Eukaryotic Transcription Factors Flashcards
(110 cards)
1
Q
What are the 3 phases of transcription?
A
Initiation
Elongation
Termination
2
Q
How many subunits make up RNA pol II?
A
12
3
Q
From what do RNA polymerases and its accessory factors take their cues about where to stop and start?
A
Sequences within the DNA.
There is a code written into DNA that tells RNA pol and its factor how to interpret where transcription units (genes) are located/begin etc.
E.g. TATA box and polyadenylation signal (AATAAA/AAUAAA).
4
Q
What is the default state of gene transcription in eukaryotes?
A
OFF
RNA pols won’t form a PIC on a given isolated promoter without being explicitly told to do so - even with the 44 GTFs in place, RNA pol still won’t do anything.
5
Q
Which is the only nuclear polymerase to recieve signals directly from the outside world instructing it to begin transcribing?
A
RNA pol III recieves signals directly from the outside world instructing the transcription of tRNA-encoding genes.
6
Q
With which nuclear RNA polymerase is most eukaryotic gene regulation associated with?
A
RNA pol II responsible for transcription of protein-coding genes.
7
Q
What is the approximate length of gene regulatory motifs/cis-acting motifs?
A
5-15bp.
8
Q
What name is given to the cluster of gene regulatory motifs found close to the promoter in higher eukaryotic protein-coding genes?
A
Upstream promoter element (UPE).
9
Q
Define the terms upstream, downstream and intergenic?
A
Upstream = everything in the region in front of the TSS. Downstream = everything after the mRNA (NOT everything after the TSS). Intergenic = everything within the gene codign region itself.
10
Q
Give some examples of gene regulatory motifs found in higher eukaryotics particularly humans?
A
UPEs, enhancer, silencer, locus control region, insulator.
11
Q
What is the function of most gene regulatory motifs?
A
These motifs are binding sites for sequence specific DNA-binding proteins: transcription factors.
12
Q
What are transcription factors?
A
Small proteins that bind to gene regulatory motifs in DNA via sequence-specific recognition.
13
Q
What is the function of transcription factors?
A
Transcription factors sense signals from inside or outside the cell and then communicate with RNA pol II to influence the initiation or elongation of transcription.
Transcription factors by binding to DNA are recruiting co-activators/co-repressors.
14
Q
Give an analogy to demonstrate how different TFs can regulate one gene?
A
Transcription factors bind different motifs and therefore, each TF can relay a different signal. Jointly, they ‘decide’ on the activitiy of the gene and therefore, transcription regulation can be thought of as a parliamentary demoncracy - the sum of yes (transcription activation) and no (transcripiton repression) votes determines the outcome (transcription).
15
Q
Are most transcription factors activators or repressors?
A
Activators.
16
Q
Describe the general structure of a transcription factor?
A
Broadly speaking, transcription factors have two really distinct structural domains, the effector/activation domain and the DNA-binding domain.
17
Q
How is transcription factor structure affected by its gene sequence?
A
Transcription factor modularity is reflected in the primary structure of the protein/the gene sequence.
If you look at the sequence, you will often find the effector domain is encoded in one bit of primary sequence, then there will be a gap then the DNA-binding domain is encoded in another region.
18
Q
How are transcription factors classified into families?
A
Transcription factors are classified into families according to the structure of their DNA-binding domains.
There are different kinds of protein folds that are able to bind to DNA and recognise sequences.
19
Q
How many transcription factors make up the repertoire of human transcription factors?
A
The human transcripiton factor repertoire consists of 1600 different proteins.
20
Q
What is the most common DNA-binding domain type in the repertoire of human transcription factors?
A
Zinc finger (750 human TFs have a zinc finger DNA-binding domain).
21
Q
Why are zinc finger DNA-binding domains thought to be the most common DNA-binding domain in mammals?
A
Zinc finger domains themselves are modular with combination of fingers decoding different short nucleotide sequences.
From an evolutionary perspective zinc fingers represent a wonderful mix-and-match DNA-binding toolkit for transcription factor design.
In being modular, you can take fingers out of a gene,mix and match them and evolution seems to do this.
