1
Q
what is epigenetic?
A
- epi means above or beyond/below
- refers to a range of chemical modification that control gene accessibility but do not alter the underlying genetic code
2
Q
what are the two main targets of epigenetic modifications?
A
- DNA modification - methylation
2. Histone modification - chemical modification of proteins that surround the DNA
3
Q
what are the characteristics of DNA methylation
A
- addition of methyl group to C-5 position of cytosine residues
- most cytosine methylation occurs in 5’-CG-3’ sequence
- occurs almost exclusively at cytosines that are immediately followed by guanine CpG dinuleotide
4
Q
what are CpG islands? where are they found?
A
- CpG sites are non-randomly distributed throughout the genome concentrated in hot spots or GpG rich regions knows as CpG islands
- they re found in 50% of promoter region of genes
5
Q
the human genome is methylated uniformly
A. true
B. false
A
B. false, human genome is not methylated uniformly, it contains regions of unmethylated segments interspersed by methylated regions
6
Q
where is 5’ methyl cytosine found?
A
it is found in approx 4% of genomic DNA primarily at cytosine-guanine dinucleotides (CpGs)
7
Q
what does DNA methylation with a. promoter region and b. CpG islands result in ?
A
a. methylation with promoter region correlates with transcriptional silencing
b. methylation of CpG islands is believed to dysregulate gene transcription - through the inhibition of TF binding either directly or indirectly via histone activation
8
Q
Define the following
a. DNMTs
b. SAM
c. SAH
A
a. DNA methylation writers are a group of enzymes refried to as DNA methyl transferases (DNMTs)
b. S-adenosyl methionine (SAM) is converted to S-adenosyl homocysteine) through methyl group transfer from SAM to SAH and is catalysed by DNMTs
9
Q
how many DNMTs are present in mammals?
A
there are 4 DNMTs
- DNMT1 - maintenance methylase
- DNMT2
- DNMT3a and DNMT3b - ‘de novo’ methylases
- DNMT3L
10
Q
what is the function of DNMT1 and requirement to produce the effect?
A
DNMT1
- maintains the pattern of DNA methylation after DNA replication
- requires hemi-methylated DNA substrate to produce the DNA pattern methylation on the newly synthesised strand
11
Q
what is the function of DNMT3a and DNMT3b?
A
- adds methyl groups to CG dinuleotides which are previously unmethylated on both strands
- re-establish the methylation pattern
12
Q
why are DNMTs required?
A
- DNMTs are required for transcriptional silencing of a number of sequence classes
- DNMT3B or DNMT1 deficient mice - embryonic lethal
- DNMT3A null mice die by 4 weeks
13
Q
what is the role of DNA methylation?
A
plays a role in
- long term silencing of genes
- silencing of repetitive elements (e.g. transposons)
- X-chromosome inactivation
- in the establishment and maintenance of genes
- supresses the viral gene expression
- supresses any deleterious elements that have been incorporated into genome of host over time
- in carcinogenesis
14
Q
how is cytosine converted into 5-methylcytosine?
A
- At CpG dinucleotides
- displaces TFs
- attracts methyl bidding proteins which are associated with gene silencing and chromatin compaction
15
Q
what is chromatin made of?
A
chromatin is made up of DNA, RNA and proteins (mainly histone and some non-histone acidic proteins)
16
Q
what is the function of chromatin?
A
- packages DNA
2. regulates DNA accessibility through chromatin structure modification
17
Q
what is the length of DNA inside and outside the nucleus?
A
outside the nucleus - 2m
inside the nucleus - 5-10 micrometer
- the chromatin packs the long DNA into a compact one found inside the nucleus
18
Q
what are the two state chromatin is found in?
A
- euchromatin
2. heterochromatin
19
Q
what are the features of euchromatin and heterochromatin?
A
euchromatin DNA - generally active - less compact (less intense staining) heterochromatin DNA - generally not active - compact (intense staining indicating tighter packaging)
20
Q
what is nucleosome?
A
- it is fundamental repeating unit of chromatin
- DNA wraps around histones to form condensed nucleosomes
- made of ‘nucleosome core particle’ and ‘linker DNA’ (54bp DNA)
21
Q
what is histone?
