Chromosome Biology (Marcin) Flashcards
Difference in chromosomes in normal vs cancer
Cancer cells usually show certain level of ‘aneuploidy’ (the wrong number of chromosomes within cells)
Cancer chromosomes contain numerical aberrations
Cancer chromosomes contain structural aberrations
Cells within the same tumour may have different karyotypes
50% of blood cancers and 90% of solid tumours show this deranged karyotype
What is the heirarchical organisation of interphase chromatin?
Structure of a chromatin loop
Contains 2 main components
CTCF (DNA Sequence Specific Binder)
- Defines the place in chromatin where the loop exists
Cohesin Complex (4 proteins complex)
- Cohesin is a ring that embraces two strands of chromatin, generating a loop
- Defines the size of the loop
How is transcription regulated within chromatin organisation?
Transcription occurs in a complex chromatin environment, not on linear DNA
Gene regulation is influenced by physical gene localization within the nucleus
Genes are organized into active and inactive compartments
- Inactive compartments: Genes are rarely transcribed
- Active compartments: Genes are highly transcribed
Changes in cell physiology may trigger transcriptional activation or silencing
Inactive genes are moved to inactive compartments, and active genes are moved to active compartments to regulate transcription
What is the role of chromatin structure in regulating DNA processes?
Chromatin switches between open (relaxed) and closed (condensed) states
Open chromatin allows access to DNA for processes like transcription and repair
Closed chromatin restricts access, silencing gene expression
DNA repair and synthesis require chromatin to open locally
Activators open chromatin for transcription; repressors close it to inhibit transcription
How do enhancers regulate gene transcription from distant locations on DNA?
Enhancers can affect transcription even if far from the gene’s promoter
Scientists discovered chromatin looping as the mechanism enabling distant enhancers to interact with genes
Chromatin loops are regulated by proteins like Cdk4 and cohesin
The loop brings the enhancer into proximity with the promoter, allowing gene activation
Activators facilitate this interaction, leading to transcription activation
Chromatin looping is critical for gene regulation, including in cancer-related processes
Importance of chromatin organisation regulation in cancer
The disruption of elements that regulate chromatin organisation may contribute or even lead to cancerogenesis
CTCF in cancer
CTCF is frequently mutated in cancer
Different CTCF mutations or abnormal CTCF levels are found in multiple cancers
CTCF/Cohesin binding sites in cancer
CTCF/Cohesin binding sites are frequently mutated in cancer
CTCF motif mutations accumulate in multiple cancers
It is a major mutational hotspot in the non-coding cancer genome
How does de-regulation of gene expression occur?
Breaking down of TAD border structure leads to de-regulation of gene expression
Mutations in loops near to each other can cause the loops to interact and gene expression is de-regulated
What is a histone?
Histones are small and highly conserved proteins which form a basic subunit of eukaryotic chromatin called a nucleosome
4 different core histones
H2A
H2B
H3
H4
Composed of histone fold and an N-terminal tail
Histone tail sits outside of the histone octamer
A nucleosome is formed from 8 histone molecules
How are histones regulated?
Via histone tail modification
Tails can be methylated, acetylated, phosphorylated or ubiquitylated
Histone code
What are some epigenetic regulation methods?
- Histone modification
- DNA methylation
- non-coding RNAs
- Histone variant exchange
Histone variants
There are some variants of histones that either contribute or stop tumourgenesis
Tumour initiation contributor:
- H3.3K27M
Tumour growth contributor:
- H2A.Z
Tumour growth inhibitor
- macroH2A
Metasasis contributor:
- H3.3
Metasis inhibitor:
- macroH2A
Are drugs being developed to target enzymes involved in epigenetic mechanisms related to cancer?
Yes, drugs are being developed to target enzymes involved in epigenetic changes
These drugs often focus on small molecules or antibodies
The goal is to reduce the activity of proteins or enzymes overactive in tumor cells
Enzymes involved in DNA methylation, histone methylation, and histone acetylation are key targets
Each epigenetic mechanism (like histone modification) has a dedicated field of drug development
Targeting these miss-regulated enzymes can help inhibit cancer progression
What are oncohistones? How do mutations in histone proteins affect chromatin structure and lead to cancer?
