GENE 7: Expressing the genome Flashcards
1
Q
What is gene expression?
A
The process by which the information from a gene is used to synthesise a functional gene product, which is either a protein (if the gene is a protein coding gene) or a functional RNA (if it is a non-protein coding gene).
2
Q
from 5’ to 3’ list the order of the structure of a protein coding gene
A
5’ - Upstream enhancers > Promoter > TATA box > %’ UTR > Exon 1 > Intron 1 > exon 2 > Intron 2 > exon 3 > 3’ UTR - 3’
3
Q
What are the three stages of gene expression?
A
Initiation, elongation and termination
4
Q
Where do basal transcription machinery assemble on the DNA during initiation?
A
The promoter
5
Q
What are the three common features of promoters for protein-coding genes?
A
- start site
- TATA box
- sequences bound by transcriptional regulators
6
Q
What does the basal transcription machinery comprise of?
A
RNA polymerase II
5 multi-subunit general transcription factors
7
Q
What are the 5 multi-subunit general transcription factors called?
A
TFIIB, D, E, F and H
Boris Didn’t Eat French Ham
8
Q
TFIID comprises the TAT-binding protein (TBP) and around ___ TAFs (TBP-associated factors)
A
11
9
Q
What is the first component to bind to the promoter?
A
TFIID
10
Q
What forms the initiation complex factors?
A
TFIIB, E, F, H and RNA polymerase II
11
Q
Describe the processing of primary RNA transcripts
A
co-transcriptionally
5’ capping as soon as transcription has been initiated
splicing and editing while the transcript is still being made
3’-polyadenylation as an inherent part of termination mechanism for RNA polymerase
12
Q
What is 5’-capping?
A
The addition of 7-methylguanine
13
Q
Define transcription factor
A
Sequence-specific DNA binding proteins that bind at or close to the core promoter and influence the efficiency of transcription initiation
14
Q
Define DNA helicase
A
A subunit of TFIIH that uses energy from the hydrolysis of ATP to open up the DNA double helix; allowing RNA polymerase II to have access to the template strand
15
Q
Define transcriptome
A
The total complement of mRNA molecules (or transcripts) produced in a specific cell or the population of cells comprising a tissue
16
Q
Name three methods of measuring gene expression
A
- qPCR
- gene expression microarrays
- RNA-Seq (RNA sequencing)
17
Q
What can qPCR do?
A
Can quantify a specific transcript whose cDNA primers have been designed to amplify
18
Q
What can gene expression microarrays do?
A
can simultaneously detect and quantify transcripts for thousands of genes within a particular mRNA sample. Fluorescently labelled RNA hybridises to transcript-specific oligonucleotides arrayed on a solid support.
19
Q
What happens in RNA-Seq (RNA sequencing)?
A
is a ‘next generation’ technique where a cDNA library is made and sequenced. The sequences tells us which genes are expressed within a sample and the number of reads indicates expression levels.
20
Q
When does RNA polymerase II bind to the TATA box?
A
Only after TFIID, A, B have bound to the TATA box. RNA pol II will only bind after and when attached to TFIIF, followed by further transcription factors
21
Q
What information is required for qPCR?
A
Oligonucleotide PCR primers based on known transcript sequences
22
Q
When studying the transcriptome using RNA-Seq, why is each transcript’s exact RNA sequence not needed to be known?
A
While the genome sequence is required to identify which gene each read maps to, RNA-seq reads can pinpoint novel splice junctions and fusion genes.
23
Q
What is the most common DNA modification in mammals?
A
DNA methylation
24
Q
Where does DNA methylation occur? What does this produce?
A
5th carbon of cytosine ring, produces 5-methylcytosine.
CpG sites
5’-C-phsophate-G-3’
25
What does DNA methylation do to the structure of DNA? What does this lead to?
Distort the DNA helix, which inhibits transcription
26
How many CpG islands are there in the human genome?
