Epigenetic Technologies Flashcards
What is the definition of Epigenetics?
Chemical modifications to the DNA and DNA-associated structures that do not change the DNA sequence, but can regulate gene expression and can be inherited across cell division
What are two most commonly studied epigenetic mechanisms?
DNA methylation
Histone modifications
What are other epigenetic mechanisms?
‣ Non-coding RNA-mediated gene-silencing
‣ Transcription-factor binding
‣ RNA modifications
‣ Mechanisms involved in generating and maintaining heritable chromatin structure and attachment to the nuclear matrix.
How do epigenetic changes affect gene expression?
Regulate chromatin structure
What are two chromatin structures?
Euchromatin: Relaxed DNA. Loosely organised chromatin thus many transcription factors are able to bind to the DNA and initiate transcription
Heterochromatin: Supercoiled DNA. Tightly packaged.
What are three epigenetic processes?
- DNA methylation: Methyl groups attached to the CPG islands regulate gene activity
- Histone modifications: Groups attached to histone tails determine activity of DNA wrapped around them
- Chromatin remodelling: Chromatin remodelling makes certain regions of the DNA available for transcription
What are three ways in which non coding RNA mediated gene silencing can occur?
MicroRNAs
Long non-coding RNAs (lncRNA)
Proteins and enzymes involved in how chromatin is attached to nuclear lamina
What are the key epigenetic modifications in gene regulation?
DNA methylation
Histone marks
Chromatin structure
Promoter & Enhancer binding
Non-coding RNAs
RNA modifications
Position relative to nuclear lamina
Chromatin interactions
How many histone modifications/marks exist?
Up to 20 different marks have been assayed genome-wide to date and are available within publicly available resources
How are known histone modifications assayed?
What is this method also used for?
Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq) is the main approach used to detect known (e.g. H3K4me1) histone modifications genome-wide
Also used for detection of Transcription Factor binding Chromatin
How are novel histone modifications detected?
Mass-Spectrometry
How is ChIP sequencing pursued?
Fragment sample
Add antibody
Immunoprecipitation
Sequence
Analysis
What is the main approach used to detect ‘open’ vs closed chromatic regions genome wide/ chromatin accessibility assay?
DNase I hypersensitive sites sequencing (DNAse- seq)
What steps are involved in DNAse-seq?
Fragment sample
Immunoprecipitation
Sequence
Analysis
What other methods are used to estimate chromatin accessibility and exact nucleosome position?
FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements) is another approach to detect chromatin ‘openness’ conformation
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) also assesses chromatin accessibility
MNase-seq (Micrococcal nuclease (MNase)-assisted isolation of nucleosomes sequencing) is used to establish where nucleosomes are positioned
What is the main approach used to detect gene expression levels genome-wide?
RNA-sequencing (RNA-seq)
What is used for detection of RNA-based epigenetic changes?
RNA-seq
What detects absolute expression levels, novel transcripts, alternative splicing, intron/exon boundaries, SNP variants, but can have transcript-length and reference-mapping bias.
RNA-seq
How is Non-coding RNA-mediated gene silencing detected?
RNA Seq used to measure levels of non-coding RNAs, but need different library preparation for small RNAs
How are RNA modifications detected?
RNA sequencing and Bisulfite Sequencing to detect RNA methylation
RNA sequencing detects what ?
Non-coding RNA mediated gene silencing and RNA-modifications
What steps are involved in RNA sequencing?
Purify RNA
Prepare libraries
Sequence
Analysis
How can long-range chromatin interactions be measured?
A number of methods have been applied. One is the Hi-C method.
What steps are involved in the HI-C method?
Crosslink DNA
Cut with restriction enzyme
Fill ends and mark with biotin
Ligate
Purify and shear DNA, pull down biotin
Sequence using paired ends
What use does analysing different epigenetic modifications in gene regulation have?
When combined with cell type what does this allow for?
Allow us to interpolate more precise gene regulation states using genome-wide profiles of different epigenetic processes
Build Epigenome Atlas
What are two
Epigenome Atlas’?
The ENCODE project
NIH Epigenome ROADMAP Project
What does using a reference epigenome ‘Atlas’ data allow for?
General understanding of biology of gene regulation across cell types and developmental stages
Integrative computational analysis of epigenomic signatures across reference datasets has resulted in chromatin state domain identification, e.g. using ChromHMM.
Some information in ChromHMM= Active promotor, poised promoter, repressed promoter, active enhancer
What is DNA methylation?
The addition of a methyl group/CH3 onto the C5 position of the cytosine to form 5-methylcytosine in mammals
One of the most common and stable epigenetic marks
How does DNA methylation occur?
Enzyme methyltransferase transfer methyl group from donor molecular (SAM) onto fifth position of the cytosine ring
Enzymes involved in DNA methylation include…
Methylation Methyltransferases (DNMTs)
- DNMT1, DNMT2
- DNMT3a, DNMT3b
- DNMT3L
De-Methylation
TET (ten-eleven translocation) family of enzymes (TET1, TET2 and TET3)
What are the four different forms of methylated cytosines?
Which of these is the most stable form in the process of methylation?
5mC, 5hmC, 5fC, 5caC
5mC
In mammals 5mC occurs primarily in what CpG context?
What is key for function?
