Regulation of Expression 1 Flashcards
What is the function of the core promoter element?
Allows transcription initiation complex to assemble
What are the steps of transcription initiation?
1) TFIID complex binds to core promoter element
2) TFIIA and TFIIB bind
3) RNApolII and TFIIF bind
4) TFIIE and TFIIH bind
5) DNA strands separate with the use of ATP
6) Transcription bubble forms
7) rNTPs are added to 3’-OH end as RNA elongates
What is the TFIID complex composed of?
TBP (TATA-binding protein) and approx. 13 TAFs (TBP-associated factors)
What does TFIID bind to?
TATA box or acetylated histones
What do TFIIA and TFIIB bind to?
BRE (TFIIB recognition element) sequence and TFIID
Which component of the transcription initiation complex has ATP hydrolase activity?
TFIIH
Why are the genes typically switched “off” in eukaryotes?
Due to the inherent barrier properties of chromatin
What are the two main ways in which the amount of produced RNA is regulated?
By regulating rate of initation, elongation and termination; by regulating RNApolII recycling
At which stage of the transcription cycle is the state of expression for a particular gene determined?
Initiation
What are the 4 main elements that compose core promoter?
1) TATA box
2) BRE (TFIIB recognition element)
3) Initiator
4) Downstream promoter element
What is the basal transcription apparatus composed of?
RNA polymerase II and 6 general TFs (D, A, B, F, E, H)
What is are the two main roles of TAFs?
1) Assist TBP in binding to DNA
2) Recognise TATA-less promoters through modified histone binding
What is the main difference between proximal promoter and enhancer sequences?
Enhancer sequences are location independent, can be both upstream and downstream of the gene at up to 1 Mb away
What is the main similarity between proximal promoter and enhancer sequences?
Both contain mixes of elements required for TF binding, such as TATA box, GC box, CAAT box and octamer motif
What is the function of gene-specific transcription factors?
Recognise a specific consensus sequence in the promoter or enhancer, then recruit basal transcription machinery
What are the 3 ways in which gene-specific transcription factors carry out their function?
1) Directly recruit general transcription factors
2) Recruit co-activators that facilitate promoter opening
3) Facilitate chromatin modifications or conformational changes required for transcription activation
What are the 4 factors that determine tissue-specific differences in TF function?
1) Expression
2) Cooperativity
3) Intracellular localisation
4) Activation
What are the two main techniques for identifying TF binding sites?
Band shift assay (EMSA - electrophoretic mobility shift assay)
ChIP (chromatin immuno-precipitation)
How does band shift assay (EMSA) work?
Reagents: a protein (TF) and a candidate DNA sequence with a radioactive probe
Method: DNA and protein are mixed and loaded on a non-denaturing gel
Readout: if there is interaction between TF and sequence, two bands (or heavier band) will be seen as some of the DNA will be bound to protein and therefore retained on the gel. If there is no interaction (or no protein added as control), only one band will be seen at the size of naked DNA.
How does ChIP work?
Reagents: cells, formaldehyde, antibody against protein of interest, DNA isolation kit
Method: 1) crosslink proteins to DNA using formaldehyde
2) lyse cells, isolate chromatin, sonicate to shear DNA into mononucleosomes
3) add anti-POI antibody (e.g. anti-TF of interest)
4) isolate Ab/chromatin complexes
5) reverse the crosslink and isolate DNA
6) analyse DNA via a) qPCR (for a single DNA locus) or b) high-throughput sequencing
7) analyse sequencing reads against the genome
What 4 questions can ChIP answer?
1) Where is the TF bound?
2) What genes does the TF regulate?
3) What is the relative binding affinity of the TF?
4) What are the partners of the TF?
What is the function of mediator complex?
Connect activators at promoters/enhancers with the basal transcription machinery
Which two proteins control chromatin looping?
Cohesin and CTCF
What is the function of cohesin?
Extrudes loops by moving one DNA fiber along another
What is the function of CTCF?
Determined cohesin anchor points on DNA
What is the function of insulator elements?
Form a complex with cohesin and CTCF that prevent enhancer/promoter binding
How many subunits typically form a mediator complex?
Approx. 30
What are the 2 physical reasons why DNA in eukaryotes is inaccessible?
1) tight looping around chromatin
2) electrostatic interactions with histones
What are the 3 ways in which chromatin can be regulated to modulate DNA access?
1) Posttranslational modification of histones (methylation, acetylation)
2) Nucleosome repositioning, assembly, disassembly
3) Altering nucleosome properties via histone variants
On which region of the histone are PTMs normally found and why this region?
The N-terminal domain as it sticks out
What is the main technique used to map chromatin modifications?
ChIP-seq, using an antibody against modified histone
Which 3 histone modifications are associated with transcription start site?
H3K9ac
H4K16ac
H3K4me3
Which 2 histone modifications are associated with mid-gene?
H3K36me3
H3K79me3
Methylation status of which histone residue can be used to differentiate between promoter and enhancer?
H3K4
What functional domain can bind acetylated lysine?
Bromodomain
What are the two ways in which acetylation promotes transcription activation?
1) by providing an acetylated Lys for activators and coactivators to recognise and bind
2) by neutralising the positive charge on Lys which weakens electrostatic interactions and loosens chromatin structure
What is the function of HATs?
Transfer an acetyl group from acetyl-CoA to a lysine residue
What are the direct and indirect mechanisms of HAT recruitment?
Direct - chromatin recognition; indirect - activator recognition
What is the function of HMTs?
Transfer 1, 2 or 3 methyl groups from S-adenosyl methionine onto Lys or Arg residues
What are the two protein classes can recruit HMTs to promoters?
