18.01.20 Epigenetics

Question 1

Give an overview of epigenetics.

Answer 1

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.

Question 2

Give three different mechanisms of epigenetic gene regulation.

Answer 2

1. DNA methylation
2. Histone modification
3. Non-coding RNA

Question 3

Give two types of histone modification

Answer 3

acetylation/deactylation

methylation/demethylation

Question 4

Give an overview of DNA methylation

Answer 4

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)

Question 5

How are genes methylated?

Answer 5

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.

Question 6

Which enzymes are involved in DNA methylation?

Answer 6

DNMT1
DNMT3A
DNMT3B

Question 7

Describe the role of DNMT1 and its associated proteins.

Answer 7

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

Question 8

Describe the role of DNMT3A and DNMT3B and their associated proteins.

Answer 8

De novo methylase: adds initial pattern of methyl groups. Expressed mainly in early embryo

Associated with: Histone methyltransferases; histone deacetylases

Question 9

What is the role of DNMT3L?

Answer 9

non-functional DNMT but does help stimulate de novo methylation and is thought to be required for the establishment of maternal genomic imprints.

Question 10

What is the role of TET1-3?

Answer 10

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.

Question 11

When do rapid changes in DNA methylation occur (draw diagram)?

Answer 11

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.

Question 12

How does the position of DNA methylation influence gene control?

Answer 12

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.

Question 13

Describe Methyl-CpG-binding proteins.

Answer 13

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).

Question 14

What is histone modification and its effect?

Answer 14

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.

Question 15

What is acetylation/deacetylation?

Answer 15

Histone modification

Acetylation/Deacetylation – adds/removes an acetyl group (COCH3) to free amino groups of lysines or arginines.

Question 16

What is the effect of acetylation/deacteylation on transcription?

Answer 16

Lysine acetylation almost always correlates with increased transcriptional activity.

Deacetylation is generally associated with heterochromatin and represses transcription.

Question 17

How is acetylation/deacetylation catalysed?

Answer 17

Acetylation is catalyzed by histone acetyltransferases (HATs)

Deacetylation is catalyzed by histone deacetylases (HDACs).

Question 18

What is histone methylation/demethylation/?

Answer 18

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.

Question 19

Give two classes of non-coding RNA.

Answer 19

Long and short

Question 20

Give an example of a short non-coding RNA and its function.

Answer 20

MicroRNA

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.

Question 21

Give an example of a long non-coding RNA

Answer 21

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.

Question 22

Give examples of heritable epigenetic changes. What is their effect?

Answer 22

Heritable from cell-daughter cell, not parent-child

1. X-inactivation
2. Imprinting

Both result in monoallelelic gene expression.

Question 23

Give examples of epigenetic changes associated with disease.

Answer 23

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
5. PWAS
6. Sotos syndrome: NSD1 (5q35.3) is a histone methyltransferase that can negatively and positively influence transcription

Question 24

What is the association between DNA methylation and cancer?

Answer 24

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

Question 25

What is the effect of loss of methylation and the link with cancer?

Answer 25

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

Question 26

What are CpG islands?

Answer 26

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.

Question 27

What is the association between CpG islands and cancer?

Answer 27

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.

Question 28

Other than gene silencing, what other effect can a change in methylation have in cancer patients?

Answer 28

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.

Question 29

Give an example of when it is useful to perform methylation studies in cancer?

Answer 29

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.

Question 30

Why are epigenetic changes a target for drug therapy? Which are the most popular?

Answer 30

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.

Question 31

Give an example of epigenetic therapy that targets DNA methylation and the disease they are used for

Answer 31

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.

Question 32

Give an example of epigenetic therapy that targets histone modification and the disease they are used for

Answer 32

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

valproic acid.

Have shown significant anti-tumour activity and some have been approved for use, but interest is also increasing in neurological and neurodegenerative diseases.

Question 33

Why should epigenetic therapy be used with caution?

Answer 33

Caution in using epigenetic therapy is necessary because epigenetic processes and changes are so widespread.

To be successful, epigenetic treatments must be selective to irregular cells; otherwise, activating gene transcription in normal cells could make them cancerous, so the treatments could cause the very disorders they are trying to counteract.

Despite this possible drawback, researchers are finding ways to specifically target abnormal cells with minimal damage to normal cells, and epigenetic therapy is beginning to look increasingly promising.