IT6: How is DNA accessed? Flashcards
What’s the difference between specific and non-specific TF binding in terms of contacts made with the DNA?
All non-specific interactions are with the sugar phosphate backbone on the outside, whereas specific interactions involve contact with the bases.
How do transcription factors find their binding site in DNA? Give specific examples.
- Docking geometry
The SV40 enhancer contains a cluster of cell-specific regulatory elements that has overlapping half-sites. All factors can form interactions with one another, and various docking geometries can alter such interactions. - Cooperativity (AND logic)
Sp1 and OTF1 act in a cooperative manner such that even when OTF1 is mutated to be low affinity, transcriptional activity of the two is still 83% and not the 7% predicted. - 3D-facilitated diffusion
What is transcription factor clustering?
Transcription factor (TF) condensates or clusters are dynamic and reversible assemblies of TFs that are formed through liquid-liquid phase separation.
The formation of TF condensates is thought to be driven by weak, multivalent interactions between intrinsically disordered regions (IDRs) of TFs, such as electrostatic interactions, hydrogen bonding, and van der Waals forces. These interactions create a high concentration of TFs within the droplets, which can recruit other transcriptional machinery, such as RNA polymerases and coactivators, to facilitate gene transcription.
What are the dynamics of transcription factors within the nucleus? How do TFs increase their occupancy at a certain DNA motif?
Eukaryotic gene expression is inherently ‘noisy’ due to random fluctuations of chromatin remodeling, nutrients, binding dynamics, etc.
The residence time of TFs at binding sites is usually very short (except histones), but this can be increased by binding the DNA at higher frequencies (i.e., higher k-on) or increasing residence time itself (i.e., 1/k-off).
What is the impact of chromatin on transcription factor behaviour? Give an example of some TFs that are able to overcome these interactions.
Nucleosomes restrict the occupancy of most TFs by reducing binding and accelerating dissociation.
Pioneer factors overcome this through high affinities to nucleosome-embedded sites and facilitation of nucleosome displacement. i.e., they don’t have better binding rates, but do have better dissociation rates.
e.g., the Yamanaka factors OCT4 and SOX2. The degree of DNA distortion imposed on the nucleosome depends on the position of the motif. Position also dictates whether the two TFs will bind cooperatively or not.
Give 2 examples of Group I helix-turn-helix transcription factors and what their roles are.
- Lac repressor - conformational modulator of IPTG/allolactose
- CAP/Crp activator: conformational modulator of cAMP
Give 2 examples of Group II zinc-coordinating transcription factors and what their roles are.
- Nuclear hormone receptor - binds a hormone and gets transported to the nucleus to bind the DNA.
- Gal4 - positive regulator for galactose-induced genes.
Give an example of Group III basic leucine zipper domain transcription factors and what their roles are.
AP1 fos/jun heterodimer: oncogenes that regulate cell proliferation and are members of the early immediate genes.
Give an example of Group III basic helix-loop-helix ZIP domain transcription factors and what their roles are.
Myc/Max heterodimer: activates target genes involved in metabolism - inhibited by Mad replacing Myc
Give 2 examples of Group IV alpha-helical transcription factors and what their roles are.
- MADS: e.g., serum response factor
- HMG: chromatin remodeling
Give an example of Group V B-sheet transcription factors. How does it bind the DNA?
TATA binding protein
Binds into the minor groove using a wide B-sheet to bend the DNA.
Give 2 examples of Group VI B-hairpin/ribbon transcription factors and what their roles are.
- IHF - role in sigma54 activation
- Tus protein - acts as a counter-helicase to block DNAP progression and aids in replication termination
Give 2 examples of Group VII (other) transcription factors and what their roles are.
- Rel family (can bind as homo- or heterodimers)
- STAT family: mediate responses to cytokines and growth factors, requiring phosphorylation for function
What _ mechanisms do transcription factors use to bind DNA?
- TFs generally bind in the major groove as dimers
- Improving the consensus matching improves binding kinetics
3.
How are eRNAs involved in the activation of globin genes?
In the case of globin genes, the locus control region (LCR) is an enhancer that regulates the expression of multiple globin genes.
