1
Q
Define epigenetics
A
The changes in gene transcription which is not brought about by alterations in DNA sequences
2
Q
Define chromatin
A
A complex of DNA and proteins (mainly histones)
3
Q
What is the function of chromatin?
A
- prevention of DNA damage
2. regulation of DNA replication and gene expression
4
Q
Define histones
A
Basic (positively charged) structural proteins on which genomic DNA is wrapped around
5
Q
Briefly describe the structure of a histone peptide chain
A
A histone peptide chain has an N-terminus tail and a conserved Histone Fold Domain (HFD) at the C-terminus.
6
Q
What is the function of the HFD in histones?
A
The HFD is responsible for the binding of histones into heterodimers
7
Q
List the core histone monomers
A
- H2A
- H2B
- H3
- H4
8
Q
Which bases are capable of methylation?
A
Cytosine and Adenine
9
Q
Name the phenomenon in which heterochromatin spreads to nearby euchromatic regions
A
Positive effect variegation
10
Q
Briefly describe the DNase I HS assay
A
- DNase I hypersensitive sites (HS) are accessible regions of the chromatin
- These regions are digested by DNase I, leaving little DNA for sequencing or hybridisation with probes.
- With gel electrophoresis and Southern Blotting, we are able to determine “naked” regions of DNA.
11
Q
Briefly describe the ATAC-seq assay
A
- ATAC-seq stands for Assay for Transposase-Accessible Chromatin using sequencing
- Hyperactive mutant Tn5 transposase is used to insert sequencing adapters into open regions of chromatin.
- Tagmentation is the simultaneous fragmentation and tagging of DNA by Tn5 preloaded with sequencing adapters.
- Tagged DNA fragments are purified, PCR amplified, and sequenced
12
Q
What are the advantages of ATAC-seq?
A
- low/no requirements for biological samples
- no sonication or antibodies required
- no sensitive enzymes (DNase I) involved
- fast, can be completed within three hours
13
Q
State the histone modifications associated with euchromatin
A
H3K4me3 and H3K27ac
14
Q
State the histone modifications associated with heterochromatin
A
H3K27me3 and H3K9me3
15
Q
What is H3K4me3 associated with?
A
Transcriptionally active promoters
16
Q
What is H3K9me3 associated with?
A
Constitutively repressed genes
17
Q
What is H3K27me3 associated with?
A
Conditionally repressed genes
18
Q
What is H3K36me3 associated with?
A
Actively transcribed gene bodies
19
Q
What is H3K9ac associated with?
A
Actively transcribed promoters
20
Q
What is H3K27ac associated with?
A
Active enhancers (super-enhancers)
21
Q
Give three examples of writers
A
Acetyl-transferases, methyl-transferases, and kinases
22
Q
Give three examples of erasers
A
Deacetylases, demethylases, and phosphatases
23
Q
What is the function of EZH2?
A
EZH2 is a component of PRC2 that methylates H3K27 and H3K9
24
Q
Give three examples of HDAC inhibitors
A
- Trichostatin A
- Valproic acid
- Virinostat (SAHA)
25
What is a chromodomain?
Chromodomains are interpretation motifs found on readers, allowing them to recognise epigenetic marks.
26
Briefly describe the ChIP assay
ChIP is used to study DNA-protein interactions
1. Cross link bound proteins to DNA
2. Isolate chromatin and shear DNA
3. Precipitate chromatin with protein specific antibody
4. Reverse crosslink and digest proteins. The bound DNA will be isolated.
27
Why is DNA wrapped around histones?
1. To condense the large DNA strands so that it can fit inside the nucleus
2. To regulate DNA transcription
28
What are DNA translocases?
DNA translocases are a catalytic ATPase subunit present in all remodeling complexes, that can slide DNA relative to histone cores.
29
State the three basic mechanisms of chromatin remodelling
1. Histone reconstruction
2. Histone covalent modification
3. Histone repositioning
30
Describe the structure of a nucleosome
A nucleosome core particle consists of height histone proteins (two each of H2A, H2B, H3, H4) and 146 bp of dsDNA
31
What are bromodomains?
Bromodomains are a protein domain that specifically recognises acetylated lysine residues.
