Transcriptional regulatory networks 2 Flashcards
Chromatin immunoprecipitation
Isolation of DNA sequences bound to proteins such as transcription factors
On a western blot, what level of protein abundance would be detected in WCE/IP from wildtype?
Wildtype levels of protein
On a western blot, what level of protein abundance would be detected in WCE/IP from WT (beads only)?
Can’t detect band because protein isn’t precipitated down
On a western blot, what level of protein abundance would be detected in WCE/IP from amorph?
No band because gene is gone
On a western blot, what level of protein abundance would be detected in WCE/IP from hypomorph?
Less protein (less fluorescence) but band is still present
On a western blot, what level of protein abundance would be detected in WCE/IP from hypermorph?
More fluorescent band (more protein)
On a western blot, what level of protein abundance would be detected in WCE/IP from ubiquitin mutant?
More protein (brighter band) because less degradation
What is the procedure for ChIP-chip?
- Add formaldehyde (crosslinks TF to DNA fragment) and sonicate DNA to ~1 kb
- Create two separate treatments: Control treatment with no antibody/IgG (nonspecific) antibody which allows for DNA regions to be represented equally. Experimental treatment: add specific antibody (e.g. myc antibody in Myc-tagged TF strain) and immunoprecipitate antibody.
- Reverse cross links and purify DNA (detach TF from DNA)
- Add fluorescent dye to control group DNA (Cy3) and immunoprecipitated DNA (Cy5)
- Hybridize equal amounts of Cy3 and Cy5 labelled DNA onto tiling microarray
If the immunoprecipitated DNA in a ChIP-chip is Cy5 and the control DNA is Cy3, what would a red well indicate?
Indicates that promoters in this well are strongly bound by TF
If the immunoprecipitated DNA in a ChIP-chip is Cy5 and the control DNA is Cy3, what would a green well indicate?
Indicates that promoters are not bound by TF
In the ChIP-chip procedure, what type of microarray is used and why?
* on exam
A tiling microarray is used, not an ORF microarray because TF binds to the promoter which is not part of the open reading frame
What is the enrichment ratio in a ChIP-chip?
Ratio of the promoter region detected by the antibody pulldown/IP versus detected in input DNA with no antibody/nonspecific IgG antibody (control)
What is ChIP-Seq?
Combines chromatin immunoprecipitation with next generation sequencing.
- After chromatin immunoprecipitation, isolated DNA binding sites are sequenced using a high-throughput sequencer. Using this data, the binding site is then mapped onto the chromosome
In ChIP-Seq, what do the peaks on the binding site graph indicate?
The peaks on the graph indicate enrichment of promoter region as determined by number of reads detected from the IP DNA versus the input DNA
What are three advantages of ChIP-Seq?
- Resolution: 1 nucleotide versus 30 nucleotides for microarays
- Higher sensitivity: less enriched bound DNA fragments can be detected
- Less amount of ChIP-DNA required: 10-50 nt vs 1-2 ug
What ChIP procedure is typically used to validate ChIP-chip results?
- Describe how
ChIP-qPCR
REverse cross-linked DNA can be used as template DNA for qPCR (using SYBR green) to confirm the TF binds to a specific promoter
- If you pull down the wrong promoter, primers won’t bind
- If the TF binds strongly to a promoter, there will be more template DNA in IP DNA versus input DNA so more PCR amplification (higher enrichment ratio)
In yeast, it was found that (more/less) promoters were bound by one transcription factor, and (more/less) promoters were bound by many transcription factors
More, less
Describe and draw an autoregulation regulatory motif
* on exam
Gene produces its own transcription factor that activates its own transcription (like a cycle)
Describe and draw a multi-component loop regulatory motif
* on exam
Transcription factor 1 binds a gene that produces transcription factor 2. transcription factor 2 binds Gene 1 which produces transcription factor 1 (like a cycle)
Describe and draw a feedforward loop regulatory motif
* on exam
Transcription factor binds a gene and another gene further down the same pathway
Describe and draw a single input regulatory motif
* on exam
Transcription factor binds multiple genes
Describe and draw multi-input regulatory motif
* on exam
Multiple transcription factors bind a group of the same genes
Describe and draw a regulator chain regulatory motif
* on exam
Chain of sequential transcription factor binding
e.g.
Trancription factor A binds gene B. Gene B transcribes Transcription factor B which binds gene C and so on
Describe the topology of the transcriptional-regulatory network in general
The network is highly connected and span several related biological processes
What can ChIP-chip/seq of histones determine? Describe how
* on final
Can determine histone occupancy and histone modification patterns (acetylation and methylation) in the genome
- a more negative enrichment ratio of histones means that the histone is less likely to bind to the chromosome (less protein binding to the DNA, less DNA pulled down during ChIP)
- ChIP enrichment ratio can be plotted against transcription rate of downstream gene
- Shows that highly-transcribed genes have less histone occupancy and vice versa
Why would mitochondrial DNA have a small enrichment ratio for H4-bound DNA?
* on final
Not eukaryotic DNA so it doesn’t have chromosomes (which use histones to pack into the DNA)
Why would rRNA have a small enrichment ratio for H4-bound DNA?
