WEEK 9: COMPLEX DISEASES II EPIGENETICS Flashcards
What can epigenetic mehcanisms be affected by?
- Development (in utero, childhod)
- Environmental chemicals
- Drugs/pharmaceuticals
- Aging
- Diet
In histone modification, where do the epigenetic factors bind to?
- The histone tails
What are the health endpoints of epigenetic mechanisms?
- Cancer
- Autoimmune disease
- Mental disorders
- Diabetes
What do actively transcribed genes carry high levels of?
- Active modifications such as acetylations and methylation
What does genome wide chromatin accessibility suggest in terms of cancer?
- Suggests molecular mechanisms for cancer associated inherited variants and somatic mutations in the NON CODING genome
What effect does increased DNA methylation have on tumour suppressor genes?
- It turns them OFF
What effect does decreased DNA methylation have on Oncogenes?
- Turns them ON
What is imprinting in complex diseases confounded by?
- Environmental factors
What are miRNAs involved in?
- Multiple important roles in gene regualtion (development) and implicated in some cancers
What are endogenous short interfering RNA (endo siENA) incolved in ?
- Derived from pseudogenes , inverted repeats etx\c. and involved in gene regulation in somatic cells and in regulating some types of transposon
What are long non coding RNAs involved in?
- Regulating gene expression
- Monoallelic expression (X-inactivation, imprinting) and/or has antisense regulators
What is involved in the RNAi mechanism?
- Ds RNA binds to the protein DICER
- This CLEAVES dsRNA into smaller fragments
- One of the RNA strands is loaded onto a RISC complex
- Links the complex to the mRNA strand by basepairing
- mRNA is cleaved and destroyed –> no protein can be synthesised
What is epigenetics?
- Any modification NOT directly related to DNA sequence
- Epi= “on top of” or “in addition to” DNA sequence and traditional inheritance
Are all epigenetic changes heritable?
- NO
- can also be from environment
Heterochromatin has ____ levels of methylation and ____ levels of acetylation
- Higher levels of methylation and lower levels of acetylation
Eurochromatin has _____ levels of metylation and _____ levels of acetylaton.
- Lower levels of methylation and higher levels of acetylation
What are 3 examples of histone modifications?
- Acetylation
- Methylation
- Phosphorylation
Can more than one histone modification occur at same time?
-YES
Why is residue H3K9 important?
-Because it contains 3PTMs!!! trimethylation (Constitutive heterochromatin), Dimethylation (Faculatative heterochromatin), Acetlyation (Euchromatin)
Can histone modifications have strong effects on mRNA transcription/expression?
- YES
Can different histone modifications at the same residue have opposite effects?
- YES
What are the histone modification detection methods used to identify?
- Used to identify DNA sequences associated with histone and other modifications resulting from INCREASED DNA accessibility (bc. actively transcribed) + can be applied to SCREENING the WHOLE GENOME
What are the two methods for detecting histone modifications?
- DHS (DNase I Hypersensitive Assay)
- cHIp (chromatin immunoprecipitation)
What does DHS invovle in detection of histone modifications?
- Crossliniking TFs to DNase
- Transcriptional activity in areas that are open/prone to degradation by DNase
- Used to mark regulation sites
- (Not really used anymore)
Wat does chIP involve in the detection of histone modifications? (3 things it is used for)
- Used to identify TFs bound to SPECIFIC DNA sequence motifs
- Used to characterize DNA sequence motifs recognised by TFs
- Used to study the EFFECT of polymorphisms (SNPs)
- It is immunoprecipitation combined with high throughput screening
- It identifies locations in the genome BOUND BY PROTEINS
What does ChIp stand for?
- Chromatin ImmunoPrecipitation combined with high throughput Sequencing
What is ENCODE?
- ENCyclopedia Of DNA Elements
- Whole genome approach for a map of human genome activity
What kinds of histone modifications are involved in human disease and which types of diseases are these?