32
Briefly discuss the phenomenon of nucleosome repositioning.
1. Nucleosome repositioning allows for the binding of a regulatory factor to its site on nucleosomal DNA.
2. Remodelers are necessary to provide rapid access to nucleosomal DNA by sliding the octamer along DNA
3. Alternatively, the site may be accessed through the generation of a DNA wave or through histone octamer ejection
33
State the four effects of histone repositioning
1. Nucleosome sliding
2. Histone exchange
3. Nucleosome eviction
4. DNA wave (altered nucleosome structure)
34
State the four families of chromatin remodelers
1. SWI/SNF
2. CHD
3. ISWI
4. INO80
35
What are non-coding RNAs?
ncRNAs are functional RNA molecules that is transcribed from DNA but not necessarily translated into proteins.
36
Briefly classify the various types of ncRNA
ncRNAs can be divided into two main groups: housekeeping and regulatory ncRNAs
Housekeeping ncRNAs include rRNAs, tRNAs, snRNAs and snoRNAs.
Regulatory ncRNAs include miRNAs, siRNAs, piRNAs and lncRNAs
37
State the main function of siRNA
Silences transcription (targets dsRNA)
38
State the main function of miRNA
Silences transcription (targets mRNA)
39
State the main function of piRNA
Transposon repression, DNA methylation
40
State the main function of lncRNA
Genomic imprinting, X-chromosome inactivation
41
Detail the process of silencing by siRNA
1. dsRNA elicits a cellular response mediated by Dicer, which cleaves dsRNA into multiple siRNA fragments
2. siRNAs are loaded into RISC and one strand is discarded and degraded (passenger strand). The other strand (guide strand) acts as a template.
3. The guide strand assembles into a functional siRNA-RISC complex, which contains the siRNA bound to the Argonaute-2 protein (Ago-2)
4. Target mRNAs are recognised by Watson-Crick base pairing and bound by the siRNA-RISC complex
5. mRNA degradation is induced
42
What is RISC?
RISC stands for RNA-induced Silencing Complex
43
State the three mechanisms by which mRNA can be silenced
1. Cleavage of mRNA (degradation)
2. Destabilisation through the shortening of the poly-A tail
3. Less efficient translation of mRNA into proteins
44
miRNAs exhibit high variation among species, allowing for the targeting of a wider variety of mRNAs. TRUE or FALSE?
FALSE. Many miRNAs are evolutionary conserved.
45
Detail the canonical pathway of miRNA biogenesis
1. pri-miRNAs are cleaved into pre-miRNAs by Drosha in the nucleus.
2. pre-miRNAs are exported into the cytoplasm by exportin 5.
3. pre-miRNAs are then cleaved into small dsRNA by Dicer
4. RISC mediates the recognition of the mRNA to be targeted
46
Detail the non-canonical pathway of miRNA biogenesis
1. Drosha cleavage is substituted with splicing. Pri-miRNAs are processed to pre-miRNAs by the spliceosome machinery and a debranching enzyme
2. the RNA product of splicing then adopts a pre-miRNA like form and is transferred to the cytoplasm via exportin 5 to continue with the canonical pathway.
47
What are mirtrons?
mirtrons are a type of miRNA that are located in the introns of mRNA-encoding genes
48
State the structure of pre-miRNA
pre-miRNA adopts a hairpin loop structure, before it is cleaved by Dicer to form dsRNA
49
Describe the two fates of mRNA after binding with miRNA.
miRNAs have a seed sequence which determines their binding to target mRNA as well as the effects exhibited on target mRNA.
Perfect binding leads to degradation, while imperfect binding leads to translational inhibition.
50
Briefly explain how 3' UTR reporters can be used to study miRNA
1. miRNAs typically bind the 3' UTRs of target mRNAs.
2. The 3' UTR of the gene of interest will be cloned downstream of a reporter gene (e.g. luciferase)
3. Interaction between 3' UTR and miRNA will result in degradation or translational inhibition of the reporter gene.
51
State the differences between miRNAs and siRNAs
1. miRNAs are derived regions of RNA transcripts that fold back on themselves to form short hairpins, while siRNAs are derived from longer regions of dsRNA
2. siRNAs require perfect of near-perfect base pairing with their mRNA targets while miRNAs only require partial base-pairing (as few as 6-8 nucleotides can be used for recognition
52
What is a seed region?