(Co-)Activators and actively transcribing RNApolII
Example of histone methylation that is associated with active promoters?
H3K4me3
Example of histone methylation that is associated with repressed promoters?
H3K27me3
What are bivalently marked promoters and what genes are they commonly associated with?
Promoters that have both active and repressed markers, making them “poised” to start transcription rapidly. Commonly associated with developmental genes in embryonic stem cells.
Which sequence motif can mediate TATA-independent transcription initiation?
CpG island
What are the two mechanisms by which CpG islands recruit TFIID?
1) H3K4me3 recognition by TAF3
2) acetyl recognition by TAF1
What are the differences between transcription intiation from TATA-containing and TATA-less promoters and which genes are they associated with?
TATA-containing promoters initiate transcription from a defined point. Associated with tissue-specific gene expression.
TATA-less promoters initiate transcription from many start sites. Associated with ubiquitous gene expression.
What is the main technique used to map all DNA regions associated with nucleosomes?
Footprinting assay
How does footprinting assay work?
1a) isolate chromatin from cells OR 1b) assemble genomic DNA and purified histones in vitro
2) hydrolyse the accessible DNA between nucleosomes using micrococcal nuclease
3) digest histones with proteinase
4) purify DNA previously associated with nucleosomes
5) analyse with HT sequencing (or Southern blotting/qPCR if analysing a single locus)
Describe the nucleosome positioning pattern at promoters
5’NFR (nucleosome-free region), flanked by strongly positioned -1 and +1 nucleosomes that typically contain H2AZ histone variant. Nucleosome positioning becomes “fuzzier” towards mid-gene.
How two factors determine nucleosome positioning?
1) intrinsic sequence properties
2) chromatin remodeller activity
What intrinsic sequence factors determine nucleosome positioning?
Poly-A or poly-T tracts resist winding around histones and are commonly found at promoter
Nucleosome-favouring sequence is found at strongly positioned nucleosome region and has a pattern of A/T dinucleotide every 10bp as well as G/C dinucleotide every 10 bp in antiphase to A/T dinucleotides
How are chromatin remodellers identified experimentally?
Through genetic screens, looking for mutants with altered positioning, occupancy or phasing of nucleosome pattern
Name 5 mechanisms of chromatin remodellers
1) nucleosome repositioning
2) nucleosome ejection
3) DNA unwrapping
4) dimer exchange
5) dimer ejection
Name 4 main families of chromatin remodellers
1) SWI/SNF
2) ISWI
3) CHD
4) INO80
What 3 domains are found in chromatin remodellers?
1) ATPase
2) Regulatory
3) Histone-interacting
Do chromatin remodellers act alone or as part of complexes?
Part of large protein complexes that confer additional specificity
What is the composition of yeast SWI/SNF complex?
1x catalytic subunit (SWI2/SNF2) - a DNA-dependent ATPase
12x other subunits that mediate interactions with DNA and protein, conferring specificity
What are the two primary mechanisms of SWI/SNF mediated nucleosome remodelling?
Nucleosome repositioning or ejection
What are the steps of SWI/SNF mediated nucleosome repositioning?
1) SWI/SNF binds and disrupts histone/DNA contacts
2) ATP is hydrolysed to pull extra DNA from one side and form a bulge
3) Once DNA is loosened, the loop is propagated around the nucleosome as it
4a) slides with respect to DNA 4b) is ejected
What are the direct and indirect mechanisms of SWI/SNF complex recruitment?
Direct - via bromodomain-mediated recognition of acetylated chromatin
Indirect - via interaction with activators
What is the primary mechanism of INO80-mediated nucleosome remodelling?
Histone exchange
Is INO80-mediated remodelling activatory or suppressory?
Either, depending on context
Which histone variant is primarily associated with promoters at active genes?
H2AZ
What is the main difference between canonical H2A and non-canonical H2AZ?
H2AZ interacts less strongly with H3-H4 dimer, therefore nucleosomes are less stable and DNA is more accessible
What is required for H2AZ delivery?
Specific histone chaperones like Chz1, NAp1
What is required for H2AZ incorporation and removal?
INO80 family chromatin remodellers
What are two direct and one indirect mechanisms of INO80 recruitment?
Direct histone binding or nucleosome-free region recognition
Indirect recruitment via TF binding
What are the 3 functions of H2AZ?
1) When part of nucleosomes, “primes” promoters for activation
2) May enhance turnover of entire nucleosomes
3) May promote binding of other TFs, e.g. HAT Brd2 in humans
Describe main steps of PHO5 induction via PHO4 in phosphate starvation
1) PHO4 binds at upstream activation sequence 1 - a low-affinity, always available binding site
2) Additional TFs, including SWI/SNF complex, are recruited
3) SWI/SNF complex repositions an adjacent nucleosome, freeing upstream activation sequnce 2 - a high-affinity binding site
4) Additional PHO4 binds and further recruits TFs and SWI/SNF complex in a positive feedback loop
5) Promoter is freed and available for transcription machinery assembly
What is the phosphate-based mechanism by which PHO4/PHO5 pathway is induced?
At high phosphate, PHO4 is phosphorylated and unale to bind UAS1. At low phosphate, PHO4 is dephosphorylated and able to bind UAS1 recruiting additonal TFs and opening UAS2, eventually allowing expression of PHO5. PHO5 scavenges phosphate, increasing intracellular phosphate concentration until PHO4 becomes phosphoryalted and repressed again.
Are TATA-less or TATA-containing promoters more dependent on HATs? Why?
TATA-less, because they require acetylation of histone tails to be recognised by TAF1 for TFIID recruitment.
