Transcription factors and target genes Flashcards
What distinguishes a transcription factor from a transcription regulator?
A transcription factor (TF) is a DNA-binding protein that contributes to the expression regulation of a gene.
A transcription regulator (TR) is a protein that also contributes to the expression regulation of a gene, but normally does not bind directly to DNA. TRs interact with TFs to regulate gene expression.
Transcription factors as regulators
- TF is a protein which is produced in Cytoplasma and migrate to nucleus when needed
- Activation of TF through endogenous or exogenous signals
- activated TF enters Nucleus and binds to specific DNA sequence
- A group of TFs (TF2) directs polymerase 2 to the start site. Cis element and TF are the key to eukaryotic gene expression.
How to find target genes of a transcription factor?
Finding direct target genes of TFs is (often) difficult and requires a combination of methods to be successful.
- coexpression analysis
- TF expression coupled to transcriptome analysis (Transient ox in cells, constitutive ox in cells, inducible ox in transgenic organism)
- reading paper: similar phenotype
- ChIP-seq
- DAP-seq
- By yeast one-hybrid (Y1H) analysis
coexpression analysis - what are potential problems/limitations?
Idea:
Genes with similar expression patterns might be regulated by the same TF
–> Comparison of many expression profiles obtained by RNA-Seq and micro-arrays
Potential Problem:
* Not all similarly expressed genes are regulated by the same upstream TF
* Typically a gene gets turned on by a signal after TF that controls it.
Use a Transcriptome and then Co-Expression analysis
Transient overexpression of a transcription factor in cells (e.g., plant protoplasts or animal cells)
- Pure DNA (Promotor and TF-gene) are introduced into cells, after incubation RNA is isolated and the expression analyzed.
Potential problems:
* high quality, concentrated DNA needed
* Isolated cells not in natural environment (may lead to artefacts)
Advantage:
* fast (no transgenic organisms needed)
* can be done with different TFs parallel (microtiter plate)
Constitutive overexpression of a transcription factor in a transgenic organism, followed by global transcriptome analysis (e.g., via RNA-seq)
Keeping the activity of a transcription factor consistently high in a genetically modified organism is called constitutive overexpression. This is done by placing the gene under a strong, constant promoter.
- Transgenic Organism (organism is genetical modified to express teh additional transcription factor)
- Global Transcription alanalysis (RNA-seq) High-throughput sequencing of RNA to assess gene expression changes.
Method:
* extracting RNA,
* converting it to cDNA
* sequencing to quantify and analyze RNA transcripts.
* Identifies differentially expressed genes (DEGs) between the transgenic and control organisms.
Purpose: To understand how sustained elevation of the transcription factor affects the global gene expression profile of the organism.
Problem: many genes may be far
downstream of the TF (same problem for knock-out lines)
ChIP-seq
Chromatin immunoprecipitation
–> investigating DNA-Protein interactions
If combined with NGS you can determine the exact binding site which the protein of interest (TF) is bound to
Principle:
* Fixation: Using formaldehyde to fix proteins (TFs) to DNA
* Generate smalle DNA fragments. Fragmentation to 300-500 bp long Fragments
* Using a specific Anitbdy that target Protein of interest
* Purification: Seperate target DNA-Antibody complex from other ones. Antibody has magnetic beads so you can do it by using a magnet
* Reverse cross-linking: proteins are removed from DNA (DNA need to be isolated fpr NGS)
- Sequencing or PCR
–> ChipSeq: Use NGS to sequence all fragments and find all targets of TF
PCR
–> Chip-qPCR: Using primers for the Gene of interest, to confirm TF binding and binding site (primers to amplify promotor region containing TF binding site vs. others)
Advantage: Experiment can be performed over time (different time-points) –> TF Interactions over time
Problems:
- TF antibody needed (high specificity)
- Not always easily available
–> Developing a transgenic organisms that expresses tagged version of the TF and use a commercially available Antibody against Tag can take long
- Common Tags: myc,HA, GFP (GFP can be seen, however can impact biological function because of size –> can be testes when TF-GFP can rescue WT phenotype in TF-knock down mutant)
EMSA: how does it technically work? Which controls should be included in the
experiment
Electrophoretic mobility shift assay
–> (In Vitro)
Principle:
If the TF binds to the labeled probe (dsDNA fragment from the target gene including the promotor region) the complex will move slower and a band will appear.
Ingredients:
- Native dsDNA Fragment from Target Gene including Promotor labeled with infra-red dye
- TF (expressed in E-Coli and purified or expressed in vitro) native
Controls:
1. Free Probe Control:
–> Demonstrates the mobility of the labeled probe without protein binding.
- Non-specific Competitor Control:
–> Tests binding specificity using excess unlabeled, non-specific DNA or RNA. Specific binding should remain unaffected. - Specific Competitor Control:
–> Confirms specificity by introducing excess unlabeled, specific DNA or RNA that competes for binding. - No-Protein Control:
–> Serves as a negative control, ensuring observed shifted bands result from protein binding and not artifacts, by omitting the protein of interest.
Yeast one-hybrid analysis
Purpose:
Study protein-DNA interactions, specifically transcription factor and target DNA interactions.
Components:
* Bait Protein: Transcription factor fused to a DNA-binding domain.
* Target DNA Sequences: Contain the binding site for the transcription factor, typically upstream of a reporter gene.
Principle:
If the bait protein binds to the target DNA, it activates the reporter gene.
Applications:
* Identify and characterize transcription factors.
* Study binding affinity to different DNA sequences.
* Investigate gene expression regulatory mechanisms.
