Transcriptional regulatory networks 1 Flashcards
What four things make up a simple eukaryotic transcriptional unit?
- Transcription factor
- DNA motif
- Preinitiation complex assembly (contains RNA pol II, general transcription factors, mediator)
- Gene
Transcription factors are composed of what two things?
- DNA-binding domain
- Activation domain
Function of DNA-binding domain on TF
Binds a short nucleotide sequence (motif) up to 1000 bp upstream of start codon (zinc fingers, helix-turn-helix, basic domains)
What does the binding specificity of the DNA-binding domain (DBD) depend on?
Depends on amino acid sequence and structure of DBD, multimerization (homodimers/heterodimers)
Function of TF activation domain
Recruits RNA polymerase to promoter region
- very specific in activation
What two additional structures may be found in complex metazoan transcriptional control modules?
- Multiple enhancer and silencer elements containing clustered DNA-binding sites for transcription factors
- Elements can be located 10-50 kb upstream or downstream of the promoter
- Transcription factor binding can result in looping of DNA to bring regulators in proximity (so enhancer can still regulate transcription initiation even if they’re many bps away)
What 4 things make up the structural organization of transcriptional-regulatory networks? Go from simplest to most complex
- Basic unit (single TF binds single target gene/binding site)
- Motifs
- Modules
- Transcriptional regulatory network
Single-input motif (SIM)?
- Draw it
One transcription factor binds multiple different target genes
Multiple-input motif (MIM)?
- Draw it
Two transcription factors bind their target genes but can also swap target gene binding
Feed-forward loop (FFL) motif?
- Draw it
TF can activate target gene either by indirectly activating another TF that binds the target gene or by directly binding to the target gene.
Describe how transcriptional-regulatory networks can be organized into modules
One module = one tissue
- TFs can cross into other tissues to activate different target genes, depending on how close the tissues are
True or false: transcriptional regulatory networks are constant
False
- They show the network at one time point
- There is a gradual change in phenotype over time due to gene regulation
What is the goal with transcription factor networks? What is needed for this?
Goal is to identify interactions between transcription factors and motifs of their gene targets so that we can assemble the network
- Need high-throughput reliable methods to accomplish this (thousands of interactions in network)
What establishes a gene expression microarray profile?
Profiles are established by which transcription factors are turned on
Why are there more genes than transcription factors?
Many transcription factors act as “master regulators”
What does elucidation of transcriptional events involved in specifying a tissue type during development allow for?
Undoubtedly increases our understanding of developmental biology and the underlying basis behind many diseases
What are the 3 main characteristics of target genes regulated by a given TF?
* on the final
- There must be a common DNA motif on the promoter
- All of the target genes have to be co-expressed/regulated
- The TFs must have a common function
True or false: most TF targets have been characterized
False
- Only some targets identified for 50% of yeast and <10% of mammal TFs
What are 4 obstacles involved in identifying direct transcriptional targets?
- Noise from microarray data (if the cell is not happy, lots of genes will be upregulated/downregulated unrelated to the TF
- Motif detection: few nucleotides long and presence of degeneracy
- Intergenic regions containing motifs are very large: like looking for a needle in a haystack
- Complexity of circuit (feedback/feed-forward loops)
Describe the hypothetical way to conduct microarray profiling of transcription factor mutants and the expected results
- Extract RNA from wildtype TF expression, reverse transcribe and dye the cDNA green (Cy3)
- Extract RNA from TF mutant and dye with Cy5 (red)
- Combine WT and mutant RNAs on microarray
Expected result: Should see just green on microarray
What is a major obstacle of microarray profiling of transcription factor mutants and what are the actual results of microarray profiling of transcription factor mutants?
What must be done before microarray profiling to avoid this?
The conditions that activate most transcription factors are unknown and therefore expression profiling mutant TF strains in rich media yields little information on the targets
- No change seen between wildtype and the mutant expression in microarrays (yellow)
- Transcription factors must be activated prior to microarray profiling- chemical activation, construction of artificial transcriptional activators, gain of function alleles and overexpression of transcription factors
Since microarray profiling of transcription factors is not practical for deletion mutants, what can be done instead?
Create a TF overexpression mutant and combine the dyed RNA from the overexpression mutant with the dyed RNA from the wildtype. Should see more dyed RNA from OE mutant
Describe how overexpression mutants can be created using the GAL1 promoter
GAL1 promoter is off when glucose is present in media (no transcription), but on when galactose is present (high transcription rate)
- Place TF gene in front of GAL1 promoter in vector and place cell in high galactose media to get TF overexpression
What is a yeast TFOE array?
An array of 175 yeast strains, each overexpressing a unique gene