Oncohistones are mutated forms of histones that contribute to cancer development by altering chromatin structure and gene regulation
Mutations in histone proteins can cause dysregulation of chromatin without killing the cell
These mutations may occur at sites that are normally modified, such as methylation
Example:
- Mutation of histone H3 at lysine 36 prevents methylation, disrupting the recruitment of the SetD2 complex
Such mutations alter chromatin structure and gene transcription
This dysregulation can lead to inappropriate recruitment or loss of factors at the chromatin
While cancer is primarily a genetic disease, epigenetic changes like histone mutations also contribute to cancer development by altering gene regulation
What is a centromere?
- A constricted region on a chromosome that joins sister
chromatids - The site where kinetochore is formed
- Specialised fragment of DNA, which allows sister chromatids to segregate
What is a kinetocore?
- A multi-protein (~200 proteins) complex that forms at a centromere
- Specialised structure, which allows sister chromatids to segregate during cell division
- Site on a chromosome where microtubules attach
What are the key features of centromeric chromatin and its role in centromere function?
- Centromeric chromatin is distinct from euchromatin and heterochromatin, with a unique set of histone marks and centromeric proteins
- Centromeres are defined by CENP-A (a histone H3 variant) which acts as a centromeric marker and is loaded by different mechanisms than other histones
- Most eukaryotic cells centromeres are defined epigenetically
- Centromeric chromatin (CENP-A-containing arrays of nucleosomes) recruits several multiprotein complexes called Constitutive Centromere Associated Network (CCAN) to centromeres. These proteins play a role in proper functioning of centromeres and kinetochores
Is it possible to locate the centromere during interphase?
Yes, locating CENP-A (CenH3) will locate the centromere
Centromeric (CCAN) components are at centromeres throughout the cell cycle
Kinetochore components are at xcentromeres only during mitosis
How do kinetochores assemble?
Kinetochores assemble on centromeric chromatin and bind microtubules
Kinetochores are assembled on centromeric chromatin in the beginning of mitosis
What are kinetochores major functions?
Three major functions of the kinetochore:
1. Capturing microtubules to form a connection between chromosomes and mitotic spindles
2. Identifying incorrect attachments and repairing them
3. Harnessing the force to generate movement of chromosomes during anaphase
What drives kinetochore assembly?
Kinetochores are assembled on centromeric chromatin in the beginning of mitosis
Mitotic kinases CDK1 and Aurora B are involved in the process of kinetochore assembly
What is the structural core of the kinetochore?
Structural core of a kinetochore is called the KMN network
KMN network:
- KNL1/Spc105 complex
- Mis12 complex
- Ndc80 complex
The KMN network forms a physical connection between centromeres and microtubules of the mitotic spindle via 2 separate pathways
The affinity of Ndc80 complex to microtubules is regulated by phosphorylation via Aurora B kinase
Subunits of the KMN network form a binding platform for many regulatory proteins (including surveillance and correction mechanism components)
Spindle Assembly Checkpoint (SAC) components bind to kinetochores that are not attached properly to microtubules of the mitotic spindle
Centromere/Kinetochore in cancer
Centromere (CCAN) and kinetochore genes are misregulated in many cancers
Authors found that the overexpression of some centromere/kinetochore genes correlate with increased levels of genomic instability and several specific adverse tumour properties, and prognosticate poor patient survival for breast and lung cancers, especially early-stage tumours
They also found that the levels of the overexpression can help to forecast patient response to adjuvant chemotherapy or radiotherapy
How does the mitotic spindle form?
Formation of the mitotic spindle may be achieved using different pathways, but finally chromosomes should reach metaphase plate where they are attached to the plus-ends of kinetochore microtubules emanating from opposite spindle poles (bi-polarity)
Many different motor proteins contribute to this state, which is accomplished by the “trial and error” approach
Importance of mitotic spindle microtubule attachment to chromosome?
Incorrect attachments are not stable and do not last
Correct attachment becomes “locked” in space
How are incorrect mitotic spindle -> chromosome attachments fixed?
