Eukaryotic Chromosome Structure and Function Flashcards
What is the name given to the factors that mediate communication between transcription factors and RNA pol II?
Co-activators/co-repressors.
What is the function of co-activators?
Co-activators mediate communication between transcription factors and RNA pol II to facilitate gene activation/’ON’.
What is the function of co-repressors?
Co-repressors mediate communication between transcription factors and RNA pol II to facilitate gene repression/’OFF’.
What are some of the ways in which co-activators/co-repressors mediate communication between transcription factors and RNA pol II?
Some co-activator/co-repressor complexes provide direct physical interfaces bewteen transcription factors and RNA pol II, many others function by remodelling chromatin structure to indirectly modulate RNA pol II activity.
How is DNA actually found in vivo?
DNA is not a straight line, eukaryotic DNA is complexed with histone proteins to form nucleosomes which repeat like ‘beads-on-a-string’.
DNA is 50% protein and 50% DNA.
Describe the structure of the nucleosome?
A nucleosome is comprised of an octameric protein core which contains two copies each of four different histones called H2A, H2B, H3 and H4. These core histones are structurally similarwith a 3 alpha-helix 'histone fold' and an unstructured tail region. The octamer is assembled in a very specific way, a H3/H4 tetramer sits in the middle and then 2 H2A/H2B dimers come together to form a semi-symmetrical octamer structure. Then 147bp (1.7 turns) of DNA wraps around this core structure.
At what different levels do co-activators and co-repressors manipulate chromatin?
There are two fundamentally/functionally distinct levels at which chromatin can be manipulated…
- Local-level remodelling at gene-regulatory regions (promoters/UPEs/enhancers).
- Domain-level remodelling over large chromosomal regions.
What is the consequence of nucleosome presence in vitro?
In vitro, the presence of nucleosomes acts as a physical barrier to transcriptin factor and PIC binding to DNA, therefore, the formation of nucleosomes inhibits activation and transcription of a DNA template.
Nucleosomes are refractory to the process of transcription, they are barriers to RNA pol II transcriptional elongation.
Why can’t we say nucleosomes are a barrier to all transcription?
Some RNA pols such as bacteriophage T7 enzyme are able transcribe nucleosomal DNA in the test tube.
It is possible eukaryotic RNA pol II may have evolved this characteristic of being unable to transcribe in the presence of nucleosomes as a method for regulating eukaryotic transcription elongation.
What is meant by local chromatin remodelling?
A major function of some co-activators and co-repressors is to be brought to DNA to manipulate individual nucleosomes to allow transcription factor and RNA pol II access to DNA.
What are the two different types of activity performed by co-activators and co-repressors?
- Nucleosome positioning.
2. Nucleosome structure.
Give some examples of the enzyme activities performed by chromatin-remodelling enzymes - co-activators and co-repressors?
Some use energy from ATP hydrolysis to modify nucleosomes - these enzymes do physical work on nucleosomes.
Some covalently modify histone protein amino acid residues within nucleosomes.
What is the key reagent in chromatin-seq?
Micrococcal nuclease (MNase).
Briefly describe the process of chromatin-seq?
Take the micrococcal nuclease and add it to cells.
MNase will not cleave DNA if the DNA is wrapped around nucleosomes.
The presence of the histone octamer protects DNA in a nucleosome from MNase cleavage leaving ~150bp of undigested DNA.
MNase will also not cleave DNA when bound to transcription factors leaving 20-50bp of undigested DNA that can be sequenced!
Purify and sequence the resulting 150bp MNase-resistant DNA fragments, map them back to the genome and count the number of sequence reads.
How does MNase-seq differ from DNase-seq?
Whereas DNase-seq is looking at DNase accessibility, we are looking at nuclease protection.
The bits of DNA that you sequence are the ones that were wrapped around nucleosomes as opposed to those that weren’t bound to transcription factors.
What is meant by nucleosome positioning?
If nucleosomes were wrapped randomly on DNA sequences, chromatin-seq experiments would yield random distribution of sequence reads throughout a genome. In fact this turns out to be completely wrong.
In real chromatin, we find cells work really hard to put nucleosomes in very specific positions.
