GEN 7`: Expressing the Genome Flashcards
1
Q
Observe the learning outcomes of this session
A
2
Q
What is gene expression?
A
- the process by which the information from a gene is used to synthesise a functional gene product
- either a protein or a functional RNA
3
Q
Label the structure of a protein-coding gene
A
4
Q
Fill the missing gaps about gene expression
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5
Q
Define transcription factor
A
- a sequence-specific DNA binding molecule (typically a protein) that binds at or close to the core promoter and influences the efficiency of transcriptional initiation
6
Q
What is 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
7
Q
What is the transcriptome?
A
- the total complement of RNA molecules (or transcripts) produced in a specific cell or in a population of cells comprising a tissue
8
Q
Recap some ways to measure gene expression?
A
9
Q
Before RNA polymerase II can be recruited to express a gene, what must happen to chromatin first?
How does that happen?
A
- the region of chromatin must first become accessible
- this is partly achieved by chromatin-modifying enzymes
- in mammals, the most common DNA modification that contributes to the regulation of transcription is DNA methylation, which occurs at the five carbon position of the cytosine ring, resulting in 5-methylcytosine.
10
Q
How do methyl groups affect DNA transcription?
A
- methyl groups distort the DNA double helix, inhibiting transcription
11
Q
What do CpG sites stand for?
A
- They stand for 5’—C—phosphate—G—3’, simply referring to a cytosine residue immediately upstream of a guanine.
- The human genome contains ∼30,000 CpG islands (CGIs), which are stretches (0.5–2 kb) of DNA with a greater frequency of CpG dinucleotides than the rest of the genome.
12
Q
How much of the human genomic DNA is 5-methylcytosine?
A
- approx. 1.5%
13
Q
What happens when a CpG island is methylated or unmethylated?
A
- When a CpG island in the promoter region of a gene is methylated, expression of the gene is usually silenced.
- Conversely, many CGIs occur at gene promoters, and their DNA nearly always remains unmethylated, thus allowing gene expression to occur.
14
Q
Observe this image of CpG dinucleotides and C-G base-pairs
A
15
Q
Compare CGI and non-CGI genomic sequences
A
16
Q
Observe this diagram of how CGI methylation affects gene expression
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17
Q
What is genomic imprinting?
A
- genomic imprinting is a form of epigenetic inheritance
- this is where DNA methylation ensures only one parental allele is expressed
- when the paternal allele is expressed, the maternal copy is silences and vice versa
18
Q
Describe Prader-Willi Syndrome, a disorder involving genetic imprinting
A
- is often caused by deletion of a region on chromosome 15 that includes the gene SNRPN.
- This occurs in the paternal chromosome after the maternal SNRPN allele has already been silenced by imprinting, leaving no expression of SNRPN.
- In PWS, patients 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.
19
Q
Describe Angelman syndrome
A
- caused by 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
- In this syndrome, loss of the gene UBE3A results in disorder of the nervous system characterised by developmental disabilities, seizures, speech deficits, and motor oddities.
20
Q
Look at the diagram of histone H3 and summarise the key modifications on it
A
- acetylation of lysine 27:
- promotes gene transcription
- methylation of lysine 27:
- or commonly tri-methylation, suppresses transcription and results in large regions of inactive chromatin
- methylation of lysine 9:
- di- or tri-methylation of H3 lysine 9 silences gene promoters and prevents transcription
- methylation of lysine 4:
- tri-methylation of histone H3 lysine 4 results in active promoters and gene expression
21
Q
What can chromatin folding do?
A
- it can enhance transcription
22
Q
Explain the chromatin loop
A
- 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 hundreds of kb away from their core promoters
23
Q
Look at this diagram and explain what TAD is
A
- they are Topologically Associating Domains (TAD)
- they 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
24
Q
Observe this diagram and explain what chromosomal compartments are
A
- chromosomes display a non-random organisation within the nucleus, influenced by their gene density and transcriptional status
- the exist in active ‘A’ compartments towards the interior of the nucleus or in inactive ‘B” compartments towards the periphery
25
What are chromosomal territories?
- chromosomes segregate into distinct territories
26
What methods can we use to determine genomic architecture?
- in other words, how can we find out where a given transcription factor is bound to DNA, or in which regions of the genome a given histone modification is found?
- ChIP-Seq (Chromatin immunoprecipitation coupled with high-throughput sequencing)
- Chromosome conformation capture
27
Describe ChIP-Seq (Chromatin immunoprecipitation coupled with high-throughput sequencing)
- Chip-Seq is an experimental method that identifies where specific proteins bind to genomic DNA.
- It involves cross linking proteins that are bound to DNA so they are fixed.
- A specific protein, such as a transcription factor, is then isolated using an antibody.
- Any bound DNA remains fixed to it, and can then be sequenced, telling us where that transcription factor was bound in the genome.
- A similar but simpler approach (ChIP-qPCR) uses qPCR instead of high-throughput sequencing to analyse just a small region of the genome.
