Eukaryotic Chromosome Structure and Function Flashcards
(242 cards)
1
Q
What is the name given to the factors that mediate communication between transcription factors and RNA pol II?
A
Co-activators/co-repressors.
2
Q
What is the function of co-activators?
A
Co-activators mediate communication between transcription factors and RNA pol II to facilitate gene activation/’ON’.
3
Q
What is the function of co-repressors?
A
Co-repressors mediate communication between transcription factors and RNA pol II to facilitate gene repression/’OFF’.
4
Q
What are some of the ways in which co-activators/co-repressors mediate communication between transcription factors and RNA pol II?
A
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.
5
Q
How is DNA actually found in vivo?
A
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.
6
Q
Describe the structure of the nucleosome?
A
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.
7
Q
At what different levels do co-activators and co-repressors manipulate chromatin?
A
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.
8
Q
What is the consequence of nucleosome presence in vitro?
A
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.
9
Q
Why can’t we say nucleosomes are a barrier to all transcription?
A
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.
10
Q
What is meant by local chromatin remodelling?
A
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.
11
Q
What are the two different types of activity performed by co-activators and co-repressors?
A
- Nucleosome positioning.
2. Nucleosome structure.
12
Q
Give some examples of the enzyme activities performed by chromatin-remodelling enzymes - co-activators and co-repressors?
A
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.
13
Q
What is the key reagent in chromatin-seq?
A
Micrococcal nuclease (MNase).
14
Q
Briefly describe the process of chromatin-seq?
A
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.
15
Q
How does MNase-seq differ from DNase-seq?
A
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.
16
Q
What is meant by nucleosome positioning?
A
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.
17
Q
Describe the chromatin state of a gene that is not transcribed?
A
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.
18
Q
What are pioneer transcription factors?
A
Transcription factors that are able to bind their motifs when they are wrapped on the surface of a histone octamer.
19
Q
Describe the chromatin state of a gene that is being actively transcribed?
A
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.
20
Q
How does the degree of nucleosome positioning vary between species?
A
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.
21
Q
Why are nucleosome-free regions found in gene regulatory DNA?
A
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.
22
Q
How are nucleosome-free regions created at gene regulatory DNA?
A
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.
23
Q
What are chromatin remodelling ATPases?
A
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
24
Q
How do SWI/SNF ATPases act to remodel chromatin?
A
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.
25
By what mechanism does SWI/SNF generate NFRs?
Histone eviction
26
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.
27
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.
28
What kind of sequence motifs appear to exclude nucleosomes/constrain nucleosome binding?
Short tracts (5-20bp) of As (common in gene regulatory sequences).
29
What do chromatin remodelling co-activators and co-repressors manipulate?
Nucleosome positioning
| Nucleosome structure
30
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.
31
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.
32
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.
33
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.
34
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.
35
How many different types of post-translational modifications exist?
There are at least 60 reversible post-translational modifications.
36
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.
37
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.
38
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.
39
Give some examples of histone acetyltransferase co-activators?
SAGA, CBP/p300, Tip60.
40
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.
41
Give some examples of histone deacetylase co-repressors?
RPD3, SMRT and NCoR.
42
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.
43
What kind of histone post-translational modifications do bromodomains recognise?
Acetyl-lysine.
44
What kind of histone post-translational modifications do chromodomains recognise?
Methyl-lsysine.
45
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.
46
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.
47
Give some histone post-translational modifications associated with nucleosomes in non-transcribed regions of the genome?
H3K9me2/3
H4K20me3
H3K27me3
48
Is H3K27me3 a repressive or activating mark?
Repressive
49
Is H3K9me2/3 a repressive or activating mark?
Repressive
50
Is H4K20me3 a repressive or activating mark?
Repressive
51
Which complex lays down the H3K27me3 mark?
Polycomb repressive complex 2.
52
Is H3K4me3 a repressive or activating mark?
Activating
53
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.
54
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.
55
Is H3K9ac a repressive or activating mark?
Activating
56
Is H3K27ac a repressive or activating mark?
Activating
57
Generally speaking is acetylation an activating or repressive mark?
Activating
58
Give some examples of pioneer transcription factors in humans?
Oct3/4
| FoxA
59
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.
60
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.
61
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.
62
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.
63
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.
64
Give some examples of variations of 3C technology?
3C, 4C, 5C, Hi-C, ChIA-PET
65
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.
66
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.
67
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.
68
How is 3C data often plotted?
3C data is often plotted as a contact probability map.
69
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.
70
What are the 3 rules of reading a contact probability map?
1. 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.
2. 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.
3. 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.
71
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.
72
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.
73
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.
74
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.
75
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.
76
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.
77
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.
78
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.
79
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.
80
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.
81
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.
82
With the exception of mitosis, in what two forms are chromosomes found within the nucleus?
Condensed heterochromatin.
| De-condensed euchromatin.
83
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.
84
Which type of microscopy technology allows visualisation of individiual nucleosomes?
Cryo-electron microscopy.
85
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.
86
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.
87
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.
88
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.
89
Name two repressor proteins that keep heterochromatin compacted?
Polycomb repressors.
| Heterochromatin protein 1.
90
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.
91
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.
92
What histone mark does HP1 recognise?
HP1 recognises H3K9 methyl residues.
93
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.
94
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.
95
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.
96
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.
97
What are locus control regions?
Locus control regions are specialised enhancers that recruit co-activators which mediate domain-wide spreading of gene-activating histone PTMs.
98
How does pioneer transcription factor binding to LCRs facilitate the conversion of heterochromatin to euchromatin?
Pioneer transcription factors are able to bind their cognate sequence specific motifs even when inaccessibly wound up in a nucleosome and these motifs are commonly found in locus control regions.