A
- it is the largest protein in chromatin
- highly conserved
- assembled into octameric complexes
22
Q
what does the nucleosome core particle consists of?
A
histone octamer (146bp) + histone 1
23
Q
what is composition of nucleosome?
A
nucleosome = core of 8 histone molecules + H1 histone + DNA
- histone octamer
- A 147bp DNA segment wrapped around histone octamer
- adjacent nucleosome connected via linker DNA (~60bp)
- histone H1 at the base of nucleosome near DNA binding to linker DNA
- space between the beads in 14nm
24
Q
what is the histone core composed of?
A
1. two copies of each four histones x2 H2A x2 H2B x2 H3 x2 H4 2. structures domain called histone fold and unstructured 'N-terminal tail'
25
what is the function of nucleosome?
1. acts as signalling hub by providing scaffold for the binding of chromatin enzymes
2. displays a combinatorial array of post translation modifications (PTMs)
3. PTMS further regulated the recruitment of chromatin enzymes and tunes both -
a. nucleosome stability
b. higher order compaction of chromatin
26
what is the role of epigenetic writers, readers and erasers in DNA modification?
1. epigenetic writers introduce the chromatin modifications
2. epigenetic erasers remove these modifications
3. epigenetic readers - recognise the modifications and allow epigenetic regulator to bind to them at specific regions to facilitate gene transcription
27
what is the role of chromatin modifications in regulating DNA?
chromatin modifications play an important role in DNA regulation by regulating transcription, repair and replication
28
what are the features if 'switched on' and 'switched off' genes? in terms of
1. activity of chromatin
2. methylation state of cytosine
3. acetylation state of histones
```
switched on -> transcription possible
1. active (open) chromatin
2. unmethylated cytosine
3. acetylated histone
switched off -> transcription impeded
1. silent (condensed) chromatin
2. methylated cytosine
3. deactivated histone
```
29
what are the features of histone tail?
1. forms 30nm fibre by forming H bonds with adjacent nucleosome tails
2. histone tails provide site for covalent t modifications like phosphorylation, methylation, acetylation etc
3. such modifications determine the interaction of histones with other proteins which in turn regulate the chromatin structure and transcription etc
30
what are the types of histone modifications?
1. phosphorylation
2. methylation
3. acetylation
4. ubiquitination
5. biotinylation
31
acetylation is catalysed by which enzymes?
HAT - histone acetyltransferase (addition acetyl)
| HDAC - histone deactylase (removal of acetyl)
32
how does acetylation reduce affinity of tail or what impact does it have on chromatin structure?
1. it removes the +ve charge of the histone tail thus reducing the affinity for -ve charged phosphate groups of DNA
2. this relaxes the high order chromatin structures
3. it also increases the access of transcription factors to DNA through structural changes in nucleosome or nucleosomal arrays
33
what is a bromodomain?
- they are found in TFs
| - BD recognise acetylated lysine through HATs residues on histone tails
34
what is methylation and it is carried out by which enzymes?
- methylation is the addition of methyl functional group to lysine or arginine of the histone tail
- it is carried out by "histone methyltransferase (HMT)"
- arg can be methylated once or twice but lys can be methylated once/twice or thrice
35
a. what is a non-coding RNA?
b. what is its prevalence in humans?
c. what is its role?
- non-coding RNA (ncRNA) is a RNA molecule that is not translated into protein
- in humans 97-98% of transcriptional output is ncRNA
- its function is to regulate gene expression at transcriptional and post-transcriptional level
36
what are the two types of ncRNA? what is it role?
1. short ncRNA <200 nts (nucleotides)
2. long ncRNA >200 its
- they both play a role in heterochromatin formation, DNA methylation targeting, histone modification and gene silencing
37
a. what are microRNAs?
b. its function? and
c. how does it mediate this?
a. small ncRNAs ~22 its long
b. function
1. repress gene expression
2. involved in development, differentiation, proliferation and apoptosis
3. assemble into RISC
c. it acts by base-pairing to the complementary mRNA sequences
38
what is RISC? and what does it consist of?
```
its is RNA induced silencing complex
it consists of
1. DICER (endonuclease that cuts RNA into short segments)
2. TRBP (Tar RNA binding protein)
3. argonaute protein family
```
39
is microRNA expression tissue specific?