28
Describe Chromosome conformation capture
- Chromosome conformation capture is a method for identifying physical contacts between different genomic sequences
- Initially, CCC used PCR analyses to detect interactions involving specific regions of the genome. The most powerful use of this approach (Hi-C), however, uses high throughput sequencing to detect interactions between any two region in the genome. Hi-C data are represented graphically as contact maps such as those in the next figure, which also show how Hi-C reveals the presence of TADs, Compartments and Chromosome Territories.
- first pic description:
- Basis of chromosome conformation capture (CCC) methods.
- Formaldehyde fixes interacting regions of chromatin in the nucleus. DNA is then digested and fragments that remain associate are ligated. After removing crosslink, the DNA is analysed.
29
What is ENCODE and what is the regulome?
- ChIP-seq and CCC methods can reveal how chromatin organisation differs between cell types and changes with gene expression.
- They have generated vast amounts of data that can be freely accessed and are central to The Encyclopedia of DNA Elements (ENCODE) (https://www.encodeproject.org/help/project-overview/).
- ENCODE is an international consortium of research groups which aims "...to build a comprehensive parts list of functional elements in the human genome, including elements that act at the protein and RNA levels, and regulatory elements that control cells and circumstances in which a gene is active."
- Collectively, these regulatory elements are sometimes referred to as the regulome.
- Defining the regulome is clearly necessary for a proper understanding of the genome.
- It is also clinically important as the majority of disease-associated genetic variants identified to date by GWAS are located in non-coding regions
30
How can chromatin organisation be considered as layers of a hierarchal structure?
31
What are the effects of mutations in enhancer-promoter 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.
- This occurs in diseases including T-cell acute lymphoblastic leukaemia (T-ALL), asthma and heart diseases.
32
What are the effects of disruptions of stable TADs?
- Disruption of stable TADs occurs in some inherited diseases e.g. F-syndrome and sex reversal.
- 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.
33
What are the effects of disruption of chromosomal territories?
- 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.
34
How is gene expression controlled?
- Gene expression can be controlled by both intra- and extra-cellular signals.
- A number of highly conserved signalling pathways have evolved, and are used in different tissues and processes.
- 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.
- Each pathway involves key signalling proteins such as Wnt.
35
Describe the Wnt signalling pathway
- Wnt proteins are secreted glycoproteins that activate different intracellular signal transduction pathways.
- They regulate cell proliferation and are required for proper embryonic development.
- Mis-regulation of Wnt signalling can result in various diseases, including cancer.
- The Wnt signalling pathway is involved in 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.
36
How do we maintain epigenetic marks in dividing cells?
- DNA methyltransferases (DNMTs) are a family of enzymes that have an important role in the inheritance of epigenetic markers.
- DNMT1 maintains DNA methylation by identifying hemimethylated DNA: CpG dinucleotides that are methylated on the original DNA strand but not the newly synthesised strand. By methylating the unmethylated cytosine at such sites, the original mark is copied as illustrated below:
37
Describe DNA demethylation
- DNA demethylation, the removal of a methyl group from DNA, is just as important as DNA methylation.
- Demethylation of DNA can be through passive and active mechanisms.
- 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.
- Passive DNA demethylation usually takes place on newly synthesised DNA strands in the absence of DNA methylation maintenance.
38
When does de novo DNA methylation occur?
- During development, DNA methylation much be acquired de novo in order to promote appropriate gene expression and differentiation into different cell types.
- de novo DNA methylation is carried out by DNMT3A and DNMT3B
- Crosstalk between DNA methylation and histone modification suggests that histone modifications, such as methylation at lysine 9 of histone H3, initiates heterochromatin formation and subsequent DNA methylation ensures stable silencing of the promoter.
39
What is the concept of an 'epigenetic landscape'?
- In 1957, Conrad Waddington proposed the concept of an 'epigenetic landscape' to represent the process of cellular decision-making during development.
- Waddington imagined cells as pebbles rolling down a mountain with hills and valleys taking a number of specific trajectories, leading to different outcomes or cell fates.
40
What is induced pluripotency?
- Induced pluripotency defines the laboratory process by which somatic cells can be converted into induced pluripotent stem cells (iPSCs), with features similar to embryonic stem cells.
- iPSCs were originally generated through expression of four transcription factors: OCT4, SOX2, KLF4 and MYC.
- 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!
41
What is direct reprogramming?
- Direct reprogramming refers to the conversion of fully differentiated cells to other cell types, bypassing an intermediate pluripotent stage.
- The diagram below shows the transcription factors capable of direct reprogramming when expressed in various cell types:
42
Is reprogramming an easy thing to do?
- On the basis of Waddington's model, somatic cells in differentiated states maintain their own cell fate and do not normally change from one differentiation pathway to another, although nuclear reprogramming can alter cell fate.
- Because reprogramming is working against the usual course of cell differentiation, even optimised protocols are inefficient, with only a small proportion of cells successfully reprogramming.
- The reprogramming process has been depicted as climbing a mountain, because it is much harder to achieve than differentiation, which is a spontaneous process, as sliding down a hill (see diagram below).
- Inducing pluripotency requires a series of epigenetic changes, including global DNA hypomethylation.
43
What types of epigenetic changes are required to induce pluripotency in reprogramming?
- Inducing pluripotency requires a series of epigenetic changes, including global DNA hypomethylation.