This pioneer transcription factor binding to the LCR usually brings with it/recruits histone acetyltransferases that are co-activators and function to open up a domain of active euchromatin.
99
What is the function of locus control regions?
Locus control regions are specialised enhnacers that recruit co-activators which mediate domain-wide spreading of gene-activating histone PTMs.
Therefore, LCRs function to create large domains of general histone acetylation encompassing an entire activated gene and its regulatory regions.
Pioneer transcription factors bind to LCRs and recruit histone acetyltransferases and chromatin remodelling co-activators which are able to bind their own remodelled product and remodel the next region thus induce the spreading of euchromatin and facilitate the opening up of an entire chromosomal domain.
This histone acetylation is decreasing internucleosomal interactions at the domain level fluffing up individual nucleosomes and chains of nucleosomes generally making the region more accessible.
Other LCR chromatin remodellers include histone demethylases that remove repressive histone methyl post-translational modifications that were recruiting HP1 and polycomb repressors etc.
100
What stops euchromatin and heterochromatin states from spreading forever?
The current working model is that these euchromatin and heterochromatin states don't spread forever, instead there's another gene regulatory element that restricts where a domain of chormatin can spread to and this is the insulators.
101
What are insulators and what is their function?
Insulators act as the boundary elements between chromatin domains and therefore stop euchromatin or heterochromatin states from spreading forever.
Insulators also restrict inappropriate interactions between enhancers and promoters and prevent the enhancer from one gene accidentally activating the promoter of another gene.
102
What is the main insulator binding protein in mammals?
A zinc finger factor called the CTCF binding factor.
103
How do we identify insulators in the genome?
Insulators are bound by CTCF which is the human insulator binding factor, so clusters of CTCF motifs close together is generally a good indicator that a particular sequence will act as an insulator.
104
What is CTCF and what is its function?
CTCF is the human insulator binding factor, it is sometimes a transcription factor (a zinc finger TF) but also acts as an architectural factor that tethers insulators to each other and to intranuclear structures thus physically isolating the LCRs, enhancers and promoters.
105
Besides CTCF, what else binds insulators?
Insulators can be binding sites for cohesin molecules which help insulators stick together.
106
What happens to the chromatin environment a the protein-coding region of a gene during transcription?
When a gene is being transcribed, the whole protein-codign region lights up and is modified by a variety of very, very specific histone modifications.
These specific modifications are being carried out by RNA polymerase itself.
107
What is the chromatin remodeller laying down highly specific histone modifications at the protein-coding region of an actively transcribed gene?
RNA pol II itself.
The transcriptional elongating complex (TEC) (Elongating RNA pol II) is a potent chromatin-remodeller in its own right.
There are a variety of chromatin remodelling co-activator complexes that bind RNA pol II as elongation factors and use covalent and ATP-dependent nucleosome modifying activities to help RNA pol II elongate through nucleosomes because it cannot do this alone.
108
Is RNA pol II just recruited to UPE promoters?
No. Most active gene-regulatory regions recruit RNA pol II to cryptic promoters and intiate transcripts called eRNAs.
Enhancers, LCRs and UPEs are all regulating promoters but all recruit RNAP II which transcribes from all these points in the genome.
109
What are enhancer RNAs (eRNAs)?
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Enhancer RNAs (eRNAs) are transcripts which arise from enhancers/LCRs etc. which have cryptic promoters and therefore recruit RNAP II to this area of DNA and this results in the production of a transcript.
These eRNAs spread all over the genome and they are actually long non-codign RNAs.
```
110
What is the function of eRNAs?
eRNA transcription may be contributing to opening up the chromatin of the ACH rather than producing a functional transcript.
The eRNAs are a form of long non-coding RNAs that ultimately get degraded, by in transcribing from these cryptic promoters at enhancers/LCRs, RNA pol II is being brought to the necessary regions and its powerful chromatin remodelling activity is being recruited to open up active euchromatin domains - it isn't the transcript itself that is important or holds a function, simply the process of eRNA transcription is functionally important in opening up chromosomal domains.
111
How are separated gene regulatory regions able to interact with each other to form active chromatin hubs?
Flexible chromatin domains are generated encompassing activated genes which allow separated gene regulatory regions to interact with each other to form active chromatin hubs.
Each activated gene regualtory DNA region recruits local chromatin-remodelling co-activators and RNA pol II and chromatin opening is enhanced by the production of unstable long non-coding RNAs such as eRNAs.
112
If the whole region of an active gene is being transcribed into different kinds of transcripts, what is special about the mRNA?
Higher eukaryotic pre-mRNA transcripts have introns which are removed by the splicing machinery and the mRNA is modified to make the mRNA stable.
These eRNAs are ultimatley just degraded by RNases, they aren't protected or modified or processed.
The splicing machinery is a complex of proteins that declare the mRNA to be special and stabel and confer its exportation from the nucleus.
113
What are the 3 general types of organisaiton of human gene regulatory regions?
1. Upstream promoter elements only - only 10% of human genes fall into this category of having only a small cluster of transcription factor binidng motifs.
These tend to be genes that have relatively simple signalling information being integrated.
2. UPE plus widely separated enhancers/LCRs.
The ehnacers and LCRs are often quite a distance from the UPE and simply the intevening DNA is looped out to allow these regions to interact with the UPE promoter.
3. UPE plus super-enhancers.
Super-enhancers aren't that common and simply occur where it seems lots of little enhancers have all been put together in one region of the genome.
114
Approximately what percentage of human genes have just upstream promoter elements?
10%.
115
What kind of genes tend to only have upstream promoter elements as their organisation of gene regualtory regions?
