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Y3 (2) Epigenetics in Development and Disease > Lecture 6 - Diseases, environmental factors, mechanisms, approaches (part 1) > Flashcards

Lecture 6 - Diseases, environmental factors, mechanisms, approaches (part 1) Flashcards

1
Q

How can both genetics and epigenetics lead to cancer?

A

Genetics

Genome and mutation , natural selection, evolution

Epigenetics

Epigenome and environment, adaptive response, specific aquired trait (Lanmark)

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2
Q

What epigenetic mechanisms have been linked to cancer?

A
  • DNA methylation
  • Histone modification
  • Non coding RNAs (H19 is an oncogene encoding for catalytic mRNA: maternally important in development, growth and cancer)
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3
Q

What genes encode DNA methylation enzymes?

A
  • Maintenance methylation (Dnmt1)
  • de novo methylation (Dnmt3a/b)
  • demethylation (dMTase)
  • passive loss through replication, one round = hemimethylated (can be remethylated by maintenance methylases) or lost after two
  • These enzymes can also play a role in other functions
    • Dnmt1 can act as de novo enzyme
  • Enzymes are essential (mutants are embryonic lethal)
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4
Q

Where is methylation of chromatin on the genome normally?

A

In normal cells

  • some regions heavily methylated, some devoid
  • heavy methylated regions need genome stability of to silence a gene (TE)
  • methylated close to the centromeres in the pericentromere region (this region needs to be stabilised)
  • chromosome has TE that decrease stablility
    • must be lots of selective pressure to keep methylation as:
    • M-c can be deanimated into thymidine, introducing a point mutation into the chromosome
  • In the telomeres any genes present are silenced by methylation
  • Locus-specific silencing
    • oncogenes silenced with methylation
  • CpG islands (could have a heavy methylated gene but still gets transcribed if open promoter)

Context of methylation important

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5
Q

Outline DNA methylation in cancer development

A

Hypomethylation

  • ubiquitous even in early benign tumors
  • Global DNA hypomethylation leads to chromosomal instability
    • areas normally stabilised by methylation (methylation lost), these areas more prone to chromosomal breakages nad recombination e.g. T-cell lypmhoma in mice don’t get Dnmt1 expression, chromosome becomes instable
  • activation of oncogenes
    • LTR need to be repressed, often oncogenes, viral genes

Hypermethylation

  • more common in advanced tumours
  • inactivation of tumor supressor genes, repair genes etc.

Balance has been compromised

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6
Q

What is the cancer epigenetics paradox?

A

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

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7
Q

What is the experimental evidence that hypomethylation is widespread in tumour cells?

A
  1. Extracted DNA from tissue (normal and cancerous)
  2. Digested with methylaiton sensitive restriction enzymes
    1. MspI cuts 5’C CGG 3’, but wil cut regardless of methylation status (control)
    2. HpaII cuts at 5’C CGG 3’ (methylation sensitive)
    3. Hhal cuts at 5’ GCG C 3’
    4. looked at y-globin and human growth genes
  3. southern blot with different probes against different genes
    1. By HpaII and HhalI see normal cells have large fragments of FNA and smaller fragments in cancer patients (lost methylation)
    2. Control: digesting with MspI cuts regardless, get same pattern WT/cancer
  5. Also looked at patients with colon, lung or liver cancers
  6. Saw same pattern - global methylation decreases in cancer.

This has now been vlaidated with modern technology.

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8
Q

What is DNA hypermethylation in cancer development?

A

Get locus specific gain of methylation

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9
Q

What does a CpG island hypermethylation profile of human cancer show?

