19.01.20 Epigenetics/DNA methylation across the genome

Question 1

What is epigenetics?

Answer 1

• Heritable and transient changes in gene expression that do not alter the primary DNA sequence - Epigenetic effects persist throughout an organism’s lifetime and are passed on to multiple generations - Switches genes on/off - Determines how proteins are transcribed - Contributes to variable expression in different cell types - Initiated and sustained by at least three mechanisms: 1) DNA methylation 2) histone modification 3) RNA-associated silencing

Question 2

Why is DNA methylation important?

Answer 2

• 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 - Involved in imprinting, X-chromosome inactivation, suppression of repetitive elements and carcinogenesis - A signal that regulates gene expression (generally suppresses)

Question 3

How are genes methylated?

Answer 3

• 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). • Added methyl group acts as a signal that is recognised by specific MeCpG-binding proteins. • High proportion gene promoter CpGs (CpG islands) stay unmethylated → less prone to deamination.

Question 4

What enzymes are involved in DNA methylation?

Answer 4

3x DNA methyltransferases 1) DNMT1 2) DNMT3A 3) DNMT3B

Question 5

What is the role of DNMT1?

Answer 5

• called the ‘maintenance methylase’ - copies the methylation from hemimethylated DNA to its new partner strand after replication - throughout life of organism

Question 6

What is the role of DNMT3A and DNMT3B?

Answer 6

• called the ‘de novo methylases’ - both add initial pattern of methyl groups. Expressed mainly in early embryo

Question 7

What are the changes in DNA methylation during development?

Answer 7

• Methylation of imprinting centres (ICs) is erased more slowly than rest of genome in primodial germ cells - It is then re-established with different kinetics in male and female germ cells - After fertilization, the maternally and paternally derived genomes are widely demethylated (but ICs remain differentially methylated)

Question 8

What happens if methylation process goes wrong?

Answer 8

• ICs normally evade embryonic wave of epigenetic reprogramming
• Failure to protect ICs can lead to hyper and hypo methylation
• Some patients with multi-locus imprinting disturbance (MLID) have been shown to have genetic variants which affect embryonic methylation

Question 9

What are methyl-CpG binding proteins?

Answer 9

• The added methyl group acts (5MeC) as a signal that is recognised by specific MeCpG-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.
• LoF of MECP2 causes Rett syndrome (X-linked).

Question 10

What is histone modification?

Answer 10

• Histones are proteins that are the primary components of chromatin
• 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 more relaxed form it is active and the associated DNA can be transcribed.
• If chromatin is condensed (inactive), DNA transcription does not occur.

Question 11

What are the two main ways histones can be modified?

Answer 11

1) Acetylation/Deacetylation
2) Methylation/Demethylation

Question 12

Describe the Acetylation/Deacetylation histone modification process?

Answer 12

• adds/removes an acetyl group (COCH3) to free amino groups of lysines or arginines
  1) Acetylation is catalyzed by histone acetyltransferases (HATs)
  2) Deacetylation is catalyzed by histone deacetylases (HDACs)
  3) Lysine acetylation almost always correlates with increased transcriptional activity
  4) Deacetylation is generally associated with heterochromatin and represses transcription

Question 13

Describe the Methylation/Demethylation histone modification process?

Answer 13

• adds/removes a methyl group to free amino groups of lysines or arginines.
  1) Methylation is catalyzed by histone methyltransferases (HMTs)
  2) Demethylation is catalyzed by histone demethylases (HDMs).
  3) Effect depends upon which residue is methylated and gene in which the modified histone is found

Question 14

Name two types of non-coding RNA

Answer 14

1) Short (i.e. microRNA)
2) Long (i.e. lncRNA)

Question 15

Describe microRNAs

Answer 15

• 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.
• Multiple genes can be targeted by a single microRNA, making potential therapeutic use difficult.
• A single gene can be targeted by multiple microRNAs.
• The ‘seed site’ refers to nucleotides 2-7 from the 5’ end of the microRNA and is often evolutionarily conserved.
• 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. There is, however, some evidence of microRNA targeting and binding to the coding regions and 5’UTRs.

Question 16

Describe lncRNAs

Answer 16

• 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.

