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EXMD504 > 6. Aberrant Gene Expression in Cancer > Flashcards

6. Aberrant Gene Expression in Cancer Flashcards

1
Q

what colour are HIGHLY expressed genes in a gene expression heat map?

A

red

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2
Q

2 key features of cancer (in relation to genes)

A
  1. aberrant gene function
  2. altered patterns of gene expression
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3
Q

what 2 things ultimately lead to abnormal gene expression?

A

genetic and epigenetic alterations

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4
Q

why is it helpful to stratify patients based on aberrant gene expression in cancer?

A

for treatments –> i.e. ER+ tumours

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5
Q

what is the purpose of epigenetics?

A

allows cell with same DNA/set of chromosomes to be programmed differently to express different genes –> i.e. allows differentiation

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6
Q

epigenetic changes are:

A

epigenetic changes are all phenomena that produce heritable changes in genome function without affecting DNA sequence

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7
Q

expression state of a gene is determined by: (5)

A
  1. packaging/accessibility of regulatory regions
  2. promoters, enhancers
  3. chromatin
  4. TF
  5. chromatin-modifying enzymes
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8
Q

accessibility of chromatin to transcriptional regulation is controlled by: (2)

A
  1. modification of the DNA itself
  2. modification/rearrangement of nucleosomes (histones)
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9
Q

describe NUCLEOSOMES

A

2 turns of DNA wrapped around histone octamer –> N-terminal tails protrude out and can be post-translationally modified

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10
Q

what is the octamer made of?

A

2 subunits of 4 diff H proteins

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11
Q

what signifies the status of the chromatin?

A

the pattern of histone modifications

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12
Q

4 regulating enzymes and their roles

A
  1. writers (ADD modifications)
  2. erasers (REMOVE modifications)
  3. readers (READ modifications)
  4. movers (remodel chromatin by moving nucleosomes, allowing gene transcription)
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13
Q

HISTONE VARIANTS
- what are they?
- which histones do they affect?
- when/how are they produced?

A
  1. minor variants can replace histone proteins
  2. for histone 2A and 3
  3. produced in INTERPHASE and inserted into previously formed chromatin by a chromatin-remodeling complex
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14
Q

what does the chromatin-remodeling complex do?

A

recruits specific binding proteins to change chromatin status

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15
Q

how can we detect functional elements in the genome?

A

histone modifications in non-coding regions label the functional elements

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16
Q

how are promoters often labeled (2)?

A
  • trimethylation
  • H3K27 acetylation
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17
Q

how can we map the epigenome?

A

with ChIP-Seq

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18
Q

what does ChIP-Seq tell us?

A

antibody pulls histone modifications and can sequence the DNA attached –> tells us where specific histone marks are located

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19
Q

what indicates the cell-type specificity of non-coding elements?

A

histone modifications

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20
Q

how can we map the open chromatin regions?

A

ATAC-Seq

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21
Q

how does ATAC-Seq work?

A

uses transposases that preferentially insert into open regions –> then sequence these regions to know where the open regions are

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22
Q

what do chromatin accessibility profiles reveal?

A

distinct molecular subtypes of cancer

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23
Q

2 ways that epigenetics change accessibility of chromatin to transcriptional regulation

A
  1. DNA modification
  2. rearrangement of nucleosomes/histones
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24
Q

describe methylation patterns

A

methylation patterns generally vary between diff cell types and diff stages in development

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25
Q

role of DNA methylation?

A

SILENCE gene expression

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26
Q

3 roles of methylation

A
  1. genomic imprinting
  2. X-chromosomal inactivation
  3. suppression of retrotransposons
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27
Q

what is genomic imprinting?

A

diff expression of maternally and paternally inherited alleles –> methylation shuts down genes from 1 parent

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28
Q

what is X-chromosomal inactivation?

A

one chromosome is shut down –> for sex-specific genes

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29
Q

where does methylation occur on DNA?

A

occurs at CYTOSINE in context of CG/CpG islands

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30
Q

Describe DNMT1 and its role

A

maintains existing DNA methylation patterns

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31
Q

Describe DNMT3A and DNMT3B and their role

A

de novo methyltransferases that methylate CG dinucleotides

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32
Q

which enzymes are methylation writers?

A

DNA methyltransferases (DNMT)

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33
Q

which enzymes are methylation erasers?

A

TET

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34
Q

how is DNA methylation a source of mutations?

A

NORMAL: Cytosine can be deaminated to produce uracil –> but uracil DNA glycosylase can remove the uracil and fix the problem

MUTATION: Cytosine can be methylated, then deaminated to produce thymine –> makes a TG mismatch that is a mutation

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35
Q

what are CpG islands?

A

high concentrations of G+C bases and dinucleotide CpG

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36
Q

what % of CpG islands are associated with known transcriptional start sites?

