Dr. Robert Weinzierl (TF Focus) Flashcards
Definition of a core promoter?
A core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator‑independent (basal) transcription by RNAP II in vitro
Core promoters typically extends approximately 40 bp up‑ and down‑stream of the start site of transcription (+1) and can contain several distinct core promoter sequence elements
Are core promoters very diverse in Higher eukaryotes?
Core promoters in higher eukaryotes are highly diverse in structure and each core promoter sequence element is only found in a subset of genes
What does an ideal TATA-box promoter sequence look like?
TATA-box is a core promoter element that is typically located 25-30 bases upstream from TSS and is defined by a A/T rich consensus that is flanked by G/C rich sequences (emphasising the position of the TATA box)
Example shown is derived from adenovirus major late promoter –> One of the best examples of highly active TATA-boxes
What is one of the main problems around using a TATA box element as a core promoter element in higher eukaryotes?
Eukaryotic genomes are huge in comparison with bacterial genomes
Hence, given that a TATA motif can arise by chance every 4 Kb, it is too short/weak to confer specificity
Thus, normally TATA boxes are accompanied by other core promoter cis-regulatory elements + TATA boxes are only found in a small proportion of core promoters
What protein complex recognises the TATA box motif?
The TATA-Box is specifically recognised by the TFIID protein complex.
TFIID contains a TATA-binding protein (TBP) that recognises the DNA motif
How large is TFIID and how does it recognises the TATA box?
TFIID is a large multiprotein complex (~10-12 different subunits; species-dependent)
Only one of these subunits is responsible for recognizing the TATA box: the ‘TATA-Binding Protein’ (‘TBP’)
Hence, TBP can specifically bind to TATA-boxes on its own –> none of there other proteins are involved in TATA recognition and binding
Outline the structure of TBP and what properties allow it to bind to DNA.
TBP is a crescent/U-shaped protein that is able to wrap around the DNA double helix
TBP contains +ive charged Lys and Arg residues which interact with the -ive charged backbone + conserved phenylalanine residues are placed in the DNA minor groove - allowing it to make hydrophobic contacts (reasonably strong binding)
Hydrophobic interactions cause the DNA to kink by 80o
Is TBPs saddle structure highly conserved across species?
Sequencing of TBP-encoding genes from many organisms has shown that the ‘saddle’ structure is highly conserved
This saddle structure is present in TBPs from all species
On the DNA level → in the saddle region we see a drop in % conservation in distant species whereas the N-terminal region tends to be be more divergent.
In higher eukaryotes there is a substantial N-terminal extension present that may contain poly-glutamine (poly”Q”) stretches
Why do bacteria promoters tend to be A/T rich?
In bacterial promoters the –10 element is also AT-rich, but this element is not functionally equivalent to eukaryotic TATA boxes
The bacterial –10 region facilitates the localized DNA strand-separation (‘promoter melting’; ‘transcription bubble formation’) to allow RNAP to initiate transcription. The eukaryotic TATA-box is located much further away from the transcription start site (-25 to –30!) and this part of the promoter is never part of the transcription bubble!)
What is the functional importance of the TATA (A/T rich region) box in the promoter region?
In eukaryotic TATA boxes the deformability of the minor grove width in regions of A/T base-pairs is important
This increase deformability allows for more intimate contacts between the TBP saddle and minor groove → lack of protruding -NH2 in G bases allows for this
What does physical data on DNA bending at TATA boxes reveal to us?
TATA region already has a tendency to bend
Oligo DNA strand with TATA region will bend on its own without the presence of TBP → upon addition of TBP the bending angle increases
Recap - What are the two main factors that are important for TBP binding?
TATA-boxes work on two levels:
- Sequence-specific contacts between TBP and the TATA-box consensus sequence
- TATA-boxes on their own influence the higher-order structure of DNA by bending it slightly. This localised bend attracts the binding of TBP to the TATA-box (matches conformation).
Note that pre-bending can be achieved with a variety of A/T rich sequences, thus explaining (at least to a certain extent) the high degree of sequence heterogeneity of eukaryotic TATA-boxes
What important fact should you always keep in the back of your mind when thinking about TFIID/TBP binding to a TATA sequence?
Always important to consider DNA accessibility which depends on the chromatin environment
Most TATA-like sequences are ‘hidden’ by chromatin structures and are thus never accessible to TBP
After TFIID binding to the TATA-box, what other two players bind to the protein complex?
TFIIB and A bind either side of TFIID → stabilising the complex (synergistic effect)
What does X-ray crystallographic data reveal about TFIIB binding to the TFIID complex/DNA?
TFIIB is a protein made from a single polypeptide that folds to form two similar domains (Note - TBP is a dimeric protein fused together)
TFIIB contacts DNA either side of the TATA box → conveys additional specificity as it recognises GC elements upstream and downstream of the TATA box called BRE – B recognition elements upstream
Evidence → structural and biochemical data
How do TFIIA and TFIIB interact with TBP?
How is TBP activity regulated within the cell?
TBP negatively auto regulates it’s accessibility to promoters through homodimerization, meaning…
- TBP monomers can dimers together forming a homodimer that does not bind to DNA in turn regulating activity levels → dimerisation defective yeast mutants show high levels of activator independent gene exrpession
- Furthermore, TBP in it’s monomeric state is more vulnerable to degradation
Hence, dimerisation prevents excessive levels of TBP binding/activity and also protects it from degradation in the cell (safe storage)
What is an example of a TBP antagonist?
Some transcription factors prevent formation of a productive transcription initiation complex
NC2 (Dr1-DRAP1), a negative cofactor, inhibits transcription initiation via direct interactions with the TBP-DNA binary complex
Function → prevents pervasive transcription (stops random transcription of genes) → streamlining the transcription machinery
What is the structure of NC2 and how does it prevent the transcriptional activity at the ?
NC2 is composed of small two subunits (alpha and beta) that are conserved among eukaryotes
NC2 bindings to TBP to form a stable complex → NC2 blocks binding and assembly of TFIIB (required for RNAP binding) in turn blocking RNAP binding → preventing initiation complex formation
What does the textbook description of RNAPII transcription initiation via a TATA motif?
Do most promoters have TATA boxes?
Bioinformatic analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past, and that the majority (>75%!) of core promoters do not contain TATA boxes
Many of the TATA-motifs even deviate form the consensus
What are examples of other promoter elements can be present in the promoter region?
>80% of RNAPII-transcribed genes lack TATA-boxes
There are further consensus motifs that may be present to various extents in different promoters:
Initiator Element (‘Inr’)
Downstream Promoter Element (‘DPE’)
Motif 10 Element (‘MTE’)
TCT motif (polypyrimidine initiator; in ribosomal protein gene promoters)
These other promoter elements are even less clear than the TATA box
What does promoter positional information reveal about TATA, Inr, DPE and Dref elements?
TATA and Inr → conserved dense peak at a specific position → conserved location
DPE and Dref (drosophila) → broader peak
Summary picture of the proximal promoter elements?
MTE – minus ten element
Review - Kadonaga, J.T. (2012). Perspectives on the RNA polymerase II core promoter. Wiley Interdiscip. Rev. Dev. Biol. 1, 40-51.
What roles do other proteins in the TFIID complex play?
