Epigenetics

Question 1

Give an example showing the flexibility of the epigenome.

Answer 1

-Twins genetically identical
-DNA methylation pattern, label
pattern from one twin in red and other one in green
-Then, digitally superpose them, so yellow if overlap
-Initially, DNA methylation pattern relatively similar
-DNA methylation pattern start to diverge significantly over time (50 year old twins have more epigenetic tags in different places than 3 year old twins do), due to being in different environments over time

Question 2

Identify the mechanisms of epigenetic gene silencing.

Answer 2

1. DNA methylation
2. Histone modifications
3. Nucleosome remodelling

Question 3

Define epigenetics.

Answer 3

Heritable modifications of DNA that do not alter the primary sequence (e.g. me-C at CpG), but result in altered gene expression (e.g. gene silencing).

Question 4

Describe the process of DNA methylation and its link to gene silencing.

Answer 4

• Covalent modification of a methyl group to cytosine at position C5 to make 5-methylcytosine.
• Most genes have GC rich areas of DNA in their promoter regions (=CpG islands, )
• Methylation of the C residues within the CpG islands leads to gene silencing (unmyelination correlates with gene expression) because inhibits gene transcription by preventing the binding of transcription factors to the promoter and inhibiting transcription by converting chromatin from an open to a closed conformation
• Occurs mainly in mammalians (e.g. not in yeast/drosphila)
• DNA methylation is heritable
• DNA methylation is aided by enzymes which can catalyse de novo methylation reactions (on unmyelinated strand) or maintenance myelination (if one strand methylated, recognise that and put a methyl group on other strand as well)
• Methyl CpG binding proteins contain a methyl binding domain that specifically recognizes methylated CpGs, and further favour compact chromatin by recruiting other proteins such as histone deacetylases which remove acetyl groups.

Question 5

What are possible methylation states of DNA strands ?

Answer 5

Unmethylated
Hemimethylated (one strand methylated only)
Fully methylated (both strands)

Question 6

Identify and describe diseases arising from abnormal epigenetic regulation.

Answer 6

1) Prader–Willi syndrome
– Symptoms: mental retardation, obesity
– Underlying cause: Inheritance of two maternal chromosomes 15 (paternal deficiency) or deletion of segment containing active Prader-Wili gene and imprinted Angelman gene.
-Associated with genomic imprinting

2) Angelman syndrome
– Symptoms: mental retardation, “happy puppet” syndrome, jerky movements + inappropriate laughter
– Underlying cause: Inheritance of two paternal chromosomes 15 (maternal deficiency) or deletion of segment containing active Angelman gene and imprinted Prader-Wili gene
-Associated with genomic imprinting

NB: On maternal chromosome, gene for Prader Wili normally imprinted (silenced) and Angelman gene active.

On paternal chromosome, Angelman gene normally imprinted (silenced) and Prader-Wili gene active.

3) Also: 
• Beckwith–Wiedemann (BWS) syndrome (overgrowth) 
• Fragile X syndrome
• Myotonic dystrophy (congenital)
• Wilms’ Tumour
-All associated with genomic imprinting

4) Rett Syndrome
-Due to defects in DNA methylation machinery
-dominant X-linked, neurodegenerative disorder
-affects 1:10,000-15,000 (females only, embryonically lethal for males )
-Caused by a mutation in the gene encoding Methyl-CpG-binding protein 2 (MeCP2),
which in turn leads to loss of gene silencing at many loci.

5) Cancers
- Tumour suppressor genes silenced by DNA methylation in cancer

Question 7

Distinguish heterochromatin from euchromatin.

Answer 7

Both higher order structure of chromatin

HETEROCHROMATIN

• Highly condensed in interphase
• Transcriptionally inactive (contains few genes)
• Replicates late in S phase

EUCHROMATIN

• Organized in 30nm fiber during interphase
• Transcriptionally active
• Replicates early in S phase

Question 8

How are heterochromatin and euchromatin relevant in epigenetics ?

Answer 8

POSITRON EFFECT

• Spreading (by natural events) of heterochromatin into euchromatic regions
• Causes cell to cell variability in gene expression (silencing of genes which are usually active in euchromatin)
• May occur due to translocation of certain genes (=rearrangement of parts between nonhomologous chromosomes), where area of euchromatin from one chromosome translocates next to area of heterochromatin of another chromosome, regardless of any boundaries between euchromatin and heterochromatin
• These expression states are stably inherited in the daughter cells (lack of gene expression maintained)

Question 9

Describe the higher order of chromatin in telomeres.