It has been proposed that the LCR produces eRNAs which can interact with specific TFs, such as the mediator complex, which is a coactivator that bridges enhancer-bound TFs and RNA polymerase II at the promoter region.
Describe the anatomy of an enhancer. How do they activate genes?
- Core enhancer sequence for TF binding
- Proximal vs distal regulatory elements
- Open chromatin
The sequential model states that co-activators and Mediator form direct interactions captured by a cohesin loop to bring the elements together.
Local high concentrations of phase-separated assemblies provides a general regulatory mechanism.
What is the role of chromatin remodeling in transcription factor binding?
Chromatin remodeling factors can modify the chromatin structure to allow transcription factors to bind to the DNA sequence.
These factors can modify the chromatin structure by sliding, ejecting, or repositioning nucleosomes.
Describe the key steps in gene activation, starting from recruitment of histone modifiers.
- Recruit histone modifiers e.g., HATs, to destabilize nucleosomes.
- Recruit nucleosome remodelers to move or remove nucleosomes e.g., SWI/SNF
- Recruit general TFs (the basal transcription machinery) to form the PIC, alongside the co-activator Mediator
- Ser5 phosphorylation of RNAPII CTD by TFIIH.
- Recruit elongation factors e.g., pTEFb, to phosphorylate Ser2 and initiate elongation.
What are the main characteristics of immediate early genes?
How are the IEGs SRF and ELK1 regulated to produce Fos?
- Short with fewer exons
- High prevalence of TATA boxes and CpG islands, making promoters strong
- Enriched for multiple specific TF binding sites
The promoters of IEGs are accessible even before activation, contributing to rapid activation. In the absence of mitogen signaling, ELK1 recruits a HDAC to maintain low transcription levels.
SRF acts as a platform for ELK1 to bind to the promoter.
Activation of ELK1 by mitogens results in activation of a HAT to modify the chromatin and recruit Mediator. Mediator undergoes a phosphorylation-dependent interaction with ELK1.
Fos is then transcribed and translated, going on to activate secondary response genes for cell proliferation or survival.
Describe the basic structure and pathway organisation of heat shock genes.
How do they respond so quickly?
- Helix-turn-helix DNA binding domain
- Binds as monomer or trimer depending on the binding sequence
- Intrinsic thermo-sensing via the temperature sensing domain to trigger oligomerization
Heat shock factor is responsible for turning on HSGs, but is normally sequestered by heat shock protein. Heat shock causes the release of HSF (HSPs are diverted to engage with unfolded proteins instead), allowing it to trimerize and enter the nucleus.
HSGs tend to encode HSPs causing negative feedback by inhibiting HSFs.
The response is so fast because RNAPII is held at the promoter-proximal pause, waiting for HSF to bind.
What’s the difference between type I and type II nuclear receptors? How do they bind DNA?
Type I:
- cytoplasmic and bound to heat shock proteins
Ligand binding releases them from HSPs to enter the nucleus and bind heat response elements.
Type II:
- retained in nucleus
- always bound to DNA response element, even without ligand presence
- bind as heterodimers, usually with retinoid X receptor
The C-terminus binds the ligand and causes flipping of an alpha-helix. This flipping results in the N-terminus to go from disordered to ordered, allowing the protein to bind DNA.
Describe the use of long-range gene activation at the globin locus.
In the context of the globin locus, long-range gene activation involves the interaction of distal enhancers with the promoter region of the globin genes. These enhancers can be located many kilobases away from the promoter region, but are brought into close proximity through the formation of DNA loops facilitated by protein complexes such as the locus control region (LCR).
The LCR is a cluster of enhancers located upstream of the globin genes that is necessary for the proper regulation of globin gene expression.
What are the two major classes of repetitive sequences, and how can they be formed? What is the importance of repetitive sequences in complex genomes?
Constitutive heterochromatin assembles over repetitive DNA sequences.
- Tandem repeats
- Dispersed repeats
Formed via:
- Replication slippage
- Unequal crossing over
- Type I retrotransposon (LTR retrotransposons and non-LTR retrotransposons) - vertebrates
- Type II DNA-only retrotransposons via transposase proteins - prokaryotes
How were variegation modifiers identified in mice?