TBP is responsible for the sequence-specific binding to TATA-boxes
Some of the other subunits of the TFIID complex, ‘TBP-Associated-Factors’ (‘TAFs’) recognise the additional sequence elements TAF2, TAF6, TAF9
TAFs making sequence specific contacts with MTE and DPE
Inr element binding is not fully understood
What are the two main types of transcription initiation patterns?
The terms “focused” and “dispersed” refer to opposite ends of a continuum of transcriptional patterns
Focused initiation, transcription starts from a single nucleotide or within a cluster of several nucleotides → associated with regulated genes (on/off)
Dispersed initiation, there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides → housekeeping genes
Picture of the two different models of transcription initiation patterns?
How does chromatin structure influence the transcriptional state (stable & labile)
Nucleosome positioning/density influences the type of transcription
Labile → focused transcription → all the core elements are recognized
Stable → dispersed → requires gene-specific transcription factors to drive transcription
How does TBP poly-glutamine expansion change over evolution?
TBP Polyglutamine expansion takes place as move through the vertebrate evolutionary lineage → more complex organisms = longer Polyglutamine repeat tail
Evolution progresses → Polyglutamine expansion
What types of diseases are associated with the TBP polyglutamine tail?
What is cerebellar ataxia?
What does DNA analysis of the TBP polyglutamine tail reveal about the healthy and pathological forms?
CAG - codes for glutamine
Most of us have around 35-36 glutamine residues at the TBP N-terminus
Some people have a pathological amount → corresponding to 47-55 residues
The number of CAG repeats is inversely correlated with the age of onset for cerebella ataxia → Meaning higher repeat number corresponds to a younger age of onset
Under the microscope, what does cerebella ataxia pathology look like?
Main message - TBP aggregates accumulating in cells.
- TBP containing an expanded polyQ stretch is expressed at the same level as its normal counterpart and forms neuronal intra-nuclear inclusions containing other proteins involved in protein folding or degradation
- polyQ expansion reduces in vitro binding of TBP to DNA
- polyQ expansion causes abnormal interaction of TBP with the general transcription factor TFIIB
Note - SCA17 is another gene that is responsible for driving disease progression
What do mouse models reveal about polyglutamate expansion and cerebella ataxia pathology?
All WT mice survived
But mice with glutamine expansion had significantly reduced lifespan → correlated with the level of polyglutamine expansion
How does TBP binding differ between a healthy and pathological state (cerebella ataxia)?
What link was found between TBP and depression?
CAG repeat sizes in the TBP gene were investigated in two well-characterized Dutch cohorts → including 2165 depressed and 1058 non-depressed individuals aged 18–93 years
A CAG repeat length exceeding the median in both alleles was associated with an increased risk for lifetime depression → showing us that repeat polymorphisms act as complex genetic modifiers of depression
What are Super-core promoters?
Using knowledge on pre-existing promoters we are able to construct artificial ‘Super-core promoters’ that drive high levels of gene expression (higher than strongest natural promoters i.e. viral promoters)
Mediated by introduction of expression vectors → useful for a wide range of scientific and biotechnological applications
What does the in-vitro assay using the detergent Sarcosyl reveal about super-core promoters?
In vitro transcription reactions can be limited to a single round of transcription by adding the detergent Sarcosyl → Sarcosyl prevents further initiation but does not interfere with transcript elongation (1 round of initiation - no re-intiation)
The super core promoter is ‘stronger’ than the two other promoters because it supports a higher frequency of transcript initiation → ~40% of available DNA templates are used in the in vitro assay (this is very efficient!)
Likewise, Footprinting studies show that the super core promoter bind TFIID with higher affinity than other promoters
What does the construction of the super-core promoter reveal about the nature of promoter regions?
Shows us that all of the core promoter motifs contribute to the strong binding of TFIID to the super core promoter → meaning higher specificity of each TF to the promoter elements contributes to an overall stronger interaction (additive)
Example → Super core promoter (SCP1) compared to viral promoters (CMV and AdML) in a Luciferase assay
Do the number of RNAP’s differ between species? Do mitochondria and chloroplasts have their own RNAP?
Eukaryotes
RNAP1 → exclusively transcribe ribosomal RNA
RNAP2 → very diverse → mRNA product
RNAP 3 → more diverse than 1 but still not as diverse as RNAP1
Archaea - RNAP similar to RNAP 2
Bacterial - RNAP – most distinctive → divergent evolution
What does the following image highlight about the evolution of RNAPs?
When examining the different RNAP structures we can start to understand how closely related they are based on structural similarities
Takeaways…
LUCA – Last universal common ancestor + Color categorizes similar subunits
Bacteria RNAP branched off earlier → evolved separately relative to Archaea and Eukaryotes
Even though bacteria subunits evolved separately there are still several homologs (as shown by color)
Characteristics of Eukaryotic RNAPII?
Outline the 3D structure of yeast RNAPII?
What are the two main conformations for RNAPII?
RNAPII has a ‘open’ and ‘closed’ clamp conformation.
The conformation is found prior to DNA binding whereas the closed conformation correlates to the DNA bound state.
X-ray crystallographic data suggests that there is a highly localised hinge mechanism required for RNAPII activity.
What does the electrostatic potential of the RNAPII surface show us about it’s interaction with DNA?
Inside of RNAP is positively charged whereas outside is negatively charged → favours the bound state of RNAP to the negatively charged DNA
Outline how RNAPII catalyses the synthesis of a novel mRNA molecule?
- Downstream DNA enters RNAPII
- Inside the DNA molecule is unwound forming ssDNA, exposing the template strand for RNA synthesis
- Nucleotide triphosphate enter into RNAPII via the pore and binds to the complementary base present on the DNA template
- Mg2+ ion catalyses nucleophilic addition of the nucleotide to the growing RNA strand → results int the formation of a short (10B.p) DNA-RNA hybrid
- As RNAPII moves along, the nascent RNA strand dissociates and exists the protein complex + the template and non-coding strand bind back together
What happens to the angle of the DNA strand as it moves through the RNAPII complex?
DNA that goes in and out is bent by 90 degrees → Strand separation allows the bending to be energetically favorable (difficult if it were DS)
What is a problem with the conventional NTP funnel that feeds the nucleotides into the RNAP active site?
Problem with the classical funnel structure that leads up to the catalytic site → Pore is very narrow (1 nucleotide wide) → if the wrong nucleotide diffuses into the active site (¼ chance of being correct) it would have to diffuse back out before another nucleotide can move in → inefficient
What is the alternative model for NTP entry into the RNAPII active site?
Additional channel → wider & exposes 3 + 4 nucleotides at a time to incoming NTPs
What structure in RNAPII is responsible for the translocation of RNAPII across DNA?
Remember translocation is an active process that needs to be directed
Who’s responsible → Long alpha-helix - bridge helix → pushes RNA-DNA hybrid through the protein complex
Outline the bridge helix trigger loop translocation mechanism for RNAPII.
Bridge helix (green) bends (kinks), which pushes the DNA-RNA hybrid out of the catalytic site → subsequently, it returns to relaxed conformation
Trigger loop (blue also involved)
Further reading?
Are large quantities of abortive transcripts produced during transcription intiation?
The process of RNAPII breaking free from the initiation complex appears to be a difficult process → evident by the high levels of abortive (short) transcripts that are produced
1-5% of initiated transcript will end up as full length RNA but once RNA breaks free from the initiation complex the % of successful transcripts increases
What has FRET analysis revealed regarding the cause of the large number of abortive transcripts?