Describe the chromatin state of a gene that is not transcribed?
If a gene is switched off and not being transcribed, on the whole, the gene regulatory regions are covered with nucleosomes. However, within the linker region between the two nucleosomes there are binding motifs for pioneer transcription factors which are able to bind their motifs when they are wrapped on the surface of a histone octamer.
Nucleosomes aren’t well-positioned in these regions.
What are pioneer transcription factors?
Transcription factors that are able to bind their motifs when they are wrapped on the surface of a histone octamer.
Describe the chromatin state of a gene that is being actively transcribed?
When a gene is activated, gene regulatory DNA and promoters become exposed as nucleosome-free regions (NFRs). This NFR is flanked by strongly positioned nucleosomes.
How does the degree of nucleosome positioning vary between species?
Different organisms differ in the extent to which they position their nucleosomes.
In yeast, 80% of nucleosomes are precisely-positioned.
In humans, only active gene-regulatory sequences show positioning.
Why are nucleosome-free regions found in gene regulatory DNA?
Nucleosome-free regions
(NFR) in gene-regulatory DNA allow access for TF binding.
By putting a NFR over the gene regulatory DNA, the chromatin in that region is being opened making the DNA accessible so that transcription factors can bind.
How are nucleosome-free regions created at gene regulatory DNA?
The creation of NFRs at gene-regulatory DNA is largely driven by recruitment of chromatin-remodelling ATPase co-activators.
Energy from ATP hydrolysis drives non-covalent changes in nucleosome structure, the manipulation of nucleosome position is the job of the chromatin-remodelling ATPases.
What are chromatin remodelling ATPases?
Chromatin remodelling ATPases are complexes that contain a core ATPase subunit, plus other proteins that modulate and target their activity.
There are multiple classes of chromatin remodelling ATPases, but they all have a subunit which is an ATPase - a kind of motor whch can grab hold of DNA and move DNA from one place to another. The activity of the ATPase is often regulated by other subunits
How do SWI/SNF ATPases act to remodel chromatin?
SWI/SNF ATPases can evict/displace histones from the DNA to remove nucleosomes from underlying sequence.
The evicted histone components are handed on to histone chpaerones such as ASF1.
SWI/SNF can grab hold of a nucleosome and pop it off DNA and hand a histone octamer core either onto another DNA molecule or onto a histone chaperone thus creating a nucleosoem-free region by physically removing the nucleosome.
By what mechanism does SWI/SNF generate NFRs?
Histone eviction
By what mechanism does ISWI generate NFRs?
Nucleosome sliding.
These remodelling ATPases translocate the DNA molecule over the histone core allowing the core to move relative to the underlying DNA sequence.
This generates an open region of DNA for transcription factors and RNA pol II to be able to bind and activate gene transcription.
Is all nucleosome manipulation actively driven by chromatin remodelling ATPases?
No, whilst a lot of nucleosome manipulation comes fromes from these active ATPases, there is some nucleosome positioning hardwired into DNA sequences. Some gene regulatory DNA has particular sequences favourable for nucleosome wrapping which accounts for some nucleosome positioning.
There are also particular tracts of AAAA which don’t wrap around the histone octamer well and thus act as barriers to nucleosome formation.
What kind of sequence motifs appear to exclude nucleosomes/constrain nucleosome binding?
Short tracts (5-20bp) of As (common in gene regulatory sequences).
What do chromatin remodelling co-activators and co-repressors manipulate?
Nucleosome positioning
Nucleosome structure
What is nucleosome destabilisation?
There’s a SWI/SWF ATPase known to destabilise nucleosomes that, it doesn’t evict them or slide them out the way it somehow higgles the nucleosome so the DNA wrapping around the octamer is much more labile and accessible to DNA-binding proteins.
This helps transcription factors gain access to motifs even if the histone octamer isn’t entirely removed.
Briefly describe how histone variants affect nucleosome structure?
Reversible replacement of normal histone proteins with a histone variant is another possibility for altering nucleosome structure.
Whilst most histone octamers are comprised of 2 H2A/H2B dimers and a H3/H4 tetramer, there are variant histones occassionally incorporated into particular nucleosomes for particular reasons.
