods for Epigenetics Research Bisulfite conversion - for DNA methylation analysis  Sodium bisulfite chemistry for discrimination of C and 5’-mC  Downstream analysis: PCR, qPCR, sequencing, HRM, etc.  Time to results: 1 to 2 days

6. Bisulfite-Modification of Genomic DNA Bisulfite is a mutagen, it converts cytosine to uracil 5-methyl-cytosine is resistant to bisulfite and is not converted to uracil

7. Common Methods for Epigenetics Research Bisulfite conversion - for DNA methylation analysis  Sodium bisulfite chemistry for discrimination of C and 5’-mC  Downstream analysis: PCR, qPCR, sequencing, HRM, etc.  Time to results: 1 to 2 days Chromatin Immunoprecipitation (ChIP) – for DNA binding protein analysis  Antibody-mediated enrichment of specific protein:DNA complexes  Downstream analysis: PCR, qCPR, sequencing (ChIP-seq), microarray (ChIP-chip)  Time to results: 1 to 5 days

8. Chromatin IP Workflow

9. Chromatin Structure and Accessibility Heterochromatin Transcriptionally silent Inaccessible to transcriptional machinery Euchromatin Transcriptionally competent Accessible to transcriptional machinery The EpiQ Chromatin Analysis Kit Epigenetic Events DNA methylation (●) Histone modification (●●)

10. A Novel Research Tool for Epigenetic Analysis Heterochromatin Transcriptionally silent ► inaccessible to transcriptional machinery Inaccessible to nuclease ► no DNA digestion ► available for qPCR ► minimal C q shift Euchromatin Transcriptionally competent ► accessible to transcriptional machinery Accessible to nuclease ► DNA digestion ► unavailable for qPCR ► large C q shift We can cut the DNA here We can not cut the DNA here Proper amplification, no Cq shift Not proper amplification, Cq shift

11. EpiQ Workflow Only 3 hours

12. EpiQ Analysis of Reference and Control Genes

13. Hela PC3 LN H15 99% Accessible HelaPC3 LNCaPHCT15 mRNA gDNA Analysis of ACTB (  -actin) 99% Accessible 98% Accessible 96% Accessible

14. Hela PC3 LN H15 HelaPC3 LNCaPHCT15 mRNA gDNA Analysis of ACTB (  -actin) 99% Accessible 98% Accessible 96% Accessible

15. Constitutively Expressed Genes

16. Analysis of CDH1 (E-cadherin) Hela PC3 LN H15 mRNA

17. Hela PC3 LN H15 mRNA 25% Accessible 69% Accessible 95% Accessible 99% Accessible HelaPC3 LNCaPHCT15 gDNA Analysis of CDH1 (E-cadherin)

18. RNA Hela PC3 LN H15 mRNA HelaPC3 LNCaPHCT15 gDNA Analysis of CDH1 (E-cadherin) 25% Accessible 69% Accessible 95% Accessible 99% Accessible

19. Epigenetically regulated genes - Summary ?

20. Analysis of an Outlier

21. Hela PC3 LN RNA Unmeth Meth DNA Methylation Analysis of GSTP1

22. Hela PC3 LN Unmeth Meth Hela LNCaP RNA

23. DNA Methylation Analysis of GSTP1 Hela PC3 LN Unmeth Meth PC3 Unmeth Meth Hela LNCaP RNA

24. DNA Methylation Analysis of GSTP1 Hela PC3 LN Unmeth Meth PC3 Unmeth Meth Hela LNCaP Findings: In PC3 cells the GSTP1 promoter exists in two distinct states: 1. Unmethylated and accessible and 2. Methylated and inaccessible RNA

25. Analysis of an Outlier Findings: The GSTP1 promoter in PC3 cells exhibits monoallelic DNA methylation, a rare form of epigenetic regulation

26. EpiQ Assay Conclusions Small changes in chromatin structure results in large gene expression changes (10% change = 3 to 4 fold change) Intermediate states of chromatin structure are real and act to fine tune gene expression EpiQ chromatin analysis kit provides useful information and insights into epigenetic gene regulation

27. EpiQ Chromatin Kit Data Analysis Tool

28. Data Input Data Summary % chromatin accessibility Assay Quality Checks Transfer Cq values

29. EpiQ Guides & Support Tools Available at: www.bio-rad.com/epiq

30. Product Positioning & Target Customers EpiQ is a complementary epigenetics research tool, expanding beyond standard techniques used for DNA methylation and histone analysis. It provides quantitative information about chromatin accessibility and results correlate very strongly with patterns of gene expression. EpiQ differs from bisulfite conversion and ChIP by measuring the functional state of chromatin which is regulated by epigenetic events such as DNA methylation and histone modification. Any epigenetics researcher engaged in understanding gene expression regulation is a good target customer. Cancer research, stem cell biology, developmental biology.

31. Questions to Ask Your Customers Are you currently performing epigenetic analysis? Are you studying DNA methylation, histone modifications, or both? Would you like to know whether these epigenetic events are impacting gene expression through chromatin state changes? Are you interested in a kit that determines the level of chromatin accessibility for target genomic regions? One that requires very few cells and delivers results in less than 6 hours?

32. Questions Your Customers May Ask How is the EpiQ kit different from ChIP? EpiQ provides quantitative information about chromatin accessibility for genomic regions of interest. It does not determine the specific interactions between histones and genomic DNA, but rather interrogates the impact of these interactions on chromatin state. Will my existing primers for ChIP work with EpiQ? Yes, refer to the EpiQ Assay Design and qPCR Optimization Guide for information on using existing ChIP assays. The nuclease dose may need to be optimized to work effectively with ChIP assays which typically amplify shorter fragments between 75 and 200 base pairs. Can I use the EpiQ Chromatin SYBR Supermix for my ChIP or DNA methylation analysis? Yes, this supermix is able to amplify difficult genomic regions with high efficiency and can be used for ChIP-qPCR and bisulfite-qPCR/melt curve analysis.

33. Thank You For Your Attention!