Answer 9

Heterochromatin

Question 10

Describe the process of X-chromosome inactivation.

Answer 10

• PROBLEM: Discrepancy of 1 X-chromosome in males (XY) but 2 X-chromosomes in females (XX)
• SOLUTION: Females need to silence one X-chromosome early in embryonic development- X-chromosome inactivation (Lyonization)
• At 64 cell stage, random event in cells of embryo causes inactivation of one X chromosome. Condensation of randomly selected X chromosome and mechanism of silencing is initiated by Xist (X-inactive-specific-transcript), which ‘marks’ inactive X: only expressed from inactive X-chromosome and codes for an RNA (~17kb in humans). No protein product and RNA remains in the nucleus. Followed by DNA methylation of the whole chromosome (the entire chromosome is silenced).
• Then, direct inheritance of pattern of condensation (and hence inactivation) for cell lineage occurring subsequently
• As a result, clones of cells inactivating one specific X chromosome make up a certain area of tissue then clones of cell inactivating the other specific X chromosomes will make up other areas of tisse

Question 11

Does DNA methylation ever reset ?

Answer 11

Yes, upon fertilisation

Question 12

Give an example of X chromosome inactivation.

Answer 12

• Calico (tortoiseshell) cats are female
• Random X-chr inactivation manifested
in coat colour (The paternal or the maternal X-chromosome express either orange or black coat-colour gene; white is autosomal)

Question 13

Describe genomic imprinting.

Answer 13

• ~80 imprinted genes on autosomes
• Imprinted genes only expressed from one allele (other allele is silenced)
• Mechanism is likely DNA methylation (because mechanism must be somatically stable and reversible during gametogenesis)
• Dependent on parental origin (could be either maternal or paternal that is expressed)
• Imprinting resets on passage through germline (in gametes, all imprints are erased and rewritten: in the sperm, rewritten with the paternal pattern, even the genes from the mother. In the egg, vice versa)

Question 14

Describe the evidence for genomic imprinting.

Answer 14

1) MOUSE EMBRYO MANIPULATIONS
• At early point in fertilisation, before sperm nucleus fuses with egg nucleus, destroy one pronucleus and replace
• When replace female pronucleus with another male pronucleus (Androgenetic, only chromosomes from male parent):
- poor growth of embryo
- large placenta
- non-viable
• When replace male pronucleus with another female pronucleus (Gynogenetic, maternal chromosomes only)
- embryo OK
- small placenta
- non-viable
• Conclusion: The difference is the genomically imprinted genes

2) HUMAN TUMOURS
• Hydatidiform mole (2 x ♂)
• Ovarian teratoma (2 x ♀)

3) MOUSE CHIMERAS
• Normal + androgenetic = growth enhanced
• Normal + gynogenetic = growth retarded

4) CHROMOSOMAL IMBALANCES
• Uniparental disomy = Inheritence of two chromosomes from either the father or mother (rather than one from each)

“Overall, it seems that maternal and paternal effects are complementary here, each genome contains different viable and necessary properties”

Question 15

Describe uniparental disomy.

Answer 15

• Both copies of a chromosome are inherited from the same parent (so missing the chromosome from one of the parents)
• Expression altered of imprinted genes on affected chromosome
• Result: chr 15, PWS/ AS
chr 11, Wilms’ tumour

• Non-disjunction in meiosis II –> uniparental isodisomy
Non-disjunction in meiosis I –> uniparental heterodisomy

Question 16

Identify a possible treatment to cancer, in the context of epigenetic.

Answer 16

-Sicne tumour suppressor genes silenced by DNA methylation in cancer:
-Possible treatment is to inhibit DNA methylation and re-express gene:
VIDAZA blocks DNA methylation. Used for the treatment of patients with Myelodysplastic syndrome (MDS)

Question 17

True or False: Some of the proteins that are related to whether chromatin open or closed can be used as biomarkers to distinguish survival of patient with disease.
Give an example to support your answer.

Answer 17

True.
-Brg1 “opens” chromatin
-Low Brg1-expressing tumours have a worse outcome