Where are H2A.Z containing nucleosomes often found?
ChIP-seq with an antibody to histone variant H2A.Z reveals active genes will often have an NFR with a positioned nucleosome either side of that NFR and that positioned nucleosome will contain the H2A.Z variant. NFRs associated with yeast and nhuman promoters are flanked by nucleosomes containing the H2A.Z variant.
What is the function of the H2A.Z variant?
SWR ATPases use ATP hydrolysis to open up a nucleosome and exchange a histone protein out and exchange it with the variant H2A.Z. This makes the nucleosome jigglier and more accessible for RNA pol II and transcription factors.
What is meant by histone post-translational modifications?
A whole load of co-activators and co-repressors alter the histone code, they add or remove chemical groups from the histone tails and these are called post-translational modification.
These are reversible covalent modifications.
How many different types of post-translational modifications exist?
There are at least 60 reversible post-translational modifications.
Where on the histone tails are post-translational modifications made and what type of modifications can be made?
The lysines and arginines on the histone tails can be acetylated, serines and threonines can be phosphorylated, arginines can be methylated and prolines can be isomerised.
What is the function of histone acetyltransferase co-activators?
Histone acetyltransferase co-activators add acetyl groups to multiple lysine residues in the N-terminal tails of histones H2B, H3 and H4.
How does acetylation of lysine residues in histone tails alter nucleosome structure?
Acetylated nucleosomes are more accessible to transcription factor binding and PIC formation than un-acetylated nucleosomes.
Addition of acetyl groups to histone tails destabilises the DNA wrapped around the histone octamer, it neutralises the charges and makes DNA more accessible to RNA pol II and transcription factors.
This histone acetylation removes a positive charge and therefore there is reduced affinity between histones and DNA meaning the DNA and histone tails are bound less tightly = greater accessibility.
Give some examples of histone acetyltransferase co-activators?
SAGA, CBP/p300, Tip60.
What is the function of histone deacetylase co-repressors?
Histone deacetylase co-repressors remove acetyl groups from the lysine residues in the N-terminal tails of histones. This shuts gene transcription down by making the chromatin locally less accessible to RNA pol II and transcription factors.
Give some examples of histone deacetylase co-repressors?
RPD3, SMRT and NCoR.
What are the functions of histone post-translational modifications?
Histone PTMs alter nucleosome stability.
PTMs create a protein binding site.
These histone modifications write a code marking a region of chromatin - this is called the epigenetic code.
What kind of histone post-translational modifications do bromodomains recognise?
Acetyl-lysine.
What kind of histone post-translational modifications do chromodomains recognise?
Methyl-lsysine.
How do ChIP-seq signals compare for TF binding or histone PTMs?
A genome browser comparison of ChIP-seq peaks shows ChIP-seq signals are highly localised for transcription factors with specific binding motifs but more dispersed for histone post-translational modifications. When you do ChIP with histone-specific antibodies you will see huge regions of DNA being pulled out.
Generally, describe the histone post-translational modifications made to nucleosomes in non-transcribed regions of the genome?
They have methylated histones PTMs such as H3K9me2/3, H4K20me3, H3K27me3.
Give some histone post-translational modifications associated with nucleosomes in non-transcribed regions of the genome?
H3K9me2/3
H4K20me3
H3K27me3
Is H3K27me3 a repressive or activating mark?
Repressive
Is H3K9me2/3 a repressive or activating mark?
Repressive
Is H4K20me3 a repressive or activating mark?
Repressive
Which complex lays down the H3K27me3 mark?
Polycomb repressive complex 2.
Is H3K4me3 a repressive or activating mark?
Activating
What is meant by a poised state of chromatin?
Poised chromatin is used as a priming mechanism for sites that need to be activated/repressed quickly, there’s both active (H3K4me3) and repressive (H3K27me3) marks.
Generally, describe the histone post-translational modifications made to nucleosomes in active gene regulatory regions of the genome?
Activated state (ON) gene regulatory region nucleosomes typically found flanking a nucleosome-free region (NFR) generally become modified with acetyl groups including H3K9ac, H3K27ac and H3K4me2/3.