A

50%

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37
Q

describe methylation of CpG islands

A

CpGs associated with transcriptional start sites are UNMETHYLATED even when gene is not being transcribed –> therefore these don’t determine gene expression

CpGs FURTHER from the transcriptional start site are METHYLATED to determine whether a gene is silenced or expressed

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38
Q

why are CpG islands significant?

A

represent areas of genome that were protected from mutating properties of methylation over time

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39
Q

how does DNA methylation change in cancer? consequence?

A
  1. hypomethylated CpG island at transcriptional start site becomes HYPERMETHYLATED
    - allows for direct mutagenesis of 5mC-containing sequence by deamination
  2. the rest of the genome that is normally hypermethylated becomes HYPOMETHYLATED
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40
Q

SUMMARY - what are changes in chromatin structure dependent on? (3)

A

changes in:
1. DNA methylation
2. histone modification
3. positioning of nucleosomes

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41
Q

ONCOGENES
- recessive or dominant?
- how are they overexpressed?
- how are they activated?

A
  • Dominant-acting
  • Overexpressed due to gene amplification (copy number variants)
  • Activated by structural variants that reposition a silenced gene so that it is activated by active regulatory elements (enhancer hijacking)
42
Q

TUMOUR SUPPRESSORS
- recessive or dominant?
- how are they activated? (4)

A
  • recessive
  • activated by mutations, deletions, DNA methylation, or combination
43
Q

what are driver mutations?

A

mutations important in cancer development that are positively selected

44
Q

what are passenger mutations?

A

everything that is not a driver mutation

45
Q

describe the accumulation of mutations throughout lifespan

A

always accumulating mutations from intrinsic processes, environment, and germline –> may get driver mutations that give proliferative advantage

46
Q

describe the detection of SNVs

A

sequence tumour and normal samples to find variants not found in the normal genome

47
Q

what does it mean if sequencing shows that only half the tumour sample reads have mutations and the normal sample doesn’t have mutations?

A

since the normal sample doesn’t have mutations, it is NOT GERMLINE and therefore a HETEROZYGOUS mutation

48
Q

what does SNV sequencing tell us?

A

only tells us that there are more mutations in the gene than expected, but this doesn’t tell us the gene’s function

49
Q

what do recurrent somatic mutations tell us?

A

recurrent somatic mutations can help us identify cancer driver genes

50
Q

difference in mutations creating oncogenes vs tumour suppressors

A

oncogenes = missense mutation, gain function

tumour suppressors = truncating mutation, lose function

51
Q

why are most mutations in oncogenes the same?

A

only so many ways for a gene to gain function

52
Q

why are mutations in tumour suppressors more variable?

A

there are many ways to lose function via diff truncations

53
Q

what can IDH1 mutation induce? how do we know?

significance?

A

IDH1 mutation can induce HYPERMETHYLATION

look at heat map of DNA methylation:
- IDH1 mutation corresponded to high methylation at CpG islands (abnormal)
- normal IDH1 corresponded to low methylation at CpG islands (normal)

THEREFORE, mutations have an effect on the epigenome and gene expression

54
Q

why is it unusual that IDH1 affects epigenome?

A

IDH1 is not epigenetic protein –> involved in metabolism/TCA

55
Q

how does IDH1 mutation induce abnormal hypermethylation at CpG islands?

A

IDH1 mutation causes alphaKG to become 2-hydroxyglutarate and block TET enzymes –> therefore hypermethylation

56
Q

2 types of copy number variants

A
  1. GAINS
  2. DELETIONS
57
Q

example of protein with GAIN copy number variant

A

MYCN is highly amplified in tumours

58
Q

where are extra copies of genes found?

A

on circular extrachromosomal DNA, not in genome

59
Q

what is it called when there are extra copies of a gene in the genome?

A

tandem duplications

60
Q

why is it important to look at patients with gain of function?

A

this copy number gain could indicate driver mutations of oncogenes

61
Q

why is it important to look at patients with loss of function?

A

this copy number loss could indicate driver mutations of tumour suppressors

62
Q

how can we detect cancer driver genes?

A

calculate the background copy number rate and look at its recurrence across many samples –> can look at gains/losses at the chromosomal level

63
Q

prevalence of epigenetics in cancer

A

about 50% of human cancers have mutations/alterations in epigenetic proteins

64
Q

malignant cells exhibit:

A
  1. genome-wide alterations in DNA methylation
  2. chromatin structure
  3. regulatory element activity
  4. deranged developmental program –> differentiation block or epigenetic reprogramming
65
Q

common mutations in histone proteins

A

In histone H3.3, mutations in K27, K36, and G34 are common

66
Q

what do the K27 and K36 mutations in H3.3 do?

A

REDUCE methylation

67
Q

what does the G34 mutation in H3.3 do?

