Eukaryotic Transcription Factors

Question 1

What is the default state of transcription from a gene?

Answer 1

“OFF”

Question 2

Which RNA pol is signalled directly to initiate at its promoter?

Answer 2

RNA pol III

Question 3

How is RNA pol II signalled to initiate at its promoter?

Answer 3

Short gene regulatory sequence motifs that occur in proximity to RNA pol II transcription units direct gene regulation

Question 4

What is the cluster of gene-regulatory motifs found close to the promoter in higher eukaryotic protein-coding genes called?

Answer 4

The Upstream Promoter Element (UPE)

Question 5

What is the role of a transcription factor?

Answer 5

To sense signals from inside or outside the cell and communicate with RNA pol II to influence the initiation or elongation of transcription

Question 6

What are the two types of transcription factors?

Answer 6

Activators and repressors

Question 7

Describe the structure of a eukaryotic transcription factor.

Answer 7

Modular: containing a DNA-binding domain and an effector domain

Question 8

How are TFs classified into families?

Answer 8

TFs are classified into families according to the structure of their DNA-binding domains

Question 9

What is the most common family of TFs in mammals?

Answer 9

Zinc-finger DNA-binding domains (they themselves are modular with combinations of fingers decoding different short nucleotide sequences)

Question 10

What are the three different functions of effector domains?

Answer 10

1) Transduce signals
2) Mediate protein-protein interactions with other factors
3) Enzyme activity

Question 11

How is the gene metallothionein regulated?

Answer 11

The MT UPE drives transcriptional activation of the gene when toxic metal ions are present in the cell and when the cell is stressed (cell produces metallothionein proteins to chelate heavy metal ions such as Cu, Cd, Zn and Hg and prevent toxicity).
USF, AP2 and SP1 are bound constitutively.
GR and Mtf1 bind in response to signals.
The glucocorticoid receptor (GR) is expressed in most cells but normally remains as an inactive monomer in the cytoplasm, stress hormones such as cortisol bind the factor causing it to release from its cytoplasmic parters, dimerise and translocate to the nucleus where it binds its motif and regulates transcription.
The Metal-responsive TF (Mtf1) is a zinc finger TF. The DBD of Mtf1 only fold properly and forms a functional structure in the presence of heavy metal ions.

Question 12

How do we discover and characterise TFs?

Answer 12

1) Biochemistry
2) Genetics
3) Genomics

Question 13

What are the advantages of using yeast as a model eukaryote?

Answer 13

1) Simple propagation via standard microbiological techniques: petri dishes etc.
2) Complete control of genetics: easy mutation, can grow cells as haploid, mating and meiosis too
3) Genetic manipulation techniques: plasmids, easy gene replacement via recombination
4) Functional genomics: easy/cheap because genome is small and simple

Question 14

What has been the most powerful genetic system to explore eukaryotic transcription using in vivo reporters?

Answer 14

Budding yeast

Question 15

How is a yeast transcription factor genetic screen carried out?

Answer 15

1) Make mutations that inactivate the reporter; select mutants according to phenotype on petri dishes
2) Create a gene library from normal yeast (cut the yeast genome into fragments with a restriction nuclease, and ligate into yeast plasmids)
3) Introduce library into mutant yeast and look for complementation (a plasmid containing a normal version of the mutated gene complements the original mutation)
4) Isolate and sequence the plasmid = clone of TF

Question 16

What is a hybrid transcription factor?

Answer 16

The modular nature of TFs means that domains can be swapped between TFs using recombinant DNA methods to create hybrid factors. Since the basic mechanisms of eukaryotic transcription are conserved, human effector domains will often activate transcription in yeast whilst tethered to DNA by a yeast DBD, allowing us to identify TFs in species other than yeast.

Question 17

What are the steps of a yeast one hybrid screen

Answer 17

1) Make in vivo reporter
2) A KO mutation is created in a gene encoding a yeast TF which would normally activate via a motif in the reporter gene
3) Make a one-hybrid plasmid library: human protein-coding DNA fragments are cloned next to the DBD of our TF to create a library of plasmids expressing hybrid fusion proteins - most hybrids will have no ED-like function however ones with a TF effector domain will and these will bind the BAIT motif via the DBD and the prey ED will activate the reporter

Question 18

What is an EMSA?

Answer 18

Electromobility shift assays are a general method for exploring interactions between DNA sequences and DNA binding proteins. They use “native” polyacrylamide gels to separate molecules according to size/molecular weight under physiological-like conditions

Question 19

What is an EMSA probe?