- Mutations in histone acetyl trasnferases HAT and protein methyltransferases PMT
- Only in RARE SEVERE CONDITIONS
e. g. MLL gene in Myeloid or Mixed -lineage leukemia chromosomal rearrangements (infant, pediatric, adult and therapy induced acute leukemia)
What is involved in DNA methylation?
- Addition of a CH3 group to C5 of a Cytosine residue –> from the action of DNA methyl transferases (DNMTs)
- Taking place at level of Cytosines followed by Guanine base–> CpG di nucleotides
What % of CpG sites are Ch3’d (methylated) in humans?
70%
Where are CpG rich regions found?
- “islands”
- Found UPSTREAM of lots of human promoters and generally HYPOMETHYLATED
- Methylation status at CpG islands correlates with GENE EXPRESSION
Are DNA methylation sites tissue specific?
- YES!
Can DNA methylation play a role in cancer?
- YES
- e.g. De novo mehtylation can occur in cancer cells–> gene silencing –> early events in tumorigenesis
What are the two different methods for detecting DNA methylation?
- MeDIP (methylation dependent immunoprecipitation)
- bisulfite treatment
What does MeDIp involve in the detection for DNA methylation?
- Igs SPECIFIC for methyl cytosine
- Allows for the QUANTIFICATION of DNA methylation (RELATIVE QUANTIFICATION)
Which two pathways can you use for the detection og DNA methylation through MeDIP?
- Array hybridization and high throughput screening
What does the bisulfite treatment involve in terms of detection of DNA methylation?
- DNA treated with sodium bisulfite
- Causes ONLY unmethylated CYTOSINES to convert to URACIL
- Sequencing then reveals where the methylated cytosines remain in the genome
What is the process for EWAS (epigenome Wide association study)?
- Recruit cases and controls into study (aim for whole (epi) genome analyses)
- Select tissue of interest
- Extract, quantify, QC DNA
- Sodium bisulfite treatment
- From this can EITHER hybridise to microarrays OR detect by NGS (next generation sequencing)
How is EWAS involved in cancer?
- Took 6010 tumour samples from 23 DIFFERENT cancer types (TCGA)
- Identified aberrant DNA methylation and associated changes in RNA expression
- Has a pan cancer amp of aberrant DNA methylation to inform therapeutic studies
Which pathways are the KEY to DNA methylation instability?
- Chromatin remodelling and Wnt signalling pathways
What are the limitations of EWAS in complex diseases ?
- It is reversible –>unstable changes
- Quantitative (small) differences
- Tissue heterogeneity/composition
- Genotype-dependent effects
- E.g. people who smoke have changes in DNA methylation patterns compared to smokers who quit –>BUT not all black and white…complex
What role does DNA methylation have in complex diseases?
High variability observed:
o In different individuals
o In different tissues
o At different timepoints (age–> different methylation profiles with different age)
What occurs in DNA methylation with relation to imprinting?
- One allele is transcriptionally INACTIVE (silenced) depending on the parent it was inherited from
- Selective gene expression impacts on the phenotypic expression
- Silencing takes place in early development and is transmitted across generations –> “reprogramming”
Are imprinted alleles usually HEAVILY METHYLATED?
-YES–> They have chromatin or histone modifications –>“epigenetic changes”
What is an example of imprinting (silencing) having different phenotypes?
- Prader-Willi syndrome–> Expressed form paternal copy (amternal silecned)
- Angelman syndrome–> Expressed from maternal copy (paternal silenced)
What are 6 characterisitcs of miRNA?
- found in animals and plants
- from endogenous genes
- Ss, stem loop structure
- Partial match with target genes (3’UTR)
- Often MUTLIPLE targets
- INHIBITION of translation
What are 6 characterisitcs of siRNA?
- found in LOWER animals and plants
- From Exogenous genes (i.e. viruses + endo-siRNAs)
- Double stranded
- PERFECT match with target genes
- Usually ONE target (same gene)
- mRNA cleavage
What do miRNAs play a role in (in humans)?
- The immune system and disease (e.g. IBD)
Which processes are long coding RNAs involved in?
- gene specific transcription
- Imprinting
- X-chromosome inactivation