A. True
B. False
True, it is highly regulated according to the cell's development lineage and stage
40
outline the steps involved in biogenesis of miRNAs
1. transcription in nucleus results in pri-miRNA formation
2. the pri-miRNA is processed to pre-miRNA through Drosha processing
3. pre-miRNA is then exported into the cytoplasm through exporting-5
4. mature miRNA is then attached to RISC through DICER processing
5. the pre-miRNA then directs the miRNA to mRNA binding for degradation or inhibition of translation
41
what is the mechanism of action for miRNAs?
mature miRNAs binds to the 3'UTR of mRNA and removes the protein coding region of target gene mRNA binding and thus inhibits translation
42
what is uniparental disomy (UPD)?
patients who inherit both homologous chromosomes from the same parent is called UPD
43
what happens in
a. normal chromosome
b. paternal UPD
c. maternal UPD
a. normal - 2 copies of homologous chromosomes (x1 from each parent)
b. maternal UPD - 2 copies of homologous chromosomes, both are maternal alleles and no expression of paternal alleles
c. paternal UPD - 2 copies off homologous chromosomes from paternal alleles and no expression of maternal alleles
44
how does UPD cause diseases?
it causes developmental and neurological diseases due to changes in epigenotype and disruption of genomic imprinting
45
which mechanism results in genomic imprinting?
DNA methylation results in genomic imprinting, it is established in parental germ line and not removed from the zygote
- de novo methylate starts methylation at new sites and acts on CG sites
46
why is the imprinted loci hemizygous in genomic imprinting?
DNA methylation is carried out after DNA replication however, it is replicated only on one strand (hemi-methylated)
47
what is the parental conflict theory?
Father promotes growth and development of fetes by
1. IGF2 - on
2. IGF2R - off
Mother impedes the growth of fetes by reducing the effect of IGF2
1. IGF2 - off
2. IGF2R - on
48
deletion of what factors result in
1. large offspring,
2. dwarf offspring and
3. normally sized offspring
1. large offspring - deletion of IGF2R from mother
2. dwarf offspring - deletion of IGF2 from father
3. normal sized - deletion of fathers Igf2 and mothers Igf2R, the imprints of Igf2 and Igf2R cancel each other
- changing the imprint of one copy of gene has a dramatic effect on the size of the offspring
- this result supports the genetic conflict hypothesis
49
what are ligers (very large) and tiglons? and what is the reason between their high size difference?
- ligers is the hybrid lion as father and tiger as mother while tiglons is the hybrid of tiger as father and lion as mother
- the ligers are very huge
- the difference is size is due to the different imprinted gene between the mother and father
50
what is X inactivation?
it is the inactivation of either of the parents genes; Xp (paternal gene) or Xm (maternal gene) before the mitosis
51
out line the steps involved in X inactivation of mammals?
1. in mammals, the early embryo paternal chromosome, Xp is inactivates in all the cells
2. Xp is imprinted for inactivation however, at the blastocyst stage (prior to the implantation) it is re-activated
3. but later, either Xp or Xm is randomly inactivated and genes are silenced
52
what are the two types of inactivation?
1. imprint - Xp is selectively inactivated in all early embryo mammals, at blastocyst stage (prior to the implantation) it is reactivated
2. random - either Xp or Xm in the female embryo and this X inactivation stays silent for all subsequent cell generations
53
What is an Xist?
X-inactive specific transcript, is the non-coding gene and it is necessary but not sufficient for initiation of x-inactivation
- it is complementary non-coding RNA that mediates gene silencing
54
how does Xist mediate its effects?
