IT1 Flashcards
1
Q
How could Y chromosomes have degenerated?
A
Gain of a sex determining allele on one pair of equivalent autosomes that selected for male beneficial/female antagonist mutations on the chromosome. It would then be advantageous to retain this allele, so recombination suppression over all or most of that chromosome is selected for. The absence of recombination results in the accumulation of deleterious mutations, caused by Muller’s Ratchet, leading to degeneration of the chromosome.
Purifying selection also cannot occur, and so there’s an accumulation of transposable elements.
2
Q
What is a nucleoid?
A
Meaning nucleus-like, it contains the genetic material of prokaryotic cells.
3
Q
What is the structure of B-form DNA?
How does under-winding or over-winding impact the structure?
A
~10.5 bp per turn
Right-handed double helix
Under-winding: negative supercoils (right-handed)
Over-winding: positive supercoils (left-handed)
4
Q
What leads to nucleoid formation in prokaryotes?
A
- Formation of negative supercoils by topoisomerases, such as DNA gyrase.
- Histone and nucleoid-associated proteins that organize the DNA into a compact structure that’s more resistant to damage.
5
Q
What is a plectoneme?
A
A structural feature of super-coiled DNA, forming a twisted structure that resembles a series of loops.
[Think of when you over twist some rope and it forms individual loops coming off the central strand.]
6
Q
Which nucleoid-associated proteins function to bridge and loop DNA?
Describe the structure and function of each.
A
Histone-like nucleoid structuring proteins:
- short protein
- binds AT-rich DNA via C-terminus
- N-terminus contains self-associating domains to form ‘daisy chain’ filaments which stiffen the DNA
SMC:
- 2 SMC monomers and a Kleisin subunit form a ring
- The ring captures DNA to enable looping
- Head domains have ATPase activity to pull DNA through the ring
7
Q
Which nucleoid-associated proteins function to bend DNA?
Describe the structure and function of each.
A
Factor for inversion stimulation (FIS):
- Dimer
- Binds major groove of DNA
- Bends DNA between 50-90 degrees
Integration host factor (IHF):
- Heterodimer of alpha and beta subunits
- Prefers to bind AT-rich DNA
- Beta-ribbon arms protrude into the minor groove to kink the DNA 160 degrees.
8
Q
Which nucleoid-associated proteins have functions beyond packaging DNA? What are these functions?
A
IHF can bend the DNA upstream of promoters to allow DNA elements bound by TFs get closer to the gene promoter - GENE REGULATION.
SMC complexes are loaded onto newly replicating daughter chromosomes to support chromosomes individualization and segregation into daughter cells - CHROMOSOME SEGREGATION.
9
Q
How was chromatin discovered? What is its structure?
A
The term “chromatin” was first coined by German anatomist Walther Flemming in 1880, who observed it under a microscope while studying cell division in salamander larvae. Flemming noticed that the nucleus of the cell was stained differently from the surrounding cytoplasm, and he named the material that made up the nucleus “chromatin,” from the Greek word “chroma,” meaning color.
Negative stains showed a ‘beads on a string’ configuration, with particles of ~70A that had 15A gaps between them.
10
Q
What experiment confirmed the presence of histones in chromatin?
A
Liver cells were treated with DNase before the DNA was isolated and separated on a gel. The gels showed a consistent band size of ~200bp at a time, and as the digestion went on for longer, the smaller species became more intense.
If the DNA was purified before DNase was added, this patterning wasn’t observed. This suggests that there’s some repetitive spacing of sites that are accessible to DNA, but would normally be protected.
Other work on purified DNA showed the presence of 5 distinct histone proteins, and that these formed the basic scaffold for chromatin assembly.
11
Q
What was Roger Kornberg’s 1974 model of chromatin? How was this work tested?
A
- Chromatin is composed of a repeating unit of 2 of: H3, H4, H2A and H2B, and 200bp of DNA.
- Chromatin fibres consist of many of these units to form a flexibly jointed chain.
EM indicated a native chromatin configuration could only form when all of these parts were present, and adding H1 compacted the structure further. This lead to the coining of the term ‘nucleosome’ to describe the DNA/histone particles.
12
Q
What did the atomic structure of the nucleosome reveal about its structure? Explain for the low-resolution, 3.1A, and 2.8A resolution structures that were solved.
A
LOW RESOLUTION:
There are ~1 3/4 turns of a left-handed coil of double-stranded DNA around a disk shape histone octamer.
The nucleosome has a dyad axis of rotational symmetry.
A further 3.1A structure was resolved to show histones are made up of 3 alpha helices that are linked by 2 short loops and have flexible N-terminal tails. H2A also has an extended C-terminal tail.
The 2.8A structure provided a detailed view of interactions where histone tails were positioned with respect to the DNA.