Generally, the 10% of human genes that only have a small cluster of transcription factor binding sites around the promoter tend to be the genes that have relatively simple signalling information being integrated, e.g. the Metallothionien gene.
116
What is a super-enhancer?
An extended array of transcription factor binding motifs formed when lots of enhancers have all been put together in one region of the genome.
117
Give an example of a human gene that has a super-enhancer?
The RUNX1 gene has loads of mini-enhancers stuck together into a big block approximately 170,000bps long.
118
What happens if we use 3C technology to look at the whole genome/whole chromosomes not just a single gene/few thousand base pairs.
If we move up to the scale of 50,000bp approx. the promoter-enhancer interactions disappear into background noise but other interactions again off the diagonal become apparent.
If we zoom out even further, to the level of the whole chromosome, we see even more interactions now at a larger scale.
These interactions are at the level of millions of base pairs - this indicates two regions of the genome, millions of base pairs apart interacting together.
From this we conclude that regions of chromosomes interact at Mbp scales - these are termed topologically assciated domains - these interactions aren't as tight as interactions between promoters/enhancers but are definitely interactions nonetheless.
119
What region of a chromosome cannot be mapped by 3C technology and why?
Centromeres cannot be mapped using 3C technology becasue they are just repeat DNA and we don't know where all the bits of DNA attach.
120
What sets TAD boundaries?
TAD boundaries are set by insulators that bind CTCF and cohesin.
121
What different types of TADs exist?
Transcriptionally active and transcriptionally inactive.
122
What is a topologically associated domain?
This is a name given to the >1Mbp regions of chromosomes that are interacting with each other as revealed by 3C experiments.
123
What are active TADs?
Active TADs exhibit gene-active histone post-translational modifications and consist of active genes and generally replicate early in S-phase = 'euchromatins'.
124
What are inactive TADs?
Inactive TADs exhibit silent-gene histone post-translational modifications and consist of inactive genes or gene-poor regions and replicate late in S phase = 'heterochromatins'.
125
What is the correlation between TAD organisation of chromosomes and chromatin states?
The TAD organisation of chromosomes also maps onto the 'colours-of-chromatin' model.
A particular TAD will contain only sequences with a particular type of euchromatin or heterochromatin for example, all the genes in a given inactive TAD may have HP1 heterochromatin but this correlation is very tight.
126
Roughly how many genes are contained with a TAD?
Transcriptionally-active TADs specifically often contain several genes or several active chromatin hubs, perhaps 20 genes.
However you can also get tiny TADs comprised only of a single gene.
127
Can TAD formation and DNA:DNA interactions take place across chromosomes.
TAD formation and DNA:DNA interactions generally only happen between sequences on the same chromosome.
When looking a two different chromosomes with 3C technology, we don't tend to see interactions between different chromosomes.
However, this isn't an absolute law.
A variation of 3C methodology called Dip-C is highly sensitive and can be applied at single cell resolution and with this we see some inter-chromosomal specific interactions with TADs from one chromosome interacting with TADs from another chromosome.
However, this isn't extended to homologous chromosomes which largely do not interact.
128
Which variation of 3C technology is highly sensitive and can be applied to single cells?
Dip-C.
129
What do we learn from Dip-C technology about the scale of DNA:DNA interactions?
With the highly sensitive 3C technology called Dip-C we actually see some inter-chromosomal interactions occuring which often aren't seen with standard 3C technology.
130
When we look at contact probabilites from 3C experiments at the level of an interphase nucleus, what do we learn?
Shockingly, when the chromosomes decondense in an interphase nucleus, they don't mix randomly, they actually stay in very particular positions in the nucleus.
We actually see that chromosomes never fully decondense after mitosis and they maintain certain cell-specific inter-chromosomal contacts.
131
What is meant by the phrase 'chromosomes are territorial'?
We know from 3C and fluorescent in situ hybridisation experiments that chromosomes remain relatively distinct in the interphase nucleus, meaning when Chr1 decondenses all the DNA sequences of Chr1 stay roughly together in what we call a territory.
132
How does the location of euchromatin and heterochromatin TADs differ?
Euchromatin TADs are found in the interior of the nucleus whereas heterochromatin TADs are found at the nuclear periphery.
133
What are lamina-associated domains?
Lamina-associated domains (LADs) are TADs that are really intimately stuck to the nuclear lamina.
134
Describe the hierarchy of structure in the nucleus?
The nucleus is split into chromosomal territories which contain topologically associated domains (TADs) and active TADs may contain multiple active chromatin hubs and within this ACH we get down to the gene sequences (the As, Cs, Ts and Gs).
135
What is the current working model for the formation of order within the interphase nucleus?
Rather than following a particular mechanism or design, it is thought that order emerges in the interphase nucleus based on simple biophysics, this is the so-called emergent property.
Order is said to be emergent.
This order is thought to be the result of simple processes like phase separation (e.g. oil separating from water).
Things with particular biological/biochemical properties will go together and therefore active genes will clump together in one phase and inactive genes together in another phase.
These factors e.g. all the activating histone PTMs have common biophysical properties that cause nucleosome chains to condense into distinct chromatin phases - we see this order emerging in the form of ACHs and TADs.
136
What is meant by the emerging property of nuclear organisation?
It is thought, that the order in the interphase nucleus is brought about by simple separation and processing based on biochemcial properties.
Processes like phase separation are actually hugely powerful in the emergence of order in the interphase nucleus.
This applies at the level of separation of activating and inactivating histone PTMs.
It makes sense that genes with active PTMs and activating TFs bound etc. are likely to associate and it is based on this common organising principle that structure emerges in the interphase nucleus.
This kind of model is actually thought to apply to a huge range of other biological scenarios.
137
Is TAD organisation stable?
