Gene Expression Flashcards
What is a transcription factor?
Any biochemical factor involved in achieving appropriate transcription levels
What are gene-specific transcription factors?
Transcription factors that bind to specific gene.
Which TFs are there and what do they do?
1) Basal TFs: required for level of basal transcription from naked DNA template
2) General TFs: required for transcription of every protein-coding gene in vivo
3) Co-factors: required to achieve appropriate regulation but not in themselves required for specificity.
4) Gene-specific TFs: proteins that regulate transcription through direct binding of specific sequences.
What is the preinitiation complex and what does it consist of?
Complex of TFs to start transcription. There is stepwise assembly of PIC on core promoter.
Consists of at least RNA Pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH.
What is TFFID?
TFFID = TBP + TAFS TBP = TATA binding protein TAFS = TBP associated factors
What is DNA looping?
The formation of a DNA loop when a protein of complex of proteins simultaneously binds to two different sites on DNA.
DNA loop can be responsible for transcriptional repression or activation.
For example, activation by causing that enhancer comes closer to the promoter.
What is the general build-up of GSTFs?
1) DNA binding domain (DBD)
Generally bind to major groove and recognizes 4-8bp motifs through large number of individually weak intermolecular interactions.
2) Activation domain (AD) (Also called ligand binding domain (LBD) )
Binding site for other proteins. Possible two have more than 1 AD in 1 TF.
3) Dimerisation domain
Domain to form dimer with other TF
What happens when TF binds to other TF through dimerisation domain?
1) Increase affinity
2) Increased specificity
Partner can for example bind to minor groove to increase specificity (example: CTE)
3) Combinatorial control
4) Negative regulation (inactive partner)
Why do GSTFs bind mostly in the major groove?
1) More space
2) More specific chemical groups that allow better distinction between different base-pairs
What are position weight matrices?
Representation of the “frequency distribution” of bases at different positions.
GSTF can bin can bind at DNA that is closely related to the most preferred DNA sequence.
What determines the structure of the ligand binding domain and how can this influence the transcription?
- Ligand binding determines the LBD structure
1) If a ligand agonist binds then the conformation changes in such a way that it creates new places for protein-protein interactions and attracts co-factors. –> favours transcription.
2) If a ligand antagonist binds then the conformation changes in such a way that there is a poor protein-protein interaction or that interactions are even blocked. –> unfavorable for transcription.
What is a reporter assay and how does it work?
Test that investigates whether a protein can activate or repress expression of the gene using a reporter protein (example Luciferase).
1) Take piece of DNA with promoter region
2) Clone that part of DNA
3) Hook promoter region to reporter
4) Transfect cells with plasmid
3) Production of reporter protein shows whether protein activates or represses expression.
What is the limitation of the reporter assay?
Does not represent chromatin very well.
What is squelching?
Liganded transcriptional activator (for example neuronal receptors) can inhibit the expression of another gene by inhibit the activity of another liganded transcription activator. Probably since it the interaction between these activators disrupt the biochemical pathways.
How does a coregulator/cofactor work?
1) Cofactors is a non-DNA binding protein
2) Interacts with both gene specific and basal TFs (“bridging”)
3) Interacts ligand-dependently
4) May harbour enzymatic activity
Can be coactivator or corepressor.
Example: Coactivator of nuclear receptor interacts with TFs through short alpha-helical motifs
Explain the activation of the TFs Nuclear receptors.
When NR TF is inactive: 1) Histone deacethylation 2) Corepressor NCoR/SMRT + HDAC is bound to NR When NR TF becomes active: 3) Ligand binds to NR --> folding of NR changes and NCoR/SMRT falls off 4) Coactivator SRC1+CBP/p300 binds NR 5) Histone acetylation --> gene opens up 6) Activation
What does the nucleosome consist of and how does it look like?
Octamer of H2A, H2B, H3 and H4
DNA is wrapped around it twice
N-terminal tails protrude away from core particle
Why can histone exchange be useful?
(1) Variant of histone can have an altered function.
H2AZ: stabilizes octamer, but prevents oligomerization which leads to less condensation so more ‘chromatin transcription’
(2) Alternative to de-modification
Getting rid of modified tails can be done enzymatically but also by exchanging a histone.
What is a poised enhancer/gene?
An enhancer or gene that is in between active and repressed genes.