Each finger seems to recognise 2bp sequence so 3 fingers can recognise a unique 6bp sequence which can be changed by swapping that finger out in evolution.
In being modular, zinc fingers allow evoltuion to explore different sequences.
22
Q
How many transcription factors make up the repertoire of yeast transcription factors?
A
209
23
Q
What is the function of the DNA-binding domain of a transcription factor?
A
Interacts with the gene regulatory DNA motifs.
24
Q
What is the function of the effector domain of a transcription factor?
A
Effector domains make protein-protein contacts with other factors - the effector domain can be viewed as an interface for protein interactions.
They are often targets for signals - they transduce external signals/cues by directly communicating with the DNA-binding domain directly.
Effector domains may have a catalytic activity.
25
Describe the regulation of the metallothionein (MT) gene?
Heavy metals (e.g. Cu, Cd, Zn and Hg) are toxic as ions in free solution.
Cells fight back by producing metallothioneins - small proteins that chelate heavy metal ions preventing their toxicity.
MT genes are relatively simple, they have a promoter, a protein-codign region, a relatively simple upstream promoter element.
The MT genes are sensing two related cues and integrating that information.
External signals are transduced regarding the presence of heavy metal ions and tissue stress.
The MT UPE drives transcriptional acitvation of the gene when toxic metal ions are present and when the cell is stressed.
USF, AP2 and SP1 are consistutively bound to the MT UPE, GR and Mtf1 bind only in response to signals.
GR (glucocorticoid receptor) is expressed in most cells but normally remains as an inactive monomer in the cytoplasm.
In a case of tissue stress, stress hormones like cortisol directly bind to the GR causing its release from cytoplasmic partners Hsp70/90 and FKBP 52 allowing it to dimerise and translocate to the nucleus where it can bind its motifs (glucorticoid response elements) and regulate transcription.
The GRE is therefore a regulatory region to respond to cellular stress.
The DNA-binding domain of Mtf1 cannot form a functional structure without the presence of heavy metal ions, when present, the DNA-binding domain folds correctly and the TF translocates to the nucleus and can bind its motifs the metal responsive element (MRE).
26
Which of these transcription factors are constitutively bound to the MT promoter and which bind in response to signals: USF, AP2, SP1, GR, Mtf1?
USF, AP2 and SP1 are constitutively bound to the MT promoter.
GR and Mtf1 bind in response to external signals.
27
What are the 3 strategies by which eukaryotic transcription factors were discovered and characterised?
1. Biochemistry.
2. Genetics.
3. Genomics.
28
What are in vitro reporters?
DNA constructs which work in a test tube.
29
What are in vivo reporters?
DNA constructs carried on plasmids or integrated into a genome which work within a host cell.
30
What is the difference between in vitro and in vivo reporters?
In vitro reporters are DNA constructs which work in a test tube, in vivo reporters are DNA constructs carried on plasmids or integrated into a genome which work within a host cell.
31
What is meant by the term reporter gene?
A reporter gene is something with a codign region that ultimately produces a transcript/protein that can be easily scored e.g. it mays a colour or something measurable in the lab.
Then using recombinant DNA technology, this 'reporter gene' is stuck next to a promoter of interest to allow the study of a single/group of transcription factors.
32
What was the main method of transcription factor discovery during the 20th century?
Biochemistry was used to purify and reconstitute mRNA transcription using in vitro reporters.
33
Why is biochemistry not really used for transcription factor discovery anymore?
It is really difficult and laborious.
It requires growing cells, making a nuclear extract, using columns to purify indivdiual proteins and idenitfy new transcription factors that way.
34
How were RNA pol II, the GTs and many activating transcription factors identified?
A standard reporter that measures the incorporation of radioactivity into an RNA, was generated by cloning a reporter sequence next to a promoter and UPE and adding a combination of purified proteins to this DNA
Any RNA synthesis from the promoter is captured usign radiolabelled nucleotide incorporation and hte resulting transcript is detected after gel electrophoresis.
This type of biochemistry is difficult and expensive!
35
What are the advantages of yeast as a model eukaryote?
