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