~30,000 (aprox. 15.% of human genome)
27
What does CpG islands mean?
stretches of 0.5-2 kb of DNA with a greater frequency of CpG dinucleotides than the rest of the genome
28
When a CpG island in the promoter region of a gene is methylated, what happens to the expression of the gene?
It is usually silenced
29
Many CGIs occur at ____ _____
gene promoters
30
What is genomic imprinting?
A form of epigenetic inheritance, where DNA methylation ensures only one parental allele is expressed. When the paternal allele is expressed, the maternal copy is silenced and vice versa.
31
How does Prader-Willi Syndrome (PWS) come about?
Genetic imprinting disorder: which is often caused by deletion of the paternal allele in the region on chromosome 15 containing the gene SNRPN. This occurs after the maternal allele has already been silenced by imprinting, leaving no expression of SNRPN.
32
What happens to PWS patients?
They suffer from extreme feeding problems, including hyperphagia, or extreme, insatiable appetite and obsession with food. Affected children are also developmentally delayed for motor skills due to decreased muscle tone.
33
What happens in Angelman syndrome?
Loss of expression in a region of chromosome 15. In this case it is usually caused by loss of the maternal allele when the paternal allele has been silenced by imprinting. Loss of the gene UBE3A results in disorder of the nervous system characterised by developmental disabilities, seizures, speech deficits, and motor oddities.
34
In addition to DNA base modifications, what else is used to regulate gene expression?
Post-translational modifications of histones proteins. These can influence the activity status of the chromatin
35
Which modifications of Histone H3 promote gene transcription?
- Acetylation of Lysine 27
| - Tri-methylation of histone H3 lysine 4 results in active promoters and gene expression
36
Which modifications of Histone H3 silence gene transcription?
- Di or tri methylation of H3 lysine 9 silences gene promoters and prevents transcription
- Methylation or often tri-methylation of lysine 27 suppresses transcription and results in large regions of inactive chromatin
37
What structural change can enhance transcription?
chromatin folding: 3D arrangement of chromatin in the nucleus
38
What is chromosomal looping?
Part of the chromosomal folding process and allows contact to be made over large genomic distances between regulatory sequences (enhancers) and gene promoters. Tissue specific enhancers are evolutionary conserved regions (ECRs) that are often located hundred of kb away from their core promoters
39
What are topologically associated domains?
Topologically Associating Domains (TAD) are self-interacting genomic regions. The genes within a TAD are brought together in a cell-specific manner to confer specific gene expression patterns that characterise phenotype. They comprise the majority of characterised enhancer-promoter pairs. They are conserved among species, cell types and tissues, highlighting their biological relevance
40
What are chromosomal compartments?
Chromosomes display a non-random organisation within the nucleus, influenced by their gene density and transcriptional status. They exist in active ‘A’ compartments towards the interior of the nucleus or in inactive ‘B’ compartments towards the periphery.
41
What are chromosomal territories?
Chromosomes segregate into distinct territories
42
Explain how ChIP-Seq can be used to determine genomic architecture
(chromatin immunoprecipitation coupled with high-throughput sequencing) is an experimental method that identifies where specific proteins bind to DNA. It involves chemical cross-linking between DNA and bound proteins. The DNA is then fragmented and a specific protein of interest (POI), such as a transcription factor, is isolated using an antibody along with any DNA cross-linked to it. Cross-links are then removed to release the DNA which can then be sequenced, telling us where that transcription factor was bound in the genome.
43
Explain how Chromosome conformation capture (3C) can be used to determine genomic architecture
A method of identifying physical contacts between different genomic regions. Chromatin strands > crosslink with formaldehyde > Cut DNA with restriction endonuclease > Ligate the ends > break cross-links
44
What is the purpose of formaldehyde in 3C?
Fixes the interacting regions of chromatin. DNA is the digested and DNA fragments that remain are ligated, After crosslinking the DNA is analysed
45
What are ChIP-Seq and 3C particularly helpful is understanding the role of?