Yeast are hugely powerful for genetic engineering.
Yeast exhibit simple propagation via standard microbial techniques meaning you can easily grow them on Petri dishes.
We can have complete control over yeast genetics, it is easy to mutagenise them, you can grow the cells as haploid, you can look at meiosis and mating.
Yeast are amenable to genetic manipulation techniques meaning you can put circular plasmids into them, you can easily replace genes via recombination, you can easily integrate bits of DNA into their genome.
36
How does the LacZ reporter work?
When the LacZ reporter is transcribed, cells turn blue.
The LacZ+ phenotype is blue yeast when cells are grown on X-GAL substrate on a petri dish.
This gives a very powerful blue, white colour selection.
37
What are the main steps of a yeast transcription factor genetic screen?
1. Make mutations that inactivate the reporter, select mutants according to phenotype on petri dishes.
We grow some blue (LacZ+) yeast and mutagenise them and select the white yeast and hopefully they contain mutations in transcription factors.
Then you perform complementation cloning.
2. Make a gene library from normal yeast.
Take a normal yeast and take every single normal gene from a yeast cell and put them onto plasmids and make a gene library.
Then take the white mutant and add the library.
3. Introduce the library into mutant yeast and look for complementation.
You simply look for colonies where the reporter is reactivated again so yeast go from white to blue and you pick that mutant and look to the plasmid inside it.
4. Purify and sequence the complementing plasmid to identify the gene encoded - probably a transcription factor.
38
What was the first zinc finger protein to ever be isolated?
SWI5.
39
Can yeast genetics be used to identify human TFs?
Yes! This is why they are such powerful tools.
40
How can yeast genetics be used to identify human TFs?
Because eukaryotic TFs are modular in structure, domains can be swapped using recombinant DNA technology to generate hybrid factors.
E.g. you could use this technology to attach a huamn effector domain to a yeast DNA-binding domain.
Because the basic mechanisms of eukaryotic trnascription are conserved, human effector domains will often activate transcription in yeast whilst tethered to DNA by a yeast DNA-binding domain.
The production of hybrid transcription factors using recombinant DNA methods provides the basis of one-hybrid genetic screens.
41
What are the main steps to producing a yeast one hybrid screen?
1. Make an in vivo reporter.
Make a knockout mutation in a gene encoding a yeast transcription factor which activates via a motif in reporter gene.
So, we have a binding motif which is the 'bait' motif which acts as a trap.
2. Make a one-hybrid plasmid library.
Human protein-coding fragments are cloned next to the DNA-binding domain of our transcription factor to create a library of plasmids with expression hybrid fusion proteins.
Effectively, you're putting random bits of human gene which make random bits of human protein next to a yeast DNA-binding domain, in the majority of cases, nothing happens, we see no reporter activation.
However, one of these many plasmids will happen to be a human TF effector domain which becomes attached to the yeast DNA-binding domain and can now potentially activate transcription.
3. When present in the reporter strain, hybrid transcription factors with bind the bait motif via the DNA-binding domian and the prey effector domain (human) will activate the reporter.
4. If you can clone a novel human effector domain, some simple plasmid sequencing will allow you to identify entire transcription factors from the genome sequence.
You isolate the plasmid, sequence the bit of human DNA that activate dthe reporter and go back to the human genome and potentially, a new transcription factor has been identified.
42
What is EMSA?
Electrophoretic mobility shift assays are a general method for exploring interactions between DNA sequences and DNA binding proteins.
43
What kind of gel is used in an EMSA?
EMSAs use 'native' polyacrylamide gels to gently separate moelcules according to size/molecular weight under physiological-like conditions.
44
What is the function of EMSAs?
EMSA allows visualisation of protein:DNA interaction.
45
What is an EMSA probe?
A short fragment of DNA containing a putative binding motif for a transcription factor or another DNA-binding entity.
Typically, an EMSA probe is 50-100bp in size and contains some motifs for DNA-binding proteins of interest.
The probe is zillions of identical molecules in solution.
The probe must also be labelled e.g. with a radiolabel or fluorescent dye.
46
Give some examples of EMSA probes?
