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Introduction to Epigenetic Regulation: How Can the Epigenomics Core Services Help Your Research?

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Introduction to Epigenetic Regulation: How Can the Epigenomics Core Services Help Your Research? Introduction To Epigenetic Regulation: How Can The Epigenomics Core Services Help Your Research? Maria (Ken) Figueroa, M.D. Core Scientific Director Gene expression depends upon multiple factors Gene Transcription Ch3 Ch3 Ch3 Ch3 DNA Sequence DNA Methylation Histone Modifications GENETIC REGULATION EPIGENETIC REGULATION Genetics and Epigenetics EPIgenetic information .... A T C G .... DNA - genetic information Inherited across generations Transmitted by mother to daughter cells Required for life Comparing genetic and epigenetic codes EPIgenetic code Hundreds of proteins and chemical modifications Cytosine Histone Writer/Reader/Eraser modifications modifications proteins .... A T C G .... DNA - genetic code 4 nucleotides Three layers of epigenetic regulation Cytosine modifications Histone Writer/Reader/Eraser modifications proteins Epigenetic vs Genetic information EPIgenetic information has plasticity .... A T C G .... Genetic information is constant Conception Death Epigenetic plasticity is required for complexity Genetic Content Stem Cell Epigenetic Content Epigenetic plasticity is required for cellular adaptation and reprogramming Genetic Content Cancer Chronic inflammation Response to stress Stem Cell iPSC Differentiation Environmenta l effects Epigenetic Content Epigenomic assays uncover novel biology in cancer Acute Leukemias Gliomas Epigenetic deregulation in non-malignant disorders Epigenetic Content 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 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. Who we are Core Scientific Core Bioinformatics Director: Director “Ken” Figueroa M.D Maureen Sartor Ph.D. Core Manager Computational biologist: Claudia Lalancette Ph.D. Ana Rodrigues Ph.D. 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. Assistance with assay and experimental design III. Library preparation IV. Raw data of guaranteed quality or repeated for free V. Bioinformatic support for data processing and comparative analysis Epigenetic Assays Genome-wide and site-specific Cytosine methylation and hydroxymethylation Cytosine modifications Genome-wide Writer/Reader/Eraser distribution of histone proteins Histone and epigenetic modifications modifier Services provided A) Genome-wide DNA methylation: -WGBS, ERRBS, meDIP-seq, Illumina Infinium arrays B) Genome-wide DNA hydroxymethylation: -hmeDIP-seq 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 Bisulfite Conversion of modified cytosines CONVERSION DEAMINATION DESULPHONATION (or 5-hydroxymethylcytosine) 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) 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 Choosing the right assay Specific CpGs or regional Genome-wide information? vs. single/few Cost loci restrictions? What assay is best for me? 5mC or 5hmC? Limited cell Histones? numbers/ DNA? DNA quality: good or poor? 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 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 DNA methylation assays Massarray/ WGBS ERRBS mERRBS meDIP-seq Pyroseq. Whole Whole Coverage Few loci ~3-4M CpG. ~3/4M CpGs genome genome Quantitative Yes Yes Yes Yes semi Base-pair res Yes Yes Yes Yes No ¼ -½ for > # of lanes N/A 3 to get >10x 1 for > 10x 1-2 10x Non-CpG N/A Many Very few Very few None reads 10ng/ DNA required 250ng 2.5-25ng 75ng 500ng-1ug amplicon Cost $-$$ $$$-$$$$ $$ $ $$-$$$ Sample Processing Workflow Consultation Request service Assay-specific interview to in iLabs and sample processing design submit samples and library experiment to core preparation Epigenomics core submits samples to DNA sequencing core Deliver Raw/ Data quality control, Sequencing core Processed Data to alignment and analysis delivers raw data to users (if requested) Epigenomics Core Where can you find us? MSRB II 2568 8:30-4:30 research.med.umich.edu/epigenomicscore .
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