Is H3K9ac a repressive or activating mark?
Activating
Is H3K27ac a repressive or activating mark?
Activating
Generally speaking is acetylation an activating or repressive mark?
Activating
Give some examples of pioneer transcription factors in humans?
Oct3/4
FoxA
How does local chromatin remdoelling explain the detection of DNase HS at active gene regulatory DNA/open chromatin?
Gene regulatory regions are active and open chromatin, there are lots of nucleosome-free regions, lots of jiggling nucleosomes that are being acetylated and this is why we see promoters/enhancers etc. becoming generally DNAse I hypersensitive because they’re being opened up and actively remodelling so are readily exposed to DNase I activity.
What happens when the repetoire of transcription factors are brought to their cognate binding motifs in gene regulatory DNA?
They in turn recruit a repertoire of different co-activators/co-repressors, some of which simply stick/physically attach RNA pol II at the promoter so it can start elongating.
Many of them however manipulate nucleosome structure and positioning involving nucleosome sliding, histone eviction, replacement of histone variants etc. chemical modification of histone variants by post-translational modification of histones. This is all happening at the local chromatin level.
Does local chromatin remodelling occur exclusively at a gene promoter/UPE?
No! Local chromatin remodelling occurs during activation of transcription at more distant gene-regulatory regions.
Enhancers are located hundreds/thousands of bps away from the promoter and they too are doing local chromatin remodelling as well.
How can local chromatin remodelling influence both the promoter and enhancers when they are so separated in sequence?
Activated eukaryotic gene regulatory regions are often separated by a large distance in sequence in the nucleus but not in space - these active gene regulatory regions physically interact with each other - the intervening DNA is looped out.
What is 3C technology?
3C = Chromosome Conformation Capture technology.
3C technologies use NGS to map interactions between DNA regions.
They are measuring the conformation and physical space of chromosomes and trapping the interactions of separated DNA sequences in the space of the nucleus.
Give some examples of variations of 3C technology?
3C, 4C, 5C, Hi-C, ChIA-PET
What are the key reagents required for 3C technologies?
Formaldehyde is requried which creates protein-DNA and protein-protein crosslinks.
We have an endonuclease that can cut DNA within DNA molecules, a sequence-specific endonuclease and a DNA ligase.
Briefly describe the process of 3C?
Cells are treated with formaldehyde to trap local physical interactions between sequences in the nucleus - e.g. imagine the enhancer and UPE promoter interacting together in an activated gene, this interaction gets trapped.
The DNA is then cleaved using the restriction endonuclease - DNA is cleaved at sites 500bp-2kb apart.
^For this step, we cut with a nuclease that doesn’t cut frequently like DNase or MNase we cut every 500bp or so. This is removing the loop of intervening DNA from these interactions.
Having treated this DNA to release the loops and intervening DNA, we add a DNA ligase - this is called proximity ligation.
The newly created DNA ends are then treated with DNA ligase - sequences such as promoters and enhancers being held in proximity by formaldehyde now become directly joined together.
Novel synthetic DNA junctions are created effectively capturing sequences which were close in nuclear space but separated in sequence.
Now we have a bit of sequenceable DNA and we can sequence it can see the promoter is attached to the enhancer for example.
DNA junctions are purified and sequenced.
What is ChIA-PET and what is it used for?
ChIA-PET = chromatin interaction analysis by paired end tag sequencing.
ChIA-PET is a 3C variant in which a ChIP step is added to enrich for gene-regulatory sequences using specific co-activators or transcription factors.
We know that with the crosslinked complexes there will be all the transcription factors and co-activators/co-repressors, antibodies can be used to pull out particular transcription factors or to enrich for gene regulatory events.
How is 3C data often plotted?
3C data is often plotted as a contact probability map.
What is a contact probability map?
A contact probability map is a 3D graph where the length of one region of DNA is plotted on both X and Y axes and the frequency of types of 3C junction sequence reads is plotted as a colour intensity on the Z axis.
In this Z axis/third dimension we are plotting as a coloured heatmap the frequency of all those different sequence interactions.
What are the 3 rules of reading a contact probability map?
- Ignore the signal forming the central diagonal line.