A seed region is a region on miRNA that determines their binding to target mRNA as well as the effects exhibited on target mRNA.
53
What are piRNAs?
piRNA stands for Piwi-interacting RNA.
54
How do piRNAs exhibit silencing?
Via the formation of a RISC complex
55
Where are piRNAs mainly expressed?
Germ cells
56
What is the function of Piwi protein?
Piwi silences the homeobox gene by binding to genomic PcG response elements together with PcG proteins.
57
State the minimum length of lncRNAs
200 nt
58
State the factors affecting the cellular function of lncRNA
1. nucleotide sequence
2. subcellular localisation
3. tertiary structure
59
Briefly describe the effect of subcellular localisation on the effect of lncRNAs
Nuclear lncRNAs tend to interact with chromatin remodeling enzymes, while cytosolic lncRNAs tend to be mRNA sponges/decoys
60
State the three main classes of lncRNAs
1. Guide lncRNAs
2. Scaffold lncRNAs
3. Decoy lncRNAs
61
What are pseudogenes?
Pseudogenes are genes that have lost their protein-coding abilities due to accumulated mutations. Pseudogenes are capable of being transcribed to decoy lncRNAs
62
State an example of cis-acting lncRNA
XIST
63
State two examples of trans-acting lncRNA
HOTAIR, RMST
64
It is possible for an X chromosome to "escape" from X inactivation. TRUE or FALSE?
TRUE. Escape genes are located on the peripheral region of the heterochromatin
65
State the lncRNA associated with PRC2
HOTAIR
66
State the role of HOTAIR in regulating gene expression.
HOTAIR suppresses gene expression.
1. HOTAIR binds to PRC2 and also interacts with a second complex containing LSD1, coREST and REST that catalyses H3K4 demethylation.
2. The HOTAIR/PRC/LSD1 complex suppresses gene expression via multiple mechanisms at the same time.
67
Briefly describe the steps involved in RIP
RIP: RNA immunoprecipitation is used to study RNA-protein interactions
1. Crosslink proteins to RNA and DNA
2. Sonicate cells and treat with DNase I
3. Add antibody against protein of interest
4. Immunoprecipitate the antibody of interest and elute RNA
5. Treat with DNase I to remove any residual DNA
6. Analyze RNA using RT-PCR
68
Briefly describe the steps involved in ChIRP
ChIRP: chromatin isolation by RNA purification is used to study chromatin-RNA interaction
1. Tiling (non-overlapping) oligonucleotides are allowed to bind to lncRNA by complementary base pairing
2. These oligos are labelled with biotin and can be pulled down with streptavadin beads
3. Isolate DNA, protein, and lncRNA involved
69
What are ASOs?
ASOs are antisense oligonucleotides that can alter the original function of the target RNA through different mechanisms:
1. prevention of 5' cap formation
2. modulation of RNA splicing
3. modulation of polyadenylation
4. RNase H1-mediated degradation
5. translation inhibition
70
What is SMA?
SMA, spinal muscular atrophy, is an autosomal recessive disorder caused by genetic defect on the SMN1 gene
71
Briefly describe SMA in relation to the SMN1 and SMN2 gene
SMN1 produces full length SMN protein. SMN2 gives rise mostly to non-functional SMNΔ7 protein and a small amount of full length SMN.
Therefore, the more the copies of SMN2, the less severe the SMA disease
72
Name the drug used to treat SMA
Nusinersen (Spinraza)
73
How does Nusinersen work?
Nusinersen is an ASO-based drug to correct SMN2 splicing.
Within intron 7 of the SMN gene, a the intronic splicing silencer (ISS-N1) recruits splicing repressors hnRNP A1 and A2 to exclude exon 7, resulting in non-functional SMNΔ7.
Nusinersen hybridises to ISS-N1 to block hnRNP recruitment, resulting in the inclusion of exon 7 and the production of full length SMN2
LSM3235 - Epigenetics
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