Chromosome Passenger Complex (CPC) (or Aurora B complex) is involved in correcting improper attachments
CPC has 4 sub units:
- Aurora B Kinase
- INCENP
- Survivin
- Borealin
CPC is involved in mitosis and cytokinesis (it moves away from
inner centromeres towards the mid-zone of the central spindle
for cytokinesis)
There is an intense crosstalk between CPC and other key regulators of the cell division, for example Plk1/Polo or Haspin
kinases
Where is the Chromosome Passenger Complex located?
There is a section of the centromere called the inner centromere
This is where the passenger complex is located
It also contains some Cohesin (cohesin is responsible for keeping chromatids together)
How does Aurora B kinase inside the Chromosome Passenger Complex help to correct improper attachements?
Kinetochore has spring-like properties
Outer kinetochore may move away from the centromere or may come closer to it under the higher or lower tension, respectively
When kinetochores are not properly attached, the tension is low and the outer kinetochore is closer to the inner centromere where CPC is localized
Aurora B phosphorylates Ndc80 protein what destabilises binding of the Ndc80 complex to microtubules
When a kinetochore is properly attached to microtubules high tension removes Ndc80 from the reach of Aurora B kinase and the attachment becomes stable
Aurora kinase roles
They are a family of aurora kinases in vertebrate cells
- Aurora A kinase is localised primarily to centrosomes and it controls centrosomal activities, e.g. mitotic spindle formation
- Aurora B kinase is a component of CPC and its localisation changes from inner-centromeric to microtubules of the central spindle and midzone. It participates in chromosome condensation, segregation and cytokinesis
- Aurora C is involved in meiosis.
What happens when aurora B kinase is depleted in cells?
Normal metaphase plate alignment is disrupted.
Chromosomes are not correctly aligned on the metaphase plate.
Some chromosomes are displaced due to incorrect attachments.
Aurora B kinase is essential for correcting chromosome
attachments.
Without aurora B kinase, the cell cannot fix incorrect chromosome attachments, leading to misalignment.
Aurora kinases in cancers
The expression of all 3 aurora kinases were found to be elevated in different cancers, which may be related to the incorrect number of chromosomes in cancer cells
Result of overexpression of aurora B in cells
Overexpression of aurora B kinase is often seen in cancer cells.
Elevated levels disrupt normal chromosome segregation.
This overexpression leads to chromosome segregation errors.
Result:
- Increased likelihood of chromosomal aberrations and instability, contributing to cancer progression.
How can aurora kinases be used in cancer treatment?
Researchers have explored inhibiting aurora A and B kinases as a cancer treatment.
Small molecule inhibitors can be used to target these kinases.
Successful inhibition can lead to cell death, targeting cells that overexpress aurora A or B.
Specificity is a challenge because aurora kinases share similar domains, making it difficult to target A or B exclusively.
Broad-spectrum inhibitors can impact all three kinases, which may be beneficial or detrimental depending on the therapeutic goal.
Inhibiton of aurora a kinase affect?
Progression through mitosis
Incorrect centriole separation
Chromosome misalignment
Abnormal spindle formation
G2/M arrest
Cell death by apoptosis
Inhibition of aurora kinase b affect?
Progression through mitosis
Defective chromosome-spindle attachment
Cytokensis failure
Polyploidy (P53 dependant)
Cell death via apoptosis
What are anti-mitotic drugs, and how do they target cancer cells?
Anti-mitotic drugs are a group of cancer inhibitors that aim to reduce cell division rates in cancer cells.
They prevent mitosis, aiming to induce cell death in cancer cells.
Some examples of targets:
- Microtubules, as major components of mitotic spindle (stabilisers, de-stabilisers)
- Kinesins, as major regulators of microtubule dynamicity
- Mitotic kinases, as major regulators of cell cycle and cell division (CDKs, PLKs, Aurora kinases, Wee 1 kinases)
Goal is to induce cell death, a primary aim for cancer treatments using anti-mitotic drugs.
Aurora kinase inhibiton is anti-mitotic
What is the structure of cohesin, and how does it function?
Cohesin is formed by two SMC proteins: SMC1 and SMC3.
Each SMC protein has:
- ATPase domains at each end – provide energy by hydrolyzing ATP, which is crucial for cohesin’s functions.
- A hinge domain in the middle, which allows the proteins to dimerize, forming a ring-like structure.