A
  • Looked at CpG island methylation states of known tumour supressors (repair genes, involved in the stability of nucleus) in a variety of cancers
  • saw hypermethylation of at least 112 tumour supressor genes in total in all cancers
  • at least one of two tumour suppressors are hypermethylated (not active) in cancer
  • sarcomas - WRN gene
  • stomach cancer - BRAC1 gene
  • link between tissues exposed to the environment and not
    • more methylations where encounter more toxins
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10
Q

Describe DNA hypermethylation in cancer development and aging, by the spreading of hypermethylation

A
  • epigenetic hypermethylation can spread over 1Mb of the genome
    • especially if acting where shouldn’t - no insulating marks
    • get further chromatin supression
    • get loss of heterogeneity by hypermethylation (seen in many cancer cell lines)
  • this can supress the expression of important genes:
    • Hypermethylation can cause silencing of P16 (tumour supressor) in breast cancer, prostate cancer, renal cancer and colon cancer [also occurs with increasing age]
    • ER (estrogen receptor gene) is hypermethyalted in colons by increasing age
    • IGFII is hypermethylated in cancer and aging

Hypermethylation can introduce C to T mutation

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11
Q

Outline the involvment of DNMTs and cancer

A
  • reduced activity of DNMT1 (maintenance methylase) has been linked to cancer
    • transgenic mice with significantly reduced DNMT1 activity (10% of WT) developed agressive T cell lymphoma, due to genome instability as a result of substantial global hypomethylation
    • DNMT3 (de novo enzymes) family: mutants show hypomethylation of the satellite sequences in the pericentric regions of the chromosome
      • sequencing cancer genomes identified mutations in DNMT3a in 25% of patients with AML
      • however, DNMT overexpression leads to hypermethylation of known tumour supressors
      • balance important
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12
Q

What is the role of histone modification in cancer development?

A
  • epigenetic make up partly determined by histone modification
    • two extreme forms of euchromatin and heterochromatin
  • key is DNA accessibility to RNA polymerase
    • can lead to activation or repression of a gene
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13
Q

Describe euchromatin and heterochromatin

A

Euchromatin

  • Accessible and plastic
  • stem cells, young cells, tumour cells

Heterochromatin

  • Innacessible and restricted
  • Commited, old and normal cells
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14
Q

Give examples of active and repressive histone modifications

A

Acetylation

  • of lysine (activation):
    • H4K16ac

Methylation

  • activating mark:
    • H3K4me3
  • repressive mark:
    • H3K9me3
    • H3K27me3
    • H4K20me1
    • H4K20me3
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15
Q

What is the nature and mechanism of histone modifications?

A
  • C terminus: positively charged, basic, DNA (negatively associated) wraps round, electrostatically good combination
  • N terminus: unfolded tail can be modified
  • Acetylation: histone acetyltransferases neutralise + charge, DNA lets go of histones
    • electrostatically less favourable, recruits other modellers which open up the chromatin
    • these are typically activators
  • Deacetylases: remove acetylation
  • Methylation: more complex. both activators and repressors
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16
Q

Describe a map of histone modifications in a normal cell and in a cancer cell

A

If in a Normal cell you have…

  • Gene rich region: high histone acetlyation (active) and open K4 methylation (active)
  • Subtellomeric and satellite repeats: low acetylation (active) and high K27, K9, K20 (repressive) methylation marks

Then in a Cancer cell...

  • Gene rich region: no acetylation, high K9 and K27 (repressive) methylation marks
  • Subtellomeric and satellite repeats: increase acetlyation (active) and open K4 methylation (active) marks

the context of histone modifications goes wrong in cancer

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17
Q

What are the histone lysine methylation systems

A
  • highly selective enzymes
  • 5 major methylatable position in H3 and H4
    • H3-K4
    • H3-K9
    • H3-K27(EZH enzyme)
    • H3-K36
    • H4-K20
  • can have mono-, di- or tri- methylation
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18
Q

How does histone acetylation and methylation status change in cancer and what are the methods by which this could happen?

A
  • Global loss of acetylation
    • global loss of H4K16ac mediated by the overexpression of HDACs in some cancers e.g. prostate and gastric (major route)
    • or reduced activity of HATs (p300) in leukemia
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19
Q

How are histone modifiers involved in cancer?