A genetic screen using a male mice line that had variegating GFP transgenes encoded in them. ENU was used to generate point mutations, and the mice were then bred to study the offspring, looking for differences in the normal variegation patterns.
This lead to the discovery of Momme’s genes, many of which were identified to be genes encoding proteins known to play a role in epigenetic processes e.g., DNA methyltransferases and HDACs.
What is position effect variegation and what two mechanisms can trigger it? What did experiments into this reveal?
PEV results when a gene normally in euchromatin is rearranged or transposed into a region of heterochromatin.
- Gene becomes integrated into heterochromatin
- Formation of multi-copy arrays triggers heterochromatin formation
Genetic screens for mutations that modify PEV uncovered su(var) and e(var), leading to the suggestion of a dosage model where heterochromatin formation is dependent on modifier concentrations.
How does heterochromatin spread (Drosophila model)? How is the spreading blocked?
SUVAR3-9 deposits H3K9me3 which HP1 binds. Hence, these two proteins interact to form a read-write feedback mechanism that mediates the spread of heterochromatin.
Barrier elements can prevent heterochromatin spreading by:
- high gene expression
- histone modifications
- tRNA genes
- targeting of demethylases
What diseases are associated with heterochromatin disfunction?
FSHD muscular dystrophy
Triple repeat expansion disorders e.g., Huntington’s
Answer the following questions with regards to RNAi in S. pombe:
1. Where does the dsRNA come from?
2. What sort of siRNAs are produced?
3. How does this result in silencing?
- bidirectional transcription and RdRP activity
- heterochromatic and inducible siRNAs
- heterochromatic siRNA is incorporated into the RITS complex which recognizes complementary sequences to recruit chromatin repressive enzymes and modifiers.
How can PEV be modified in Drosophila and mice?
Drosophila: su(var) and e(var)
Mice: Momme’s proteins
Describe the mating type screens used to study PEV in S. Pombe.
These screens take advantage of the fact that the fission yeast genome contains two mating-type loci, called mat1-P and mat1-M, which are silenced by heterochromatin in the opposite mating-type cell.
- Strain generated with a reporter gene located near the mat1 locus (usually a selectable marker).
- Cross the reporter strain with a strain containing a mutation in heterochromatin formation. This mutation should result in a loss of heterochromatin and de-repression of the reporter gene.
- Isolate and characterize progeny that exhibit variegated expression of the reporter gene.
What is RNAi and how is it involved in establishing heterochromatin?
RNAi is a mechanism by which cells silence gene expression through the use of small RNA molecules.
Small RNAs can recruit the RNA-induced transcriptional silencing (RITS) complex, which contains chromatin modifiers such as the histone H3 lysine 9 methyltransferase (HMT), a mark that is associated with heterochromatin formation.
The RITS complex can also recruit other proteins that modify chromatin structure, leading to the establishment of a repressive chromatin environment that is inaccessible to transcriptional machinery.
Describe the RNAi pathway in plants.
PolIIV and PolV are dedicated polymerases which transcribe repeat regions. This produces dsRNAs, processed by Dicer to generate siRNAs.
These can initiate heterochromatin formation, which in turn promotes more polymerase activity in a positive feedback loop.
Is heterochromatin transcriptionally inactive?
No - transcription of heterochromatin at low levels is essential for RNAi-dependent heterochromatin formation and piRNA biogenesis.
What is piRNA and 22G-RNA? How are they involved in both germ cells and transgenerational inheritance in C. elegans, respectively?
piRNA is a type of small ncRNA that protects the germ line genome from transposon activity.
22G-RNAs are another class of small ncRNAs that play a role in gene silencing, as well as the maintenance of chromatin structure and epigenetic inheritance. They direct the deposition of repressive histone modifications (e.g., H3K9me3) at target loci to form heterochromatin.
piRNAs/dsRNA generates 22G-RNAs which interact with a germline nuclear Ago, HRDE-1 to recruit H3K9 methyltransferases.
Give 2 examples of where the Polycomb system can be ‘hijacked’ to carry out specialised roles.
