Lecture 6 Flashcards by andrew fleming

What are the three key epigenetic mechanisms linked to cancer development?

1) DNA methylation
2) Histone modifications
3) Non-coding RNAs

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Why is the pericentric region methylated?

To stabilise the area around the centromere

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Outline methylation state changes during tumourogensis

1) Loss of methylation around pericentric regions
2) 5-mC to T point mutations
3) Methylation of CpG islands of genes that should be on (e.g. tumour suppressor)
4) Loss of methylation of repeats

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What is the relationship between hypomethylation and cancer development?

Ubiquitous even in benign tumours
Global DNA hypomethylation leads to chromosomal instability
Activation of oncogenes

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What is the relationship between hypermethylation and cancer development?

More common in advanced tumours

- Inactivation of tumour suppressor and repair genes

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What is the cancer epigenetic paradox?

Cause of cancer linked to global loss of DNA methylation in addition to locus-specific gain in methylation

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How was hypomethylation shown to be widespread in tumour cells?

Extracted DNA from normal and cancerous tissue
Digested with methylation sensitive restriction enzymes; which wont cut in presence of methylation, and control enzymes
Southern blot with probes for human growth hormone
Fragments cut with control enzymes were the same size
Cancer cells had smaller fragments when cut with methylation sensitive enzymes indicating loss of methylation
Has been shown in patients with wide range of cancers

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How was hypermethylation of tumour suppressor genes shown?

A CpG island hypermethylation profile of human cancer showed that at least on or two tumour suppressor genes are hypermethylated in cancers

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Which types of cancers had more of these hypermethylated tumour suppressors?

Ones in areas which are exposed to the environment

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How can hypermethylation lead to the loss of heterozygosity seen in many cancers?

Hypermethylation can spread over 1Mb of the genome

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Hypermethylation of which tumour suppressor increases with age?
Which cancers is it commonly methylated in?

p16

Breast (33%), prostate (60%), renal (23%) and colon(92%) cancers

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What happens to mehtylation state as an organism ages?

As in cancers there is a global loss of methylation

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What gene is commonly hypermethylated in aging?

Estrogen receptors in the colon

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What gene is hypermethylated in both cancer and aging? What mutation can it introduce?

IGFII

Can introduce a C to T mutation

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What effect will a null mutation of the Dnmt’s have?

Organism cannot survive

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How was reduced Dnmt1 activity shown to be linked to cancer development?

Transgenic mice that had significantly reduced Dnmt1 activity (~10%) develop aggressive T cell lymphoma due to genome instability

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What effect does reduced Dnmt3a/b activity have on cancer development?

Hypomethylation of satellite sequences in pericnetric regions of the genome

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Which cancer commonly has mutations in its Dnmt3a?

Acute myeloid leukaemia

Occurs in 25% of patients

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What is the effect over expression of Dnmt’s?

Hypermethylation of tumour suppressors

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What is the key driver between a gene being active or repressed?

DNA accessibility due to its packaging

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What are the acetylation marks of histones?

What carries out acetylation? What removes acetylation?

H4K16ac - active mark
Histone acetyl transferases (HATs)
Histone deacetylases (HDACs)

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What are the methylation marks of histones?

What carries out methylation?

Active mark - H3K4me2
Respressive marks - H3K9/27me3 and H4K20me1/3
DNA-methyltransferases

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How is the global loss of acetylation achieved in cancers?

Over expression of HDACs (e.g. prostate)
Reduced HATs (e.g. leukaemia

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Which nucleosome remodelling complex is known the inactivated in cancers? What is the effect?

SWI-SNF

Decreased expressiong of p21 and p16

Which HAT is commonly reduced in leukaemia?

p300

What is the H3K27 methyltransferase? | What genes are commonly modified in cancers by this HMT?

EZH2 | Genes that repress grwoth and promote apoptosis

How is epigenetic therapy achieved? | Where has this been successful?

Use of small molecules that rearrange the tags in the chromosomes Haematological malignancies

What types of DNMT inhibitors exist?