Answer 19

A short fragment of DNA, containing one or more putative binding motifs for a TF or another DNA-binding entity (probe must be labelled with either a radiolabel or a fluorescent dye)

Question 20

What sort of things can you find out using an EMSA?

Answer 20

1) TF:DNA sequence specificity
2) TF:DNA sequence affinity
3) TF:TF competition
4) TF:TF co-operativity

Question 21

If you have a TF but don’t really understand the motif it binds to what can you use?

Answer 21

Systematic evolution of ligands through exponential enrichment (SELEX): uses the EMSA principle to isolate a DNA motif together with the protein it binds

Question 22

Describe the steps of SELEX.

Answer 22

1) Synthesise a DNA probe containing a region of random sequences that is flanked by two know sequences to which we can apply PCR (we are making a probe that is a mixture of all possible DNA sequences)
2) Add DNA-binding protein of interest and a label
3) Separate any probe and TF complex by EMSA, affinity columns or magnetic beads
4) Purify the protein and DNA complex from the gel but cutting it out of the gel
5) PCR the DNA to make more of a new less random/”motif-enriched” probe
6) Add the TF to the new probe mix
7) Repeat 3-6
8) Sequence the enriched probe mix to discover the motif expressed as a sequence logo/position weight matrix

Question 23

What divisions of genomics are needed to understand gene-regulatory motifs at the genomic level?

Answer 23

Basic genomics, comparative genomics and functional genomics

Question 24

What can basic genomics tell us?

Answer 24

If we have characterised a motif and its TF interactions based on reporters, EMSA etc. at one gene we can use genome sequence to spot similar motifs elsewhere

Question 25

What is comparative genomics?

Answer 25

The study of differences and similarities in genome sequence and organisation within and between species

Question 26

What do regions of genome similarity between species imply?

Answer 26

Evolutionary constraint and therefore function

Question 27

What are conserved non-coding elements (CNEs) and what is their significance to development?

Answer 27

Gene regulatory sequences upstream of a gene that are conserved. CNEs change during evolution as alteration in gene regulation drives developmental innovation

Question 28

What are HARs?

Answer 28

Human accelerated regions: CNEs uniquely present or absent in the human genome relative to other primates

Question 29

What technology is described as being at the heart of functional genomics?

Answer 29

NGS

Question 30

Describe the steps of NGS.

Answer 30

1) Isolate cells
2) Make genomic DNA
3) Fragment gDNA prepare adaptor library
4) NGS
5) Assemble the sequences into the correct order to reconstruct the chromosome sequence

Question 31

What are the key TF and gene-regulatory region mapping technologies that drive functional genomics?

Answer 31

ChIP-seq; DNase-seq; ATAC-seq

Question 32

What are the key reagents of ChIP-seq (chromatin immunoprecipitation with sequencing)?

Answer 32

Cloned/purified TF protein and TF-specific antibodies

Question 33

Describe the steps of ChIP-seq.

Answer 33

1) Cross link the DNA and protein within cells with formaldehyde
2) Fragment the cells using ultrasonication to produce fragments of DNA+protein and cell debris
3) Immunoprecipitate TF+genomic motifs
4) Purify DNA
5) NGS and the frequency of reads is plotted versus genomic location - peaks are taken to infer positions of TF binding in a genome

Question 34

What are the advantages of ChIP-seq?

Answer 34

1) The antibody is specific so you get unambiguous identification of specific TF location in genome
2) NGS read counts are focussed in specific locations so only a few million reads required per experiment - cheap

Question 35

What are the disadvantages of ChIP-seq?

Answer 35

1) Positional accuracy is low (+/- 300bp)
2) The generations of high-quality antibody is technically demanding and thus expensive
3) Requires pure TF for antibody generation
4) Only yields data for the one factor under analysis
5) ChIP protocol is technically demanding

Question 36

What is the key reagent involved in DNase-seq?

Answer 36

Sequence non-specific endonuclease (usually DNase) - acts as a molecular probe

Question 37

Describe the steps of DNase-seq?

Answer 37

1) Living cells + detergent + endonuclease leads to DNase digestion of chromosomes in vivo
2) Purify DNA to get fragments of digested DNA
3) NGS to get sequences

DNase-seq allows in vivo nuclease accessibility mapping: the nuclease enzyme enters cells and creates random cuts int he chromosomal DNA unless there are proteins, like TFs, in the way. TF-bound motifs appear as gaps/footprints in DNase-seq accessibility datasets.

Question 38

What are the advantages of DNase-seq?