Name an example of one modification that activates and one the represses.
Activates: H3K27ac
Represses: H3K9me2/3
What is a writer and name an example of a writer
An enzyme that puts a modification on a histone tail.
Example: HAT
Often a writer can also be a reader
What is a eraser and name an example of a eraser
An enzyme that removes a modification of a histone tail. –> Eraser is often a complex of 2 enzumes
Example: HDAC
How does acetylation activates gene expression?
Lysine in the histone tail has a positive charge. This causes the tail to ‘stick’ to the DNA. If the lysine is acetylated then the positive charge will be neutralized causing the histone tail to stick out more and do not bind as tight to the DNA. This loosens up the wrapping of DNA around the nucleosomes.
What are readers?
Proteins that bind to the acetyl or methyl groups on the histone tails. Each protein needs a specific pattern of acetyl and methyl groups this forms kind of a barcode for the binding of proteins.
A reader can activate or repress other modifications.
Often a reader can also be a writer
On which group in the AA chain binds acetyl and on which group binds methyl?
Acetyl: only lysine
Methyl: lysine or arginine
How can methyl groups activate or repress gene expression?
The methyl groups attract certain proteins (readers) which can either activate or repress gene expression.
What is the histone code and what does it allow for?
The histone code is the code on histone tails made up from proteins.
Allows for:
- Sequential recruitment of epigenetic/chromatin factors.
- Differential recruitment (mono/di/trimethylation)
- Mutually exclusive recruitment (Kme/Kac)
What are bivalent histone modifications and what are their functions?
Bivalent histone modifications are histone modifications that are in close proximity from each other. The function is to allow a quick response since the cell is already ‘half way’ for activation and ‘half way’ for repression.
What is facultative heterochromatin?
Chromatin that is highly dynamic and flexible. –> can switch between active and inactive easily.
What are three important epigenetic mechanisms?
1) Histone variants
2) Histone modifications
3) DNA methylation
What can histone modifications be used for?
To identify genes, promoters or enhancers
What is illumina sequencing and explain how it works.
Illumina sequencing is a technique used to determine the series of base pairs in DNA.
1) Library preparation
Add adaptors to the end of DNA fragments
2) Cluster generation
- Adaptor modified DNA strand hybridized to oligonucleotide anchor
- Cluster generated by bridge amplification
- Denature and cleave
- Sequencing of forward strand
3) Sequencing
- Fluorescence marker on nucleotide
- Nucleotides are bound to DNA
- Fluor is cleaved and terminator is removed
- Elongation with new fluor nucleotide
–> Gives fluorescent signal for each nucleotide
4) Data analysis
Reads are generated that represent the fragments
What is RNAseq and what does it give you?
RNAseq measures the levels of mRNA (by measuring transcriptome) and gives a feeling of activity of expression and stability of mRNA.
It does not give the introns since these are spliced right after transcription.
In RNAseq you measure consequence of ongoing transcription + half-life of mRNA (=stability of mRNA)
What is GROseq and how does it work?
Global run-on sequencing measures the nascent RNA.
1) Isolate nuclei to get rid of mRNA in cytoplasm
2) Incubate nuclei with brUTP in presence of sarkosyl (prevent attachment of RNA Pol to DNA - only transcription that is ongoing at that moment)
–> This will cause that new transcripts are labeled with BrU.
3) Capture labeled transcripts with anti-BRU antibody labeled beads
4) Convert transcripts to cDNA
GROseq measures ongoing transcription
What are DHS regions and how can this be mapped?
DNAse I hypersensitive sites are regions of chromatin that are very sensitive to cleavage of the DNAse I enzyme.
Mapping of accessible chromatin in genome:
1) DNAse I makes a lot of dsb where DNA becomes accessible
2) Purify open fragments created by DNAse I
3) Fragments start to accumulate at positions where DNA was hyper accessible –> map this to reference genome
4) Do Illumina sequencing
What is ATAC-seq and how does it work?
Assay for Transposase-Accessible Chromatin
1) Hypersensitive transposase enzyme will fragment accessible chromatin and simultaneously tags the accessible DNA with adaptor sequences.
2) DNA with adaptor is sequenced.
What are the methods the map accessible chromatin in the genome?
DNAse I hypersensitive site mapping and ATAC-seq
What are the methods for sequencing the genome?
Illumina sequencing