CpG Islands (CGI), CGI Shores, CGI Shelves and ‘Open Sea’.
Genomic context: promoter vs gene-body; CGI vs Open Sea
What do DNA methylation detection technologies include?
Microarrays: Illumina GoldenGate/27K/450K/EPIC, CHARM, etc - hybridization to array, discrete number of CpGs/regions, different technologies to detect CpG-methylation.
Sequence-based:
(i) Bisulfite Sequencing (BiSeq, RRBS, WGBS, etc)
- Bisulfite Treatment followed by sequencing.
(ii) Capture-based (MeDIP-seq, MeCAP-seq, MBD-seq, MIRA-seq, etc)
- Extract methylated sections of the genome by antibody or protein binding; followed by sequencing.
(iii) Restriction enzyme based (MRE-seq, HPA-seq, etc)
- Restriction enzyme based detection of (un)methylated DNA; followed by sequencing
What is considered the best method for DNA methylation assay?
What is a disadvantage of this?
Whole Genome Bisulfite Sequencing (WGBS)
- Very costly
- Bisulfite conversion methods are also unable to differentiate between 5mC and 5hmC (hydroxymethylation).
- Certain genomic regions are difficult to sequence: what is the necessary sequencing depth for good coverage? Alignment can be difficult, methylation quantification difficult and read coverage of C is
What are the steps involved in Illumina 27K methylation array?
Bisulfite Conversation of DNA keeps the methylated cytosine as cytosine but converts unmethylated cytosine into uracil which is then interpreted as thymine. Thus effectively introducing a base pair change and this reflects methylations status of cytosine
Hybridize to chip: Each CPG site is then assayed by two bedes on the array and the methylated Beads hybridize to methlayed part of DNA and unmethylated beads hybridise to unmethylated part of DNA
Genotype
Single base extension
Methylation at one locus:
β = M / (M + U)
27,758 CpG-sites in promoters of ~13,000 genes
Probe QC: remap&exclude probes w/ variants: 22,290 probes
Data QC: outliers, missing data, quantile normalize, principal component analysis of methylomes, covariates vs PCs.
What steps are involved in Illumina 450K methylation array?
All steps involved in Illumina 27K however genotyping with 2 probes
Describe the methylation signal of Illumina 450K methylation array
β=M/(M+U)
485,000 CpG-sites target gene regions, incl Illumina 27K CpGs
The Infinium Assay for Methylation detects methylation status at individual CpG loci by typing bisulfite-converted tion protects C from conversion (left), whereas unmethylated C is converted to T (right). A pair of bead-bound pr detect the presence of T or C by hybridization followed by single-base extension with a labeled nucleotide.
Probe QC: remap&exclude probes w/ variants: ~450,000 probes Data QC: outliers, missing data, quantile normalize, principal
component analysis of methylomes, covariates vs PCs.
Approaches to harmonise data from 2 types of probes
Describe the methylation signal of Illumina EPIC methylation array (2016 - present)
β=M/(M+U)
850,000 CpG-sites, incl Illumina 450K CpGs
Target gene structures, but also ENCODE enhancers (~400,000) Probe QC & Data QC similar to Illumina 450K.
Commonly used R-packages for Illumina methylation data processing and normalisation: Methyl, ENmix, SWAN, BMIQ, etc
What steps are involved in MeDIP-seq (Methylated DNA ImmunoPrecipitation sequencing)?
Sonication
5mC Antibodies ImmunoPrecipitation
Ligate adaptors PCR amplifications
Sequencing (paired-end reads)
Read alignment to reference genome
Quantify reads to methylation scores
What are limitations of MeDip?
Not at single CpG resolution, and local CpG density affects the efficiency of antibody binding and immunoprecipitation.
Methods of quantifying MeDIP-seq data into methylation scores can take or not take CpG density into account. Methods include:
- Relative Methylation (RM) scores: Methylation scores represent read coverage over the region
- Only for comparisons of multiple samples within 1 genomic region.
- Absolute Methylation (AM) scores: Methylation scores represent read coverage taking into account CpG density of region. This controls for non- specific binding of the antibody to unmethylated CpGs, e.g. in genomic regions with high density of unmethylated CpGs and low density of methylated CpGs.
- Allow for comparisons across different genomic regions
MeDIP-seq&MRE-seq jointly perform better than either alone
True or false
True
What is the key difference between illumina arrays?
How many C’s can be measured
What is the future focus of epigenetic methodologies?
Single-cell analysis
What is the idea behind Epigenetic editing?
Use a DNA-targetting system to recruit epigenome-modifying factors to the genomic site of interest
What are options for DNA-targetting system (all for genome editing too):
CRISPR-Cas9 system: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-Associated Protein 9
TALENS: Transcription Activator-Like Effector Nuclease Zinc Finger Nuclease
CRISPR-Cas9 system gives opportunities for what?
High-throughput genetic screens and multiplexing targets
How is DNA methylation edited with CRISPR?
DNA targeting module (CRISPR - inactive Cas9) is connected to chromatin/DNA-modifier enzyme domains (epigenetic effectors), such as DNA methyltransferases (DNMTs) or demethylases (TETs)
Successful applications in cell lines & animal studies at bp-level
System can also be applied for histone modifications
Many potential future therapeutic applications