The Xist binding leads to histone modifications resulting in gene silencing
55
how does Xist gene lead to X linked disorders?
in rare cases, changes in the Xist gene can cause skewing of X-inactivation
56
Give an example of X-inactivation
Calcio cats have black and orange fur
the black fur (presenting black hair allele) is due to the inactivation of orange hair allele and orange fur is due to the inactivation of black hair allele
57
which period is most susceptible to environmental influences and why?
the embryogenesis and perinatal period due to
1. DNA synthesis rate is high
2. it is the period when chromatin and DNA methylation pattern is established
58
through which cells are epigenetic changes inherited mitotically?
epigenetic changes are inherited mitotically down stem cell lineages
59
give an example of epigenetic changes influencing the phenotype
- The agouti mouse
- despite being genetically identical, DNA methylation results in two phenotype
- the difference is caused by DNA methylation at the agouti viable yellow (Avy) gene
- the Avy locus is actually retrotransposon (i.e. jumping gene) that is inserted upstream of agouti gene
- they methylation can be manipulated through mother diet
60
what are the phenotypic changes you see post the histone modification Avy gene?*
1. LTR hypomethylated (Yellow mouse) - high risk of cancer, obesity and diabetes; Reduced life span
2. LTR hypermethylated (Agouti mouse) - lower risk of cancer, obesity and diabetes; prolonged life
61
what are the other factors influencing the epigenome?
Diet, medication, climate, hormones, psychosocial factors, radiation, pollutants, toxins, drugs of abuse
62
what is transgenerational inheritance?
1. epigenetic adaptations result in transcription dysregulation in grandparents (1st generation)
2. epigenetic dioders in2nd generations (parents
3. the 3rd generation as offsprings and 4th generation as reproductive cells
63
what are the limitations for poor epigenetic research?
1. small numbers
2. inappropriate tissues/cells
3. candidate gene focused
4. sub-optimal study designs
64
what are the three types of DNA methylation in cancer and what does it result in?
1. Hypermethylation -> promoter silencing; silencing of tumour suppressing genes
2. Hypomethylation
Global -> genomic instability
De novo promoter -> activation of proto-oncogenes
3. deamination -> mutation of meCpG to TpG
65
which tumour suppressor gene hypermethylated in familial colon cancer?
MLH1
66
which genes get mutated in acute myeloid leukemia?
1. DNMT3A - in 6%-36% of AML
- they act as dominant negative phenotype as compared to WT DNMT3A
- AML cells with R882H mutation have reduced methyltransferase activity
2. TET2 - 8%-27% of AML patients
- these mutations have reduced 5hmc and TET2
- confer poor prognosis in intermediate risk AML
67
what is the role of epigenetic in portage cancer?
hypermethylation in multiple genes in CaP pathway
68
what is CIMP? and what are its features?
CIMP refers to those cancers with high degrees of methylation since their multiple sites are prone to mutations
- it was identified in colorectal cancer
- CIMP associated cancers have distinct epidemiology, histology and molecular features
- CIMP subtype has been documented in several additional cancers like prate, glioma, leukaemia and breast
69
why CIMP clinically relevant?
it correlates with clinical pathological features and therefore, is clinically relevant
70
why DNA methylation is an excellent cancer biomarker?
1. it is common in all carcinogenesis
2. easy to detect with high degree of sensitivity
3. more stable than RNA and protein based markers
71
DNA methylation is used in which cancer? and what are its features in the following
1. specificity
2. sensitivity
3. detection
4. others
A. prostate - GSTP1
B. lung - SHOX2
C. colon - Sept9
72
what is circDNA and why is it useful in cancer diagnosis?
- circDNA is the cell free circulating DNA released from the tumour in the blood
- it reflects a range of molecular alterations in tumour cells such as DNA mutation, methylation
- it has been observed in most cancer types (colorectal, breast, lung, pancreatic and ovarian)
73
what are the three main ncRNAs? and their function?
1. long coding RNA (lncRNA) - reveal diverse gene expression. profiles in benign and metastatic tumours
2. sRNA or miRNA - capable of reprogramming the oncogenic cascades by mediating their silencing effects therefore, are useful target agents
74
which two are the most clinically advanced therapies in oncology?
hypomethylating agents
1. DNMTi (broad reprogrammers)
2. HDAC inhibitors
75
which drug is most commonly used as DNMT inhibitor for epigenetic therapy?
5-Azacytidine/ 5-aza-2'-deoxycytidine/ zebularine
76
how does 5-azcytodine mediate its function?