Overall TAD organisation is stable between cell types and even between similar mammalian chromosomes.
Across different cell types, we broadly see similar TAD organisation (not identical by any means, but similar nontheless).
On the whole TAD organisation is pretty locked into higher eukaryotic genomes.
138
How can TAD organisation be dynamic?
There are instances where TAD organisation shows dynamism often with gene activation/repression.
For example, certain TADs change in parallel with genes being controlled by circadian rhythm and therefore there are oscillating changes in TAD structure.
There are also examples of genes moving between heterochromatin (inactive) TADs and euchromatin (active) TADs depending on their regulated state.
For example, Hox gene expression in mammalin embryos show co-linearity with expression changes in paralle with the anteroposterior body axis and it has been shown the genes that control anterior development (swtiched on first) get gradually extruded from the initially singular active TAD (containing all Hox genes) into an inactive TAD.
This is dynamism in line with changes in differentiation and pattern of gene expression.
This dynamism is linked to phase separation - as a gene becomes activated and recrues active chromatin marks, proteins, PTMs etc. it phase separates again and thus can change TADs.
139
How does the structure and complexity of the M-phase chromosome differ from the interphase nucleus?
Whereas the interphase nucleus appears to be really complicated (with many interactions), the topologically associated domain level of organisation disappears during M-phase chromosome condensation.
The M-phase chromosome is structurally relatively simple.
There is simply a scaffold to which loops of DNA of a particular length (approx 100,000 bps) become attached.
140
What happens to TADs during M-phase chromosome condensation?
They disappear during the M phase of the cell cycle.
This is actually a hugely catastrophic process.
Almost all transcription ceases, RNA polymerases, co-activators and co-repressors and the majority of transcription factors get evicted.
However, gene regulation information isn't completely erased, the pioneer transcription factors remain in place as do many of the histone PTMs that decorate the chromosomes.
141
Does the M-phase chromosome or interphase nucleus show topologically associated domain level organisation?
The interphase nucleus - all these TADs disappear during M phase as does all the complexity.
142
What features remain throughout formation of the M-phase chromosome?
Some of the histone post-translational modifications remain in place almost like bookmarks for future expression.
The pioneer transcription factors also remain in place.
Conversly, most TFs, RNA pol II and co-activators and co-repressors get evicted.
143
Which proteins are important for condensation of the M-phase chromosome?
It turns out M-phase chromatin proteins are required to condense the loops of DNA.
Surprisingly histones aren't really important in this process.
144
Are histone proteins important in M-phase chromosome condensation?
No - there are some changes to histone proteins and they are invovled but they are pretty unimportant.
145
Approximately what length are the loops of DNA that attach to the protein scaffold in the M-phase chromosome?
100,000bps.
146
What is the function of Aurora and Haspin kinases in formation of the M-phase chromosome?
On entry to mitosis, two cell-cycled regualted kinases called Aurora and Haspin phosphorylate H3 tails (Aurora phosphorylates H3S10 and H3S28 and Haspin phosphorylates H3T3) at serine and threonine residues which generates an M-phase-specific chromatin PTM pattern.
This phosphate patterning recruits HDACs, particularly H3S10 recruits a HDAC that deacetylates H4K16 and loss of this acetyl group promoters inter-nucleosomal interaction and can promote aggregation of nucleosome chains to drive M-phase chromosome condensation.
147
Describe the histone modifications that take place during M-phase chromosome condensation and their function?
On entry to mitosis, Aurora kinases are activated which phosphorylate H3S10 and H3S28 and Haspin kinase is also activated which phosphorylates H3T3.
These phosphate groups, particularly H3S10 recruits a HDAC which deacetylates H4K16 and loss of this acetyl group promotes inter-nucleosomal interaction and promotes aggregation of nucleosome chains to drive M-phase chromosome condensation.
148
Briefly describe the experiment that demonstrates histones aren't required for M-phase chromosome condensation
99% of histones from chromosomes are removed during spermatogenesis (because DNA is very tightly packaged into the sperm-head by protamines not huge bulky histones), then add a mouse sperm nuclei to a Xenopus metaphase-arrested egg extract, the sperm chromosomes reform into a perfect M-phase chromatid.
These chromosomes don't have any histones attached to them and yet form perfectly structurally normal condensed metaphase chromosomes.
This demonstrates that nucleosomes are actually irrelevant in the formation of the M-phase chromosome.
149
What is responsible for packaging of DNA in sperm nuclei and why?
In spermatogenesis, histones are removed and instead the DNA is packaged by tiny multiply charged molecules called protamines as opposed to huge bulky histones to try and pack the genome into the tiny sperm-head.
150
Which proteins are responsible for condensing the metaphase chromosomes if not the histones?
Two key proteins are invovled, an enzyme called topoisomerase II alpha and a group of proteins called condensins.
151
What are topoisomerases?
Topoisomerases are enzymes present in eukaryotes and prokaryotes which manipulate DNA topology to control teh presence of plectonemic chromosome supercoils.
Topoisomerases add energy to molecules like DNA and underwind or overwind the helix to form supercoils.
152
Define plectoneme?
A loop of helices (especially of a nucleic acid) twisted together.
153
What does topoisomerase II alpha do?
Topoisomerase is an ATPase that uses energy from ATP and relaxes supercoils.
However, it is also an enzyme that can grab hold of two strands of DNA and cut one and pass a strand between the cut.
This tells us it is both catalytic and structural within the M-phase chromosome scaffold.
154
What are condensin I and II?
Condensin I and II are eukaryotic members of structural maintenance of chromosomes factors.
155
Describe the activities of condensin I?
Condensins have ATPase activity, condensin I is also a topoisomerase and puts positive supercoils into DNA.