- X-inactivation
Because Xist coats the X-chromosome, it also becomes coated with a ‘blanket’ of Polycomb that maintains the silencing of X-chromosomes. - Germline gene activity
A special PRC1 complex with highly specific DNA binding activity (not common) recognizes germline gene promoters to ensure maintenance of their silencing in somatic cells.
How does the Polycomb system modify chromatin at Hox genes? What key evidence supports this?
Polycomb is a trans-acting factor that maintains hox gene repression. The Polycomb system consists of two main complexes: PRC1 and PRC2.
- PRC2: H3K27me (forms heterochromatin)
- PRC1: ubiquitination (maintains heterochromatin)
DNA-binding proteins, such as Pho and GAGA , interact with Polycomb responsive elements (such as CpG islands in mammals) to allow Pc to recognize the PRE and be recruited.
PRC2 tri-methylates H3K27, which PRC1 then binds to ubiquitinate and compact the chromatin. This is called the hierarchial model.
Other PRC1 variants are able to ubiquitinate the chromatin in a PRC2-independent fashion.
Feedback loops then allow the system to be amplified across regions, generating Polycomb chromatin domains.
Key evidence:
- CpG island binding proteins can be detected at both expressed and non-expressed CpG islands i.e., they sample the DNA
- Polycomb domains form after transcription has stopped
- Transcription inhibitors less to formation of more Polycomb domains i.e., it’s highly responsive
Describe the bistable mechanism that underlies Polycomb and Trithorax regulation.
Both systems sample the DNA for an opportunity to engage and function.
In the absence of transcription, feedback mechanisms inherent to PcG proteins leads to predominance of the repressive chromatin state to maintain gene repression.
In the presence of transcription, the TrxG system is initiated (via low levels of H3K4me3 from SET1) leading to the predominance of the permissive chromatin state to maintain gene expression.
Antagonism between the systems may further reinforce the permissive or repressive chromatin states. This feedback and antagonism enables chromatin bistability whereby transcription dictates a switch between restrictive and permissive chromatin states.
Why are bistable switches preferred in biology in reference to chromatin state?
Because of the autonomous feedback mechanisms used by TrxG and PcG proteins, it’s possible for chromatin states to be maintained over a wider range than is required to actually trigger the switch. This can help to ensure memory (or hysteresis) of transcriptional states.
Linear expression outputs are risky in stochastic systems as there can be a halfway point between the two outputs, at which point the cell has no idea which way it’s going.
How does the Trithorax system modify chromatin?
TrxG protein complexes are histone modifying or chromatin remodeling factors that act to oppose Polycomb-mediated chromatin suppression. In mammals, these complexes are:
- MLL1/2
- SET1A/B (H3K4me3 addition)
They bind at the same sites as PcG proteins (PREs) and hence act in a competitive manner. SET1 can recognize Ser5-phosphorylated RNAPII, thought to lead to H3K4me3 deposition in association with initiation.
The rest of the TrxG proteins then recognize the H3K4me3 through their PHD domains. Other reader proteins include chromatin modellers, HATs and TFIID.
This blocks PcG complex binding.
Describe the bistable switch mechanism between Polycomb and Trithorax systems.
The bistable switch mechanism is thought to involve positive feedback loops, where the presence of a particular chromatin state leads to the stabilization and maintenance of that state over time. In the case of Polycomb and Trithorax, this positive feedback loop may be mediated by the recruitment of additional PRC or Trithorax complexes to the locus, which reinforce the chromatin state and ensure its stable maintenance over multiple cell divisions.
How are monoallelic genes regulated in parental imprinting?
Monoallelically expressed genes are those that are expressed from only one of the two parental alleles.
In parental imprinting, one of the parental alleles is silenced during gametogenesis, resulting in the expression of the gene from only one parental allele in the offspring. This is achieved through methylation.
- Male vs female germline differences underlie parental imprinting
What is the IGF-2-H19 paradigm?
The IGF-2-H19 paradigm refers to a classic example of parental imprinting in mammals that involves the insulin-like growth factor 2 (IGF-2) and the H19 genes located on chromosome 11p15.5.
In this paradigm, the IGF-2 gene is maternally silenced and paternally expressed, while the H19 gene is maternally expressed and paternally silenced. This is achieved through CTCF blocking the IGF2 enhancer in maternal, but the CTCF binding site is methylated in paternal which prevents it binding.