13
Q
Describe the histone-fold structure and how this is used to form histone dimers.
A
The histone-fold structure consists of three alpha helices separated by two loops, and it is highly conserved across different histone proteins and species.
The skewed u-shaped folds of the histone monomers allows for them to slot together via a ‘handshake’ to form dimers.
14
Q
How is DNA bent around the histone octamer? How is this stabilized?
A
DNA is bent around the histone at AT rich sequences through narrowing of the minor groove.
- It’s sequence-independent.
- Uses hydrogen bonds.
This is stabilized by the high-degree of positive charge in the histone octamer, coming from the >20% lysine and arginine residues.
15
Q
How do prokaryotes and eukaryotes utilize DNA supercoiling to compact their DNA?
A
Prokaryotes use DNA gyrases to negatively supercoil their DNA, forming the nucleoid with plectonemes.
Eukaryotes use supercoiling to wrap their DNA around histone octamers and compact the DNA 6-7 fold.
16
Q
What is the structure and role of histone H1 in the histone octamer?
A
Structure:
- Flexible N-terminus
- Intrinsically-disordered C-terminus that’s highly basic
- Lacks histone fold
- Globular domain sits on the dyad and interacts with linker DNA where it exits the nucleosome.
Function:
- Holds DNA in a more rigid and compact conformation
- Can be rapidly exchanging with other H1 molecules to alter chromatin structure of function, aiding in chromatin dynamics
17
Q
How are new nucleosomes synthesized and deposited on DNA?
A
- HSP70/Hsc70/NASP promote folding and dimerization of H3/H4, whilst ASF1 binds H3 to prevent tetramerization.
- MCM2 shields DNA binding interface..
- Delivery to CAF-1 in the nucleus allows binding to the PCNA and supports tetramerization and deposition on the DNA.
- NAP1/FACT deposits H2A/H2B dimers on the tetrasome.
- Chromatin-remodelling enzymes wrap the rest of the DNA around the nucleosome.
H1 deposition is less well understood.
18
Q
What is the chromatosome?
A
Nucleosome core particle + H1
19
Q
How has the positioning of histones on DNA been studied? What did this reveal about histone occupancy across the genome.
A
EM images and digestion assays show homogeneous and uniform coverage of DNA…
Nucleosome mapping (e.g., DNase seq) showed nucleosome occupancy is high, with regular arrays that lack defined phasing. But some regions have more regular phasing with low or no occupancy at TSS.
The same can be said for other non-gene elements, such as enhancers, insulators and origins of replication.
20
Q
What dictates nucleosome phasing and organization?
How was this shown?
A
- AT-rich sequence of DNA for nucleosome binding and bending. Shown via modeling predictions and testing these patterns.
[Other gene promoter sequences can also influence phasing.] - Chromatin-remodeling enzymes.
The models weren’t enough, but adding ATP to the patterns made them much more like in vivo patterns, suggesting ATP dependent processes…aka chromatin remodeling enzymes.
Deletions of the genes encoding these enzymes in yeast resulted in far less-defined nucleosome phasing. - DNA binding factors.
High occupancy of DNA binding factors competes with nucleosome occupancy, mostly at promoters, enhancers, insulators and origins. e.g., CTCF in mammals which binds insulators and causes high levels of phasing.
21
Q
What are chromatin remodeling enzymes? Describe their structure.
A
Chromatin remodeling enzymes are a class of proteins that use the energy from ATP hydrolysis to alter the structure and position of nucleosomes on DNA.
There are several families of chromatin remodeling enzymes, each with their own unique structure and function.
- 2 conserved lobes for movement along the DNA
- ATPase domain + auxilliary domains
- ATPase domain powers movement (like 2 hands walking along the DNA)
22
Q
How do remodeling enzymes move nucleosomes?
A
They use ATP to translocate or slide DNA over the surface of the nucleosome:
They anchor themselves to histones where the ATPase can then grab onto the DNA and translocate it towards the nucleosome dyad.
This process disrupts local histone-DNA interactions and as the DNA is pushed towards the dyad, the distortion is translated around the nucleosome to cause DNA translocation.
23
Q
Why might defined phasing at certain regions of the genome be important?
A
TRANSCRIPTION/TRANSLATION:
Nucleosome occupancy can block access to the DNA.
Gene promoters have evolved features that create nucleosome-free regions which may be important for GTF and RNAPII binding.
24
Q
How is phasing/occupancy of nucleosomes related to the function of the genome?
A
Nucleosome occupancy usually blocks transcription from occurring.
To overcome this, pioneer factors can recognize partial binding sequences on top of the nucleosome to recruit chromatin remodeling enzymes that displace the nucleosomes for transcription.
This is referred to as assisted loading and is likely key to how DNA sequences are unveiled for new functions.