These sequences are directly adjacent in DNA sequence and therefore, most likely represent religation of the original restriction site. - Look for signals ‘off the diagonal’ - increases in sequence frequency that lie off the diagonal.
These graphs are symmetrical so ‘off diagonal’ sequences always come in pairs. - Read horizontally from one axis to hit a signal then read vertically up to the other axis.
This tells you which two bits of DNA were interacting with one another.
What key piece of evidence do 3C experiments provide in terms of gene regulation?
3C experiments give us evidence that gene regulatory DNA wehn activated, things like enhancers and promoters really do interact with one another physically in the space of the nucleus despite being often separated by huge amounts of DNA sequence.
How are enhancers and promoters etc. held together in physical space in the nucleus despite often being separated by huge amounts of DNA sequence?
This is a job co-activators.
The general co-activator Mediator functions to hold many promoter-enhancer interactions together and can interact with multiple transcripiton factors at the same time.
What is the function of the Ldb1 complex?
The Ldb1 complex is required for late erythroid cell gene expression which links erythroid-specific gene promoters/enhancers/LCRs via interactions with GATA1 transcription factors.
What are cohesins?
Cohesins are ring-like proteins which normally function to hold chromatids together during mitosis and meiosis. These are also involved in holding enhancers, promoters, gene regulatory DNA etc. together.
What is the name given to the structure that forms when the various regions of gene regulatory DNA coalesce together?
The promoter, UPE, enhancers, LCRs even the termination region of the gene gather together in a physical space, held together by co-activators and potentially a cohesin ring, leaving an intervening loop of DNA which includes the coding region etc. this coalescing of these sequences forms an active chromatin hub.
Why are is the termination region of a gene physically contacting the promoter of a gene being actively transcribed?
This means when RNAP II has finished producing an mRNA transcript, it can hop off the terminator and go straight back to the promoter promoting repeated rounds of transcription in a gene for strongly expressed genes.
Why does an active chormatin hub form?
The coalescing of transcription factors at distant enhancers/locus control regions and their recruited co-activators/repressors combines signalling inputs and activities into one concentrated physical location in the nucleus.
This facilitates the integration of all information in a combinatorial ‘blob’ for a particular gene.
What kind of chromatin is required for the formation of an active chromatin hub?
In order for gene regulatory regions to form an ACH, the chromatin in which they are embedded in must be flexible.
If randomly embedding a promoter and reporter gene into the genome, what is required for strong well-regulated transcription in all chromosomal locations?
Just a promoter and reporter gene integrated into the genome results in no transcription/activation of the reporter gene, of course you need transcription factor binding sites.
However, even if you add the upstream promoter elements, you only get moderate transcriptional regulation in some chromosomal regions and not others.
To get strong well-regulated transcription from most reporter genes, enhancers were required as well - but not in all locations.
To get strong, well-regulated transcription in ALL chromosomal locations, locus control regions are required.
Which gene regulatory sequence is able to promote strong-well regulated transcription in all chromosomal locations?
Locus control regions are able to dominate chromosomal environments and allow strong, well-regulated transcription in all chromosomal locations.
No matter where you put a reporter gene, with an LCR, this will force the region to become supportive of transcriptional regulation.
At what two functional levels to eukaryotic chromatin remodelling co-activators/co-repressors act?
Local-level remodelling at gene-regulatory regions.
Domain-level remodelling over large chromosomal regions.
With the exception of mitosis, in what two forms are chromosomes found within the nucleus?
Condensed heterochromatin.
De-condensed euchromatin.
What is the problem with the textbook view of euchromatin and heterochromatin?
A nice, simplistic model of euchromatin and heterochromatin is proposed in the textbooks which suggests euchromatin is some sort of ‘beads-on-a-string’ chromatin and that heterochromatin is a hierarchically coiled fibre of nucleosomes which get coiled up and ultimately compacted. These comes from in vitro experiments in the 1970’s in which manipulating salt concnetrations revealed lovely chains of beads-on-a-string chromatin, this turned to simply be an artefact of the experiment.
Actually, this proposed set of hierarchically coiled fibres simply doesn’t exist at all.