156
How do condensins and topoisomerase II alpha interact to condense the M-phase chromosome?
Condensins and topoisomerase II alpha manipulate supercoils causing DNA loops to form supercoiled structures which are then trapped by these proteins.
The condensins probably create and encircle and trap supercoils within M-phase chromosome DNA loops to drive chromosome condensation.
Condensin and topoisomerase II may balance each other and oppose each others topoisomerase activity.
157
Why are there two condensins involved in compaction of the M-phase chromosome?
It is currently thought there is a two step condensation process going on for M-phase chromosome compaction...
1. Linear compression by formation of loops around scaffold.
2. Axial shortening to provide compact structure.
The two condensins are localised in different places in the early stages of M-phase chromosome condensation and this generates a two-step compaction model whereby condensin II which is localised in the nucleus in this early phase is responsible for the first step of the condensation reaction and then condensin I which is localised in the cytoplasm until the nuclear envelope breaks down comes in for the second step.
This working model would suggest why there are two different condensins.
158
Compare the location of condensin I and condensin II in the early stages of M-phase chromosome condensation?
In the early stages of M-phase, condensin I is cytoplasmic and only condensin II binds DNA in the nucleus.
However, at the point when the nuclear envelope breaks down, both become able to bind to chromosomes.
159
Describe the two step model of M-phase chromosome compaction?
Because in the early stages of M-phase chromosome condensation, condensin I is cytoplasmically localised and only condensin II binds DNA in the nucleus.
However, at the point of nuclear envelope breakdown, both condensins become able to bind to chromosomes.
This generates a two-step compaction model suggesting condensin II does the first step of the condensation reaction and then condensin I comes in and does the second step and this explains why two condensins exist.
160
Where does Ki67 bind chromosomes?
Ki67 binds at the periphery of chromosomes.
161
What is the function of Ki67 in M-phase chromosome compaction?
Ki67 binds to the periphery of metaphase chromosomes and appears to act as a capsule/surfactant to keep chromosomes separate from each other.
162
For what processes is chromatin a substrate?
Transcription
DNA replication
DNA repair
163
How is replication of a linear eukaryotic chromosomes initiated?
Replication of lienar eukaryotic chromosomes intiates from multiple replication origins.
Replicating a eukaryotic chromosome simply from a single origin would take too long.
Therefore, multiple origins initiate bi-directional replication forks which eventually meet up to complete the replication process.
164
What is sort-seq?
Sort-seq is a technology which looks at cells, sequences their genome in the S phase of the cell cycle and based on the presence or not of a replication origin, there will be double the number of sequences from that region compared to a region that hasn't started replicating yet.
165
How do origins of replication differ in euchromatin and heterochromatin TADs?
The origins that occur within euchromatin TADs in the interior of the nucleus fire and start replicating really early in S phase, they are the first thing to get replicated during S phase. Origins that are found in active euchromatin are always associated with active chromatin marks e.g. H3K27ac.
In heterochromatin the origins in these TADs start firing very late in S phase.
Late-firing origins associated with heterochromatin are generally associated with repressive histone PTMs.
166
How do we locate origins of replication?
Origins in mammalian cells do not have conserved sequences but occur clsoe to or within activated or repressed gene-regulatory regions.
Oris therefore cannot be identified based on sequence, we can only empirically measure where they are.
Origins are almost exclusively associated with gene regulatory DNA.
167
How do yeast origin of replications differ from human oris?
Yeast origins of replication do have conserved sequences.
168
What is the name of yeast origins of replication?
Autonomously replicating sequences (ARS).
169
Where are yeast origins of replication located?
They are invariably found within gene-regulatory regions, they are essentially co-localised with gene regulatory DNA.
However, interestingly, these yeast ORIs are found to be evolving within gene regualtory DNA whether the gene be turned on or off.
Yeast ORIs are found in nucleosome-free regions and are often close to binding motifs of the Abf1p transcription factor.
170
Why are yeast ORIs found within gene-regulatory DNA?
ORIs are evolving to be within gene regulatory DNA whether turned on or not because they are piggy-backing on chromatin remodelling activities.
An origin will put itself next to a bit of gene regulatory DNA because this means it will end up in a nucleosome-free region/accessible region.
171
What are origin-associated chromatin remodellers and what are they doing?
As well as exploiting the local chromatin remodelling enzymes to place themselves in accessible regions of the genome, ORIs have their own ORI-binding factors such as the ORC complex which directly recruit replication-specific chromatin-remodelling HAT complexes to promote chromatin accessibility.
172
What is HBO1?
HBO1 is a histone acetyltransferase that doesn't work in transcription, it is only involved in replication and is recruited by the ORC complex.
173
What is the ORC complex?
The ORC complex is an ORI-binding factor that is always bound to origins of replication and is specifically able to recruit HBO1 a histone acetyltransferase to add acetyl groups to fluff up nucleosomes and make them generally more accesible.
174
How is the elongating RNA polymerases AKA the transcriptional elongation complex (TEC) a chromatin processign machine?
Elongatign RNA pol II (the TEC) is a huge chromatin process machine that acts by remodelling, removing and replacing nucleosomes as it moves through chromatin making RNA.
There are a load of accessory proteins/co-activators/repressors etc. that ride along the phosphorylated CTD of elongating RNA pol II that modify nucleosomes and make them labile for transcription elongation.
175
Besides RNA pol II, give another multi-molecular complex that also functions as a chromatin-remodeller?
The elongating replicase.
176
How is the replicase a chromatin processign machine?
The replicase, in addition to replicating DNA is remodelling chromatin, removing and replacing histones and nucleosomes as it moves through the chromosomes.
177
What else is replicated by the replicase besides DNA?