Initiation → DNA comes together as shown by FRET distance decrease (scrunching) which leads to the creation of DNA Loops. This creates tension in RNAP which results in two outcome which are that RNAP continues or aborts
Outline the domains in TFIIB and their importance in transcription initiation?
- The N-terminal Zn-ribbon domain of TFIIB interacts with the RNAPII (via the ‘dock domain’ on RNAPII)
- Helps to open up the DNA at the start site using the B-linker (N-terminal region of TFIIB enters the catalytic cleft of RNAP) → The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the ‘B-reader’ that approaches the active site (important for start site recognition and positioning in active site)
- Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger TFIIB release and elongation complex formation
Do we understand how the transition from the intuition to elongation complex takes place?
What id the CTD tail in RNAPII and why is it important?
CTD grows with evolution → longest stretch in humans
2x Proline in consensus – high density → bad news for secondary structures → meaning that it doesn’t form regular structure (flexible tail)
Function → Y, S and T contain free -OH groups → targets for Post-translational modifications –> phosphorylation, ubiquitination → provides an opportunity for additional regulation i.e. phosphorylation by TFIIH kinase
Is the CTD tail present in all RNAPs?
The CTD is only present in eukaryotic RNAPII, not in RNAPI, III, IV and V!
Prokaryotic (bacterial, archaeal) RNAPs also lack an equivalent structure
What phosphorylation states of the CTD tail are associated with initiation and elongation? What molecular processes is the phosphorylation of CTD associated with?
Degree of phosphorylation changes during the process of transcription → hypo to hyper
TFIIH is a versatile factor – helicase activity for DNA melting, kinase activity for phosphrylation of CTD)
How does DNA melting during transcription initiation take place?
Outline the structure of TFIIH.
Very large complex (multiple polypeptides) → uses ATP hydrolysis to unwind DNA start site
The TFIIH core forms a crescent-shaped complex spanning from Ssl2 to Rad3
Ssl2 binds downstream DNA consistent with its role in DNA opening → Ssl2 uses ATP hydrolysis to translocate on DNA. If the Ssl2 location is fixed, Ssl2 action results in a reeling of DNA into the active centre
What does a ribbon structure of the initiation complex look like?
Huge molecular machinery → provides a decent explanation to why there is a high degree of abortive transcripts → a lot going on
What other things are happening alongside transcription elongation that play an important role in mRNA formation?
RNAPII transcription and pre-mRNA processing are coordinated events (downstream processes are equally as complicated)
5’ Capping, splicing and 3’ poly-adenylation are occurring during transcription and RNAPII (and the CTD particularly) plays a role in the regulation of these events
Why is it important to understand the various experimental techniques for transcriptional assays?
To obtain insights into gene expression mechanisms a number of different technical approaches need to be applied → Biochemistry, molecular biology, structural methods, cell biological methods etc.
Every method has inherent limitations and artefacts, so a coherent picture can only be built up by looking for consistencies emerging from a number of techniques
What are eukaryotic In-vitro transcription assays?
This is when purified DNA templates are transcribed in the test tube with nuclear extracts and/or highly purified transcription factors
The ultimate goal is the re-construct regulated gene expression from recombinant (i.e.completely defined) transcription factors
But! This goal has up to now not been achieved with any eukaryotic system → For example, human RNAPII and TFIIH cannot currently be assembled from recombinant subunits in active form!
Hence, they need to be purified from eukaryotic cell line instead
What does the overall strategy for in-vitro transcription assays look like?
Basically you are trying to get all the components required for transcription into a test tube so that you can create an experimental system whereby you can manipulate variables (i.e. presence of specific TFs).
Hence, it would be ideal if you all the required proteins using a recombinant system → not that this had not been achieved with RNAPII and TFIIH (large complexes).
How were the different core TFs in the basal initiation complex named?
Nomenclature for names come come from the original experiments
Ion-exchange chromatography and increasing salt conc was used to remove different subunits from the phosphocellulose column → systematic names given based on the time they exited the column
Things to consider when performing in-vitro transcription assays?
What are the two most common ways of quantifying the level of transcripts produced from in-vitro transcription assays?
Requirement for techniques that allow quantitation of RNA originating from a specific region of the DNA template
Two major methods are used for quantitating in vitro transcripts: primer extension assay & ‘G-less Casette’ assay
What is the primer extension assay?
A radiolabelled DNA oligonucleotide primer is hybridized to the RNA and extended by reverse transcriptase
This will result in the appearance of a single-stranded, radiolabelled cDNA product of a specific size
Gel electrophoresis allows quantitation + allows identification of transcription start site (Reverse transcriptase will polymerise in the 5’ direction to the start of the DNA strand)
What is the G-less Cassette assay?
Theory
G-less cassette → template strand has all C removed (mutagenesis) → RNA will not have any G nucleotides
Hence, when we introduce RNase T1 which requires a G residue to cleave → all other RNAs that initiated at other sites will be removed (removing background transcription) → giving us an accurate idea of the desired transcript which can be quantified after running on a gel
What are the dis- & ad-vantages of using a in-vitro assays?
How are in-vivo transcription assays performed?
In-vivo transcription assays → carried out inside living cells (cell line, organism, organdie, etc.)
Basics → Transfer DNA of interest into cell → see whether it is transcribed by coupling our gene of interest to a reporter gene to tell us that the DNA is being transcribed (GFP, luciferase, etc. )
Luciferase → sensitive + good for quantification
Can be transient or stable assays
What is the difference between in-vivo transient and stable assays?
Transient vs stable assay
Transient → introduction of plasmid à resulting in rapid expression – quick
Stable → transfection with stable integration into the genome
Advantages and disadvantages of transient in-vivo transcription assays?
What is an example of a commercial plasmid used for transient transfection?
pGL3-Control Vector
How can these plasmid constructs be used for in in-vivo transcription assays?
Both cases we are examining the ability of a given inserted DNA sequence in promoting/enhancing levels of luciferase expression
Looking for promoter sequence (Left) → Insert DNA into restriction site → if the inserted contains a DNA promoter then elevated levels of Luc are expressed which can be visualised/quantified
Looking for enhancer sequence (Right) → No enhancer → we have a low basal level of expression but we can introduce our DNA and if expression increases, we know that we have an enhancer element
What do we need to perform stable transfection assays?
Early stages identical to transient transfection assay (transfection using plasmid), but plasmid must contain a dominant selectable marker → creates selective pressure for integration in the genome
Example: Drug resistance gene (G418 drug resistance)
Result - DNA becomes stably integrated into genome and is expressed in chromosomal environment
Note → process of stable integration takes much longer than transient transfection assay
Advantages and disadvantages of in-vivo transcription assays?
How could one use performing identification of control sequences in in-vivo systems?
Basically, incorporate gene (with reporter) into in-vivo system and examine the gene is expressed
If proper expression/regulation is obtained → methodically delete regions of the sequence and repeat
How could one use performing identification of control sequences in in-vivo systems?
Basically, incorporate gene (with reporter) into in-vivo system and examine the gene is expressed
If proper expression/regulation is obtained → methodically delete regions of the sequence and repeat
What experimental method is used to investigate the human transcriptome?
Transcriptome → collection of all the transcripts in a cell → use RNAseq
Counting frequency → indicates abundance in samples
RNAseq → can be used to detect transcript isoforms
Why is analysing the transcriptome of cancer useful? What problem are we faced with when trying to do so?