Which type of microscopy technology allows visualisation of individiual nucleosomes?
Cryo-electron microscopy.
What is the current working model for chromatin structure?
At the level of individual nucleosomes, both euchromatin and heterochromatin are best modelled as disordered chains simply with subtle differences in nucleosome density. This is called the disordered chain model for chromatin states.
Describe the disordered chain model for chromatin states?
The disordered chain model for chromatin states suggests there is no hierarchy of coiled structures, just different densities of general aggregation.
At the scale of individual nucleosomes, chromatin is relatively disordered but within this is an amazing level of organisation.
This is disorder, but it is also fractal - there is order at multiple levels.
In this model, multiple forms of both heterochromatin and euchromatin appear to exist these are defined not by hierarchical compaction but instead by histone post-translational modifications and the factors recruits by these PTMs.
In the paper pioneering the disordered chain model for chromatin states, how many different types of chromatin were identified?
5 chromatin states were identified in total, 3 types of heterochromatin and 2 types of euchromatin.
What is the chromatin environment of non-transcribed genes?
Non-transcribed genes exist within domains of heterochromatin created by self-re-enforcing cycles of co-respressor-mediated histone post-translational modificiation.
These non-transcribed genes, the surrounding gene regulatory regions and their general chromosomal domains are marked repressive histone post-translational modifications (mostly methylations). This is one of the classes of chromatin, this is uniformly methylated - methylated H3 and H4 lysine residues in silent chromatin domains recruit heterochromatin repressors complexes.
The structure of this chromatin is compacted (but not into coils) and held together by repressor proteins such as polycomb repressors and heterochromatin protein 1.
Name two repressor proteins that keep heterochromatin compacted?
Polycomb repressors.
Heterochromatin protein 1.
What is the function of H3 and H4 lysine methylation in silent chromosomal domains?
Methylated H3 and H4 lysines in silent chromatin domains recruit general heterochromatin repressor complexes such as HP1 and polycomb factors.
What is the function of repressor proteins like polycomb repressors and heterochromatin protein 1 in silent chromosomal domains?
Chains of nucleosomes in heterochromatin are drawn together by binding of accessory factors such as HP1 and polycomb which make inter-nucleosomal links.
What histone mark does HP1 recognise?
HP1 recognises H3K9 methyl residues.
How does HP1 promote chromatin compaction in silent chromosomal domains?
HP1 recognises and binds H3K9 methyl residues (repressive marks) on various nucleosomes and therefore draws together lots of nucleosomes in a disordered manner.
HP1 then recruits K3K9 methylases and therefore helps create the very PTM that directs its own binding to chromatin.
This promotes a positive feedback loop whereby HP1 binds H3K9me1-3 which in turn recruits a histone lysine methylase that creates more H3K9me1-3 which binds more HP1 etc. etc. etc.
This causes spreading of this repressive state across teh chromosome in a self-re-enforcing positive feedback loop.
Describe how transcriptionally silent heterochromatin can be self-maintaining?
Repressive H3K9me1-3 marks are bound by HP1 which recruits H3K9 methylases which lay down more H3K9me1-3 marks on the adjacent histone tails lacking this methyl modification. This faciltates the recruitment and binding of more HP1 and thus a runaway, self-enforcing positive feedback loop is generated.
This repressive, transcriptionally silent heterochromatin is also characterised by DNA methylation which promotes the binding of methyl-domain binding proteins which are also able to recruit H3K9 methylases and promote the acquisition of repressive H3K9me1-3 marks.
How is gene activation and the conversion of heterochromatin to euchromatin initiated?
The conversion of heterochromatin to euchromatin is initiated by transcription factors that bind to locus control regions (LCRs).
Pioneer transcription factors such as FoxA and Oct3/4 are able to bind to their motifs even when they are inaccessible and tightly wound up in nucleosomes and their binding motifs are often located in LCRs.
The LCRs then reverse this silent self-maintaining heterochromatin spreading and open up chromosomal domains.
Where are pioneer transcription factor binding motifs found?
Many pioneer transcription factors bind to specific motifs in locus control regions that then facilitate the conversion of heterochromatin to euchromatin.