- Long, rod-like structures made of coiled coils that extend from each end.
Non SMC proteins associated near ATPase domains:
- Scc1 (Rad21)
- Scc3 (Stag1/Stag2)
Dimerization occurs via the hinge domains, allowing cohesin to encircle DNA.
Function:
- Cohesin binds to DNA, holding sister chromatids together.
- Regulatory subunits (non SMC proteins) bind near the ATPase domains to assist in cohesin’s role in chromosome cohesion.
Cohesin mechanism of action
It is loaded on chromosomes during G1 phase, after DNA replication it holds sister chromatids together
Along with CTCF it defines borders of chromatin units during interphase
Its release from chromosome arms in prophase coincides with the axial compression of chromosomes during mitosis
What are the multiple functions of cohesin?
Mitosis
- Sister chromatid cohesion (at centromeres)
- Holding together sister centrioles
Meiosis
- Pairing of homologous chromosomes during meiosis
- Assembly of the axes of synaptonemal complex in meiosis
- Coordination of sister kinetochores during first meiotic division
Interphase
- Sister chromatids cohesion (entire chromatin)
- Repair of DNA breaks
- Assembly of DNA replication factories during S phase
- Regulation of transcription
- Organisation of chromatin loops and TADs
What is non-cohesive cohesin?
In vertebrates cohesin is loaded onto DNA just after mitosis in a “non-cohesive” form.
Cohesin loading factor Scc2 is required for this step.
Eco1 (Esco1/Esco2) acetylate cohesin during S phase to establish “cohesive” cohesin that holds sister chromatids together
Cohesin is removed completely from chromatin during cell division
What are the two main pathways for cohesin removal in cell division?
Prophase pathway:
- Removes cohesin from chromosome arms
Metaphase (Separase) pathway:
- Targets remaining cohesin at centromeres, allowing for chromatid separation
How does the prophase pathway remove cohesin from chromosome arms?
Kinases like Plk1 and the protein Wapl facilitate removal of cohesin from chromatin
This pathway removes 90-95% of cohesin from chromosomes, but a portion at the centromere remains intact
Why do chromosomes appear X-shaped in metaphase?
Cohesin is absent on chromosome arms but persists at centromeres, creating an X-shaped structure
The centromeric cohesin resists the pulling force of microtubules attached to kinetochores
How does centromeric cohesin maintain tension during metaphase?
Microtubules pull on sister chromatids towards spindle poles.
Centromeric cohesin holds chromatids together, counteracting the pull and generating high tension when correctly attached
What role do shugoshin and protein phosphatase 2A play in cohesin protection?
Shugoshin and Protein Phosphatase 2A protect centromeric cohesin from removal by the prophase pathway
They do not prevent cohesin removal by the metaphase pathway
How is centromeric cohesin removed during anaphase?
In the metaphase pathway, the enzyme separase cleaves centromeric cohesin
This cleavage is essential for sister chromatids to separate synchronously, initiating anaphase
What is the spindle assembly checkpoint’s role in mitosis?
Ensures all kinetochores are attached to spindle microtubules before anaphase
Prevents anaphase until proper attachment, maintaining accuracy of chromosome segregation
What are some of the functions of cohesins?
Cohesion-dependant functions:
- DNA replication
- Homologous recombination-mediated repair
- Chormosome biorientation
Cohesion-independant function:
- Genome compartmentalization
- Transcription regulation
- DNA replication
How can we envision the outcomes of aneuploidy in a population of cells?
In a normal population of cells, a mutation or process may lead to one or a few cells becoming aneuploid.
This selection within the local environment can lead to three potential outcomes for the aneuploid cell:
- Negative Selection: The aneuploidy is detrimental in the specific environment, causing the affected cell to die out. It will not proliferate or contribute to the population.
- Neutral Selection: The aneuploid cell neither gains nor loses an advantage in the environment, allowing it to persist at low levels within the population.
- Positive Selection: The aneuploid cell possesses traits that confer a competitive advantage over other cells, allowing it to proliferate and outgrow its neighbors. This positive selection drives the development of the aneuploid population.
This process illustrates how aneuploidy can impact cell populations and contribute to the evolution of cellular traits.