DNA that is non-coding
46
What was the purpose of ENCODE?
The Encyclopaedia of DNA Elements: to determine the role of the remaining DNA, previously thought to be 'junk'. Using approaches such as ChIPseq and 3C, and collating datasets form many laboratories, the ENCODE project provides information for general access on the regulome i.e. the noncoding genomic regions have elements significant for gene regulation.
47
The non-coding genomic regulatory regions significant for gene regulation include what? What do these elements do?
promoters, transcriptional regulatory sequences and regions of chromatin and histone modification, which modulate the activity and expression of the protein-coding genes as well as other functions.
48
3C identifies what?
Long range genomic DNA contacts
49
A-type TADs are located where?
towards the interior of the nucleus
50
What are the four main layers of 3D organisation of the human genome?
Chromatin loops, TAD arrangements, compartments and territories
51
Are chromosomes organised in a random fashion in the nucleus?
No, they display a non-random nuclear organisation. They exist in A or ‘active’ compartments towards the interior of the nucleus or in B, ‘inactive’ compartments towards the periphery.
52
How can chromatin organisation be considered as layers of a hierarchical structure?
- Heterochromatin/euchromatin
- A & B compartments
- TADs and chromatin loops
53
How can pathological alterations on these layers occur?
through mutation of genes that encode the proteins responsible for maintaining chromatin organisation, such as cohesin which is crucial for forming chromatin loops.
54
What happens in diseases including T-cell acute lymphoblastic leukaemia (T-ALL), asthma and heart diseases to chromatin loops?
The formation or disappearance of chromatin loops between enhancers and promoters will lead to the gain or loss, respectively, of enhancer function and can alter transcription factor binding in the genome and contribution to disease progression.
55
Disruption of stable TADs occurs in some inherited diseases e.g. F-syndrome and sex reversal. How does this happen?
TAD boundary deletions can induce rewiring of promoter enhancer interactions, allowing enhancers from neighbouring domains to ectopically activate other genes, causing aberrant gene expression and disease.
56
The emergence and dissolution of compartments and chromosomal territories is seen in several cancers e.g. chromosomal translocations in breast cancer and prostate cancer.
When translocations bring the coding sequence of one gene into the regulatory environment of another genomic region, its transcription can be aberrantly activated or silenced.
57
Explain an outline for the control of gene expression intra and extra cellularly
These pathways transduce signals from receptors on the cell membrane to the nucleus where they modulate co-activator and co-repressor complex formation to alter gene expression patterns.
58
What are Wnt proteins?
secreted glycoproteins that activate different intracellular signal transduction pathways. They regulate cell proliferation and are required for proper embryonic development.
59
Mis-regulation of Wnt signalling can result in what?
various diseases, including cancer.
60
What is the Wnt signalling pathway involved in? Explain the pathway
The regulation of β-catenin in both normal stem cells and in cancer. In the absence of Wnt, β-catenin is phosphorylated and constitutively degraded. When an extracellular Wnt protein binds to one of its cell-surface receptors (the Fz family), however, β-catenin is de-phosphorylated and stabilised so it can translocate to the nucleus. Nuclear β-catenin binds a transcription factor Tcf, activating transcription of a set of Wnt target genes, which in turn regulate stem cells and tumorigenesis.
61
Do epigenetic factors lead to lasting change?
Cellular phenotypes established through DNA methylation patterns, and other epigenetic marks, are stabilised and inherited through successive cell cycles within a specific lineage. This inheritance through cell cycle is essential: it ensures, for example, that when an epithelial cell divides, its daughter cells express the genes needed to be an epithelial cell.
62
What do DNMTs stand for an what do they do?
DNA methyltransferases are a family of enzymes that have an important role in the inheritance of epigenetic markers.
63
What does DNMT1 do and how does it function?
DNMT1 maintains DNA methylation following differentiation by identifying hemimethylated DNA. It methylates the unmethylated cytosine at such sites, causing the original mark to be copied. DNMT in active in cell division thereafter.