A

reduces levels of H3K36me on the same or nearby chromosomes

68
Q

what is the most common epigenetic mutation in tumours?

A

SWI/SNF mutations

(>20% of all cancers have this)

69
Q

what is SWI/SNF? how does it work?

A

CHROMATIN REMODELING COMPLEX

Uses energy from ATP hydrolysis to reposition, eject, slide, or alter the composition of nucleosomes –> allows DNA-binding proteins and transcriptional machinery to access DNA and affect gene expression

70
Q

is the SWI/SNF mutation always the same?

A

not always the same protein is mutated –> 9 diff proteins can be mutated

71
Q

does the mutated SWI/SNF have oncogenic or tumour-suppressor activity?

A

TUMOUR SUPPRESSOR activity –> allows TF function for cell differentiation

72
Q

what is the relationship between SWI/SNF and PRC complexes? why?

A

antagonism –> would be synthetic lethal if both had mutations

73
Q

why are not all SWI/SNF proteins mutated?

A

would be synthetic lethal!

74
Q

what can noncoding mutations in regulatory elements lead to?

A

noncoding mutations in regulatory elements lead to aberrant expression of oncogenes, TFs, and chromatin regulators –> causing aberrant gene expression

75
Q

why are most mutations outside of the coding region? what does this mean for cancer mutations

A

only 2% of the genome is coding, the rest is non-coding –> therefore, usually in regions not active in tumour, BUT mutations in histone modifications can be cancer promoting

76
Q

do non-coding regulatory elements have more or less somatic mutations than coding regions?

A

non-coding regulatory elements have MORE somatic mutations than coding regions

77
Q

what type of mutations do non-coding elements typically have?

A

DRIVER mutations

78
Q

what was the first major non-coding mutation to be discovered?

A

recurrent driver mutation in TERT promoter

79
Q

what region of a transcriptional site are mutation-rich?

A

promoter regions are mutation-rich

80
Q

are non-coding mutations recurrent?

A

yes

81
Q

what do oncogene amplifications involve? +examples

A

the oncogene AND non-coding elements (enhancers, regulators, etc.) that can transcriptionally activate oncogenes

82
Q

how are enhancers amplified relative to their respective oncogenes?

A

enhancers can be amplified WITH or WITHOUT their respective oncogenes

83
Q

describe identification of co-selected regulatory elements

A

take amplicons of the same length and randomly shuffle –> find selection for the gene AND enhancer –> therefore enhancers are amplified WITH the oncogene

84
Q

what is the technique where we identify an enhancer and its target gene?

A

Chromatin Confirmation Capture (3C)

85
Q

How does 3C work? 5 steps

A
  1. ligate proteins interacting with DNA to DNA
  2. use restriction enzyme to cleave loops away
  3. ligate sections together
  4. sequence the pair of enhancer + promoter to know if the enhancer interacts with the promoter
  5. if the enhancer interacts with the promoter, can use heat map to see interaction
86
Q

what are topologically associated domains?

A

regions that interact with themselves but not each other –> distinct boundaries btwn elements by insulator elements

87
Q

what does enhancer hijacking explain?

A

enhancer hijacking explains aberrant gene expression patterns

88
Q

what is enhancer hijacking?

A

non-coding SNVs allow enhancers to regulate diff gene

89
Q

how can you detect enhancer hijacking?

A

look where there are recurrent break points/alterations

90
Q

how does a GAIN copy number alteration occur for enhancer hijacking?

A

enhancer is moved to diff region

91
Q

how does a DELETION copy number alteration occur for enhancer hijacking?

A

genes are deleted so enhancer upstream can interact with promoter to increase gene expression

92
Q

what are TAD boundaries?

A

insulators btwn elements make distinct boundaries

93
Q

WT TADs

A

TADs don’t interact with each other bc of insulators

94
Q

disrupted TADs

A

lose insulator elements so TADs fuse

95
Q

what is highly recurrent in group 4 medulloblastoma?

A

SNCAIP duplication

96
Q

describe SNCAIP duplication

A

assumed to be an oncogene but epigenetic data shows chromatin interactions where TADs overlap

97
Q

what do we see alongside SNCAIP duplication?

A

increased PRDM6 expression involved in methylation

98
Q

describe SNCAIP duplication with PRDM6 expression

A

when SNCAIP duplicates, TAD boundary duplicates with enhancers which reorganizes chromatin so a super enhancer can be part of neighbouring TAD and activate PRDM6 for methylation

99
Q

4 ways that hijacking can occur?

A
  1. translocations
  2. inversions
  3. duplications
  4. deletions of insulators
100
Q

what does ecDNA allow for?

A

ecDNA allows for distal DNA interactions –> via co-amplification of enhancer elements to hijack neighbouring enhancers and incorporate diff pieces of chromosomes

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