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CMMB 461 > Transcriptional regulatory networks 1 > Flashcards

Transcriptional regulatory networks 1 Flashcards

1
Q

What four things make up a simple eukaryotic transcriptional unit?

A
  1. Transcription factor
  2. DNA motif
  3. Preinitiation complex assembly (contains RNA pol II, general transcription factors, mediator)
  4. Gene
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2
Q

Transcription factors are composed of what two things?

A
  1. DNA-binding domain
  2. Activation domain
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3
Q

Function of DNA-binding domain on TF

A

Binds a short nucleotide sequence (motif) up to 1000 bp upstream of start codon (zinc fingers, helix-turn-helix, basic domains)

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4
Q

What does the binding specificity of the DNA-binding domain (DBD) depend on?

A

Depends on amino acid sequence and structure of DBD, multimerization (homodimers/heterodimers)

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5
Q

Function of TF activation domain

A

Recruits RNA polymerase to promoter region
- very specific in activation

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6
Q

What two additional structures may be found in complex metazoan transcriptional control modules?

A
  • 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)
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7
Q

What 4 things make up the structural organization of transcriptional-regulatory networks? Go from simplest to most complex

A
  1. Basic unit (single TF binds single target gene/binding site)
  2. Motifs
  3. Modules
  4. Transcriptional regulatory network
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8
Q

Single-input motif (SIM)?
- Draw it

A

One transcription factor binds multiple different target genes

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9
Q

Multiple-input motif (MIM)?
- Draw it

A

Two transcription factors bind their target genes but can also swap target gene binding

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10
Q

Feed-forward loop (FFL) motif?
- Draw it

A

TF can activate target gene either by indirectly activating another TF that binds the target gene or by directly binding to the target gene.

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11
Q

Describe how transcriptional-regulatory networks can be organized into modules

A

One module = one tissue
- TFs can cross into other tissues to activate different target genes, depending on how close the tissues are

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12
Q

True or false: transcriptional regulatory networks are constant

A

False
- They show the network at one time point
- There is a gradual change in phenotype over time due to gene regulation

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13
Q

What is the goal with transcription factor networks? What is needed for this?

A

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)

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14
Q

What establishes a gene expression microarray profile?

A

Profiles are established by which transcription factors are turned on

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15
Q

Why are there more genes than transcription factors?

A

Many transcription factors act as “master regulators”

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16
Q

What does elucidation of transcriptional events involved in specifying a tissue type during development allow for?

A

Undoubtedly increases our understanding of developmental biology and the underlying basis behind many diseases

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17
Q

What are the 3 main characteristics of target genes regulated by a given TF?
* on the final

A
  1. There must be a common DNA motif on the promoter
  2. All of the target genes have to be co-expressed/regulated
  3. The TFs must have a common function
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18
Q

True or false: most TF targets have been characterized

A

False
- Only some targets identified for 50% of yeast and <10% of mammal TFs

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19
Q

What are 4 obstacles involved in identifying direct transcriptional targets?

A
  1. Noise from microarray data (if the cell is not happy, lots of genes will be upregulated/downregulated unrelated to the TF
  2. Motif detection: few nucleotides long and presence of degeneracy
  3. Intergenic regions containing motifs are very large: like looking for a needle in a haystack
  4. Complexity of circuit (feedback/feed-forward loops)
20
Q

Describe the hypothetical way to conduct microarray profiling of transcription factor mutants and the expected results

A
  • 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
21
Q

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?

A

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

22
Q

Since microarray profiling of transcription factors is not practical for deletion mutants, what can be done instead?

A

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

23
Q

Describe how overexpression mutants can be created using the GAL1 promoter

A

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

24
Q

What is a yeast TFOE array?

A

An array of 175 yeast strains, each overexpressing a unique gene

25
Q

A lot of the time TFs that are overexpressed can be detrimental to growth (result in smaller colonies on TFOE array). What is a hypothesis for this observed toxicity?

A

When transcription factor is overexpressed, promoter occupancy is increased by mass action and direct targets are induced inappropriately (toxicity effect) - phenotypic activation
- Hypothesize that overexpression is enough for TFs to be activated
- Genes need to only be activated at the right place in the right amount at the right time

26
Q

2/3 of transcription factors when overexpressed are not activated. Why? Give 3 potential reasons

A
  1. If the regulator of the TF is absent (e.g. kinases), the TFs aren’t activated
  2. If silencing is very strong, decreased net activation
  3. TFs can form dimers to be activated and bind to the promoter. If other protein isn’t present, TF can’t be activated (i.e. form a dimer)
27
Q

Describe the methodology for microarray profiling of overexpression TF strains (7 steps)

A
  1. Isolate mRNA from empty vector and Gal1-10-TF OE mutant
  2. Reverse transcription and label with Cy3 (green) and Cy5 (red) dyes
  3. Mix cDNA and hybridize on ORF microarray
  4. Microarray profile of TF overexpression (log ratio of OE/empty vector and find genes that are furthest from no change line)
  5. Microarray profile of TF overexpression identifies gene targets but not their promoter motif
  6. MEME finds novel motifs + TF DNA-binding domains
  7. Validate motifs with gel shifts
28
Q

What is a very good indicator that TFs are activated in TF overexpression mutants?