64
What is hemimethylated DNA?
CpG dinucleotides that are methylated on the original DNA strand but not the newly synthesised strand.
65
What are the two mechanisms of DNA demethylation?
Passive and active
66
Describe active demethylation
Active DNA demethylation occurs through an enzymatic process that removes or modifies the methyl group from 5-methylcytosines. The ten–eleven translocation (TET) family of enzymes are involved in active demethylation.
67
Describe passive demethylation
Passive DNA demethylation usually takes place on newly synthesised DNA strands in the absence of DNA methylation maintenance.
68
What is de novo DNA methylation?
DNA methylation must be acquired de novo in order to promote appropriate gene expression and differentiation into different cell types.
69
What enzymes carry out de novo?
DNMT3A and DNMT3B
70
What suggests that histone modifications such as lysine at 9 of histone H3 initiates heterochromatin formation and subsequent DNA methylation stable silencing of the promoter?
Crosstalk between DNA methylation and histone modification
71
Promoter DNA methylation is associated with what?
Gene silencing, and plays an important role in maintaining cell types
72
What are the three main types of DNA methyltransferases?
DNMT1, DNMT3a, DNMT3b
73
Following fertilisation, which DNMTs are used to allow embryonic cells to differentiate into a cell type? Through what process?
DNMT3a and DNMT3b by de Novo methylation
74
The methylation pattern in different cell type is different, this leads to what?
Different gene expression pattern
75
Each cell type has a unique DNA methylation pattern, how is this maintained?
DNMT1
76
In normal adult cells, which CpG sites are methylated or not?
Most CpG sites are methylated except for promoter CpG islands (CGI)
77
What is contained within the promoter region
Regulatory elements that control the transcription of genes
78
DNMT obtains the methyl group for methylation from where?
SAM
79
How does DNA methylation occur?
1) 5th cytosine is flipped out of the DNA strand 180 degrees
2) DNMT obtains methyl group from SAM
3) Methylated cytosine flipped back into strand
80
What does Human TET do?
DEMETHYLATION> It is an enzyme that has a role in regulating DNA methylation patterns. It adds a hydroxyl group initially to 5 methyl cytosine, forming 5-hydroxymethyl cytosine. It is also able to convert 5-hydroxymethyl cytosine back to 5 methyl cytosine through different pathways. It is thus thought to be the enzyme responsible for demethylation.
81
During fertilisation and normal embryonic development, what are the first steps that occur to the DAN?
the DNA in maternal and paternal germ cells is demethylated. This is followed by de novo methylation, that allows for the expression of appropriate genes for development and cell differentiation.
82
What is induced pluripotency?
The laboratory process by which somatic cells can be converted into induced pluripotent stem cells (iPSCs), with features similar to embryonic stem cells.
83
How are iPSC generated?
Through expression of four transcription factors OCT4, SOX2, KLF4 and MYC
84
By what two methods is maternal and paternal DNA demethylated?
Paternal: genome wide active demethylationand remains demethylated following multiple rounds of cell division
Maternal: genome wide gradual passive demethylation
This is all done by TET
Meaning the methylation pattern is essentially erased by the Morula stage
85
When is de novo methylation initiated?
Blastocyst stage
86
Why could iPSC be used in modern medicine?
Because iPSCs can be cultured and manipulated in vitro, and differentiate into any somatic cell, the ability to generate iPSCs raises possibilities of growing replacement cells/tissues/organs for a patient from a small sample of their own cells. In effect, the patient can become their own donor.
87
What does direct reprogramming refer to?
Conversion of fully differentiated cells to other cell types, bypassing an intermediate pluripotent stage.
88
What did Conrad Waddington propose in 1957?
The concept of an 'epigenetic landscape' to represent the process of cellular decision-making during development.
89
According to Conrad Waddington's model what can somatic cells in differentiated states not do?
Do not normally change from one differentiation pathway to another, although nuclear reprogramming can alter cell fate.
