lecture 30

Question 1

Objectives and outline

Answer 1

• to understand the principles of DNA metabolism in the context of chromatin – epigenetic mechanisms
• introduction – DNA as the “lifeless” blueprint
  → identical twin studies
  → lessons from differential feeding in early development: diet and epigenetics
  → DNA packaging and DNA metabolism
  → DNA damage and repair – histone variants
• DNA methylation in health and diseases
  → aberrant DNA methylation in cancer
  → DNMT inhibitors - emerging cancer therapies
• histone post-translational modifications
  → histone methylation
  → histone acetylation
  → histone deactylase inhibitors - emerging cancer therapies

Question 2

What is epigenetics?

Answer 2

• still controversial and we will develop an accepted definition throughout the lecture
• literal definition
• “genetics” = greek derived from “genesis” – birth, origin
  “epi” = greek for “above, beyond)
• derived from the virtually redundant Aristotelian word of epigenesis, which was used by the Hellenic philosopher to describe his theory of gradual and progressive developmental changes
• the term epigenetics was introduced by the British scientist Conrad Waddington in the 1940s to incorporate all of the factors controlling gene expression and cell differentiation
• proposed the concept of an epigenetic landscape to represent the process of cellular decision-making during development
• at various points in this dynamic visual metaphor, the cell (represented by a ball) can take specific permitted trajectories, leading to different outcomes or cell fates

“Heritable changes in gene expression and cellular phenotype that are independent of changes in the underlying DNA sequence)

Question 3

Who are some current authorities on epigenetics?

Answer 3

• professor adrian bird: university of edinburgh (DNA methylation; Rett syndrome
• professor tony kouzarides - the gurdon institute, cambridge (histone post-translational modifcations)
• professer john mattick – university of queensland (RNA regulation)

Question 4

What is the human genome?

Answer 4

• DNA double helix is “lifeless” - contains the blueprint but requires instructions!
• the human genome contains approximately 20,000-25,000 genes
• genes encode proteins that perform most life functions
• only 1-2% of the human genome is made up of genes
• the function of many genes and the vast remainder of sequences in the human genome are yet to be fully understood

Question 5

What is non-coding RNA?

Answer 5

• originally referred to as “junk” DNA
• regulatory sequences – critical biochemical functions
• encyclopedia of DNA elements (ENCODE)

Question 6

What is link between genetics and disease?

Answer 6

• many diseases have a well known genetic basis

- e.g. BRCA1/BRCA2 mutations in breast/ovarian cancer

Question 7

What is an example of twins and disease?

Answer 7

breast cancer

• when loretta was diagnosed with breast cancer, lorraine was in the doctor’s office with her
• loretta asked if lorraine should be checked as well
• the doctor discovered that lorraine aslso had breast cancer
• after receiving treatment, the sisters are both in good health

Question 8

What is epigenetic drift?

Answer 8

• identical twins are born with the same epigenome
• epigenetic profiles begin to diverge as they age
• differences increase as twins live longer and spend more time apart
  1. epigenetic marks are removed randomly as people age
  2. environmental influences change the pattern of epigenetic marks

epi-twin study

intra-specific variations in healthspan and lifespan:

• nature, nurture, chance
• chance dominations

Question 9

What is an example of differential feeding?

Answer 9

apis mellifera

• caste switching
• based solely on differential feeding during development (royal jelly)
• royalactin – growth factor receptors (kamakura, royalactin induces queen differentiation in honeybees)
• chromatin modifications – histone deacetylase inhibitor ? (Spannhoff et al. Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees)
• worker bee, queen bee, drone
• worker bee lives for about one season while queen bee lives for about 7

Question 10

What are classical nutritional studies?

Answer 10

methylation of the agouti gene
- classical example is the change in coat colour observed in progeny of yellow agouti mice exposed to methyl donors during pregnancy due to methylation of the agouti promoter

Question 11

What is chromatin architecture?

Answer 11

• organisation of over 3 billion DNA base pairs (2m) is facilitated by compaction and condensation into a complex structure known as chromatin

Question 12

What is the nucleosome?

Answer 12

• the fundamental unit of chromatin is the nucleosome
• DNA (146 bp) is wrapped around an octameric histone core consisting of two molecules each of H2A, H2B, H3 and H4
• further compacted into 30nm fibres – not well defined
• epigenetic regulation important for DNA metabolism, transcription and repair
• heterochromatin = highly condensed; inactive regions
• euchromatin = more opn chromatin conformation; active transcription

Question 13

What happens in DNA double-strand breaks?

Answer 13

• lethal lesions – unrepaired or erroneously repaired = loss of genomic integrity, carcinogenesis
• phosphorylation of the histone variant H2A.X (1/5 H2A) an early response to DNA double strand breaks
• DNA double-strand breaks occur preferentially in actively transcribing euchromatic
• error prone repair

Question 14

What are unique histone marks?

Answer 14

• can be found on different regions of a gene and may impart unique activities
  e. g.
• trimethylated lysine 4 on histone 3 (H3K4me3) is associated with transcriptionally active genes
• trimethylated lysine 9 on histone 3 (K3K9me3) is associated with inactive genes
• euchromatin (active transcription) and heterochromatin (inactive) is epigenetically distinguishable

Question 15

What is DNA methylation?

Answer 15

• genomic DNA may be methylated at the 5th position of cytosine, typically in the sequence of CdG; known as CpG islands
• ‘adds bulk’ to make the DNA inaccessible
• methyl groups are added by DNA methyl transferases
• heavily methylated DNA is transcriptionally inactive

Question 16

What DNA methylation patterns are seen in cancer?

Answer 16

• aberrant DNA methylation
• global demethylation – increased transcription
• focal CpG island hypermethylation – transcriptional repression
• methylation-associated silencing of tumour suppressor genes
• one of the redisposing hits in Knudson’s classical two-hit model of carcinogenesis
• epigenetic changes important in cancer because they aren’t fixed e.g. adhesion molecules, needs to turn off expression to metastasise but turn on expression to adhere to new organ etc
• can’t do this if it has lost the gene

Question 17

What are histone modifying proteins?

Answer 17

histone methylation

• histone methyltransferases
• histone demethylases

histone acetylation

• histone acetyltransferases
• histone deacetylases

writers and erasers

Question 18

What is histone acetylation?

Answer 18

• regulated by the opposing actions of histone acetyltransferases (HATs) (writers) and histone deacetylases (HDACs) (eraser)
• usually the lyseine tails that are getting acetylated
• get heavily acetylated DNA/histone tails
• more open chromatin conformation
• transcriptionally active
• HDACs cause more condensed packing
• affects many key cellular pathways
• used in treating lymphoma

Question 19

What has been revealed about histone acetylation through experimental data?

Answer 19

review of experimental data 1

• sodium butyrate - small chain fatty acid
• note: valproic acod – clini > 30 years; antiepileptic
• preferential cell death in cancer cells since they have higher rates of proliferation
• histone deacetylase inhibitor
• dietary
• metabolise through the gut

2

• prototypical HDACi - hydroxamic acid
• note: SAHA (vorinostat); used clinically for CTCL
• death in cancer cells not normal cells