25
What is the 30nm fiber, and how was it discovered?
The 30nm fiber is a compacted and more condensed form of chromatin that is formed when nucleosomes are further organized and packed together.
This was discovered using EM to visualize chromatin at higher resolution, demonstrating that increased cation charge partially shielded the DNA negative repulsion to create these higher order fibers.
26
What is the structure of the 30nm fiber?
The nucleosomes are stacked on top of each other in a zig-zag orientation with twisted tetranucleosome units.
H1 binds at the dyad and determine the trajectory of the exit or entry linker DNA.
27
How do H2A and H2B molecules interact in the 30nm fiber?
The interaction between H2A-H2B dimers in the 30nm fiber is thought to occur primarily through electrostatic interactions and hydrogen bonding between amino acid residues on the surface of the nucleosome.
This mediates stacking in the tetranucleosome unit.
28
What is the role of acidic patches in 30nm fiber formation?
The acidic patches on the face of one tetranucleosome unit interacts with the basic tail of H4 on the adjacent tetranucleosome.
29
What experiment has suggested that the 30nm fiber doesn't actually exist?
SAXS measurements on mitotic chromosomes from various cell types showed that it was ribosome contamination primarily causing the 30nm SAXS peaks.
When ribosomes were washed away, most cells showed no 30nm peak...
30
What is STORM, and how has it aided in determining chromatin organization in cells?
STORM is a super-resolution microscopy technique that uses fluorescent probes that can be turned on and off to obtain highly precise spatial information about the positions of individual fluorescent molecules in a sample.
STORM has been used to visualize the higher-order organization of chromatin, suggesting there's no evidence for a regular 30nm fiber.
31
What is chromEM tomography, and how has it aided in determining chromatin organization in cells?
Combines cryo-EM with computational methods to generate 3D models of chromatin.
It showed that chromatin is far more heterogeneous than originally anticipated, with no evidence for a 30nm fiber.
32
Define the following terms:
- Euchromatin
- Heterochromatin
- Constitutive heterochromatin
- Facultative heterochromatin
Euchromatin: 'open' chromatin
Heterochromatin: 'closed' chromatin
Constitutive heterochromatin: constantly 'closed' chromatin
Facultative heterochromatin: silenced chromatin that can be activated when needed
33
Describe the genetic screens in Drosophila that identified Su(var) and E(var) genes, responsible for variegation and dose-dependence.
In these screens, researchers searched for mutations that caused changes in the expression of genes located near chromosomal regions known as "heterochromatin". [Red vs white eye experiments]
Su(var) mutations suppressed the variegation phenotype that results from the abnormal expression of genes located near heterochromatin.
E(var) mutations enhanced the variegation phenotype in a dose-dependent manner, with the severity of the phenotype increasing with the number of copies of the mutation.
Su(var) and E(var) mutations were later discovered to impact the structure and organization of chromatin. Su(var) genes maintain condensed heterochromatin, whilst E(var) genes relax the structure.
34
Give an example of a protein encoded by a Su(var) gene. What is its function?
Su(var)3-9 encodes a histone methyltransferase that uses SAM to methylate K9 on H3 tails.
35
How do modifications to chromatin influence its function?
Give an example for each.
1. Act as a binding site for reader proteins that then effect function.
E.g., Su(var)3-9 methylation promotes HP1 binding that dimerizes to hold the nucleosomes in a more compact configuration.
2. Directly influence nucleosome structure and interaction.
E.g., acetylation neutralizes the positive charge on histone lysines, reducing the thermostability of the nucleosome (if acetylated on the tail) and its affinity for DNA (if core is acetylated).
36
How does acetylation influence inter-nucleosome interactions?
The acidic patch on the face of one tetranucleosome unit interacts with the basic tail of H4 on the adjacent tetranucleosome.
Acetylation of that H4 tail prevents the interaction from occurring, resulting in a de-compaction that is similar to removing the H4 tail entirely.
37
What is TSA, and how has it been used to study the effects of acetylation on chromatin structure in vivo?
TSA inhibits HDAC activity and leads to histone hyperacetylation.
Super-resolution imaging shows a more uniform distribution of chromatin after TSA treatment.
38
What do bisulfite-seq and ChIP-seq reveal about chromatin structure?
Bisulfite seq:
Bisulfite treatment converts C to U, but leaves methylated C alone. NGS can then show the methylation status and identify CpG islands.
ChIP-seq:
Proteins (e.g., nucleosomes) are fixed on the DNA and immunoprecipitated to allow for identification of the regions of DNA to which they bind.
39
Where can DNA methylation be found in the genome, and what does it do?
Primarily found on CpG dinucleotides within vertebrate genomes. Short stretches of CpG islands escape DNA methylation (CpG islands) and these are normally associated with vertebrate gene promoters.