Compare cancers to normal tissue → provides insight to transcript dysregulation (over/under-expression of specific proteins)
Problem → cancer cells are not homogenous cell populations - meaning that healthy and cancerous tissues are mixed together
Consequence → When performing RNAseq we end up with a mixture of transcriptomes
Ideally we need to isolate the cancer tissue → Laser capture microdisection
What is Laser capture microdissection (LCM)?
Laser capture microdissection → dissect out cells based on specific characteristics – i.e. morphology
Using IR to dissect out the cancerous cells
What needs to be performed after laser capture microdissection when analysing the transcriptome of the selected cell?
How is mRNA amplification carried out?
What are the different technologies for performing transcriptomics and their relative throughput & capacity?
Number of Samples - Capacity
Number of genes queried - throughput
RNAseq → has a high capacity & throughput
What is qPCR with Taqman?
TaqMan is a quantitative PCR technique (qPCR) → It can be used to quantitate transcripts, if coupled with RT-PCR
- In the intact TaqMan probe, energy is transferred (via FRET) from the short-wavelength fluorophore on one end (green circle) to the long-wavelength fluorophore on the other end (red circle), quenching the short-wavelength fluorescence. After hybridization, the probe is susceptible to degradation by the endonuclease activity of a processing Taq polymerase.
- Upon degradation, FRET is interrupted, increasing the fluorescence from the short-wavelength fluorophore and decreasing the fluorescence from the long-wavelength fluorophore
- The rate at which fluorescence increases is directly correlated to the initial transcript level
Does transcription elongation impact levels of gene expression?
Initially, initiation was considered the driver behind differential gene expression but it turns out that transcript elongation has also an influence (pausing/arrest site)
What are the different ‘decisions’ that can be taken by RNAPII during transcription elongation?
Outline the role of the bridge helix in RNAPII translocation.
Nano-mechanical process – using bridge helix to push DNA-RNA hybrid along
What are the two main models that are used to explain RNAPs translocation during transcription elongation?
Powerstroke → rNTP hydrolysis drives movement
Brownian Rachet → kinetic energy from aq environment provides the energy for the kinking of the bridge helix (random kinetic input) → more passive
Explain both the powerstroke model and the brownian ratchet model using the attached diagram.
Possible to have a mixture of both → Not mutually exclusive
In the Brownian model RNAP is initially quite mobile until NTP moves in favoring forward movement → then allows for condensation + pyrophosphate release
Why do elongation rates differ between in-vivo and in-vitro systems?
Using the example of c-myc explain how elongation in different stages of the cell cycle influence it’s expression?
Basically…
- C-myc (oncogene) regulates 100’s of genes for cell proliferation
- Cell needs to regulate intracellular myc expression
- How? Make it hard to transcribe the gene - excessive levels can lead to cancer formation
What does the attached image show us (relation to C-myc)?
Pausing and arrest sequences in c-myc
P (pause) → does not need Efs to continue
P/A → arrest site → requires elongation factors to continue
A/T → Arrest + possible termination
Past the first 500 nucleotides the DNA transcript lacks the P and A sites → large initial hurdle
What basal factors are able to stimulate transcription elongation?
What do the large number of stalling events near the initiation site tell us?
In drosophila → stalled sites at the +25-50 site quickly after initiation sites → prevents other RNAPs latching onto DNA and initiating transcription (blockage) → mechanism to downregulate expression
What do large scale studies in Drosophila reveal about RNA polymerase stalling?
What is required to convert stalled RNAPs into actively elongating enzymes?
What two main roles do elongation factors perform to keep the transcription party going?
What is transcriptional arrest?
What does transcriptional arrest look like diagrammatically?
RNAP takes a couple steps back once arrested (slides back) → mechanism not fully understood
Consequence? → Some of the nascent RNA hangs out of the catalytic site (into the cone shaped channel) due to the backwards movement → the 3’ end moves away from the catalytic site (not in close contact with Mg2+) → impossible for the RNAP to add RNAPs
What is the structure of TFIIS?
TFIIS has a zinc binding domain (domain III) which contains a loop with acidic residues → this slides into RNAP funnel coming in close contact with Mg2+ → moves Mg2+ atom into a slightly different position allowing it to initiate nucleolytic activity
How does TFIIS/GreB bind to RNAPII?
Universal activity in eukaryotes and bacteria – encounter similar problems
How TFIIS/GreB loop interacts with Mg2+ in RNAPII?
Are there any other transcription factors that can restart an arrested polymerase?
What are the 4 different catalytic activities that RNAPII has been shown to exhibit?
Outline the 5 different catalytic activities that can be performed by RNAPII.
Just because they can be performed does not mean that they commonly occur in-vivo → require the right conditions
Are smaller RNAPs able to read through nucleosome bound DNA?
Chromatin (i.e. nucleosome-packaged DNA) is the natural substrate for RNAPII
Nucleosomes can be displaced by RNAPII in highly transcribed genes, but are not usually removed during active expression of a gene
Single-subunit RNA polymerases from bacteriophages T7 and SP6 were used for some of the earliest studies → Surprisingly, these small polymerases could transcribe completely through a nucleosome, albeit at a reduced rate compared with free DNA
What are some examples of elongation factors that allow RNAP to read to nucleosome bound DNA?
Outline the principal for Spt6 and Fact activity?
Spt6 → associates with nucleosomes à holds on to it à returns it when it is finished
Fact → displaces H2A/H2B dimer → creates a nucleosome that is highly unstable allowing DNA to peal off from the nucleosome → once cleared Fact returns the dimer
How does FACT and Nhp6 open the chromatin structure
Increased atomic detail → but the exact information about how the mechanism works is not clear
What are pioneering RNAPs?
The nucleosome is only the first level in chromatin compaction, but is unclear whether these activities are sufficient for elongation through higher order chromatin fibers in vivo
It is possible that the first polymerase to transcribe a gene is a specialized ‘pioneer’ polymerase equipped with additional tools that break down higher-order chromatin structures
Note → This is a concept but evidence is not concrete
What experimental setup was used in order to study the mechanism of RNAPII translocation during transcription?
Technique is known as Laser Trapping
Setup have two beads that are connect by a DNA strand. One bead is fixed in position whereas the other bead is able to move in the horizontal plane → all done using lasers i.e. Laser trapping
Hence, as RNAPII moves along the DNA strand it exerts a force on the mobile bead → this can be quantified by examining the level of input laser is required to hold the mobile bead in a fixed position.
This allows us to determine the kinetics and force of transcription translocation
What does the laser trapping experiment reveal about the nature of RNAPII translocation?
The new results appear to be inconsistent with the ‘power-stroke’ model, favouring the view that RNAP translocation occurs by the ‘brownian ratchet’ mechanism
RNAP moves in steps of 3-4 A → equivalent to single nucleotide at a time
Problem à small number of nucleotides incorporated à not reflective of in-vivo experiments
What does the attached image show?
What TF binds to the SV-40 enhancer?
In terms of distance, how far can enhancer elements be from the core promoter site?
Some enhancer modules are located close to the core promoter (typically within 1 kb)
In vertebrate promoters’ ‘proximal’ enhancers often contain Sp1 sites
Other enhancers can act over long distances (10 – 1000 kb!) and may therefore be located anywhere with respect to the transcribed region
‘distal’ enhancers can be located anywhere: 5’ end, introns, 3’end
What experimental setup can be used to study the distribution of tissue specific TFs?
