Flashcards in 18.01.20 Epigenetics Deck (33)
Give an overview of epigenetics.
1. Heritable and transient changes in gene expression that do not alter the primary DNA sequence.
2. Epigenetic effects persist throughout an organism’s lifetime and are passed on to multiple generations.
3. Switches genes on/off → determines how proteins are transcribed → contributes to variable expression in different cell types.
4. Essential for development and many normal cellular processes.
5 .Changes in these factors can play a critical role in disease.
6. Initiated and sustained by at least three mechanisms: DNA methylation, histone modification and RNA-associated silencing.
Give three different mechanisms of epigenetic gene regulation.
1. DNA methylation
2. Histone modification
3. Non-coding RNA
Give two types of histone modification
Give an overview of DNA methylation
Involved in imprinting, X-chromosome inactivation, suppression of repetitive elements and carcinogenesis.
Helps maintain genome stability and prevent illegitimate recombination.
Role in determining the conformation of chromatin and holds the key to the heritability of epigenetic changes.
A signal that regulates gene expression (generally suppresses)
How are genes methylated?
Biochemical process: addition of methyl group (CH3) to the C5 position of cytosine to form 5-methylcytosine (5MeC).
Almost entirely restricted to cytosines of CpG dinucleotides - results in two methylated cytosines diagonal to each other on opposing DNA strands.
In mammals ~70% of all CpG dinucleotides are methylated.
Carried out by DNA methyltransferase (DNMT) enzymes - uses S-adenosylmethionine (SAM) as the methyl donor (results in S-adenosylhomocytosine).
Animals deficient in DNA methyltransferase activity die at various stages of development.
Added methyl group acts as a signal that is recognised by specific MeCpG-binding proteins.
Concentrated on repetitive sequences (e.g. pericentric heterochromatin and dispersed transposons), sporadically distributed in genes and intergenic sequences.
High proportion gene promoter CpGs (CpG islands) stay unmethylated → less prone to deamination.
Which enzymes are involved in DNA methylation?
Describe the role of DNMT1 and its associated proteins.
Maintenance methylase: copies the methylation from hemimethylated DNA to its new partner strand after replication - throughout life of organism.
Associated with: PCNA (replication forks), methyltransferases, HP1 (heterochromatin), methyl binding proteins
Describe the role of DNMT3A and DNMT3B and their associated proteins.
De novo methylase: adds initial pattern of methyl groups. Expressed mainly in early embryo
Associated with: Histone methyltransferases; histone deacetylases
What is the role of DNMT3L?
non-functional DNMT but does help stimulate de novo methylation and is thought to be required for the establishment of maternal genomic imprints.
What is the role of TET1-3?
TET 1-3 (Ten-eleven translocation) enzymes: converts 5MeC to 5-hydroxymethylcytosine (5hmC) in what is proposed to be first step in an active demethylation pathway. Important during development and tumorigenesis.
When do rapid changes in DNA methylation occur (draw diagram)?
1. Gametogenesis - substantial de novo methylated genomes in the sperm and egg.
2. Early embryogenesis - wave of genome-wide demethylation at the pre-implantation stage. This epigenetic reprogramming erases adult methylation patterns.
3. Post-implantation - large-scale de novo methylation by DNMT3A and DNMT3B.
How does the position of DNA methylation influence gene control?
In immediate vicinity of transcriptional start site > blocks initiation.
In gene body > does not block and may stimulate transcription elongation. May effect splicing.
In repeat regions > important for chromosomal stability.
Describe Methyl-CpG-binding proteins.
5MeC pairs with guanine in the same way as unmodified cytosine but the methyl group acts as a signal recognised by specific MeCpG-binding proteins.
These can then recruit other proteins associated with repressive structures such as histone deacetylases (HDACs) and have a role in regulating chromatin structure and gene expression.
Demethylation relaxes chromatin allowing histone acetylation and binding of transcriptional complexes.
Humans have 5 MeCpG-binding proteins: MBD1-4 and MECP2.
Loss-of-function of MECP2 causes Rett syndrome (X-linked).
What is histone modification and its effect?
Histones are proteins that are the primary components of chromatin- the complex of DNA and proteins that make up chromosomes (see notes on DNA structure).
The N-terminus of histone molecules protrude from the body of nucleosomes.
Chemical modifications of amino acids in these ‘histone tails’ are major determinants of chromatin conformation and consequently influence DNA transcription.
In a compact form it is active and the associated DNA can be transcribed.
If chromatin is condensed (inactive), DNA transcription does not occur.
What is acetylation/deacetylation?
Acetylation/Deacetylation – adds/removes an acetyl group (COCH3) to free amino groups of lysines or arginines.
What is the effect of acetylation/deacteylation on transcription?
Lysine acetylation almost always correlates with increased transcriptional activity.
Deacetylation is generally associated with heterochromatin and represses transcription.
How is acetylation/deacetylation catalysed?
Acetylation is catalyzed by histone acetyltransferases (HATs)
Deacetylation is catalyzed by histone deacetylases (HDACs).
What is histone methylation/demethylation/?
Methylation/Demethylation – adds/removes a methyl group to free amino groups of lysines or arginines.
Methylation is catalyzed by histone methyltransferases (HMTs)
Demethylation is catalyzed by histone demethylases (HDMs).
Effect depends upon which residue is methylated and gene in which the modified histone is found.
Give two classes of non-coding RNA.
Long and short
Give an example of a short non-coding RNA and its function.
MicroRNAs small strands of RNA ~22 nucleotides long, interfere with gene expression at the level of translation
Form active ribonuclear complexes with cytoplasmic proteins → have RNAase activity.