A

Reduced fitness

29
Q

Microarray analysis of ectopically-expressed nontoxic transcription factors did not produce meaningful profiles. What does this indicate?

A

Mass action per se is not sufficient for activation of these transcription factors
- Goes against hypothesis of phenotypic activation from TF overexpression

30
Q

True or false: After OE of a given TF, many of the yeast genes that are upregulated as a result are functionally enriched for the known specific functions of the TF
- What does this tell us?

A

True
- Tells us that these highly enriched genes are likely to be targets of the TF

31
Q

What do TF motif-finding algorithms such as MEME search for?

A

Statistically overrepresented motifs in promoters of target genes.

32
Q

Explain how we validate motifs using gel mobility shifts

A

Mix purified DNA-binding domain of TF with radioactive-labelled oligonucleotide containing the motif for 1 hour in test tube (in vitro) and then run on gel
- If TF binds, a band appears higher up on the gel (heavier) representing the TF/motif complex (band shifts in mobility)
- If probe is unbound, then band appears at bottom of the gel (not as heavy)

33
Q

On a gel mobility shift, why might two bands appear in a lane (one higher up and one further down on the gel)?

A

Might be due to the binding efficiency of this TF
- Some unbound probe remains, but some is also bound

34
Q

Explain how you can validate the TF targets by gene overexpression
- Use HMS1 example

A

If you know the TF function, you can overexpress the gene targets that were found in your study to see if they result in the same phenotype as overexpression of the TF
HMS1 example
- Know that HMS1 is involved in pseudohyphal growth
- Overexpress YPC1 and URA10 whih are hypothesized HMS1 targets
- Results are all pseudohyphal growth -> validates gene target results

35
Q

What is measured by reverse transcription - quantitative PCR (RT-qPCR)? What can it be used to validate?

A

Abundance of mRNA
- Can be used to validate expression of microarray data (by comparing RT-qPCR results to microarray data)

36
Q

Describe the steps of RT-qPCR (4 steps)

A
  1. mRNA is reverse transcribed into cDNA
  2. cDNA is amplified using PCR
  3. SYBR green fluoresces when bound to double-stranded DNA; does not bind to single stranded DNA
  4. Fluorescence intensity is proportional to amount of PCR amplicon/mRNA abundance
37
Q

Totipotent cells and what they’re found in

A

Give rise to an entire animal including all cell types
- Found in zygotes

38
Q

Embryonic stem (ES) cells are…
- Explain what this means

A

Pluripotent
- Able to generate any type of cells IN THE BODY

39
Q

True or false: Organisms always have a pool of stem cells
- Explain why or why not

A

True
- ES cells are developmentally arrested at the pluripotent stage and can be propagated indefinitely in vitro
- Stem cells can differentiate into different tissues to replace damaged or dead mature cells

40
Q

What is the differentiation of ES cells initiated by?

A

Growth factors, transcriptional regulation and stochastic (randomly determined) processes

41
Q

In the past, somatic cells have been dedifferentiated into pluripotent stem cells. What do these observations indicate?

A

Indicate that unfertilized eggs or ES cells contain cytoplasmic factors that confer totipotency or pluripotency to somatic cells.

42
Q

What are the cytoplasmic factors that confer totipotency or pluripotency to somatic cells?

A

Transcription factors

43
Q

How can you find the transcription factors that can reprogram somatic cells into embryonic stem cells (ESCs)?

A
  • Anticipate that these transcription factors will be highly expressed in ESCs
  • Hyperactivation/overexpression of these transcription factors in somatic cells will induce pluripotent genes and inhibit differentiation genes
  • Reprogrammed somatic cells should change the transcriptome that is now similar to ESCs.
44
Q

Describe the study that induced pluripotent stem cells (IPS) in mice and the characterization of the TFs involved in this process

A
  • Initially identified 24 candidate pluripotent transcription factors when overexpressed together in a differentiated cell can cause IPS
  • Specifically looked for resistance to an antibiotic as a reporter gene, under control of a promoter that is only turned on in ES cells
  • After overexpressing various combinations of TFs, narrow down to FOUR TFs that are sufficient to create IPS (mice and humans)
  • Looked at cell number of ES, iPS and MEF (fibroblasts that don’t divide). Saw that ES and iPS numbers increased, but MEF number plateaued due to quiescence.
45
Q

True or false: the transcriptional network is conserved between different cells
- Describe the implications of this

A

False
- The transcriptional network is highly cell type-specific
- Only 5% of all regulatory interactions are common across the 41 different cell types (probably housekeeping genes)
- Need to identify many novel TFs and potential roles in tissue specification

CMMB 461 (10 decks)

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