Using Enhancer traps
Basic concept → Make transgenic animals with reporter genes (b-galactosidase) that is linked to a known enhancer.
This is injected into mouse genome and the pattern of b-galactosidase is examined to figure out the tissues that express the specific TF of interest.
What property of DNA allows for interaction of distal enhancers with the basal initiation complex?
Long-distance action of enhancers is facilitated by the DNA’s flexibility
What experimental evidence supports the idea of DNA looping in order to bring together TFs?
What is a problem that arises when using DNA loops for long range TF interaction? What is a probable solution to this problem?
Enhancers can regulate the expression of genes over long distances by looping.
Hence raising the question, how can we get specific regulation of certain genes without interference by other enhancers? Basically, how can we ensure that our enhancer is acting on the correct gene?
Solution → insulator (barrier) proteins block long range interactions
What are the three rules for enhancer-promoter interactions that one should consider?
Proximity: When multiple genes are compatible with and relatively close to a shared enhancer, the most proximal gene is preferentially activated over the distal gene → This competitive advantage disappears when both genes are located far away from the shared enhancer
Compatibility: Enhancers ignore the ‘first-come, first-served’ rule when the proximal promoter is incompatible with the enhancer → Result: activation of the distal gene
Insulation: The presence of an ‘insulator’ can block enhancer function across its binding site and prevent a compatible gene from being activated
How do insulators block unintended interactions?
Physically block/prevent long range interactions between enhancers and core promoter’s that reside outside DNA loop
How can insulator sites be used to increase expression of transgene?
If we try to incorporate a transgene into a new organism it is possible that it randomly integrates in a heterochromatic region → in turn yielding low levels of expression
But if we were to flank our DNA segment with two insulator regions we can create a region that is transcriptional active as the insulators isolate/loop the transgene from the surrounding chromatin
How does the drosophila even skipped gene act as evidence for the modular nature of enhancers?
How were the enhancer modules of the even skipped gene in drosophila analysed experimentally?
The existence of enhancer modules can be detected by linking different portions of the eve promoter to a reporter gene (e.g. b-galactosidase encoded by E. coli lacZ) → create different constructs containing different regions of the eve promoter
These constructs are then used to create transgenic Drosophila flies → If a given enhancer module is active in a specific region it will also drive the production of the reporter b-galactosidase
Subsequently, X-gal staining (blue color) of the embryos derived from such flies will reveal the expression of the reporter gene in the pattern directed by specific enhancer modules
This technique can also be used to map the location of enhancer modules
Do enhancer modules mostly act independently of each other?
What biochemical techniques can be used to localise important enhancer motifs as well as characterisation of unknown gene-specific transcription factors?
How to study the importance of enhancer motifs for expression levels?
Use different deletion mutants in a transient transfection of cells → examine how levels of reporter gene changes
How is the Eve expression concentrated into stripes during drosophila development?
Bicoid + Hunchback expression follows a gradient
Giant and Kruppel expression repress → resulting in narrow gene expression
What function roles do gene-specific TFs need to carry out?
Apart from direct activation of the basal initiation complex, how else can gene expression be enhanced?
What are examples of true activation mechanisms (direct interaction wit B.I.C)?
What should you keep in mind when considering the different tools available to enhance gene expression?
What are the generic components of a gene-specific transcription factor?
Generic representation of a gene-specific TFs
DNA binding domain → binds to DNA
Activation domain → interacts with other proteins/TFs/RNAP etc.
Domains are modular, meaning that they are made of different independent modules that carry out specific functions
What sorts of physical and chemical interactions underpin Protein-DNA binding?
How are sequence-specific contacts made? How can TFs recognise and target particular sequences?
Electrostatic interactions between proteins and the DNA backbone provide stabilising energy, but not sequence specificity → Given the TF binding does not lead to the DNA denaturation/melting, they have to be able to read the sequence from outside the double helix
How?
Boils down to the pattern of hydrogen bond donors and acceptors on each base in both the major and minor groove.
How can Hydrogen bond donor and acceptor patterns in the DNA grooves be used for sequence specific recognition?
Absolute recognition of the four different base pairs is only possible via the major groove
Transcription factors binding via the minor groove can only distinguish (A-T; T-A) from (C-G; G-C) - AT from GC
What TF mainly look for?
- Asymmetric hydrogen donor/acceptors → pattern of Hydrogen bonds which can be recognised and use to identify the bases
- Bulky methyl group on T residues → in blue circle is often used as a point of recognition
Minor Groove - Hydrogen bonds acceptors/donors in minor groove are symmetrical but the G-C bases have an extra donor group → allowing TFs to distinguish A-T from G-C
Major Groove - Asymmetrical Hydrogen donors and acceptors + presence of methyl group allows to recognise each base pair
What is the Helix-turn-helix DNA binding domain? Where are they normally found?
Helix-turn-helix (‘H-T-H’) motifs are DNA-binding motifs used in many bacterial transcription factors
e.g. lac repressor, CAP protein, bacteriophage lrepressor
H-T-H motifs are also present in eukaryotic transcription factors that are specifically expressed during embryogenesis and determine regional differentiation along the body axis (‘homoeotic genes’)
Do H-T-H domains normally dimerise?
What is the Ubx/Exd Homoeodomain Complex (example of H-T-H)?
Ubx (‘Ultrabithorax’, red) and Exd (‘Extradenticle’, cyan) homeodomains bind in a tandem manner on opposite faces of the DNA → Ubx recognises sequence specific targets in DNA whereas Exd aids in Ubx binding
These TFs play an important role in the proper development of body sections during Drosophila development.
Extra - The dashed red line represents the disordered linker between the Ubx homeodomain and its YPWM motif. The YPWM motif reaches into a hydrophobic pocket on the surface of the Exd homeodomain.
What happens when the homeotic genes are expressed in the correct spatial-temporal order?
We get the incorrect the development of body segments - e.g. sacral vertebrate converter to lumbar in mice
Note - Homeotic genes are master regulator genes that direct the development of particular body segments or structures in a the correct spatial-temporal manner
Why is an alpha helix used to make DNA specific contacts?
The side-chains of amino acids present in the recognition helix make extensive and sequence-specific contacts with the base pairs via the major groove
Basically, good surface complementarity!
This becomes really evident when examining the Van Der Waals radii of structures.
Are there H-T-H variants?
Yes, there is a lot of variation in H-T-H motifs
Some with Beta-sheets, multi-helical bundles, etc.
But in each case we have a core recognition helix, whereas any other region is responsible making other stabilising contacts with the DNA → regardless it is still considered a H-T-H
What is the Leucine zipper domain?
Leucine Zipper Domain
- The binding of the a-helices to form the Y-shaped DNA-binding domains
- The Y shape is held together by hydrophobic interactions between regularly spaced leucine residues on both helices ( i.e. ‘leucine zipper’)
- From a structural perspective, the leucine-zipper is the simplest DNA-binding motif
What are some examples of TFs that use the leucine zipper domain?
Several important transcription factors controlling cell proliferation contain ‘leucine-zipper’ DNA-binding domains
- The yeast gene-specific transcription factor GCN4 uses a homodimeric leucine zipper motif to form a DNA-binding motif
- The c-JUN/c-FOS heterodimer (‘AP-1’) uses a heterodimeric leucine zipper motif to form a DNA-binding domain → c-jun/c-fos have been identified as proto-oncogenes, i.e. they can be involved in converting a normal cell into a cancer cell if they are mutated
On the primary structure level, what does a leucine zipper consist of?