Question 17

Example 1 of epigenetic changes in rare disease - Rett syndrome

Answer 17

• Neurodevelopmental disorder of females. Arrested development 6-18 months. Small hands/feet and deceleration of rate of head growth
• Caused by loss of function of the MECP2 gene which encodes a 5MeC-binding protein

Question 18

Example 2 of epigenetic changes in rare disease - Fragile X syndrome

Answer 18

• The most frequently inherited mental disability, particularly in males
• FMR1 gene. Full mutation caused by over 200 CGG repeats. Too many CGGs cause CpG islands at promoter region of FMR1 gene to become methylated; which turns the gene off, stopping production of fragile X mental retardation protein

Question 19

Example 3 of epigenetic changes in rare disease - Beckwith-Wiedemann syndrome

Answer 19

• Overgrowth, hemihyperplasia, macroglossia, embryonal tumours.
• Can be abnormal methylation pattern at 11p15 (maternal LOM at imprinting centre 2 or maternal GOM at imprinting centre 1). Paternal UPD of 11p15. LOF variants in the maternal inherited CDKN1C gene. Genes involved IGF2, H19, CDKN1C, KCNQ1 and KCNQ10T1.

Question 20

Example 4 of epigenetic changes in rare disease - Russell-Silver syndrome

Answer 20

• Small for gestational age with relative macrocephaly at birth, prominent forehead with frontal bossing, postnatal growth failure, triangular facies
• Paternal hypomethylation of ICR1 at 11p15.5, maternal UPD at 7q32. Dels/dels/translocations at either 11p15 or 7q32. maternal GOF variants in CDKN1C, paternal LOF variants in IGF2. Pathogenic variants in PLAG1 and HMGA2.

Question 21

Example 5 of epigenetic changes in rare disease - PWS/AS

Answer 21

• Hypotonia, poor growth, then hyperphagia, LD, sleep disturbances (PWS) or LD, ataxia, epilepsy, hyperactivity (AS)
• Related to imprinting of chromosome 15 – mat UPD causes PWS, pat UPD causes AS. Genes involved – OCA2/UBE3A

Question 22

Example 6 of epigenetic changes in rare disease - DiGeorge Syndrome

Answer 22

• Learning difficulties, characteristic facial appearance, cleft palate, conotruncal heart defects, thymic hypoplasia, susceptibility to infection due to a lack of T cells
• DGCR8 encodes a double stranded RNA-binding protein that is essential for miRNA biosynthesis in the brain (mouse model).
• DGCR8 deficiency results in cardiovascular defects and down-regulation of miRNAs in vascular smooth muscle cells, reduced cell proliferation, and increased apoptosis

Question 23

Example 7 of epigenetic changes in rare disease - Transient neonatal diabetes mellitus (TNDM - 6q24 related)

Answer 23

• Severe intrauterine growth retardation, hyperglycaemia in neonate which resolves but may recur in later life. If associated with multilocus imprinting disturbance (MLID) then addition feature are likely to be present
• Paternal UPD6, paternal duplication of 6q24, hypomethylation of maternally inherited PLAGL1 TSS alt-DMR. In patients found to have multilocus imprinting disturbance (MLID), almost half of these have biallelic pathogenic variants in ZFP57

Question 24

Why is methylation so important in cancer?

Answer 24

• Cancer cells are characterised by a massive global loss of DNA methylation
• Could be caused by:
  1) Environmental link – environmental exposure and DNA methylation association
  2) Age related link – age related DNA methylation profile changes of the human cell
• 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.
• Disruption of imprinting can cause increased risk of cancer and tumour formation
• Gene promotor CpGs (called CpG islands) usually remain unmethylated in mammals but these become hypermethylated in cancer cells causing these genes to be sileneces (genes which are normally always active)
• This process turns off tumour supressor genes (e.g. MLH1, VHL) further increasing risk of tumours

Question 25

How does methylation affect familial cancers and microsatellites?

Answer 25

• About half of the genes that cause familial or inherited forms of cancer are normally turned off by methylation
• Most of these genes normally suppress tumour formation and help repair DNA
• Hypermethylation can also lead to instability of microsatellites
• 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 (MSI) in many tumours is indicative of a defect in a DNA repair gene, particularly the mismatch repair genes
• Patients with tumour MSI without a pathogenic variant in one of the mismatch repair genes may have promoter hypermethylation (particularly of MLH1)
• However, MLH1 promoter methylation is a sporadic event, therefore if detected it is NOT consistent with a diagnosis of Lynch syndrome
• Changes in histone modification patterns are directly linked to cancer→ HAT genes have been shown to be altered in cancers leading to loss of H3 acetylation at tumour suppressor genes

Question 26

What epigenetic therapy is availble?

Answer 26

• 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
• Inhibitors of DNA methylation can reactivate genes that have been silenced
• e.g. 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

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
• 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