The replicase not only replicates DNA but also has to replicate chromatin as a substrate and therefore may play a role in replicating potentially epigenetic information such as that written in histone post-translational modifications.
There is epigenetic information held within the histone code that can be potentially replicated.
178
What kind of method of DNA replication is human DNA replication?
DNA replication/synthesis is semi-conservative.
179
What is PCNA and what is its function?
Proliferating cell nuclear antigen is the sliding clamp on which the replicase rides, but it also serves as an organising platform for auxiliary replications factors including many chromatin replication proteins.
PCNA is responsible for recruitment of DNMT1 to hemi-methylated DNA at the replication fork.
PCNA recruits chromatin-remodelling ATPases such as ISWI and INO80.com which use ATP hydrolysis to break up the structure of nucleosomes as the replicase moves along DNA.
180
What happens to the nucleosomes during DNA replication?
The nucleosomes must be bulldozed off the DNA in order for it to be replicated.
The nucleosomes are broken down and the histones and their interactions are broken down, however this isn't brought about by the replicase itself but instead by chromatin-remodelling ATPases like SWI/SNF.
181
What happens to the histone dimers and tetramers during DNA replication?
The original/parental hisotne dimers and tetramers are ultimately transferred to the FACT and Asf1 histone chaperones.
Both FACT and Asf1 appear to be attached to the replicase via interactions with the Mcm2-7 helicase.
182
Why are histones passed to chaperone proteins when transferred off DNA for DNA to be replicated?
Histone proteins themselves are actually really nasty, they're amazingly highly charged and basic and form aggregates and have lots of nasty consequences if they're hanging around in a cell and therefore, when taken off DNA to allow for DNA replication, they are transferred to FACT and Asf1 chaperones.
183
What are histone chaperones?
Histone chaperones are protein factors which associate with histone dimers or tetramers and stimulate a reaction involving histone storage, exchange or transfer without being part of the final product.
184
How does the new DNA end up with the correct histone proteins?
Following transfer of old/parental histones to histone chaperones (like FACT and Asf1) to allow for DNA replication, the old hisotnes are recycled back onto new daughter chromosomes.
However, the cell will only have half the amount of histones required and so new histones are also needed.
185
What are the two methods of synthesis of new histone proteins?
1. Replicative histones.
| 2. Replication-independent histones.
186
How are new replicative histones synthesised?
Large amounts of the canonical histones H2A, H2B, H3 and H4 are synthesised and incorporate during S-phase.
Multi-copy genes; regulated burst of transcription and translation; mRNAs have no poly(A) tail.
187
What is strange about the mRNA transcripts of replicative histones?
They have no poly(A) tail.
188
What is strange about replicative histone genes?
They don't have introns.
| They are multi-copy genes.
189
How are new replication-independent histones synthesised?
Variant histone forms are produced at modest level and incorporated into chromatin throughout the cell cycle.
These are single/low-copy genes with constituted transcription and translation and the mRNAs are polyadenylated.
190
At what point in the cell cycle do replicative histones become incorporated into chromatin?
S phase.
191
At what point in the cell cycle do replication-independent histones becomes incorporated into chromatin?
Throughout the cell cycle.
192
Give some examples of replication-independent histone variants?
H3.3, CENP-A, H2A.X, H2A.Z, macroH2A, H2ABbd.
193
How do the mRNAs of replicative and replication-independent histones vary?
Replicative histone mRNAs aren't polyadenylated whereas replication-independent histone mRNAs are.
194
How does the number of replicative histone genes compare with the number of replication-independent histone genes?
Replicative histone genes are mutli-copy, we have 61 copies of each replicative histone because we need to make lots of them! Whereas replication-independent histones are low/single-copy genes.
195
How many copies of each replicative histone gene do we have?
61
196
How are new histone dimers/tetramers assembled?
New replicative histone dimers/tetramers are assembled on and bound by histone chaperones (similar to the chaperones that hold onto the replicative histones that get recycled).
NAP-1 is a histone chaperone which binds new H2A:H2B dimers.
Asf1 binds new H3:H4 dimers and once in the nucleus, hands them to CAF-1 where they appear to associate as tetramers.
197
Which histone chaperone binds new H2A:H2B dimers?
Nucleosome Assembly Protein (NAP-1).
198
Which histone chaperone binds new H3:H4 dimers?
Anti-silencing factor (Asf1).
199
What histone chaperone are new H3:H4 dimers associated with when they appear to associate as a tetramer?
Chromatin Assembly Factor (CAF-1).
200
What mark is unique to newly synthesised H4?
New histone H4 is marked by deacetylation on K5 and K12 by the HAT1 enzyme.
This diacetyl mark is unique to newly synthesised H4.
201
Which newly synthesised histone is the diacetyl K5 K12 mark unique to?
Newly synthesised histone H4.
202
How are new histone:chaperone complexes transported into the nucleus?
New histone:chaperone complexes are transported into the nucleus at S-phase by specific karyopherins.
203
What is the function of karyopherins?
Karyopherins are molecules that move proteins in and out of the nucleus and histones have their own set of karyopherins tha tmove the newly synthesised histones into the nucleus.
Karyopherins are invovled in new histone transport, transporting the new-histone:chaperone complexes into the nucleus at S-phase.
204
Which histones does Kap114 transport into the nucleus?
H2A and H2B.
205
What histones does Kap123 transport into the nucleus?
H3 and H4.
206
Which karyopherin is responsible for the transport of H2A and H2B into the nucleus?
Kap114.
207
Which karyopherin is responsible for the transport of H3 and H4 into the nucleus?
Kap123.
208
Once synthesised, what happens to the old and new histones during DNA replication?