Regular spaced hydrophobic residues that stick out on at a given position in the helix in turn allowing for extensive non-polar contacts between two helices - periodicity of 3/4 residues for the hydrophobic amino acids
Allows for the formation of a slightly coiled super-helix with leucine residues interacting with each other
What residues are normally found near the DNA in a leucine zipper?
DNA binding → We typically find highly positively charged residues allowing for interactions with negatively charged DNA backbone
In the case for jun/c-fos binding to DNA, what other protein gets involved?
NFAT à TF that aids and stabilizes the binding of C-JUN and C-FOS → cooperative binding → increase the binding half life
What is the Helix-loop-helix DNA binding domain?
Helix-Loop-Helix → Remember that it is different to H-T-H
The H-L-H motif is a slightly more complex version of the ‘leucine zipper’ motif because it includes a loop interrupting the alpha-helices → this reverses the direction of the helix
The binding of the alpha-helices to form the Y-shaped DNA-binding domains still involves hydrophobic interactions between regularly-spaced leucine residues on both helices (-> ‘leucine zipper’)
What are some examples of TFs that use a H-L-H DNA binding domain?
Several important transcription factors acting as oncoproteins contain helix-loop-helix (‘H-L-H’) DNA-binding domains
- The c-MYC/MAX heterodimer and MAX/MAX homodimer uses a helix-loop-helix motif to form a DNA-binding domain
- Several other proteins regulating various developmental and physiological processes in cells - the bHLH-PAS proteins (PAS: Per-ARNT-SIM, the first three transcription factors containing such a domain arrangement)
Regardless, DNA binding region contains positively charged residues → e.g. Max homodimer has a high density of arginine and lysine residues
What are Zinc-finger DNA binding domains?
- Zinc finger domains contain a zinc atom that is coordinated by cysteine and/or histidine residues
- The zinc-finger motif is made from a beta-turn and an alpha-helix held together by
a Zn atom, which is coordinated by cysteine (C) and histidine (H) residues → Forms a compact DNA binding domain (zinc acts as a molecular glue between the two 2o structures) - Only found in eukaryotes, not in bacteria
- One of the most widely used DNA-binding motif!
- BUT: some zinc fingers-containing proteins bind to RNA or proteins
Why are zinc fingers a popular motif in eukaryotes?
- Short sequence/Small motif that is folded in a compact manner → Zinc acts as glue bringing the helix and B-turn together
- Can be used in tandem repeats → Each Zinc-finger helix recognises a 3-nucleotide target site in the DNA
Hence, longer & more complex DNA sequences can be recognised by arranging Zinc-fingers with different sequence recognition ability in tandem - allows for a higher level of sequence specificity!
What structure in the Zinc-finger is responsible For DNA contacts?
Are there any examples where a Alpha-helix is not used for sequence specific contacts?
Ribbon-Helix-Helix motifs places b-sheet into the major groove
Attached example → uses B-sheet to bind and interact with major groove → not as relevant in humans but present in bacteria
This functionally diverse protein superfamily regulates the transcription of genes that are involved in the….
Uptake of metals, amino-acid biosynthesis, cell division, the control of plasmid copy number, the lytic cycle of bacteriophages and, other cellular processes in bacteria.
Why is p53 TF unique?
P53 TF → no other protein that is similar to p53 → a lot of effort to put the central helix into the major groove
Complex structure that is prone to mutation for a simple helix contact
What does the attached image, derived from a paper published about p53, show us?
Paper → mapping frequency of mutations in DNA binding domains + table showing frequency of each specific mutation in tumours
High frequency of arginine mutations (DNA contacts)
Two types of mutations found:
- Structural → not direct effecting the DNA binding domain
- DNA-contact → direct mutation on DNA binding
Does DNA binding of TFs in pairs (2 or more) influence the sequence specificity of each respective TF?
Yes, binding of transcription factor pairs alters their binding specificity
Binding of two TF next two each other can change specificity → combinatorial effect
Building a sequence logo for binding independently and together we can observe some differences
It is possible that binding alters the local structure/flexibility of DNA which changes the binding recognition of the other TF
Hence, it is important not not ro view the TFs in isolation
Example → ELK1 is more conservative in the duo pair whereas GCM1 more liberal in it’s choices (recognition changes)
Do gene-specific TFs normally make direct contacts with RNAPII or basal factors?
What is the mediator complex?
What is the subunit composition of the mediator complex? Does it exhibit activity in-vitro after purification?
Mediator is a large complex consisting of ~20 different subunits
Purified Mediator has several biochemical activities in vitro:
- it enables transcriptional activation (‘coactivator function’)
- it stimulates the TFIIH-mediated phosphorylation of the RNAP CTD → stimulating the transition from initiation to elongation
What triggers the release of mediator from the RNAPII complex?
Are there other Co-activator complexes that have been identified?
Mediator → one of many examples → TFIID and Mediator probably only act as coactivators for transcription factors bound close to the transcription start site (proximal)
Other transcription factors binding further away use other coactivator complexes, such as CBP/p300, P/CAF, TRAP/DRIP/ARC
A lot of the co-activator complexes have histone acetylation activity → relax chromatin structure → open up DNA to other elements of the TF machinery
Problem → In Higher eukaryotes these complexes appear quite fragile → not stable for biochemical analysis
What are the characteristic properties of activation domains?
Activation domain in solution is unstructured but the coactivator subunits are ordered protein surfaces →
Initial association between AD and CA is mainly driven by electrostatic complementarity → results in somewhat chaotic/disorganized binding
Slower step → we obtain a more ordered structure formed driven by hydrophobic interactions
Why is this useful? Allows activation domains to interact interact with a variety of co-activators → flexibility + promiscuity of binding partners
What are some examples of common activation domains?
What message is the following image trying to portray?
Does the transcription factor Myc also exhibit a high degree of disorganisation in it’s activation domain?
Yes, Myc does exhibit a intrinsically disordered protein structure in it’s activation domain.
When observing the DNA sequence, we observe that the first 88 residues contain Myc boxes which are islands containing higher levels of conservation, in particular the bulky amino acid.
When observing the secondary structure, we see a relatively low propensity of secondary structures forming (highest is 40%) → indicating a very transient formation
In eukaryotes, what proteins show the highest level of disorder in their secondary structure?
Looking at the number of Intrinsically disordered proteins in human, yeast and E. Coli for proteins associated with binding, catalysis and transcription
When examining proteins involved in transcription regulation…
E. Coli → low levels of disordered proteins → right skew
Yeast → normal distribution
Human → left -skew distribution
This trend clearly doesn’t not hold up in catalytic proteins → desire higher levels of conservation
What is an example of a well-understood TF factor in yeast, specifically with regards to it’s activation domain?
The transcription factor GCN4 (from yeast cells) contains a central “acidic activation domain” (cAD) that binds to the MED15 (GAL11) subunit of the Mediator Complex.
Co-activator that recognizes the acidic activation domain is Gal11 → which is a subunit of the mediator complex → ABD1, 2 and 3 can bind to the activation domain on Gcn4
What conformational change does the acidic activation domain in GCN4 undergo when interacting with Med15?