Active enhancers tend to be hypomethylated.
However, removal of DNA methylation does little to impact gene expression, suggesting it doesn't primarily function to regulate gene expression. Instead, its removal can lead to increased expression of young retrotransposons. So it protects cells from retrotransposon elements jumping into coding elements, etc.
40
How are transposable elements suppressed in vertebrate genomes?
1. General methylation (prevents TF binding and recruits HDACs to maintain compaction)
2. SETDB1-H3K9me3 pathway (potentially recruits HP1 for compaction, or recruits the HUSH complex)
41
How does SETDB1 know to methylate parasitic DNA elements? How does this methylation then spread?
It relies on a family of KRAB domain zinc finger DNA binding proteins that recognize the sequences in transposable elements first. These proteins interact with an adaptor protein which recruits SETDB1 to nucleate H3K9me3.
KRAB DBPs are rapidly evolving, likely in a 'red-queen' fashion to keep up with the hundreds of new types of invasions.
SETDB1 forms an alternative complex with MMP8 that binds H3K9me3 to cause spreading of methylation.
42
What is the HUSH complex?
The HUSH complex consists of three core components: TASOR , MPP8*, and MORC2. These proteins are thought to work together to recruit additional factors that promote transcriptional repression of retroviral elements.
MORC2 is an ATPase that is thought to compact chromatin.
*MPP8 forms an alternative complex with SETDB1 to spread methylation on transposable elements.
43
How is chromatin organized and modified at gene promoters?
1. Contain histone variants H2AZ and H3.3, making the nucleosomes intrinsically less stable and are dynamic.
2. H3K4me3
44
How can H3K4me3 affect promoter function?
Give examples of 3 proteins that can bind this modification and what their functions are.
1. Affects nucleosome-DNA interactions.
2. Recruits proteins that bind acetylated histones:
SAGA acetyltransferase complex binds H3K4me3 to open up the chromatin more.
CHD1 (chromatin remodeling enzyme) helps RNAPII pass the nucleosomes by partially unwinding the upstream DNA and pumping the DNA towards the polymerase.
TAF3 is part of the TFIID complex which must bind to form the PIC containing RNAPII.
45
What is the Polycomb complex? What does it do?
A repressive complex composed of two chromatin modifying enzymes: PRC1 and PRC2.
- PRC1 is a H2A ubiquitin ligase
- PRC2 is a H3 methyltransferase
This complex binds non-methylated DNA in CpG islands that aren't actively transcribed, initiating a feedback system that leads to Polycomb chromatin domain formation and spreading. This is achieved through PRC1 and PRC2 modifications causing the recruitment of more PRC1 and PRC2 machinery.
46
How does gene body chromatin differ from gene promoter chromatin? What machinery is involved?
Gene promoter: H3K4me3
Gene body: H3K36me3
During transcription initiation, Ser5 on the CTD of RNAPII is phosphorylated, recruiting SET1. SET1 deposits H3K4me3 on gene promoters.
As RNAPII transitions into elongation, the CDT is phosphorylated at Ser2. SET2 can bind this and deposit H3K36me3 in the gene bodies.
47
What are the roles of H3K36me3 in gene body chromatin architecture and function?
1. Transcription - recruits demethylaters and HDACs.
2. DNA methylation - recruits HMTs to reinforce DNA methylation in gene bodies.
3. Splicing - recruits RNA splicing factors to the gene body, facilitating proper splicing of nascent transcripts via co-transcriptional splicing.
4. DNA repair - recruits proteins that are involved in mismatch repair and homologous recombination.
48
What histone modification characterizes enhancer elements? What protein deposits these?
H3K4me1. This modification is catalyzed by the histone methyltransferase complex MLL3/4 and is specifically enriched at enhancer elements rather than promoters.
In addition to H3K4me1, enhancer elements may also be marked by other histone modifications, such as H3K27ac. H3K27ac is caused by CBP/P300 which can help to support PIC formation and enable transition of RNAPII into elongation mode.
49
What is the role of CTCF in mammals?
A DNA binding factor that functions to insulate the genome, blocking the capacity of enhancers to activate promoters from different regions of the genome.
50
How do insulators work to regulate genes?
They can form cohesin rings that uses ATP to loop the DNA. The extrusion is then halted by CTCF interacting with a defined component of the cohesin ring.
Within regions of extrusion, communication can occur between enhancers and promoters, but CTCF blocks this communication between adjacent loops e.g., TAD domains.
51
What is the role of the centromere in cell division?
During cell division, the centromere serves as the attachment point for spindle fibers, which are microtubules that extend from opposite poles of the cell and attach to the kinetochores located at the centromere of each chromosome.
The spindle fibers then pull the chromosomes apart, so that each daughter cell receives an identical set of chromosomes.
52
How are centromeres and kinetochores distinguished? What are their basic functions?