During S phase, the new histones along with their chaperones which bind to PCNA end up stationed around the replication fork around the replicase ready to go back onto the duaghter strand.
The old and new histones descend on the replicase becoming bound to the replicase ready to be processed and reloaded.
209
What is the function of CAF-1?
CAF-1 is a histone chaperone that new H3:H4 dimers associated with when they appear to associate as a tetramer.
CAF-1 in association with NAP-1 forms the nucleosome assembly complex associated with the assembly of new nucleosomes on the daughter strand.
CAF-1 is also a chaperone for HP1 and picks up MBD1 and KMT1E from the parental DNA.
210
What is the function of NAP-1?
NAP-1 is a histone chaperone which binds new histone H2A:H2B dimers.
NAP-1 in association with CAF-1 forms the nucleosome assembly complex associated with the assembly of new nucleosomes on daughter strands.
211
What is the function of Asf1?
Asf1 is a histone chaperone which binds new H3:H4 dimers.
212
Together what do CAF-1 and NAP-1 form and what do they do?
CAF-1 and NAP-1 act as a nucleosome assembly complex.
The nucleosome reassembly/assembly of new nucleosomes on the daughter strand is catalysed by CAF-1 and NAP-1 as chromatin assembly enyzmes.
213
Which two chromatin-assembly enzymes are involved in the assembly of new nucleosomes?
CAF-1 and NAP-1.
214
How do CAF-1 and NAP-1 promote nucleosome assembly?
In vivo, CAF-1 and NAP-1 recruit chromatin-remodelling ATPases to help assembly and correctly space nucleosomes.
These two histone chaperones both require ISWI complexes NURF, ACF, CHRAC and RSF and the CHD factor CHD1 for nucleosome re-depositioin by the RNA pol II TEC.
215
Aside from histone chaperons CAF-1 and NAP-1, what other chromatin remodelling ATPases are required for nucleosome assembly?
Both histone chaperones require ISWI complexes - NURF, ACF/CHRAC and RSF.
They also require the CHD factor CHD1.
216
Is their any covalent chromatin-remodelling involved in DNA replication?
Covalent chromatin-remodelling is involved in replication, the HBO1 HAT associates with the Mcm2-7 and acts to acetylate further residues on the incoming histone H4.
HBO1 is bound to ORC at ORIs and is a covalent remodeller which modifies chromatin during replication.
217
How is histone deposition initially restricted during DNA replication?
Histone deposition is initially restricted to the (H3:H4) tetramer.
These H3:H4 tetramers are the first thing to be detected and partially wrap around DNA, they're smaller than the normal nucleosome.
Both old and new tetramers are mixed and randomly segregated between each daughter DNA strand.
218
Describe the order of histone deposition on newly synthesised DNA?
Initially histone deposition is restricted to the H3:H4 tetramer with old and new tetramers randomly distributed between each daughter strand.
These partially wrap DNA but are smaller than the normal nucleosome.
Deposition of H2A:H2B lags behind H3:H4 and also mixes new and old dimers, deposition of these H2A:H2B dimers occurs 10-100's of tetramers behind the replication fork.
219
Name two processes which are semi-conservative?
DNA replication
| Chromatin replication
220
How are DNA replication and chromatin replication semi-conservative in different ways?
DNA replication is semi-conservative in that there is one perfectly old and one perfectly brand-new strand of DNA.
Chromatin replication is semi-conservative in that there are 50:50 new and old histones however these are decorated randomly between daughter strands so they aren't semi-conservative in the same way... we don't get one strand of DNA with exclusively new histones and one with exclusively old histones, they are segregated randomly between daughter strands.
221
Does semi-conservative histone replication allow for faithful copying of the epigenetic PTM code?
Yes and no...
In theory, with chromatin-replication being semi-conesrvative, some of the information written into the epigenome in histone PTMs could be transmitted to daugther cells, but this sometimes happens and sometimes doesn't.
222
Does histone recycling provide a mechanism for a memory of chromatin PTM states?
Only for histone H3 and H4 because old tetramers are immediatley re-deposited behind the replication fork.
This H3 and H4 information goes back onto the duaghter strands and provides a memory of previous chromatin-remodelled state.
The same isn't true for H2A and H2B PTM information which gets lost becuase the old H2A:H2B dimers are deposited more slowly.
223
What histone PTM information is thought to be heritable though S-phase and why?
H3/H4 PTMs might be heritable through S-phase by both replication linked and un-linked mechanisms.
H3:H4 tetramers are immediatley re-deposited behind the replication fork on the new daughter strand and therefore the H3:H4 information is passed to the daughter strand.
Howver, H2A:H2B dimers are deposited more slowly and the H2A/H2B PTM information is lost, all teh old and new H2A and H2B histones get mixed up and the information encoded is randomised and therefore, not transmitted to daughter strands.
224
Give an example of histone information being transmitted to daughter strands during DNA replication?
The H3/H4 PTMs recycled at polycomb and HP1 heterochromatin allow re-establishment of this chromatin-spreading mechanism.
The polycomb repressive mark H3K27me3 is immediately put back onto daughter strands after DNA replication and polycomb repressor complexes can then bind this marks and lay them down in the surrounding chromatin.
The pre-existing information (H3K27me3) mark is loaded directly from parental to daughter strand and gives enough information for the chromatin-spreading process to re-establish that chromatin state.
225
Which histone mark does HP1 bind?
H3K9 methyl
226
What is meant by the CAF-1 memory module and what is its function?
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Both the replicase and CAF-1 directly bind to a recruit components of the HP1 system to the replication fork re-establishing the state of HP1 heterochromatin.
The CAF-1 memory module describes the complex of enzymes carried by the replicase that bind via CAF-1 to the PCNA loop - this memory module is riding along DNA with the replicase re-establishing a state of HP1 heterochromatin.