What are 5 possible origins of aneuploidy?
- Errors in kinetochore-microtubule attachments (e.g. merotely)
- Supernumerary centrosomes (too many centrosomes)
- Weak Spindle Assembly Checkpoint fails to delay anaphase
- Impaired sister chromatin cohesion
- Cytokinesis failure
What does chromosome missegregation look like?
At the cellular level lagging chromosomes are thought to be the reason of aneuploidy
What are the 4 major forms of attachment of microtubules to chromosomes?
- Amphitelic (Correct)
- Monotelic (Wrong, but activates SAC)
- Syntelic (Wrong, but activates SAC)
- Merotelic (Wrong, doesn’t activate SAC)
One is correct, three are not, but only two out of those three can easily activate SAC
We do not know how, but merotelic attachments are also repaired before anaphase, however these are the most difficult to repair and sometimes cells cannot cope
A failure may occur when cells are overwhelmed with the number of erroneous attachments or when the repair system is impaired.
Microscope example of merotelic attachment
A merotelic attachment can cause a lagging chromosome that may be randomly distributed, leading to one daughter cell with an extra chromosome and another with one missing (aneuploidy)
Although many aneuploid cells die, some survive and may proliferate, increasing the risk of cancerous growth due to further mutations
What is a micronuclei
Missegregated chromosomes frequently form micronuclei (a small nucleus) after mitos
What are supernumerary centrosomes?
It is when a cell contains more than the normal (2) number of centrosomes
Supernumerary centrosomes very rarely lead to multipolar divisions
Even if they do, typically cells die after such divisions
Instead, supernumerary centrosomes lead to formation of multipolar spindles, which later convert into bipolar ones, but merotelic attachments are formed and they persist
What happens when there are supernumerary cenrosomes?
Extra centrosomes lead to the formation of multipolar spindle
In most cases multipolar spindles become bipolar due to the centrosome clustering
However, incorrect kinetochore-microtubule attachments formed during the multipolar stage may persist, especially in the form of merotelic attachments
This causes higher rates of cell death, but also increases the chances of chromosome missegregation that leads to aneuploidy
SAC and premature release of chromatid in aneuploidy?
Premature release of the chromatid cohesion can lead to aneuploidy
Similarly, an impaired Spindle Assembly Checkpoint is another potential reason of aneuploidy
Cytokinesis affect on aneuploidy?
Cytokinesis defects affect the ploidy of a cell,
The tetraploid cell that is generated following a complete cytokinesis failure can serve as the ideal starting point to generate aneuploid cells
Tetraploidization can promote tumorigenesis and CIN
Structural chromosome abberations in cancer
Very high number of different chromosomal re-arrangements was discovered in many different cancers
Next generation sequencing, especially the whole genome sequencing approach, reveals the details about the rearrangements and how they may be generated.
Mechanisms of these structural variations are mostly unknown, however in many cases the very high rate of genomic instability is related to problems with major pathways of DNA repair
What is an example of chromosomal translocation involved in cancer formation
Philadelphia Chromosome
(Chronic Myelogenous Leukemia - CML)
The chromosome translocation responsible joins the Bcr gene on chromosome 22 to the Abl gene from chromosome 9, thereby generating a Philadelphia chromosome
The resulting fusion protein has the N-terminus of the Bcr protein joined to the C-terminus of the Abl tyrosine protein kinase
In consequence, the Abl kinase domain becomes inappropriately active, driving excessive proliferation of a clone of hemopoietic cells in the bone marrow
Extreme chromosome sturctural abberations
Some rearrangements are much more complex, involving many points of break and fusions along the chromosomes
But there are also examples of extreme rearrangements that change chromosomes beyond recognition
An example of one of these mechanims is called Chromothripsis
Massive genomic rearrangement by pulverising chromosome and stitching it together is one of drivers of cancer genome evolution
How can anueploidy be used to treat cancer?
Cells have a certain limit of aneuploidy that they can withstand before cell death
Researchers are exploring ways to increase aneuploidy in cancer cells so that they surpass the threshold and the cell dies
Normal cells will also become aneuploid but since they didn’t have aneuploid before, they potentially wont go over the limit