Centromere: chromatin structure under the kinetochore that specifies where the kinetochore will form.
- Segregates chromosomes
- Holds sister chromatids together
- Coordinates the above processes
Composed of DNA sequence and proteins.
Kinetochore: protein complex that microtubules attach to during chromosome segregation.
53
Compare type I and II alpha satellite DNA.
Type I:
- Rich in CENP-B binding sites and CENP-A nucleosomes
- Assembly site for kinetochore
- 'open' chromatin
Type II:
- fewer CENP-B binding sites
- heterochromatic
- enriched in condensin and cohesin
54
How was centromeric DNA and proteins discovered in budding yeast? How were centromeric proteins then discovered in humans?
A genetic assay for chromosome loss made it possible to isolate centromeric DNA from yeast. The yeast genome was digested and fragments put into plasmids and transfected into cells. Only plasmids that contains centromeric DNA could replicate stably, and so these cells had their plasmids sequenced.
The centromeric DNA was then used to isolate centromeric proteins, using an EMSA gel that showed heavier DNA fragments i.e., DNA bound by protein.
Human centromere proteins were found when patients with scleroderma were shown to have antibodies that recognized the centromere proteins (CENP). Subsequent studies identified the individual proteins within the CENP complex: CENP-A, CENP-B and CENP-C.
55
What is the function of centromeric DNA, and how is it organized?
Centromeric DNA is highly organized and consists of a conserved DNA sequence called the centromere core, which is typically several kilobases in length. The centromere core is flanked by variable amounts of heterochromatic DNA that often contain transposable elements, satellite DNA, and other repetitive sequences.
The centromere core is composed of CENP-A histone H3 variants.
Centromeric DNA is made of alpha-satellite DNA that forms higher-order repeat units of 171 bp monomers.
56
What is the role of centromeric alphoid DNA in centromere formation?
Centromeric alphoid DNA (alpha-satellite) is a type of tandem repeat DNA sequence that is found in the centromeric regions of human chromosomes.
It's AT-rich, and contains CENP-B and CENP-A binding domains which are needed for de novo centromere formation.
57
Describe the tandem repeat structure of centromeric alphoid DNA.
Centromeric alphoid DNA is a highly repetitive DNA sequence found in the centromere of most human chromosomes. It consists of tandem arrays of a 171 base pair repeat unit, which is composed of two subunits, the 68 base pair higher-order repeat (HOR) and the 13 base pair basic repeat unit (BRU).
The HOR is composed of two parts, an AT-rich region and a GC-rich region, that are separated by a CENP-B box, which is a binding site for the centromere protein B. The GC-rich region contains a conserved motif known as the CENP-A box, which is required for the recruitment of the centromere-specific histone variant CENP-A to the centromere.
58
What are neocentromeres, and how are they formed?
Neocentromeres are a type of centromere that forms in non-centromeric regions of chromosomes i.e., centromeric DNA is not only insufficient, but isn't actually needed to make a centromere.
Formed via:
1. Deletion or inversion of the original centromere
2. Epigenetic reprogramming to acquire centromeric features
3. Translocation of centromeric DNA to other regions
59
Describe the structure of centromeric chromatin. How is CENP-A localized?
CENP-A histone H3 variant
Other CENP proteins forms a network of interactions with the DNA and CENP-A nucleosomes.
CENP-A contains a targeting domain that binds a CENP-A specific histone chaperone (HJURP) which deposits it on the centromeric DNA. FACT is involved in its incorporation.
60
How is centromeric and pericentric chromatin organized in the nucleus?
Centromeric and pericentric chromatin are organized in a distinct manner in the nucleus. In interphase, centromeric chromatin is often located at the nuclear periphery or near the nucleolus. Pericentric chromatin is generally found clustered in distinct regions of the nucleus known as heterochromatic domains.
Human centromeric DNA is extremely long, yet there are only a few nucleosomes involved. It's thought that the DNA folds in 3D space to form a platform with centromeric proteins concentrated on one face for kinetochore assembly.
61
What is the role of pericentric domains in the maintenance of chromosome integrity and sister chromatic cohesion?
The pericentric regions are the site of kinetochore assembly, which is necessary for proper chromosome segregation during cell division.
In addition, the pericentric regions are rich in heterochromatin, which helps to stabilize chromosome structure and prevent the formation of DNA breaks or aberrations. The cohesion between sister chromatids is also maintained by proteins that are associated with the pericentric regions, including cohesin complexes and condesin.
62
How were telomeres initially discovered?
Telomeres were initially discovered by Hermann Muller in 1938 while studying the fruit fly Drosophila melanogaster. He observed that chromosomes appeared to be capped at the ends by a special structure that prevented them from fusing with other chromosomes.
63
How was telomeric DNA discovered? What is its sequence and structure?