Upon addition of GAL11 we can visualize (NMR) the transition towards a more organised structure → alpha helical domain forms
How is the GAL11-ABD1 domain structured?
The GAL11-ABD1 domain is made up of four a-helices linked by short flexible connections
There is a distinct cleft containing a “hydrophobic floor” made up of various amino acids with extensive hydrophobic side chains
(V=valine; Y=tyrosine; W=tryptophan; M=methionine; A=alanine; T=threonine)
The edges of the cleft are mostly positively charged
How does the GCN4 complex interact with Gal-11?
The initially unstructured GCN4-cAD, when combined with GAL11-ABD1, takes up a polymorphic series of a-helical structures within the GAL11-ABD1 hydrophobic groove
The authors refer to this phenomenon as a “fuzzy” interaction, because it cannot be uniquely defined other than as a family of related structures that interconvert over time
Unlike other molecular interactions, this mode of binding combines order with a degree of chaos
What residues are conserved in the GCN4 activation domain?
What experiment was performed in order to highlight the importance of hydrophobic residues in the activation domain of GCN4?
Examine importance of residues using mutagenesis studies – Examined how mutations in the AD influenced ARG3 expression
Table
Top – primary amino acids in AD → Central portion is highlighted
Wild-type → induces a 7-fold induction of ARG3 → indicating activation → in the lanes we have residues that have been substituted and their effect on ARG3 expression
E.g. Alanine → replace by hydrophobic residues → improve ARG3 expression
WT large hydrophobic residues → can NOT improve activity → Look at W, L and F in red → highlighting importance
Graph
Alpha helical propensity à correlates with stimulation propensity à stabilization of alpha helix à improve stimulation potential
What tools can be used to study molecular dynamics, both experimentally and computationally?
Why are molecular dynamic simulations very useful?
What are the principles of molecular dynamics?
Molecular dynamic → fill box with water molecules and ions (Brownian motion) to simulate real physiological conditions using coordinates + predict the forces
Basically, you start with you initial coordinates for our protein(s) of interest and give each atom a specific coordinate in our system → then we examine the forces of the moving molecules and move each atom accordingly → move time forward and repeat process → step-wise calculations of the dynamics of the system
In terms of molecular timescales, why are computational simulations extremely useful?
Molecular timescales - some events are difficult/not captured experimentally → occurring extremely quickly
Hence, it is useful to adopt MD as it allows us to simulate interactions on this smaller timescale
BUT there is an upper limit → longer timescales extremely computationally demanding
What are the computational requirements for running molecular dynamics?
What did molecular dynamics simulations of GCN4 and Gal11 reveal about the relative contribution of specific residues to binding?
Experimentally, how is DNA foot printing used to identify the sequences bound by transcription factors?
- Obtain genomic DNA
- Labelled with a fluorescent/radioactive probe/atom
- Expose some of the DNA to the TF of interest
- Expose the DNA to a restriction enzyme that cleaves DNA independently of the sequence
- Run on gel and visualise
- When comparing the control to the sample exposed to the protein, we should expect to see regions on the gel that lack DNA cleavage products → TF binding protected this region from degradation.
- Given enough data for a specific TF from foot printing analysis (different binding sites) we can align the sequences to gain a consensus.
What are sequence logos?
What does the bit-score in sequence logos tell us?
Scale in bits
Bits – unit of information
2 bits give 4 different combinations that allows us to identify 4 bases
Higher bit value → increased conservation
1 bit → half of the sequences have this specific base in the specified position?
When working with amino acids → we need 4 bits as there are more combinations possible
What does the bit-score in sequence logos tell us?
Scale in bits
Bits – unit of information
2 bits give 4 different combinations that allows us to identify 4 bases
Higher bit value → increased conservation
1 bit → half of the sequences have this specific base in the specified position?
When working with amino acids → we need 4 bits as there are more combinations possible
What is an example of an advanced sequence logo that can be included into the analysis?
What is an example of an ‘alternative’ sequence logo that is also used?
What the the terms ‘input’ and ‘output’ refer in the world of pattern recognition of sequences?
Input: a series of short DNA sequences each containing a common pattern (such as a binding site for a transcription factor)
Output: a ‘weight matrix’ or ‘scoring matrix’ description of the common sequence pattern
What are Position Specific Scoring Matrices (PSSMs) or PWMs?
Basically PSSMs are trained by given a input data of many different sequences which it scans through.
When scanning it counts the frequency of nucleotides at specific positions and converts the overall frequency into log odds in order to normalise the scores (correcting for the ¼ change of having nucleotides there)
This PWM can then be used to scan through sequences of interest and if a high score is produced (matching TF consensus binding site), this indicates the possibility of a potential binding site.
Example of using TF clustering to eliminate false positive results in in-silico TF binding site predictions?
Under what condition is using phylogenetic tools to identify TF binding sites most powerful?
Phylogenetic tools are likely to be most powerful if closely related (‘sensu strictu’) species are used.
Why? Go to far down the evolutionary line the sequences diverge too much → harder to pick up a signal
Example - S. Cerevisiae and S. Pombe are commonly used in labs but are quite distant in terms of evolutionary lineage → Useful to use more closely related species
What are Polyglutamine diseases?
At least 8 fatal neurodegenerative diseases are due to expanded polyglutamine tracts present in some proteins
All these diseases result in progressive loss of motor and cognitive functions due to neuronal degeneration in the central nervous system
Outline what happens in the polyglutamine exranpsion disease, Kennedy’s Disease?
Normally the androgen receptor (male hormone receptor) binds to testosterone, resulting in hormone translocation into the nucleus à where it acts as a gene specific TF
There is normally a polyglutamine stretch in the healthy copy of the gene but in Kennedy’s disease we get excessive expansion → 2/3x as long
What’s the consequence of this?
Protein’s aggregate together to form inclusion bodies, resulting in a neurotoxic phenotype.
What are the Cellular and Molecular Effects of polyglutamine expansion i.e. why is it pathological?
How does a protein with polyglutamine expansion create inclusions? What are the toxic effects of having high levels of misfolded/aggregated proteins?
PolyQ → misfolded fragments form oligomers, which associates together to form inclusion bodies
Various toxic effects
- Transcriptional alteration
- Metabolic dysfunction
- Proteasome impairment
- Stress response abnormalities
Note - Toxic effects are most apparent in neuronal cells
What is Huntington’s disease? What pathology do we observe in Huntington’s Disease?
Huntington’s disease (HD) is an inherited disorder that causes neurons to die in various areas of the brain → Caused by DNA mutation leading to CAG (glutamine) expansion
Post-mortem brain from a patient with Huntingdon’s Disease showing massive atrophy of the striatum and corresponding expansion of the lateral vesicles
How have Yeast models been useful to study the polyglutamine phenotype?
Yeast models have been a useful tool in understanding the influence of PolyQ expansion
Proteins containing 23 or 75 glutamine would be expressed in cytoplasm or nucleus (Either with or without nuclear localization sequence)
Subsequently, gene expression profiles were determined using DNA microarrays. The mutant expression profiles would be compared with the WT.
Results
Key - Dark color (decreased expression) / White/pale color (expression not significantly effected)
23 + 75 → cytoplasm → doing something but not a lot
N23 + N75 → nuclear localisation → significant downregulation in gene expression → specifically effecting genes involved in transcriptional and replicative processes → results match SAGA complex (HAT) mutants which is required to keep the chromatin structure open.