A probe might consist of a region (50-200bp) of a real gene, prepared by restriction digestion of a plasmid or by PCR amplification from a genome.
A probe can be a short (20bp) synthetic dsDNA oligonucleotide designed and synthesised for by a commerical DNA synthesis company.
47
What can be interpretted from a shifted band on an electrophoretic gel?
Any complex of probe + protein/transcription factor (which will form if the candidate protein interacts with the probe) will be larger than the probe alone and form a band on the EMSA gel which migrates more slowly and therefore form a band higher up the gel.
This phenomenon is called a gel shift/retardation and can be considered evidnece that a TF binds to a motif within the probe.
48
What is meant by super shift?
EMSAs can be used to visualise/identify multi-protein interactions.
If we add two proteins to a probe and upon addition of a second protein, we get an even larger complex = moves even slower in the gel = even greater retardation of the band, this is known as a super-shift.
49
What can be explored using EMSA by mixing various combinations and concentrations of probes and DNA-binding proteins?
TF:DNA sequence specificity.
TF:DNA sequence affinity.
TF:TF competition.
TF:TF cooperativity.
50
What is the problem with EMSA?
EMSA is only really useful when you know the sequence motif you want to investigate, however, it is common from a genetic screen to identify a transcription factor and purify it biochemically and not know the precise sequence motif that it binds.
51
What is SELEX?
Systematic evolution of ligands through exponentail enrichment.
SELEX uses the EMSA principle to isolate a DNA motif together with the protein it binds.
52
How does a SELEX probe differ from an EMSA probe?
A SELEX probe is still a piece of synthetic DNA, but it has a region of completely random DNA.
SELEX probes are approx. 60bp long containing a region of Ns (during synthesis an equal amount of A, C, T and G is added).
The ends of DNA of a SELEX probe always have the same sequence.
The random region of a probe is flanked by two known sequences to which we can apply PCR to amplify the probe.
We are making a probe that has a mixture of all of the possible sequence motifs - every binding motif a protein could want.
53
Briefly describe the SELEX process?
1. Make a DNA probe, instead of including a known motif, synthesise DNA containing a region of random sequence.
2. Add the DNA-binding protein of interest (presumably transcription factor of interest) to the random DNA SELEX probe (labelled somehow e.g. radiolabel).
In theory, the transcription factor/DNA-binding protein should find and bind to the small number of probes in the mix which match it's cognate motif.
The transcription factor is selecting for the sequences it likes.
3. Separate any probe + transcription factor complex by EMSA (or other method e.g. affinity column/magnetic beads). The shift = the TF has purified the motifs containing the TF binding motif by shifting them up the gel.
4. Purify the protein + complex from the gel.
5. Perform on the DNA from the complex to make more of the motif-enriched probe.
There's not much DNA in the band so we amplify it by PCR and now have a mixture of low and high affinity motifs.
Radiolabel this and go again...
6. Add the TF to the new probe mix and perform another EMSA.
This time a stronger shift will occur because the probe is enriched for sequences the transcription factor will bind.
7. Repeat steps 3-6 usually 5-10 times.
Repeating this SELEX process achieves the exponential enrichment of motifs that the TF of interest can bind.
8. Sequence the enriched probe mix to discover the motif.
54
What is the difference between EMSA and SELEX?
EMSA allows us to perform experiments investigating transcription factor and motif interaction.
SELEX allows us to identify motifs for known transcription factors.
55
What do you need to be able to understand gene regulatory motifs at the genomic level?
Basic genomics
Functional genomics
Comparative genomics
56
What is basic genomics?
Sequence analysis predicts genes and functional regions.
57
What is functional genomics?
X-seq experiments define functional regions.
58
What is comparative genomics?
Comparative genomics is the study of differences and similarities in genome seuqence and organisation within and between species.
Inference of genome function via sequence similarity and difference.
59
What is the problem with basic genomics for inferring transcription factor function?
If you have characterised a motif and its transcription factor interaction, you can use genome sequence to find similar motifs elsewhere in the genome.
You may assume that the transcription factor also functions at these motifs which makes sense if you find the motif in the UPE of another gene for example.