Functional cloning - yeast genome was digested and ligated to gene sequences. Only fragments that encoded telomeric DNA would make the gene sequences stable. These stable sequences could then be isolated and sequenced.
SEQUENCE:
Telomeric DNA consists of sequence repeats. In humans, this is TTAGGG, but it varies across species.
STRUCTURE:
Telomeric DNA is double-stranded, with a G-rich single-stranded 3' overhang that forms a T-loop to protect the end of the chromosome.
64
What is the end replication problem and how is it solved?
What was the evidence for this?
The end replication problem refers to the inability of DNA polymerase to fully replicate the ends of linear chromosomes. This is made worse by the 3' overhang found in telomeric DNA.
To overcome this, cells use telomerase.
EVIDENCE:
1. Tetrahymena were used to show telomere length is variable and telomeric sequences can be seeded de novo.
2. Tetrahymena telomeric DNA was stably and heritably maintained in yeast cells via the addition of yeast telomeres. Hence, telomeres are made de novo.
3. Biochemical isolation of the telomere lengthening activity. Addition of protease OR RNase abolished activity...the enzyme requires RNA.
4. The killer experiment: changing the RNA sequence alters the telomere DNA sequence i.e., the RNA is the template.
65
What is the role of telomerase RNA?
Template for the synthesis of telomeric DNA by reverse transcriptase activity of telomerase.
It also has structural roles.
66
How is telomerase expression regulated, and how does this link to cancer?
In normal somatic cells, telomerase expression is tightly regulated and telomerase activity is low or absent, leading to progressive telomere shortening with each cell division. In contrast, most cancer cells have high telomerase activity, which allows them to maintain their telomeres and continue to divide indefinitely. Upregulation of telomerase activity is therefore a hallmark of cancer cells, making telomerase an oncogene.
67
What is the end protection problem and how is this overcome?
What experiment showed this?
The end protection problem refers to the vulnerability of chromosome ends to be recognized as double-stranded breaks and activate DNA damage response pathways, which could lead to genomic instability and cell death.
- Shelterin complex
- Formation of protective T-loops
Protein fractionation was used to isolate proteins from nuclear extracts that specifically bound telomeric DNA sequences. The protein was purified and mass spec was used to identify it as TRF1.
68
What is the Shelterin complex and how does it function in t-loop formation?
The Shelterin complex is a protein complex that binds to telomeres and regulates their structure and function. It is composed of six proteins including TRF1, TRF2 and POT1.
TRF1 and TRF2 form homodimers that bind double-stranded DNA with nanomolar affinity.
POT1 binds to the single-stranded overhang of the telomere, preventing it from being recognized as damaged DNA and protecting it from degradation.
Together, these proteins form a protective structure called the t-loop, where the single-stranded overhang is folded back and base-paired with the double-stranded telomeric DNA. It's able to do this because the telomeric DNA is made up of the same repeats!
69
What is a t-loop, found within telomeres? How do we know this exists?
Its consists of a single-stranded DNA overhang at the 3' end of the telomere that invades the double-stranded telomeric DNA and forms a displacement loop (D-loop) structure. This D-loop structure is stabilized by the shelterin complex, which includes proteins such as TRF1, TRF2, and POT1. The t-loop structure is important for protecting the chromosome ends from being recognized as double-stranded breaks, which could lead to DNA damage responses and chromosomal instability.
It has been visualized using both EM and super-resolution imaging.
70
How is telomere homeostasis maintained?
The longer the telomere becomes, the less active telomerase is. When telomeres are short, then telomerase is more active.
This is mediated by Shelterin, as the more Shelterin there is, the less telomerase activity there is.
71
Describe the structure of budding yeast centromeres.
The centromere core contains 3 elements: CDEI, II and III.
CBF1 is a sequence-specific DNA binding protein that binds CDEI. CBF3 is a protein complex that binds a motif in CDEIII.
CDEII is the least conserved, but is highly AT-rich and is bound by Cse4 nucleosome.
72
What is the constitutive centromere-associated network (CCAN)?
The constitutive centromere-associated network (CCAN) is a protein complex that's responsible for various functions at the centromere, including kinetochore assembly.
It's formed of many types of CENP proteins, hence the 'alphabet soup' name!
73
What are the individual roles of the 3 human centromeric proteins?
CENP-A: histone H3-like protein
CENP-B: sequence-specific DNA binding protein
CENP-C: large DNA binding protein
74
What's the difference between point centromeres and regional centromeres?
Point: highly defined sequence recruiting sequence-specific DNA binding protein e.g., yeast
Regional: complex tandem arrays of sequences lacking specific binding factors.
75
What is the function of CENP-A and how is it inherited?
- Epigenetic mark to define centromere position.
