6. Aberrant Gene Expression in Cancer Flashcards
what colour are HIGHLY expressed genes in a gene expression heat map?
red
2 key features of cancer (in relation to genes)
- aberrant gene function
- altered patterns of gene expression
what 2 things ultimately lead to abnormal gene expression?
genetic and epigenetic alterations
why is it helpful to stratify patients based on aberrant gene expression in cancer?
for treatments –> i.e. ER+ tumours
what is the purpose of epigenetics?
allows cell with same DNA/set of chromosomes to be programmed differently to express different genes –> i.e. allows differentiation
epigenetic changes are:
epigenetic changes are all phenomena that produce heritable changes in genome function without affecting DNA sequence
expression state of a gene is determined by: (5)
- packaging/accessibility of regulatory regions
- promoters, enhancers
- chromatin
- TF
- chromatin-modifying enzymes
accessibility of chromatin to transcriptional regulation is controlled by: (2)
- modification of the DNA itself
- modification/rearrangement of nucleosomes (histones)
describe NUCLEOSOMES
2 turns of DNA wrapped around histone octamer –> N-terminal tails protrude out and can be post-translationally modified
what is the octamer made of?
2 subunits of 4 diff H proteins
what signifies the status of the chromatin?
the pattern of histone modifications
4 regulating enzymes and their roles
- writers (ADD modifications)
- erasers (REMOVE modifications)
- readers (READ modifications)
- movers (remodel chromatin by moving nucleosomes, allowing gene transcription)
HISTONE VARIANTS
- what are they?
- which histones do they affect?
- when/how are they produced?
- minor variants can replace histone proteins
- for histone 2A and 3
- produced in INTERPHASE and inserted into previously formed chromatin by a chromatin-remodeling complex
what does the chromatin-remodeling complex do?
recruits specific binding proteins to change chromatin status
how can we detect functional elements in the genome?
histone modifications in non-coding regions label the functional elements
how are promoters often labeled (2)?
- trimethylation
- H3K27 acetylation
how can we map the epigenome?
with ChIP-Seq
what does ChIP-Seq tell us?
antibody pulls histone modifications and can sequence the DNA attached –> tells us where specific histone marks are located
what indicates the cell-type specificity of non-coding elements?
histone modifications
how can we map the open chromatin regions?
ATAC-Seq
how does ATAC-Seq work?
uses transposases that preferentially insert into open regions –> then sequence these regions to know where the open regions are
what do chromatin accessibility profiles reveal?
distinct molecular subtypes of cancer
2 ways that epigenetics change accessibility of chromatin to transcriptional regulation
- DNA modification
- rearrangement of nucleosomes/histones
describe methylation patterns
methylation patterns generally vary between diff cell types and diff stages in development
role of DNA methylation?
SILENCE gene expression
3 roles of methylation
- genomic imprinting
- X-chromosomal inactivation
- suppression of retrotransposons
what is genomic imprinting?
diff expression of maternally and paternally inherited alleles –> methylation shuts down genes from 1 parent
what is X-chromosomal inactivation?
one chromosome is shut down –> for sex-specific genes
where does methylation occur on DNA?
occurs at CYTOSINE in context of CG/CpG islands
Describe DNMT1 and its role
maintains existing DNA methylation patterns
Describe DNMT3A and DNMT3B and their role
de novo methyltransferases that methylate CG dinucleotides
which enzymes are methylation writers?
DNA methyltransferases (DNMT)
which enzymes are methylation erasers?
TET
how is DNA methylation a source of mutations?
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
what are CpG islands?
high concentrations of G+C bases and dinucleotide CpG
what % of CpG islands are associated with known transcriptional start sites?
50%
describe methylation of CpG islands
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
why are CpG islands significant?
represent areas of genome that were protected from mutating properties of methylation over time
how does DNA methylation change in cancer? consequence?
- hypomethylated CpG island at transcriptional start site becomes HYPERMETHYLATED
- allows for direct mutagenesis of 5mC-containing sequence by deamination - the rest of the genome that is normally hypermethylated becomes HYPOMETHYLATED
SUMMARY - what are changes in chromatin structure dependent on? (3)
changes in:
1. DNA methylation
2. histone modification
3. positioning of nucleosomes
ONCOGENES
- recessive or dominant?
- how are they overexpressed?
- how are they activated?
- 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)
TUMOUR SUPPRESSORS
- recessive or dominant?
- how are they activated? (4)
- recessive
- activated by mutations, deletions, DNA methylation, or combination
what are driver mutations?
mutations important in cancer development that are positively selected
what are passenger mutations?
everything that is not a driver mutation
describe the accumulation of mutations throughout lifespan
always accumulating mutations from intrinsic processes, environment, and germline –> may get driver mutations that give proliferative advantage
describe the detection of SNVs
sequence tumour and normal samples to find variants not found in the normal genome
what does it mean if sequencing shows that only half the tumour sample reads have mutations and the normal sample doesn’t have mutations?
since the normal sample doesn’t have mutations, it is NOT GERMLINE and therefore a HETEROZYGOUS mutation
what does SNV sequencing tell us?
only tells us that there are more mutations in the gene than expected, but this doesn’t tell us the gene’s function
what do recurrent somatic mutations tell us?
recurrent somatic mutations can help us identify cancer driver genes
difference in mutations creating oncogenes vs tumour suppressors
oncogenes = missense mutation, gain function
tumour suppressors = truncating mutation, lose function
why are most mutations in oncogenes the same?
only so many ways for a gene to gain function
why are mutations in tumour suppressors more variable?
there are many ways to lose function via diff truncations
what can IDH1 mutation induce? how do we know?
significance?