Answer 38

1) Positional accuracy is high: +/- 1bp
2) DNase is cheap
3) DNase digestion protocol is technically easy and can be applied to many cell types
4) A low level of sequencing (i.e. cheap NGS) can be used to identify active gene-regulatory regions such as enhancers and promoters as these are more accessible to DNase so show up as DNase hypersensitive sites (peaks)

Question 39

What are the disadvantages of DNase-seq?

Answer 39

1) No information on the identity of the TF causing the footprint (no antibody)
2) To spot a footprint requires a high level of background sequencing; 2 reads per genomic bp on average - very expensive NGS

Question 40

How can be obtain all the benefits of DNase-seq but without the cost?

Answer 40

ATAC-seq: assay for transposes accessible chromatin

Question 41

What is the key reagent used in ATAC-seq, what does it do and what is is prepared with?

Answer 41

Transposes: another type of molecular probe that mediates transposition/recombination, prepared so that it is stuck half-way through a recombination reaction with an illumina NGS adaptor DNA molecule

Question 42

What is the function of NGS adaptors?

Answer 42

Illumina NGS requires adaptors that are attached to the ends of input DNA, attach DNA molecules to the illumina sequencer flow cells and support the DNA sequencing reaction

Question 43

Describe the steps of ATAC-seq.

Answer 43

1) Living cells + detergent + transposase leads to transposition within chromosomes in vivo
2) Purify DNA to get fragments of adaptor-tagged DNA
3) NGS

The transpose enzyme enters cells and transposes/recombines illumina NGS adaptors into accessible DNA however the transposase is extremely sensitive to chromatin structure and will only enter open chromatin regions in the genome.

Question 44

What is the difference between DNase-seq and ATAC-seq regarding the number of sequence reads per experiment?

Answer 44

Whilst DNase has a slight preference for open chromatin, the ATAC-seq transposase only works here thus only these regions appear in the data-set meaning fewer NGS reads required per experiment.

Question 45

What are the advantages of ATAC-seq?

Answer 45

1) Positional accuracy is as high as you want: +/-1 bp
2) NGS read counts are focussed to specific locations (open chromatin) - only a few million reads required per experiment = cheap NGS
3) Method creates its own illumina library; sample processing is simpler than DNase-seq
4) Can be applied at single cell level

Question 46

What are the disadvantage of ATAC-seq?

Answer 46

1) No information on the identity of the TF causing the footprint (no antibody)
2) Adaptor-linked transposase = expensive
3) ATAC-seq methodology is mid-way in technical challenge between DNase-seq and ChIP-seq

Question 47

How do bacterial TFs communicate with RNA pol and give an example?

Answer 47

By direct physical interaction e.g. the activator of the E.Coli lac operon CRP-cAMP interacts directly with RNA pol to stabilise its association with the promoter thereby allowing transcription initiation to occur efficiently

Question 48

What were the main components of transcription identified using yeast genetic screens?

Answer 48

1) TFs
2) PIC components
3) Co-activators and co-repressors

Question 49

Name three examples of RNA pol II co-activator complexes.

Answer 49

1) Mediator complex
2) SAGA complex
3) SWI/SNF complex

Question 50

What is the primary function of TF effector domains?

Answer 50

To make protein:protein interactions with co-activators and co-repressors, recruiting them to the gene-regulatory DNA

Question 51

The co-activator mediator functions at almost all eukaryotic RNA pol II genes, what is its primary role?

Answer 51

To stabilise binding of the RNA pol II PIC at the promoter by forming a physical bridge between TF and RNA pol II/PIC. The mediator makes multiple contacts with RNA polymerase II including the Rpb3/Rpb11 subunits and the unphosphorylated form of the Rpb1 CTD - it therefore directly selects the initiating form of RNA pol II.

Question 52

Describe the structure of Mediator.

Answer 52

Mediator has an extended globular structure; it is large enough to encircle RNA pol II and the physical complexity of mediator appears to provide a flexible interface for the interaction of many different TFs.

Question 53

How does mediator promote RNA pol I transcription?

Answer 53

By stimulating the CTD kinase TFIIH.

Question 54

How are subsequent rounds of RNA pol II transcription initiated?

Answer 54

A sub-complex of mediator and some of the GTFs sometimes remain at the promoter after initiation and may function as a scaffold to promote subsequent rounds of RNA pol II transcription.

Question 55

What is SAGA and what does it do?

Answer 55

SAGA is a co-activator that promoters PIC formation. It contains several TAFs normally found in TFIID which may substitute for part of TFIID activity in highly regulated genes.

Question 56

Some co-activator/co-repressor complexes provide direct physical interfaces between TFs and RNA pol II, how else can co-activator/co-repressor complexes function to indirectly modulate RNA pol II activity?

Answer 56

By remodelling chromatin structure.