During cell division, CENP-A is inherited in a semi-conservative manner, meaning that it is passed down from the mother and father to daughter cells. New CENP-A proteins are added through a positive feedback group, where CENP-A nucleosomes recruit CENP-C and HJURP to deposit new CENP-A nucleosomes.
76
How does t-loop formation and the Shelterin complex prevent:
- NHEJ
- HDR
TRF2 blocks ATM kinase and NHEJ.
POT1 binds ssDNA to block HDR from occurring at the 3' overhang.
[NB: DNA damage signaling kinases are actually needed to recruit and activate telomerase...]
77
What are nuclear bodies?
Specialized membrane-less structures within eukaryotic nuclei that serve diverse functions, but are mainly involved in RNA processing and ribonucleoprotein formation.
They form stochastically from 'seeds' and then sequester protein factors.
[Also known as bimolecular condensates]
78
What is Rabl's model of chromosome organization?
Chromosomes do not lose their identity, even though they're no longer visible through the microscope.
79
What is Boveri's hypothesis of chromosome organization?
Chromosomes occupy distinct terretories within the nucleus, and these are stably maintained during interphase. Changes then occur during mitosis.
80
What is Giemsa staining?
Giemsa staining is a type of histological staining technique used to reveal the chromosomal banding pattern of chromosomes.
81
What are the 3 types of chromosome bands?
1. G-bands:
Most common type of chromosome that are AT-rich and gene-poor due to transcriptional repression.
2. C-bands:
Also AT-rich, making up centromeric DNA with repetitive satellite sequences. Hence, this is gene-free.
3. R-bands:
GC-rich bands that are transcriptionally active, containing housekeeping and tissue-specific genes.
82
How did we confirm whether chromosomes would either form 'spaghetti' or 'dumplings' in cells?
UV irradiation experiments showed that chromosomes form distinct territories, as predicted by Boveri.
FISH then showed that chromosome territory positioning is non-random; gene-rich chromosomes are positioned towards the center, and gene-poor towards the periphery.
83
Give 2 examples of chromosomes that are 'exceptional'.
1. The giant polytene chromosome in flies
These are multiple copies of chromosomes that are then held together in parallel bundles.
2. Giant lampbrush chromosomes
Highly elongated chromosomes that are arranged in loops, visible as thin fibres extending from a central axis.
84
How can we analyse the spatial distribution of G and R-bands sequences inside the nucleus?
Pulse replication labeling to identify replication foci.
85
What are early- and mid-late replicating domains?
Early replicating domains (ER) correspond to regions of the genome that are replicated during the first half of S phase. These domains are characterized by open chromatin structure, high gene density, and are enriched for active chromatin marks such as H3K4me3 and H3K27ac. i.e., R-bands.
Mid-late replicating domains (MLR) correspond to regions of the genome that are replicated during the second half of S phase. These domains are characterized by condensed chromatin structure, low gene density, and are enriched for repressive chromatin marks i.e., G-bands.
86
How can we study mesoscale chromatin organization?
HiC and super-resolution microscopy.
87
Which biophysical forces are at play to shape chromatin?
1. Nucleosomal charge
2. Phase separation
3. Loop extrusion
4. Transcriptional activity
88
What are TADs?
Topologically-associated domains are self-interacting genomic regions that are thought to regulate gene expression by limiting the enhancer-promoter interactions to each TAD. These are formed by CTCF and cohesin loop extrusions.
89
What are LADs?
Lamina-associated domains are specific types of TADs that heavily interact with the lamina.
90
Describe the sex determination mechanism in Drosophila.
Females: XX + AA
Males: X + AA
Sxl is expressed on X, so only XX provides enough for it to trigger the female pathway.
SXL activates a cascade of sex determination genes, including the transformer (tra) and doublesex (dsx) genes, which are regulated by the X:A ratio signal.
91
Describe the dosage compensation mechanism in Drosophila. How is it related to sex determination mechanisms?
The Sxl gene is only expressed in females, and its protein product functions as a splicing factor that promotes the production of female-specific transcripts.
In the absence of SXL (i.e., males), MSL2 becomes un-inhibited, triggering the formation of a dosage compensation complex.
Hence, the control of sex determination and dosage compensation are linked.
92
What is the Drosophila dosage compensation complex? How does it work?
The Drosophila dosage compensation complex (DCC), also known as the male-specific lethal (MSL) complex, is a group of proteins that equalize the expression of genes on the X chromosome between males and females.
It contains 5 protein subunits that increase transcription of the genes on the single X chromosome in males. One subunit, MOF, is a HAT that acetylates H4K16, leading to a more open chromatin structure.
It also contains two non-coding RNAs that are thought to be functionally redundant, but aid in localizing the DCC to the X-chromosome.
93
How and why is chromatin structure changed in a sex-dependent way in Drosophila?