* on final
rRNA is the most abundant RNA in the cell (very highly transcribed), so it lacks histones
What other transcription related protein can ChIP-chip detect and what implications does this have?
RNA polymerase to detect regions of transcription and can potentially find new genes
What other replication-related proteins can ChIP-chip detect and what implications does this have?
DNA replication complexes can be used to detect origins of replication along chromosomes, DNA repair and checkpoint functions
What 4 things make up epigenetics?
Histone modifications (e.g. acetylation, methylation), DNA methylation, chromatin remodelling, microRNAs
How can ChIP-chip/ChIP-Seq help us with epigenetics?
Genome-wide mapping of epigenetic markers and catalyzing enzymes by ChIP-chip and ChIP-Seq have given us better understanding of how global changes in chromatin structure regulate gene expression in biological processes
When acetylation is high, methylation is ____ and vice versa
Low
In general, why are direct target genes of transcription factors difficult to identify?
Due to false positive rate from functional genomic approaches.
Why is transcriptome data (transcriptome microarrays) not always fully reliable on its own? Give 3 reasons
- Large amount of secondary gene responses (not direct target genes) because of toxicity from phenotypic activation
- Certain network motifs (e.g. feedforward loops) complicate identification
- Transcriptome data is noisy
Why is ChIP-chip data not always fully reliable on its own? Give 3 reasons
- ChIP-chip bound sites may not be active and affect gene regulation
- Promiscuous binding of transcription factor (TF binds targets that it normally doesn’t bind)
- ChIP-chip/ChIP-Seq data is noisy
Since transcriptome and ChIP-chip data is usually unreliable on its own, what may be done to better identify direct target genes of transcription factors?
Combining transcriptome, ChIP-chip and DNA motif analyses better identify direct targets
- Can also include genetic data (perturbation of TF and target genes can produce similar phenotypes)
What is a yeast one hybrid assay used to determine?
* On exam
Used to determine what transcription factors bind to the promoter of interest
Yeast one hybrid is said to be ____-centered
* on exam
Gene
ChIP-chip and overexpression techniques are _____-centered
* on exam
Protein
What are the 4 steps of yeast one hybrid?
* on exam
- Create TF library /ORFeome of TF fused to transcriptional activation domain (ADs usually from virus because they’re so powerful, TFs can be from any organism)
- Transform vectors into bacteria with promoter (DNA bait) of a reporter gene present (HIS3/LacZ)
- Phenotype before TF binding: His- phenotype, white colonies - If transcription factor binds to promoter, report gene is activated
- His+ phenotype
- Blue colonies - Select for transformants, sequence plasmids to determine identity of transcription factors
Why do we use 2 reporters in a yeast one hybrid assay?
* on exam
Often have false positives, so this gives us more confidence in our results
Why is an exogenous AD required for yeast one hybrid assay?
* on exam
AD ensures that reporter is fired up because some TFs can be repressors
- TF has its own AD as well but strong AD from virus ensures that reporter gene is being activated upon binding
The number of target genes regulated by each transcription factor follows a ____ relationship
Power-law relationship
- Many transcription factors only regulate a few genes while few transcription factors regulate many genes
- Get straight line of data if you log transform both axes
Which type of transcription factor is likely to be more important? One that regulates lots of promoters or one that regulates few promters?
One that regulates a lot of promoter because this would increase the risk of essential genes being mis-regulated which is more dangerous for the cell overall
Why is a transcription factor hub?
Hubs are global transcription regulators (many target genes) that are likely to be essential genes
What happens if you knock out a hub transcription factor?
If you knockout a hub, there are likely essential genes being misregulated
The number of transcription factors regulating a target gene (combinatorial regulation) follows a ____ relationship
- Explain why
Exponential decay relationship
- Most genes are regulated by a similar number of transcription factors (1-4)
- Due to protein and DNA contraints at the promoters thereby limiting the number of possible transcription factors to regulate the gene simultaneously
Global regulators are more likely to be (essential/nonessential) genes, while master regulators are more likely to be (essential/nonessential genes), while specifiers are more likely to be (essential/nonessential genes)
Essential, both, nonessential
Design an experiment to determine if a TF is an activator or repressor
* on exam
Can first check if TF and gene target are localized within the same tissue:
1. Fuse TF promoter with GFP and gene target promoter with GFP.
2. See if fluorescence is present within the same tissue
3. Can also see if knockdown of TF results in any abnormalities within the tissue
Check if TF is activator or repressor:
1. Fuse promoter of gene target with GFP
2. Knockdown transcription factor and look for GFP fluorescence of target gene promoter in response
- If knockdown results in less fluorescence -> TF is activator
- if knockdown results in more fluorescence -> TF is repressor
3. Can then do a motif search to see if the TF motif is similar to any other known motifs in the suspected tissue of interest
What are three experiments you can do to validate a TF motif?
- Yeast one hybrid assay
- See if TF-AD hybrid can bind and activate specific motif attached to reporter gene - Knockdown of TF, and see if similar known promoter element fused to GFP has increased or decreased fluorescence
- Knockdown TF, and see if there’s any abnormalities in the tissue where known motif similar to TF motif is present