Nucleoside and non-nucleoside inhibitors

How do nucleoside DNMT inhibitors work?

e.g. 5-Azacytosine Cytosine analogue with a N in place of a C This N means that the SAM, a co-substrate in methyl group transfers, gets stuck an cannot move away, stopf methylation of other sites and can cause apoptosis

What are the disadvantages of nucleoside DNMT inhibitors?

Requires incorporation during S phase so repeat treatments necessary Can be toxic - possible cause secondary tumours

What are the positive and negatives of non-nucleoside DNMT inhibitors?

+ve: less toxic | -ve: Effectiveness if controversial

How do non-nucleoside DNMT inhibitors work? | Give an example drug

Block the active site of DNMT or block synthesis of DNMT e.g. MG98 - an mRNA that causes double stranded RNA to be made with the transcript of DNMT which is then degraded

How can issues with histone acetylation be treated?

HDAC inhibitors

How do HDAC inhibitors work? | Give an example

Inhibit histone deactylation | e.g. Vorinostat (SAHA) which has shown good response to T cell lymphoma patients

What are the drawbacks of HDAC inhibitors?

- Short pharmacological life | - Not sure if histone deactylation is the actual target of the drug

What was shown about epigenetic drift using twins?

Significant drift in epigenetic marks between older twins that younger twins - die to lifestyle?

How was the epigenetic drift between the twins investigated?

Comparative genomic hybridisation for methylated DNA

How do cultured fibroblasts overcome the progressive loss of DNMT1 activty as they age?

Over expression of DNMT3a - however this leads to hypermerthylation - possibly of certain genes or CpG islands that should not be hypermethylated

How was DNMT expresion been shown to be linked with life in drosophila?

Over expression mutation in DNMT expands lifespan by 58% | 50% reduction DNMT mutants live 27% shorter than wt

What did Hannum investigate using methylation?

The ability the predict the age of a cell by its DNA methylation state Used whole blood count to define 71 different CpG sites can predict age to +/- 4 years with 96$ accuracy

Which diseases have been shown to be effected by hypomethylation?

Autoimmine and neurodegenerative disorders

What happens to heterochromatin as an organisms ages?

Core histone levels decrease - less packaging | Histone modifications change- global increase of methylation of some histones, and changes in acetylation

What two types of senescence exist?

Replicative - Hayflick limit is reached and cells degenerate, model for aging Oncogene induced - expression of oncogene, e.g. RAS. Good model for cancer

Which two methyl marks control the master regulators of ES cells?

H3K4/27me3

What changes are made to H3K4/27me3 marks in senescent cells?

H3K4me3 reduced in genes that need to be down-regulated, such as cell cycle genes H3K27me3 increases at genes that promote senescence

What do regions rich in H3K27me3 marks interact with?

The lamins: to form Lamin-associated chromatin domains (LADs). Whereas regions of H3K4me3 regions loop away from the lamins

What happens to LaminB1 levels during senescence?

The levels decrease

What effect does knocking out LaminB1 have?

Causes cells to enter senescence Causes locus specific remodelling of chromatin Leads to loss of nuclear integrity (chromatin leaking into cytoplasm)

In mammals what acetylation mark is lost as an organism ages?

H4K16ac

In yeast what acetylation mark changes occur as the organism ages?

Global loss of H3K56ac and global gain of H4K16ac

What is the effect of this gain of H4K16ac in yeast?

Loss of silencing at the telomers

What is Sir2?

An NAD+ dependent Histone deactylase (HDAC) that de-acetylates H4K16ac marks

How is Sir2 activated?

Decreased glycolysis leads to increased levels of NAD and activates Sir2

What did the role of NAD in Sir2 activation link together?

Metabolism and life span

How can Sir2 activity be decreased?

Grow yeast in presence of nicotinamide (a product of Sir2 activity), these individuals show accelerated ageing and loss of transcriptional silencing

How can yeast age be determined?

The number of rDNAs

What is the mammalian ortholog of Sir2?

SIRT1

Why is the role of SIRT1 complex?

Deactylates H1K26, H4K16ac and many other targets. Hypoacetylation of H4K16 is hallmark of cancer. Is both tumour suppressor and promoter