A
  • Histone modifiers (SWI-SWF complex, ATP dependent, recurited to acetylated histones) have a subunit that is a tumour supressor and its inactivation can decrease the expression of p21 and p16 and lead to transformation to cancerous cells
  • p300 reduced activity in some cancers (leukemia) leads to a global loss of acetylation
  • HDAC dysregulation (overexpression) can indicate poor prognisis
20
Q

Give an example of how histone methylation can be linked to cancer (H3K27me3)

A
  • H2K27me3 silences genes that are involved in neoplastic growth
  • some genes known to repress growth and promote apoptosis (anti-cancer) are modified with high H3K27me3
  • EZH2 (H3K27 methyltransferase) is overexpressed in some cancers (prostate and breast) and is linked to poor pronosis
21
Q

Outline epigenetic therapy as a treatment for cancer

A
  • particularly successful in treating haematological malignancies, but not known if it is useful in treating solid tumours
  • use small molecules to treat cancer
  • easier to rearrange tags than to correct mutations in chromosome (but also easier to mess up)
  • however when the drug is removed the cancer can return
22
Q

What are DMNT inhibitors and what are the two main types?

A

First line of defense against cancer, hoping to activate tumour supressors that have had supressions

  1. Nucleoside DNMT inhibitors (more effective but more toic)
  2. Non-nucleoside DNMT inhibitors: drugs that block the active site of DNMT or its expression
23
Q

Outline nucleoside DNMT inhibitors an as anticancer therapy

A
  • 5-azacytidine
  • toxic with side effects (Zebularine is a less toxic derivitive)
  • mechanism of action: degradation of DNMT
  • can induce apoptosis

Disadvantages

  • requires incorperation into DNA during S phase and therefore need repeat treatment
    • this has implication on patient quality of life
    • don’t know effects of prolonged demethyltion to healthy cells
    • possible cytotoxicity and can result in secondary tumors
  • 5-azacytidine is unstable in an aqeous solution
  • side effects
24
Q

By what mechanism do nucleoside DNMT inhibitors inhibit DNA methylation?

A

Use covelent trapping mechanism

  • during methylation cytosine converted to 5MCyt
  • MTase and SAM (methyl donor) replace an C with a Methyl group
  • If an N is present instead of a C (following treatment with 5-azacytidine) SAM gets covelently trapped and can’t move away
  • binding to DNA with an enzyme trapped can activate apoptosis
  • Can also titrate out methyltransferase as keeps getting stuck, the reduce levels of Dnmts by titrating them out of solution
25
Q

Outline non-nucleoside DNMT inhibitors as potential anti-cancer therapies

A
  • drugs that block the active site of DNMT or blcok the synthesis of DNMT
    • MG98 is an oligonucleotide that blocks mRNA
  • less toxic but effectiveness is contraversial
  • some natural compounds (such as main phenol in green tea) are in this category, thought to reduce methylation
26
Q

Outline HDAC inhibitors (non-nucleosidal DNMT inhibitors) as potential anti cancer therapies

A
  • Many were used to treat epilepsy and depression before their use in cancer was discovered
  • Used even if HDAC levels are normal, but if HAT not working efficiently enough
  • Many types:
    • Including vorinostat (SAHA)
      • inhibits histone deacetylation
      • good response in T cell lymphomas by patients with advanced disease who have not responded well to other therapies
  • Disadvantages: short pharmacological half life, not sure if histone deacetlation is the actual target of the drug
27
Q

What experimental evidence is there that the oral drug therapy suberoylanilide hydroxamic acid (SAHA) reduceds T-cell lymphomas?

A
  • Took biopsy of an enlarged lymph node from a patient with T-cell lymophoma before and after treatment
  • Using anti-AcH3 antibody (recognises acetylated histones)
  • Levels of histone acetylation were recovered
28
Q

How have inhibitors in clinical trials shown to reactivate tumour supressor genes?

