Introduction To Epigenetic Regulation: How Can The Epigenomics Core Services Help Your Research? Maria (Ken) Figueroa, M.D. Core Scientific Director

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1 Introduction To Epigenetic Regulation: How Can The Epigenomics Core Services Help Your Research? Maria (Ken) Figueroa, M.D. Core Scientific Director

2 Gene expression depends upon multiple factors Gene Transcription Ch3 Ch3 Ch3 Ch3 DNA Sequence DNA Methylation Histone Modifications GENETIC REGULATION EPIGENETIC REGULATION

3 Genetics and Epigenetics EPIgenetic information... A T C G... DNA - genetic information Inherited across generations Transmitted by mother to daughter cells Required for life

4 Comparing genetic and epigenetic codes EPIgenetic code Hundreds of proteins and chemical modifications Cytosine modifications Histone modifications Writer/Reader/Eraser proteins... A T C G... DNA - genetic code 4 nucleotides

5 Three layers of epigenetic regulation Cytosine modifications Histone modifications Writer/Reader/Eraser proteins

6 Epigenetic vs Genetic information EPIgenetic information has plasticity... A T C G... Genetic information is constant Conception Death

7 Epigenetic plasticity is required for complexity Genetic Content Stem Cell Epigenetic Content

8 Epigenetic plasticity is required for cellular adaptation and reprogramming Genetic Content Cancer Chronic inflammation Stem Cell Response to stress ipsc Differentiation Environmenta l effects Epigenetic Content

9 Epigenomic assays uncover novel biology in cancer Acute Leukemias Gliomas

10 Epigenetic deregulation in non-malignant disorders Epigenetic Content

11 Limitations to studying the epigenome ü Assays can be cumbersome and challenging to set up ü Multiple different assays available and understanding which is the correct assay for your experiment may not be so clear ü Reagents are expensive ü Experiment design can be complicated: e.g. special primers or special biological considerations ü Data analysis is not standard

12 Epigenomics Core Central Aims 1) Provide consultation services to assist in the experimental design of epigenetic studies, including assistance with platform selection, primer design and experimental setup. 2) Provide next generation sequencing methods to study the distribution of cytosine modification (5mC and 5hmC) and histone modifications in cultured cell lines and primary tissue specimens with limiting cell numbers. 3) Provide state-of-the-art single locus assays for performing cytosine and histone modification studies in restricted number of loci. 4) Provide bioinformatic support for epigenomic data sets, including data processing, quality control assessment, and comparative data analysis between experimental cohorts.

13 Who we are Core Scientific Director: Ken Figueroa M.D Core Bioinformatics Director Maureen Sartor Ph.D. Core Manager Claudia Lalancette Ph.D. Computational biologist: Ana Rodrigues Ph.D.

14 Benefits that investigators will derive from using the core. 1. Access to experiments that are otherwise restricted to a small group of specialized labs 2. Reduced cost of sample processing 3. Integrated service including: II. III. IV. Assistance with assay and experimental design Library preparation Raw data of guaranteed quality or repeated for free V. Bioinformatic support for data processing and comparative analysis

15 Epigenetic Assays Genome-wide and site-specific Cytosine methylation and hydroxymethylation Cytosine modifications Histone modifications Writer/Reader/Eraser proteins Genome-wide distribution of histone and epigenetic modifier

16 A) Genome-wide DNA methylation: -WGBS, ERRBS, medip-seq, Illumina Infinium arrays B) Genome-wide DNA hydroxymethylation: -hmedip-seq Services provided C) Genome-wide histone modifications - ChIP-seq library preparation - Coming soon: N-ChIP for a panel of validated histone Abs D) Single-locus quantitative DNA methylation - MassARRAY EpiTYPER or Pyrosequencing E) Bioinformatic services for epigenomic data sets - Data QC included in all services - Fee-for-service: Data processing, biological comparisons, integration with publicly available or user-generated datasets

17 Bisulfite Conversion of modified cytosines CONVERSION DEAMINATION DESULPHONATION (or 5-hydroxymethylcytosine)

18 Bisulfite Conversion creates a C/T SNP Creates a C/T SNP that can be: 1- distinguished through sequencing 2- Amplified differentially through different primer pairs 3- Digested differentially through specific RE 4- Differential hybridization on C/T SNP array (Illumina)

19 The 5hmC dilemma 5hmC is resistant to deamination by sodium bisulfite à cannot be distinguished from mc 5hmC is present in varying quantities in different tissues à high in ESCs and brain tissue Most tissues have very low quantities à affinity-based methods require a lot of input DNA for successful library prep HOWEVER, 5hmC is not simply an intermediary step in DNA demethylation and plays a role in transcriptional regulation 5- methylcytosine 5- hydroxymethylcytosine

20 Genome-wide vs. single/few loci Choosing the right assay Specific CpGs or regional information? Cost restrictions? 5mC or 5hmC? Histones? What assay is best for me? DNA quality: good or poor? Limited cell numbers/ DNA?

21 Bisulfite-based methods Advantages: 1. Have base-pair resolution 2. Can offer precise quantification (% methylation) Limitations: 1. Can have false positives due to incomplete conversion of unmethylated Cs 2. Does not distinguish mc from 5hmC 3. Requires very high quality DNA (bisulfite degrades DNA) 4. Bisulfite treated DNA is unstable and can t be stored 5. Bisulfite treatment further degrades DNA 6. PCR amplification needs to be very well optimized to amplify both unmethylated DNA and methylated DNA with equal efficiency to avoid false positives for methylation

22 Affinity-based methods Advantages: 1. Can be performed with lower quality DNA 2. Do not depend on cumbersome bisulfite treatment 3. Good antibodies should not cross-react with 5hmC 4. You query the whole genome without requiring to sequence the whole genome Limitations: 1. Do not offer base-pair resolution 2. Are highly dependent on CG content (> affinity for more CG dense regions) 3. Lack of signal may be due to true unmethylated CpGs or to inefficient pulldown (false negatives) 4. Ab-based methods have bad signal-to-noise ratio and variability due to Ab

23 DNA methylation assays Massarray/ Pyroseq. WGBS ERRBS merrbs medip-seq Coverage Few loci Whole genome ~3-4M CpG. ~3/4M CpGs Whole genome Quantitative Yes Yes Yes Yes semi Base-pair res Yes Yes Yes Yes No # of lanes N/A 3 to get >10x 1 for > 10x Non-CpG reads DNA required ¼ -½ for > 10x 1-2 N/A Many Very few Very few None 10ng/ amplicon 250ng ng 75ng 500ng-1ug Cost $-$$ $$$-$$$$ $$ $ $$-$$$

24 Sample Processing Workflow Consultation interview to design experiment Request service in ilabs and submit samples to core Assay-specific sample processing and library preparation Epigenomics core submits samples to DNA sequencing core Deliver Raw/ Processed Data to users Data quality control, alignment and analysis (if requested) Sequencing core delivers raw data to Epigenomics Core

25 Where can you find us? MSRB II :30-4:30 research.med.umich.edu/epigenomicscore

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