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
why is it unusual that IDH1 affects epigenome?
IDH1 is not epigenetic protein –> involved in metabolism/TCA
how does IDH1 mutation induce abnormal hypermethylation at CpG islands?
IDH1 mutation causes alphaKG to become 2-hydroxyglutarate and block TET enzymes –> therefore hypermethylation
2 types of copy number variants
- GAINS
- DELETIONS
example of protein with GAIN copy number variant
MYCN is highly amplified in tumours
where are extra copies of genes found?
on circular extrachromosomal DNA, not in genome
what is it called when there are extra copies of a gene in the genome?
tandem duplications
why is it important to look at patients with gain of function?
this copy number gain could indicate driver mutations of oncogenes
why is it important to look at patients with loss of function?
this copy number loss could indicate driver mutations of tumour suppressors
how can we detect cancer driver genes?
calculate the background copy number rate and look at its recurrence across many samples –> can look at gains/losses at the chromosomal level
prevalence of epigenetics in cancer
about 50% of human cancers have mutations/alterations in epigenetic proteins
malignant cells exhibit:
- genome-wide alterations in DNA methylation
- chromatin structure
- regulatory element activity
- deranged developmental program –> differentiation block or epigenetic reprogramming
common mutations in histone proteins
In histone H3.3, mutations in K27, K36, and G34 are common
what do the K27 and K36 mutations in H3.3 do?
REDUCE methylation
what does the G34 mutation in H3.3 do?
reduces levels of H3K36me on the same or nearby chromosomes
what is the most common epigenetic mutation in tumours?
SWI/SNF mutations
(>20% of all cancers have this)
what is SWI/SNF? how does it work?
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
is the SWI/SNF mutation always the same?
not always the same protein is mutated –> 9 diff proteins can be mutated
does the mutated SWI/SNF have oncogenic or tumour-suppressor activity?
TUMOUR SUPPRESSOR activity –> allows TF function for cell differentiation
what is the relationship between SWI/SNF and PRC complexes? why?
antagonism –> would be synthetic lethal if both had mutations
why are not all SWI/SNF proteins mutated?
would be synthetic lethal!
what can noncoding mutations in regulatory elements lead to?
noncoding mutations in regulatory elements lead to aberrant expression of oncogenes, TFs, and chromatin regulators –> causing aberrant gene expression
why are most mutations outside of the coding region? what does this mean for cancer mutations
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
do non-coding regulatory elements have more or less somatic mutations than coding regions?
non-coding regulatory elements have MORE somatic mutations than coding regions
what type of mutations do non-coding elements typically have?
DRIVER mutations
what was the first major non-coding mutation to be discovered?
recurrent driver mutation in TERT promoter
what region of a transcriptional site are mutation-rich?
promoter regions are mutation-rich
are non-coding mutations recurrent?
yes
what do oncogene amplifications involve? +examples
the oncogene AND non-coding elements (enhancers, regulators, etc.) that can transcriptionally activate oncogenes
how are enhancers amplified relative to their respective oncogenes?
enhancers can be amplified WITH or WITHOUT their respective oncogenes
describe identification of co-selected regulatory elements
take amplicons of the same length and randomly shuffle –> find selection for the gene AND enhancer –> therefore enhancers are amplified WITH the oncogene
what is the technique where we identify an enhancer and its target gene?
Chromatin Confirmation Capture (3C)
How does 3C work? 5 steps
- ligate proteins interacting with DNA to DNA
- use restriction enzyme to cleave loops away
- ligate sections together
- sequence the pair of enhancer + promoter to know if the enhancer interacts with the promoter
- if the enhancer interacts with the promoter, can use heat map to see interaction
what are topologically associated domains?
regions that interact with themselves but not each other –> distinct boundaries btwn elements by insulator elements
what does enhancer hijacking explain?
enhancer hijacking explains aberrant gene expression patterns
what is enhancer hijacking?
non-coding SNVs allow enhancers to regulate diff gene
how can you detect enhancer hijacking?
look where there are recurrent break points/alterations
how does a GAIN copy number alteration occur for enhancer hijacking?
enhancer is moved to diff region
how does a DELETION copy number alteration occur for enhancer hijacking?
genes are deleted so enhancer upstream can interact with promoter to increase gene expression
what are TAD boundaries?
insulators btwn elements make distinct boundaries
WT TADs
TADs don’t interact with each other bc of insulators
disrupted TADs
lose insulator elements so TADs fuse
what is highly recurrent in group 4 medulloblastoma?
SNCAIP duplication
describe SNCAIP duplication
assumed to be an oncogene but epigenetic data shows chromatin interactions where TADs overlap
what do we see alongside SNCAIP duplication?
increased PRDM6 expression involved in methylation
describe SNCAIP duplication with PRDM6 expression
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
4 ways that hijacking can occur?
- translocations
- inversions
- duplications
- deletions of insulators
what does ecDNA allow for?
ecDNA allows for distal DNA interactions –> via co-amplification of enhancer elements to hijack neighbouring enhancers and incorporate diff pieces of chromosomes