A
  1. Tumour supressor off: by DNA methyaltion and histone modification
  2. Treat with 5-azacytosine, zeb or Dnmt1-inhibitors to remove DNA methylation
  3. then treat with SAHA or HDAC-inhibitors to remove histone modification
  4. opens up complex and allows expression of a tumour supressor
  5. However H3K9me3 persists and therefore if the drug is removed this may signal more methylation
29
Q

What epigenetic changes can promote/supress cancer?

A
  • histone modifications and DNA methylation can act together to promote cancer
  • some genetic and dietary interventions that extend life span also tend to supress cancer
30
Q

What is the experimental evidence that as you age your epigenome shifts?

A
  • used monozygotic twins (any changes are not to do with genetics)
  • looked at % of
    • H4 acetylation (% of AcH4)
    • H3 acetylation (% of AcH3)
    • DNA methyation (of 5mC)
    • little drift in 3-year old twins
    • Significant drift in 50 year old twins
    • Epigenetic drift in aging identical twins
  • Also did a compatarive genomic hybridisation for methylated DNA
    • stained chromosome for methylation patterns
      • similar chromosome methylation patterns between tiwns
      • regions of hypomethylation in one twin compared to the other
      • regions of hypermenthylation in one twin compared to the other
    • drift as get older
31
Q

What is the involvement of DNA methylation in aging

A
  • global decrease in DNA methylation in older individuals
    • probably due to progressive loss of DNMT1 activity
    • repetitive repeat elements become less methylated over time (causing chromsome instability)
  • cell may overcompensate by overexpressing DnmT3b (demonstrated in cultured fibroblasts)
    • leads to hypermethylation of certain genes of CpG islands

[These are all also known epigenetic changes in cancer]

  • SAM (the methyl donor to most methyltrasferases) shows dysregulation in its metabolism
  • drosophila with over expressed Dnmt lives over 50% longer than the WT
    • dnmt mutants (50%reduction in activity) lived 27% shorter than WT
32
Q

How was it demonstrated that DNA methylation states can be used to predict the age of a cell?

A
  • Hannum et al used whole blood to define 71 different CpG sites
  • Used mathematical model to predict the age of the cell
  • Can predict chronological age to + 4 years and 96% accuracy
33
Q

How is DNA methylation assciated with senescence? (Aging)

A

DNA methylation represses a set of growth inhibitory genes and this repression is attenuated with the onset of senescence

Pushes cells towards proliferative arrest

34
Q

Aside from cancer, what other age-related diseases is DNA hypomethylation obsevred in ?

A

Hypomethylation is observed in som eautoimmuno and neruodegenerative disorders such as alzheimers

35
Q

What changes to heterochromatin structure occur with ageing?

A
  • Global loss of chromatin
  • Locus-specific changes
    • Core histone levels decrease with age
      • ​overexpression of core histones can increase lifespan by 50% (yeast)
      • possibly becuase more histones mean more packaging and genomic stability
    • Histone modifictions change
      • ​golabl increase in methylation of some histones, leading to transcriptional silening at promoter of active genes
        • leads to senescence
    • Acetylation and deacetylation
36
Q

What interactions can mark the boundaries of active or repressive chromain?

A
  • some evidence of interactions between chromatin and nuclear periphery proteins (e.g. lamin)
37
Q

what are the two types of senescence?

A

Replicative senesence (RS)

  • Hayflick limit: shortening of telomeres
  • appear to degenerate (model for aging)

Oncogene induced senesence (OIS)

  • expression of an oncogene (e.g.RAS)
  • restricts the progression of cancer (model for cancer)
  • prevents infinate replicative life span
38
Q

What was observed in chromatin profiling of senescent cells?