The problem is, short motifs (e.g. 6bp transcription factor binding motifs) have a habit of popping up by chance in long DNA sequences.
This is really just sequence gazing.
You find that the sequence may pop up in the middle of nowhere, not in a gene or a promoter or even near a gene, does this mean your transcription factor binds here, this is just speculation...
60
What can we infer from regions of genome similarity between species?
These regions of similarity often imply evolutionary constraint and therefore function.
It implies something is being held by evolution because it is functional.
Exons tend to be conserved because they are functional, gene regulatory DNA is often also conserved which infers i has a function - these are called conserved non-coding elements and they provide evidence of gene regulatory function.
61
What is the name given to bits of conserved gene regulatory DNA between species?
Conserved non-coding elements.
62
What are conserved non-coding elements?
Conserved bits of gene regulatory DNA between organisms, this implies they hold an important function.
63
How many conserved non-coding elements are found in the human genome?
```
3 million (>12bp long).
Equivalent to 4% of the human genome.
```
64
What % of the human genome is conserved non-coding elements?
4%.
65
What are human accelerated regions?
These are regions of potentail regulatory DNA which aren't conserved in certain primates, these are CNEs uniquely present or absent in the human genome relative to other primates and therefore, could be things that give us our unique humanness e.g. bits of gene regulation allowing us to build brains in particular ways.
66
Even with comparative genomic evidence to suggest conservation of a motif and imply TF and basic genomic sequence data confirming the presence of a TF motif in a gene regulatory region, what else do we need to prove a given transcription factor binds a particular motif?
Functional genomics - a whole set of experiments that look at the genome wide dispersal of proteins and sequences.
67
Which technology is key to all functional genomics studies?
Illumina/next-generation sequencing.
68
What are the basic steps of a functional genomics study?
1. Isolate cells.
2. Perform an experimental procedure.
3. DNA fragments are yielded as output of experiemntal procedure, prepare an adaptor library.
We have an experimental procedure which yields its output in fragments of DNA.
4. Perform NGS.
Sequence those bits of DNA using next-generation sequencing/illumina.
5. Count the short sequence reads.
Align the sequence reads back to the genome and measure the frequency of those sequence reads.
6. Infer an experimental result.
69
Give some examples of X-seq technologies?
```
RNA-seq
NET-seq
ChIP-seq
DNase-seq
ATAC-seq
```
70
What are the 3 key transcription factor and gene regulatory region mapping technologies?
ChIP-seq, DNase-seq and ATAC-seq.
71
Why are there so many different X-seq methods?
There are lots of clever scientists harnessing the power of DNA sequencing in different ways.
As great as these methods are, no one method measures everything so there's diversity.
Sometimes you have to compromise on cost - functional genomics relies on counting millions of NGS reads and plotting their frequency across the genome, the number of reads is proportional to cost, if you need to generate lots of NGS reads, the cost is high, therefore, sometimes you may need to compromise and use a technology with fewer reads.
72
What is the key reagent in ChIP-seq?
Antibodies - a monoclonal antibody against the transcription factor of interest.
73
What is the main requirement for ChIP-seq?
You must know the identity of the transcription factor you're studying and have it cloned and purified so you can raise an antibody to it.
74
Briefly describe the process of ChIP-seq?
Perform your experiment.
Take a cell sample and treat it with formaldehyde to crosslink together everything that was physically attached in vivo - anything bound to DNA will be chemically crosslinked thus solidly localised where it was bound.
Ultrasonically fragment these cells to generate fragments of DNA with the attached proteins.
Use the antibody as a grab-handle to pull out bits of DNA that were attached to the transcription factor.
The antibody binds to the TF which is crosslinked to its motif which has been ultrasonically fragmented.
Purify the DNA from this mix.
This will yield lots of bits of DNA that in theory have a binding motif for your transcription factor of interest.
Resulting DNA is sequenced and frequency of reads plotted versus genomic location - this gives you a map of everywhere in the genome that your transcription factor binds in a given cell/tissue.
75
What can we infer from peaks in ChIP-seq sequence?