MOF is a sex-dependent HAT that's only activated in male Drosophila due to the lack of SXL that normally inhibits its activity.
It deposits acetyl groups on to histones in the X-chromosome to open the chromatin structure up and allow for easier access for transcription.
This is important for dosage compensation.
94
How is the Drosophila dosage compensation complex targeted to the X-chromosome?
Give some experimental evidence that shows this.
1. The X-linked genes for the 2 roX RNAs (these coat the male X-chromosome) are exceptionally strong attractors of the DCC.
2. The complex then spreads to low affinity sites through chromatin modification interactions.
[A copy of roX on an autosome is sufficient to recruit the DCC, but not to cause spreading.]
95
Describe the sex determination mechanism in C. elegans.
Female: XX
Male: XO
X encodes a repressor of the XOL-1 (XO-lethal) gene. Males don't encode enough of the repressor, so XOL-1 is active.
XOL-1 then represses the HER-1 repressor. HER-1 promotes male differentiation.
Without XOL-1, HER-1 remains repressed, and this triggers the dosage compensation pathway.
96
Describe the dosage compensation mechanism in C. elegans. How is it related to sex determination mechanisms?
Absence of HER-1, caused by an absence of XOL-1, triggers the assembly of the dosage compensation complex.
The C. elegans DCC is related to the condensin family of proteins. This is used to reduce X-linked gene expression in hermaphrodites.
Sex determination and dosage compensation are linked.
97
How is the C. elegans dosage compensation complex targeted to the X-chromosome?
1. The DCC binds to a specific DNA sequence motif called the "rex site," which is enriched on the X chromosome, thereby targeting the complex to the entire chromosome.
2. DCC spreads to other sites through recognition of histone modifications, compacting the chromosome.
98
Describe the sex determination pathway in mammals.
In mammals, sex is determined by the presence or absence of the Y chromosome, which carries the sex-determining region Y (SRY) gene.
The SRY gene encodes a transcription factor that triggers the development of male gonads from the undifferentiated gonadal tissue. In the absence of the SRY gene, female gonads develop.
99
Describe the dosage compensation pathway in mammals.
Is it linked to sex determination?
X-chromosome inactivation occurs early in development and is controlled by a large non-coding RNA called Xist (X-inactive specific transcript). Xist is transcribed from the inactive X chromosome and coats the entire chromosome in cis, leading to the recruitment of Polycomb complexes and transcriptional silencing of most X-linked genes.
The active X chromosome, on the other hand, expresses another non-coding RNA called Tsix, which acts as a repressor of Xist expression, ensuring that only one X chromosome is inactivated.
The two pathways are not linked.
100
How is X-chromosome inactivation regulated throughout development in female zygotes?
When the sperm and egg fuse to form the zygote, both the X chromosomes that were previously inactivated become reactivated. The paternal X-chromosome is then imprinted for XCI.
In the early blastocyst stage, the paternal X chromosome is reactivated, to allow for random X inactivation.
101
What are the roles of SPEN and hnRNPK in dosage compensation in mammals?
These are Xist-associated proteins.
SPEN: recruits HDACs to histone tails.
hnRNPK: recruits PRC1 of the Polycomb complex for monoubiquitination. Ub recruits PRC2 to maintain the gene silencing.
102
What is the TAD structure of inactive mammalian X chromosomes, and what causes this?
the inactive X chromosome (Xi) is organized into two mega-domains that are separated by a boundary region called the X-inactivation center (Xic). The boundary region contains the Xist gene.
This is caused by the SMCHD1 protein (recruited by PRC1 during XCI), that removes CTCF and hence causes a loss of TAD boundaries.
103
What is a Barr body?
the heterochromatic structure formed by the inactive X-chromosome in mammals.
104
What happens to mammalian sex chromosomes in meiosis?
What can occur if this goes wrong?
X and Y chromosomes are not fully homologous and only pair at their pseudoautosomal regions.
The remaining regions of the X and Y chromosomes, which do not pair, segregate randomly into the resulting gametes.
This can result in gametes with an abnormal number of sex chromosomes, which can lead to genetic disorders such as Turner syndrome (45,X) or Klinefelter syndrome (47,XXY or 47,XYY).
105
What is meiotic sex chromosome inactivation (MSCI)?
When the sex chromosomes, especially the X chromosome in males, are transcriptionally silenced during meiosis.
The process of MSCI involves the recruitment of several chromatin-modifying proteins, such as histone variants and histone-modifying enzymes, that work together to establish a repressive chromatin environment around the sex chromosomes. It also involves DNA damage response pathways, using ATR.
This leads to the silencing of most genes located on the sex chromosomes during meiosis.
Once meiosis is completed, MSCI is reversed and the sex chromosomes become transcriptionally active again in haploid gametes.