1. the irreversible binding between the 5-azacytadine/5'aza-2' deoxycytidine residues and nucleotides analogues can lead to rapid loss of DNA methyltransferase activity
2. further, it interacts with the three most known DNMT1. (DNMT1, DNMT3a,DNMT3b) inhibiting the DNA methylation in subsequent rounds of DNA synthesis
77
how do we know epigenetic therapy is useful?
~15% of patients with myeloidysplastic syndrome or acute myeloid leukaemia (AML) respond to epigenetic therapy by reduced malignant cell burden, improved RBC count and improved survival
78
what are the drawbacks of epigenetic therapy?
1. lacks specificity
2. resistance is common
3. not successful in solid tumours
79
name four HDAC inhibitors
1. varinostat
2. beliostats
3. romidepsin
- these three for cutaneous or peripheral T cell lymphoma
4. panobinostat; used in combination with bortezomib (proteasome inhibitor)
this for multiple myeloma
80
how is the combination of DNMTi and HDACi beneficial?
1. showing increased expression of silenced genes
| 2. anti-tumour responses involving apoptosis
81
what are three main combination of epigenetic therapies with others?
1. combination of DNMTi with standard cytotoxic drugs to re-sensitise cancers to these standard agents
2. epigenetic targets and immunotherapy
3. synthetic lethality and drug development
82
what is synthetic lethality ?
synthetic lethality refers to the relationship between genes (and pathways), in which loss of either one alone does not affect the cell survival but the simultaneous loss of both cells leads to cell death
83
how is synthetic lethality used in cancer?
cancers cells that carry mutation in one of two oats of synthetic lethality is selectively killed by chemical inhibition of the second pathway while normal cells are spared as they do not carry the mutation
84
what is the hallmark of cancer?
1. global DNA hypomethylation
| 2. gene specific promoter hypermethylation of tumour suppressor genes (NDGR4,MLH1,TET2)
85
describe the steps by which DNA methylation is measured in the lab
1. DNA is treated with sodium bisulfite
2. Sodium bisulfite converts unmethylated cytosines to uracil by deamination.
3. The uracil is then converted to thiamine by PCR (or another whole genome amplification method).
4. The bisulfite converted DNA can then be used for various different sequencing / array-based technologies e.g. pyrosequencing, Illumina 450K DNA methylation arrays, bisulfite whole genome sequencing.
86
what are the three types of bisulfites based target specific sequencing?
1. direct sequencing
2. clones bisulfite PCR
3. bisulfite-pyrosequencing
87
what are the pros and cons of bisulfites-pyrosequencing?
```
Pros
1. relatively quick and cost effective
2. target specific regions of the genome
3. provides single nucleotide read out
4. reproducible
5. good validation tool for probes on the 450K
Cons
1. not high throughput (n=24-96)
2. small fragments (max=100bp)
```
88
what are the types of WGBS?
1. reduced representation bisulfites sequencing (RRBS)
2. target enrichment
3. MEDIP
4. SMRT
89
what are the pros and cons of RRBS?
Pros
1. reduces sequencing costs while increasing read depth at CpG rich regions
2. provides single nucleotide read outs
Cons
1. short fragments can create mapping problems
2. biased towards repeat sequences, CpG rich sequences
3. can miss under methylated regions
90
what are the pros and cons of target enrichment?
```
pros
1. reduces sequencing costs
2. targeting CpG rich regions of genome shores and DMRs (differentially distributed methylated regions)
3. can create custom targets
Cons
1. library preparation is time consuming
2. 3ug DNA required
3. not truly genome wide
4. automation not supported
```
91
what are the pros and cons of MEDIP?
it asses genome wide assessment of methylation
pros
1. reduces sequencing cost (5Gb required)
2. no bias to one specific sequence
3. can be adapted for different cytosine residues e.g. 5-hmC
cons
1. don't get base pair resolution (max. resolution - 150bp)
2. can't target specific regions e.g. gene body shores
3. hard to detect undermethylated regions
4. hard to detect methylation levels
92
what are the pros and cons of sequencing base modifications directly (SMRT)?
pros
1. longer reads, allowing phasing of haplotypes, repeat regions etc.
2. sequence base modifications directly
3. sequence information and base modification
cons
1. need good quality ds DNA
2. 5ug DNA required
3. X250 coverage needed for 5mC and 5-hmC
4. technology still being optimised
TMS
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