These complex of enzymes include HP1, MBD1 and KMT1E.
The CAF-1 memory module fully restores the HP1 chromatin state direclty during the passage of the replicase.
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227
What is the chaperone for HP1?
CAF-1.
228
What is the substrate for the CAF-1 memory module?
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The newly replicated HP1 heterochromatin provides an immediate substrate for the CAF-1 memory module.
Both H3K9me and methylated CpG marks that characterise HP1 heterochromatin are diluted during DNA synthesis as they are split between two daughter strands, the enzymes that comprise the CAF-1 memory module recognise this on the duaghter stand and immediately re-establish the HP1 heterochromatin.
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229
What is responsible for restoration of the HP1 heterochromatin state during passage of the replicase?
The CAF-1 memory module.
230
What is the only example of direct replication of a chromatin state?
The CAF-1 memory module re-establishes HP1 heterochromatin following passage of the replicase.
231
What are the characteristics of newly replicated chromatin?
Newly replicated chromatin for about 200 nucleosomes or so is really DNase hypersensitive indicating a labile structure and there are lots of acetyl marks put on during their processing.
The newly replicated chromatin retains synthesis-specific PTMs such as H5K5K12ac2.
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What is chromatin maturation?
Immediately after chromatin replication, chromatin is highly DNase sensitive and harbours lots of synthesis-specific PTMs, mainly acetyl marks.
After about 200 nucleosomes have gone by from the replicase, the chromatin matures.
This leaves stable nucleosomes with all those synthesis-specific acetyl marks removed and the whole thing becomes less DNase hypersensitive.
233
How does chromatin maturation occur after chromatin replication?
Chromatin maturation leaves stable nucleosomes that have lost the synthesis-specific acetyl PTMs that are retained immediatley after chromatin replication and mature chromatin is less DNase hypersensitive than newly replicated chromatin.
This process isn't passive and requires chromatin remodelling ATPases of the ISWI class to tighten up nucleosomes and space them appropriately.
Chromatin remodelling ATPases like ACF and RSF contain ISWI class ATPases which establish regular spacing between newly deposited nucleosomes.
The ISWI enzyme seems to contain a molecular ruler.
ISWI chromatin-remodelling ATPases and histone deacetylases are attached to PCNA and therefore associated with the replicase and are important in chromatin maturation.
It appears the replicase casts off PCNA molecules which are being used as nucleation sites to bind chromatin-remodellers that mature the chromatin.
234
Which chromatin remodelling ATPase has a molecular ruler and what is its function?
ISWI class ATPases appear to contain a molecular ruler which is used to establish regular spacing between newly deposited nucleosomes after chromatin replication and during chromatin maturation.
235
How is chromatin maturation indirectly linked to replication?
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Chromatin maturation involves ISWI class ATPases and histone deacetylases which are associated with the replicase via attachment to PCNA. These ISWI ATPases contain a molecular ruler that is used to establish regular spacing between newly deposited nucleosomes during chromatin maturation.
It appears PCNA molecules are cast off the replicase and used as nucleation sites for binding of chromatin-remodelling ATPases and therefore, chromatin maturation is indirectly linked to the replicase itself.
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236
Which chromatin process is directly linked to the replicase?
Restoration of HP1 heterochromatin following DNA replication is directly linked to the replicase because the it is performed by the CAF-1 memory module which is attached to the PCNA look, this complex is directly riding along with the replicase and therefore, when hemi-methylated CpG motifs are recognised in daughter strands, factors directly associated with the replicase immediatley restore the HP1 heterochromatin.
237
How are PCNA molecules invovled in chromatin maturation?
PCNA molecules are periodically ejected from the replicase, they're case off from the replicase and replaced by new PCNA complexes, particularly during lagging strand synthesis and these ejected PCNA molecules act as nucleation/recruitment sites for chormatin maturation factors e.g. histone deacetylases which remove the synthesis-specific PTMs to leave mature chromatin and ISWI ATPases which act as a molecular ruler to establish regular spacing of newly deposited nucleosomes.
238
What is the function of ISWI ATPases in chromatin maturation?
ISWI ATPases contain a molecular ruler which helps establish regular spacing between newly deposited nucleosomes.
239
What is the function of histone deacetylases in chromatin maturation?
Histone deacetylases remove the synthesis-specific PTMs that are retained on newly replicated chromatin that help conver DNase I hypersensitivity.
240
Give some examples that suggest 'epigenetic' chormatin states are actively and accuratley replicated like the DNA genetic code?
Old histone H3/H4 molecules are immediately recycled to daughter strands therefore, tagging them with parental PTMs and from this the same marks can be established on new H3/H4 replicative histones.
HP1 and other chromatin maturation factors (like ISWI ATPases) are linked to the replicase and drive direct replication of heterochromatin states.
241
Give some examples that suggest 'epigenetic' chromatin states are actively and accurately replicated like the DNA genetic code?
Gene-regulatory chromatin structures and many histone PTMs states are laid down/generated by transcription factors and co-activators/co-repressors completely independently of DNA replication.
Both HP1 and matured chromatin states can re-estbalish themselves without replication and using replication-independent mechanisms.
So, in theory, replication isn't absolutely required for these chromatin states to be transmitted, although it can help.
242
Is chromatin really epigenetic?
By strict definition, something can only be epigenetic if it is transgenerational, i.e. heritable across both mitosis and meiosis and chromatin states whilst sometimes heritable thought mitosis aren't heritable through meiosis and therefore, by strictest definition probably aren't epigenetic.
Despite not truly being epigenetic, chormatin states are relatively stable and actively maintained, just not permanently heritable.