A ncRNA (Airn) is also involved. It inhibits IGF2R expression through RNA polymerase collision (transcriptional interference).
How is imprinting restored after the cyclical erasure during development?
Specialized KRAB proteins bind to imprinted DMRs to keep the genes silent when methylation is wiped away and helps to reestablish methylation as development proceeds.
What are differentially methylated regions? What diseases are associated with these?
Differentially methylated regions (DMRs) are regions of the genome that exhibit differential DNA methylation patterns between two or more distinct cell types or developmental stages.
Imprinted DMRs are regions that are differentially methylated depending on the parent of origin.
Deletions of DMRs in either maternal or paternal genes are seen in Angelman syndrome (AS) and Prader-Willi syndrome, respectively.
What’s the role of NOS (Nanog, Sox, Oct) in X inactivation?
NOS TFs specific to early embryo cell repress the Ub ligase that inhibits REX1, and Xist to promote Tsix expression.
As their levels drop, cells pass a REX1 threshold sufficient to activate Xist RNA expression on a single allele.
How is X inactivation random, and how does it only occur in females?
X inactivation is random because it occurs in a stochastic manner during early embryonic development. Appropriate Xist patterns are then dictated by non-canonical imprinting.
The Xist enhancer is repressed by REX1 and requires removal via an E3 Ub ligase that’s transcribed just upstream of Xist. Without both X chromosomes, there’s not enough ligase to deplete REX1, so males don’t have dosage compensation.
How is olfactor receptor choice random?
The OR gene clusters are in highly heterochromatic regions that contain high densities of LINE1 retrotransposons, and this suppressive environment is key in picking just one receptor gene.
Each gene has its own promoter and enhancer, and evidence suggests selective removal of H3K9me2/3 is what allows for a single OR gene to be transcribed.
Feedback ensures that no other alleles are chosen.
What’s the difference between specific and non-specific TF binding in terms of contacts made with the DNA?
All non-specific interactions are with the sugar phosphate backbone on the outside, whereas specific interactions involve contact with the bases.
What diseases are associated with heterochromatin disfunction?
FSHD muscular dystrophy
Triple repeat expansion disorders e.g., Huntington’s
What are the 3 key features of enhancers that underlie their function?
- promiscuous (can enhance whatever promoter they’re next to)
- independent of orientation
- cis-acting
What two mechanisms underlie how multiple genes can be enhanced by a single enhancer?
- We are all winners
All genes are activated by the enhancer, so must share the ‘enhancing power’ equally. - Winner takes all
A single gene is activated at any one time e.g., olfactory receptor choice.
How can promoter-enhancer interactions be stabilized?
- TADs
- Regulatory loops (within TADs)
How does the interferon-B enhancer work?
Enhanceosome must be assembled and bind to the enhancer, as well as recruiting co-activators and chromatin remodeling enzymes. These will only be recruited if all TF binding sites are bound.
Nucleosomes that flank the enhancer and mask the TATA box are opened so that RNAPII can access the binding site.
Why is the interferon-B enhancer called a coincidence detector?
The signals that induce individual TFs aren’t sufficient to activate interferon-B transcription.
It’s only if all TFs are bound that full output is created, meaning the interferons are only expressed when absolutely necessary.
What is the locus control region and how does it regulate B-globin genes?
The locus control region (LCR) is a region of DNA located upstream of the beta-globin gene cluster that regulates the expression of the beta-globin genes.
The LCR contains several enhancer elements that bind to transcription factors and promote the expression of the beta-globin genes.
Only one B-globin gene is transcribed at any given moment, and the timing of expression depends on the distance between the LCR enhancer and the genes.
What is the locus control region and how does it regulate B-globin genes?
The locus control region (LCR) is a region of DNA located upstream of the beta-globin gene cluster that regulates the expression of the beta-globin genes.
The LCR contains several enhancer elements that bind to transcription factors and promote the expression of the beta-globin genes.
Only one B-globin gene is transcribed at any given moment, and the timing of expression depends on the distance between the LCR enhancer and the genes.