Peaks in ChIP-seq sequence read freuqency are taken to infer positions of transcription factor binding in the genome.
76
What is the resolution of ChIP-seq?
+/-100-300bp.
| This is pretty good positional information but isn't a very high resolution technology.
77
What is the main limitation of ChIP-seq?
It isn't really positionally accurate or high resolution (resolution +/- 100-300bp).
78
What are the advantages of ChIP-seq?
It gives good, unambiguous identification of specific transcription factor locations in the entire genome (specific because the antibody is specific).
NGS read counts are focused on specific locations by using the antibody as a molecular grab handle which means only a few million reads are required per experiment = cheap NGS.
79
What are the disadvantages of ChIP-seq?
Positional accuracy is low +/-300bp.
The generation of a high-quality antibody is technically demanding and expensive.
You must have the transcription factor purified for antibody generation.
It only data for the one factor under analysis (because it is so specific due to the mAB).
ChIP protocol is technically demanding and laborious to conduct in the lab - months of optimisation are required to get the antibody working in a particular way etc.
80
What is DNase-seq?
In vivo nuclease accessibility mapping.
81
What is the key reagent in DNase-seq?
Sequence non-specific endonuclease (usually DNase I) which cleaves DNA randomly.
The nuclease is a molecular probe - a molecule similar in size to the DNA + TF complex.
82
What is the difference between endonucleases and exonucleases?
Endonucleases cleave within the middle of a DNA moelcule whereas, exonucleases nibble from pre-existing ends.
83
Briefly describe the process of DNase-seq?
Take some living cells and treat them with detergent to permeabilise/punch holes in them - you don't need to crosslink them (unlike ChIP-seq).
Add DNase I to those permeabilised cells.
The nuclease enters the cells and creates random cuts in the chromosomal DNA (yielding sequencable fragments of DNA) unless there are proteins e.g. transcription factors in the way - the transcription factors therefore protect the motifs that they bind from cleavage via DNase I.
If we sequence all the resulting DNA fragments, the ends of the resulting sequences mark genomic sites of DNase accessibility - these are plotted as frequency distribution.
The transcription-factor bound motif by definition is inaccessible to the nuclease and therefore will never be in the sequences that make up that data set.
So, you map the positions of the ends of your sequences and look for where the sequence goes missing - this is called a footprint in the data where a protein is bound to DNA, this is base-pair accurate!
84
Why is DNase-seq referred to as in vivo nuclease accessibility mapping?
DNase-seq yields fragments of DNA that can be sequenceed only at sites which are accessible to DNase I.
Sites which are inaccessible to DNase I, i.e. are bound by transcription factors, or other DNA-binding proteins cannot be cleaved by DNase I upon DNase treatment and cell permeabilisation and as a consequence we are mapping accessible regions of the genome.
85
What is a DNase footprint?
This is a gap in the signal when plotting ends of sequences of fragments obtained by DNase I cleavage during DNase-seq.
These footprints indicate regions of transcription factor binding and are base-pair accurate.
86
What are the advantages of DNase-seq?
The positional accuracy is almost perfect, it is base pair accurate (compared to ChIP-seq which is only accurate +/-300bp) and therefore, you can map factors that are really close together.
DNase-seq tells you precisely where your transcription factor binding motifs are - accurate down to the single base pair.
DNase itself is cheap (unlike a mAb needed for ChIP-seq).
The DNase digestion protocol is technically easy and applicable to many different cell types.
87
What are the disadvantages of DNase-seq?
Whilst you get a base-pair accurate map of transcription-factor binding motifs in the genome - based off digital DNase footprinting, this doesn't tell you the identity of the transcription factor, just that something was bound to DNA at this precise point.
There are some transcription factors capable of binding the same motif e.g. USF and Myc can bind the same motif, so you really cannot tell what protein was bound to that motif (there is no antibody giving specificity unlike ChIP-seq).
To spot a footprint requires a high level of background sequencing - 2 reads per genomic bp on average =
You need a serious amount of NGS and this is expensive!! particularly if you want to look at different conditions/different cells etc.