A

Major expression changes in important genes can be explained by changes to H3K4/27me3

Large scale cahnge in the senescence genome for histone modifiations:

  • H3K4me3 (acitve) is reduced at genes that need to be down-regulated to stop cell proliferation (e.g. cell cycle genes)
    • in senesent cells get peaks of expression which don’t exist in proliferative cells
  • H3K27me3 (repressive) is reduced at genes that promote senesence
    • peaks in senesence (like in proliferative cells) and drops (not like proliferative cells)

Bivalent control of stem cells is mediated by PCR and trx, changes to these marks also linked to senecence

39
Q

What are LADs?

A
  • LADs: Lamin-associated chromatin domains
  • rich in heterochromatic marks such as H3K27me3
  • but regions that loop away are rich in activating marks such as acetylation and H3K4me3
  • contributes to the sability of the nucleus
40
Q

How was it determined whether disruptions to the chromatin during senescence are due to the breakdown of the nuclear lamina?

A
  • Looked at the overlap between LADs and the heavily modified regions of the chromatin with increases in H3K4me3 and H3K27me3 (mesas)
  • These regions are devoid of active genes
  • The big drop regions in H3K27me3 (canyons) tend to form adjacent to LADs and contain active genes and enhancers
  • These two marks (H3K4me3 and H3K27me3 tend to change profile during senescence and have an asscoation with the nuclear laminar
41
Q

How was the role of Lamin B1 determined in senescence?

A
  • Western blot: looked at expression of lamin B1 in proliferating and senescent cells
    • Lamin B1 levels drop during senescence (GAPDH as control)
  • Knock down of Lamin B1 in proliferating cells: causes premature senescence
    • after two round of doubling cell seneseces
    • corresponds with lamin B1 levels droping during senescence
  • Knock down of Lamin B1 causes similar chromatin changes to senecent cells
    • lamin B1 levels drop during senescence
    • knockdown of laminB1 causes cells to senesce after only two population doubligns
      • causes locus specific remodelling of chromatin (K4/K27me3 peaks and drops observed in normal senesence)
      • leads to loss of nuclear integrit and chromatin loss in to the cytoplasm
42
Q

What happens to LaminB1 levels in progeria?

A

In progeria llamin B1 is mutated, and is a key component of the disorder

Premature aging

43
Q

What chromatin changes occur in aging?

A
  • chromatin modifications
    • histone acetylation and deacetylation
      • in mammals loss of H4K16ac linked to aging
      • In yeast during replicative aging
        • global loss of H3K56ac
        • global increase in H4K16ac
        • results in loss of silencing at telomeres, H416ac is deacetylated by sir2 (HDAC)
44
Q

What is Sir2?

A
  • Sir2 is an NAD+ dependent histone deacetylase (HDAC)
  • Calorie restrcition decreases aging
  • NAD is a by-product of metabolism
  • Proposed model of activity:
    1. Decreased glycolysis
    2. increased cellular NAD levels
    3. NAD + acetyl histone
    4. Activity of Sir2 activated by increased NAD
    5. Nicotinamide + O-acetyl-ADP-ribose + histone
    6. Increasing life span
  • Yeast grown in the presence of excess nicotinamide show accelerated aging and loss of transcriptional silencing
  • Role in yeast more clear, more complex in other organisms
45
Q

How do you measure the age of a yeast cell?

A
  • When yeast cells grow, buds into mother cell and daughter cell
  • Yeast chromosome has R DNA (repetitive domains, can recombine easily and pop out of the genome)
  • when mother cell splits, retains more of the R-DNA
  • Can determine age of yeast
46
Q

What are the features of the Sir2 mammalian ortholog, SIRT1?

A
  • role in aging is contraversial and complex
  • deacetylates H1K26, H4K16ac and many other targets including p53
  • global genomic hypoacetylation at H4K16 is a hallmark of human cancer cells in cell lines and clinical samples
  • can be both a tumour supresor and promoter

Y3 (2) Epigenetics in Development and Disease (8 decks)

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