Have a base sequence that is complementary to a specific mRNA sequence, meaning that each microRNA degrades a specific mRNA.
Because the complementary RNA sequence is short, 1 microRNA can degrade multiple mRNAs corresponding to several different genes, often with similar functions.
Thus, microRNAs differ from RNAase enzymes in that the former are a targeted regulatory mechanism to reduce gene expression.
MicroRNAs work post-transcriptionally by binding to the 3′-untranslated regions of their target mRNAs, thereby inducing enzymatic degradation and preventing translation.
Give an example of a long non-coding RNA
Long noncoding RNAs (lncRNAs):
Represent another class of epigenetic mark.
These transcripts are ~200 bp long and are thought to form ribonucleoprotein complexes that interact with chromatin, regulating histone modifications and the structural transformations that distinguish heterochromatin from euchromatin.
Previously, lncRNA was thought to be a by-product of normal gene transcription, but we now know that lncRNAs show cell type-specific expression and also respond to diverse environmental stimuli, suggesting that their expression is both regulated by and responsive to the environment.
Give examples of heritable epigenetic changes. What is their effect?
Heritable from cell-daughter cell, not parent-child
Both result in monoallelelic gene expression.
Give examples of epigenetic changes associated with disease.
1. Rubenstein-Taybi (16p13.3): Mutations in the CREBBP or EP300 genes, both of which encode HATs (histone acetylation)
2. ICF syndrome (immunodeficiency, centromeric instability, facial anomalies: 60% caused by mutation in DNMT3B (20q11.21) DNA methyltransferase gene. ICF patients have 42% less global DNA methylation, especially in inactive, heterochromatin regions
3. Rett syndrome: xaused by loss of function of the MECP2 gene (Xq28) which encodes a 5MeC-binding protein
4. Fragile X syndrome
6. Sotos syndrome: NSD1 (5q35.3) is a histone methyltransferase that can negatively and positively influence transcription
What is the association between DNA methylation and cancer?
Cancer cells are characterised by a massive global loss of DNA methylation.
Environmental link – environmental exposure and DNA methylation association
Age related link – age related DNA methylation profile changes of the human cell
What is the effect of loss of methylation and the link with cancer?
As methylated genes are typically turned off, loss of DNA methylation can cause abnormally high gene activation by altering the arrangement of chromatin.
Hypomethylation causes (1) increased genomic instability; (2) reactivation of transposable elements; and (3) loss of imprinting.
Demethylation can favour mitotic recombination, leading to deletions, translocations, and chromosome instability.
Loss of imprinting of e.g. IGF2, leads to increased cellular levels of IGF2 and is associated with increased risk of liver, lung, intestinal, colon cancers.
Disrupted imprinting is associated with tumour formation e.g. Wilms tumour and Beckwith Wiedeman syndrome.
About half of the genes that cause familial or inherited forms of cancer are turned off by methylation
What are CpG islands?
Usually a region of at least 200bo of DNA with a GC% of >60%.
Usually associated with the start of a gene (~40% of promoters of mammalian genes)
Usually unmethyalted, unlike CpGs in other parts of the gene.
What is the association between CpG islands and cancer?
CpG islands become excessively methylated in cancer cells, thereby causing genes that should not be silenced to turn off.
This abnormality is the trademark epigenetic change that occurs in tumours and happens early in the development of cancer.
Hypermethylation of CpG islands can cause tumours by shutting off tumour-suppressor genes (eg. MLH1, VHL (Von-Hippel-Lindau) and RASSF1A)- may be more common in human cancer than DNA sequence mutations.
Other than gene silencing, what other effect can a change in methylation have in cancer patients?
Hypermethylation can also lead to instability of microsatellites, which are repeated sequences of DNA.
Microsatellites are common in normal individuals, usually consist of repeats of the dinucleotide CpA.
Too much methylation of the promoter of the DNA repair gene can make a microsatellite unstable and lengthen or shorten it.
Microsatellite instability has been linked to many cancers, including colorectal, endometrial, ovarian, and gastric cancers.
Give an example of when it is useful to perform methylation studies in cancer?
Methylation status of tumours can help diagnose Lynch syndrome: tumour samples that have microsatellite instability (MSI) but a pathogenic mutation is not detected in MLH1, MSH2, MSH6 or PMS2 should be tested for MLH1 promoter methylation in the tumour DNA.
However, MLH1 promoter methylation is a sporadic event, therefore if detected it is NOT consistent with a diagnosis of Lynch syndrome.
Why are epigenetic changes a target for drug therapy? Which are the most popular?
Epigenetic changes seem an ideal target because they are by nature reversible, unlike DNA sequence mutations
The most popular of these treatments aim to alter either DNA methylation or histone acetylation.
Give an example of epigenetic therapy that targets DNA methylation and the disease they are used for
Inhibitors of DNA methylation can reactivate genes that have been silenced.
Two examples of these types of drugs are 5-azacytidine and 5-aza-2’-deoxycytidine
Work by acting like the nucleotide cytosine and incorporating themselves into DNA while it is replicating.
After incorporation, drugs block DNMT enzymes from acting, which inhibits DNA Methylation
These drugs have been approved for treatment of patients with myelodysplastic syndromes.
Give an example of epigenetic therapy that targets histone modification and the disease they are used for
Drugs aimed at histone modifications are called HDAC inhibitors.
HDACs remove the acetyl groups from DNA → condenses chromatin and stops transcription.
Blocking this process with HDAC inhibitors turns on gene expression.
The most common HDAC inhibitors include phenylbutyric acid, SAHA, depsipeptide, and
Have shown significant anti-tumour activity and some have been approved for use, but interest is also increasing in neurological and neurodegenerative diseases.