88
What is the unexpected advantage of DNase-seq?
If you cannot afford the high level of background sequencing required to spot a footprint, you useful information can still be yielded from these experiments.
Generally speaking, active gene regulatory regions e.g. enhancers/promoters/regions of open chromatin are generally more accessible to DNase than background chromosomes so show up as DNase hypersensitive (DNase HS) sites in the data with just a low level of sequencing.
Instead of lookign at bp frequency/level of sequence reads, you zoom out and look at peaks and generally little peaks in the data are DNase hypersensitive sites and these denote active gene regualtory regions (enhancers/promoters/etc).
These peaks appear because these regions are more sensitive to DNase so DNase hones in on these regions and cuts them at a rate slightly above background so we get peaks.
89
What are DNase hypersensitive regions?
These are markers for active gene regulatory DNA that are produced from DNase-seq because active gene regulatory regions e.g. enhancers, promoters etc. are more sensitive to DNase I so DNase tends to hone in on them and cut them at a rate slightly above average/background so we get peaks in the trace at low resolution which denote active gene regulatory DNA - they mark the open chromatin.
90
What is the key reagent in ATAC-seq?
Transposase (based on Tn5).
This is another molecular probe still roughly the same size as a TF and DNA complex (like DNase is a molecular probe, but this again is different).
This transposase mediates recombination.
This transposase has stuck to it a piece of DNA (NGS-adaptor DNA molecule) that is a recombination intermediate, suspended in the middle of a recombination reaction so is looking for another piece of DNA to join and complete the recombination.
This piece of DNA that is stuck to the transposase is actually an NGS-adaptor.
91
Briefly describe the process of ATAC-seq?
The transposase transposes adaptors into the genome - the process is like DNase-seq, the core technology is the same - however unlike the DNase which wants to cleave, the transposase wants to complete recombination.
We take living cells and permeabilise them with detergent to punch holes in the cell membrane and then we add the transposase which enters the nucleus and starts transposing within the chromosomes and recombining illumina NGS adaptors exclusively into accessible DNA.
What this is doing is sticking illumina adaptors into the chromosomes in vivo.
Again, this only occurs where DNA is accessible, illumina adaptors are being inserted into the genome only where the genome is accessible i.e. transcription factors aren't bound to DNA.
The recombination reaction cannot be completed on a piece of DNA where the transcription factor is bound.
This transposase is extremely sensitive and only enters open chromatin regions in the genome.
Then you sequence the DNA and again map the sequences back to the genome and you get gaps in the regions where a transcription factor was bound, so transcription-factor binding motifs can be identified and located.
92
Why is ATAC-seq better to DNase-seq?
The transposase is much more sensitive to chromatin environment within the chromosome than DNase. Whilst DNase I has a slight preference for open chromatin (resulting in DNase HS sites), ATAC-seq ONLY works here = fewer NGS reads required per experiment.
The background level at which DNase will cut is quite high whereas the transposase in ATAC-seq won't enter closed chromatin so hones into regions of open chroamtin and targets all of its transposition acitvity to open chromatin regions e.g. UPEs/enhancers so is much more efficient.
This focuses all the sequencing power of ATAC-seq onto the regions where transcription factors will bind.
Ultimately this means the background goes down and so you need less sequencing = cheaper.
Another benefit is that the method constructs an illumina 'library' of fragments which are ready-to-sequence DNA.
The transposase puts the sequencing adaptors onto your molecules so you just need to clean them up a bit, amplify them and put them straight on the sequencer.
93
What are the advantages of ATAC-seq?
Positional accuracy is high +/-1bp.
NGS read counts are focussed to specific locations, exclusively open chromatin because the transposase won't enter closed chromatin and therefore, this directs all the sequencing power to regions where transcription factors bind, therefore we have lower background and need fewer reads per experiment. which lowers the overall cost.
The protocol/methodology itself creates an illumina library and therefore processing of the samples ready for sequencing is simpler than DNase-seq.
ATAC-seq can also be applied at the single cell level - there is a shift/trend in biology to single-cell resolution technologies as opposed to looking at whole tissues and therefore, this technology is relevant.