Note - Induced genes mimic a mild heat-shock response → normally happens when cells are stressed
What important protein does the polyglutamine-containing domain of huntingtin bind to? (hint- linked to transcriptional regulation)
What is an important link between Polyglutamine expansion and HATs? What consequences is this having for transcriptional regulation?
Polyglutamine-containing aggregates bind histone acetyltransferases (HATs) and inactivate them
HATs are responsible for the acetylation of histones (Lysine’s on histone tails) and basal transcription factors
The altered balance between protein acetylation and deacetylation may be a key factor underlying expanded polyglutamine-induced pathogenesis
The restoration of this balance may be possible by the genetic or pharmacological reduction of the opposing enzyme group, i.e. the histone deacetylases (HDACs) → image highlights potential HDAC inhibitors
Is there evidence to suggest that HDAC inhibitors may be effective at rescuing cells expressing polyglutamine expanded peptides?
Experiments conducted in Drosophila Eye (contains neurons)
Photoneurons shown in image - normally 7 neurons in each group
Q48 → regular neuronal patterns becomes distorted – cytotoxicity
Q48 + butyrate and SAHA → normal pattern somewhat regained
What is one practical problem with using HDAC inhibitors to tackle polyglutamine diseases?
Wide variety of HDACs (classes) present in humans → various different inhibitors → not one size fits all
How would you summarise the research on polyglutamine diseases so far?
Outline the basic mechanism by which the oestrogen receptor modulates gene expression.
- Estrogen receptors is located in the cytoplasm
- Estrogen diffuses across cell membrane (hydrophobic) → binds to the receptor
- Proteins covering (heat shock and cyclophilins) the NLS signal are displaced
- NLS drives movement of ER into the nucleus
- ER receptor undergoes PTMs (Phosphorylation) & dimerization
- ER binds to target sites in the genome → acting as a transcriptional activator
What are estrogen’s biological effects?
Outline the structure of the estrogen receptor
The estrogen receptor consists of 2 activation domains, hormone binding domain, DNA binding & dimerisation domain and the NLS signal
What are the four members of the estrogen family? Which member is most predominant?
What is tamoxifen? (Estrogen receptor)
Why can tamoxifen help prevent/delay breaks cancer occurrence?
How exactly does tamoxifen binding and interact with the ER?
Basically…
When estrogen binds it gets buried inside the estrogen receptor → placing the activation loop (‘domain’) in the active state
However, when tamoxifen binds → the signaling loop gets placed in the inactive conformation → no longer functionally active
How does tamoxifen specifically influence the two activation loops/domains in the ER?
Inhibits expression of TGF-a expression → AF-2 is no longer active → limiting cell growth & proliferation
Stimulatory effect on TGF-B3 expression → AF-1 can still bind and function as an activation domain → positive effect on bone maintenance
Changing equilibrium of Activation domains → changing TF activity
What are some problems associated with tamoxifen?
What can we do to identify patients that are suitable for anti-estrogen treatment using tamoxifen?
Tamoxifen resistance arises due to mutations in ER + loss ER expression → Hence, since the effects of tamoxifen are primarily mediated through the ER, and the degree of ER expression is a strong predictor of responses to tamoxifen, loss of ER expression (or mutations in ER) result in resistance to therapy
Hence, measurement of ER levels in breast cancer tissues (common used in practise) or sequencing for mutations in the ligand binding domain (common mutation) can indicate whether tamoxifen treatment is applicable
Highlights the importance of real-time measurements & sequencing → shed light on resistance + guide therapies (personalised approach)
What have recent advances in transcriptomic and genomic sequencing allowed us to do for human disease detection?
Large-scale integration of genetic variation data, extensive annotation of functional genomic elements, along with the ability to measure global transcription, allows the impacts of genetic variants on gene expression to be detected
Genomic variation includes…
- Single-nucleotide
- Multinucleotide variants
- Short insertions or deletions (indels)
- Larger copy number variants
- Similarly sized copy neutral inversions and translocations
But importantly there is also an abundance of cis-regulatory variation in the human genome. Regulatory variation may be the key primary effect contributing to phenotypic variation in humans
What are the two ways in which variation in gene expression can manifest itself?
Variation in gene expression can be manifested in two ways:
- Changes to transcript sequences and isoforms by coding variants
- Changes to transcript abundance by dosage or regulatory variants
What is the Duffy antigen receptor?
What mutation is found in the DARC promoter region blocks DARC expression?
What is the Hemophilia B Leyden disorder?
Factor IX (F9) is an essential serine protease that participates in the intrinsic pathway of coagulation.
The enzyme is exclusively expressed in the liver
(Rare) mutations in the X chromosome-linked gene encoding Factor IX results in hemophilia B
In a variant form, Hemophilia B Leyden, affected males have less than 1% of normal Factor IX activity until puberty, but after puberty levels rise to around 60% of normal activity - Why? → during puberty androgen receptor binds helps drive expression
All Leyden patients have point mutations located within 40 bp of the transcription initiation site (promoter region)
Where are factor 9 mutations in haemophilia B Leyden located?
Note - androgen receptor has some overlap with mutant sites but the majority lies outside
What are PAX transcription factors?
PAX factors are a highly conserved family of transcription factors containing a helix-turn-helix DNA-binding domain
‘Paired axial’ (‘Pax’) genes belong to the homeobox gene family of transcription factors → The first gene of this class, paired, was originally discovered in Drosophila where it causes segmentation defect
In higher vertebrates nine members of the PAX family have been isolated which are classified into four groups
The expression of Pax genes is temporally and spatially restricted during development of CNS and various other organs
How many PAX genes are present in the human genome? In what tissues is PAX active?
The human genome contains 9 different PAX gees. Heterozygous loss-of-function mutations in PAX genes are associated with disease phenotype.
PAX influence gene expression in a wide range of tissues/cells across the human body - these cells share a common developmental origin
How could you organise the different developmental pathways influenced by PAX activity?
- Proliferation and differentiation
- Hormonal regulation
- Organogenesis
- Cell adhesion and migration
What are the three PAX genes in humans that are known to be mutated?
Pax genes with common mutations - PAX 2, 3 and 6
What does this tell us about the other genes?
Mutation in the other genes most likely results in lethality
What is Waardenburg syndrome?
Not a serious condition → differences in pigmentation
What mutations result in Waardenburg syndrome?
Mutations in the DNA binding domain (Paired domain)
Paired domain is basically two H-T-H domains that are linked together by a flexible linker
What do mutations in PAX6 cause?
Not a serious condition → differences in pigmentation
Where is PAX6 expressed in the eye development?
Outline the organisation of the Pax6 gene?
Does Pax6 have a large & complex interaction network?
Yeahhhhh BUDDDYYYY
Where are the deleterious mutations located in the Pax6 gene?
What is an example of a functional assay that can be carried out to study WT and mutant forms of Pax6 (or any other TF really)?
Transfection of Plasmids
- One encoding Pax6 gene (or TF of interest)
- One encoding a reporter gene with the Pax6 binding site
- Examine expression levels of reporter gene (Luciferase or GFP)
Old example - Using Chloramphenicol acetyltransferase (CAT) - assay with thin-layer chromatography → more CAT reporter expression more acetylation of Chloramphenicol which appears as a separate dot during chromatography
What type of Pax6 mutation leads to Aniridia (loss of iris)?
What do homozygous Pax6 mutations result in?