94
What are the disadvantages to ATAC-seq?
Again, you get no information on the identity of the transcription factor responsible for the footprint (this isn't specific because there's no antibody) - however, this disadvantage is shared by DNase-seq.
Purchasing the adaptor-linked recombination-suspended transposase as a reagent is expensive - 12 reactions = approx £800 - this is commercially more expensive than DNase I.
Technical difficulty is mid-way between DNase-seq (not as easy to perform) and ChIP-seq (not as hard to perform).
95
Where can we find a compilation of all this information on gene regulatory DNA and human functional genomics?
Data for human functional genomics has been compiled and curated by the ENCODE project.
This can be viewed on the UCSC genome browser.
The ENCODE project collates huge amounts of ChIP-seq, ATAC-seq and DNase-seq data together in one place.
96
How does communication between transcription factors and polymerases differ bewteen eukaryotes and prokaryotes?
In eukaryotes, there is a huge amount of signalling/communication/integration of information from the environment and very few transcription factors directly contact RNA polymerases and therefore, there is a highly complex, regulated and intricate network/cascade of communication.
In contrast, in bacteria, sequence-specific transcription factors generally activate transcription by talking directly to RNA pol, they physically grab the polymerase and hold it onto promoters.
97
What was the 'weird stuff' that came out of the initial biochemistry studies and yeast genetic screens to identify transcription factors?
Time and time again, the same factors were emerging from the different yeast genetic screens suggesting they held an important general function, but they were clearly distinct from the general transcription factors.
Sequence comparison demonstrated these factors also turned out to be conserved across eukaryotes implying function.
As more genes were discovered, it became clear these novel factors formed big complexes of proteins/multi-molecular complexes.
It was actually found this 'weird stuff' was needed as well as the 44 GTFs at the PIC to get stable/strong transcription activation.
These were named co-activators and co-repressors!
These sit between transcription factors and RNA polymerase.
98
How many proteins make up the mediator complex?
24
99
Give some examples of RNA pol II coactivator complexes?
Mediator (20-25 proteins >1MDa).
SAGA complex (21 proteins, 1.8 MDa).
SWI/SNF complex (12 proteins, 2MDa).
100
How many proteins make up the SAGA complex?
21
101
How many proteins make up the SWI/SNF complex?
12
102
Is Mediator a co-activator or co-repressor?
Co-activator.
103
What is the primary role of Mediator?
To stabilise binding of the RNA pol II PIC at the promoter.
Binding of Mediator to the PIC also stimulates the TFIIH complex within the PIC to switch elongation on and start to phosphorylate the CTD via the kinase acitivty of TFIIH (sometimes referred to as TFIIK). This promotes the transition from transcription initiation to elongation.
104
With what parts of RNA pol II does the Mediator complex contact?
Multiple contacts are made between RNA pol II and Mediator including Rpb3 and Rpb11 subunits and the unphosphorylated form of the Rpb1 CTD and therefore, Mediator is directly selecting for the initiating form of RNA pol II.
105
How does Mediator select for the initiating form of RNA pol II?
By making contact with the unphosphorylated form of Rpb1 CTD.
106
How does Mediator and the GTFs act as a memory mechanism at active promoters?
After intiaition, once the RNA pol II becomes processive, not all GTFs dissociate, many stay bound to increase efficiency and prevent the necessity for re-assembly of the entire PIC at active gene promoters. A sub-complex of Mediator as well as some GTFs remains bound.
This almost provides a landing pad/scaffold on gene-regulatory DNA so that RNA pol II can reload once it has finished transcribing.
107
Does Mediator make physical contact with RNA pol II?
Yes
108
Give some examples of co-activators/repressors which provide a physical link between TFs and RNA pol II?
Mediator
| SAGA
109
Do the majority of co-activators/co-repressors make physical links between TFs and RNA pol II?
No, these kinds of co-activators/co-repressors are in the minority.
Instead most influence the chromatin environment.
110
By what mechanism do most co-activators and co-repressors affect transcription?
By influencing/remodelling the chromatin surrounding RNA pol to indirectly modulate RNA pol II activity.
