Aspects of chromosome biology Flashcards
Explain the hierarchical organisation of interphase chromatin
smallest:
Topologically Associating Domains (TADs): (DNA sequences within a TAD physically interact with each other more frequently than with sequences outside the TAD)
Compartments:(euchromatin/heterochromatin) - put into compartments either A (active) or B (inactive)
chromosome territories
Biggest^
Explain the chromatin loop
Cohesin complex holds DNA strands together forming a loop
CTCF protein binds specific DNA sequences (CTCF motifs), which are positioned in the same direction. Hence, CTCF defines the size of the loop.
Loop boundaries involve cohesin and CTCF only during interphase, but not in mitosis
What do TADs stand for?
Topology associating domain
What do TADs do?
Change the position from inactive/active compartments uppon transcriptional activators/repressors binding
e.g. of an activity that requires open chromatin
Transcription
What do long distance interactions help regulate
gene expression - looping of chromatin allows for interactions between distant regions of DNA and regulatory complexes
What is spectral karyotyping
chromosome painting
Features of spectral karyotyping HeLa cancer cells
- Cancer cells usually show certain level of aneuploidy
- Numerical aberrations
- Structural aberrations
- Cells within the same tumour may have different karyotypes
Is disruption of insulated neighbourhoods a potential way to activate proto-oncogenes?
Yes, the disruption of elements that regulate chromatin organisation may contribute or even lead to cancerogenesis
Is CTCF frequently mutated in cancer, give details
Different CTCF mutations or abnormal CTCF levels are found in multiple cancers
for example: in just under 25% of cases of uterine cancer there is a CTCF mutation (Noordermeer 2020)
Give examples of how 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
Explain how breaking down TAD border structure leads to de-regulation of gene expression
Isolated neighbourhoods (two loops that don’t interact)
one of the CTCF binding sites can become methylated, meaning CTCF doesn’t bind, causing the direct interaction between the loops, including between the enhancer and oncogene, meaning cancer occurs
Explain how Perturbation of insulated neighbourhoods’ boundaries is sufficient to activate proto-oncogenes
Insulated neighbourhoods were mapped in T cell acute lymphoblastic leukemia (T-ALL).
It was found that tumour cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighbourhoods containing prominent T-ALL proto-oncogenes.
Mutations affecting chromosome neighbourhood boundaries were found in many types of cancer.
Oncogene activation can occur via genetic alterations that disrupt insulated neighbourhoods in malignant cells.
What does chromatin consist of?
DNA, RNA and proteins
What are histones
small and highly conserved proteins which form a basic subunit of eukaryotic chromatin – nucleosome
Histone tails are…
Heavily modified: Methylation, Phosphorylation, acetylation
Histones may be modified by many different post-translational modifications (PTMs) e.g.
- Methylation
- Acetylation
- Phosphorylation
- Ubiquitylation
- ADP-ribosylation, etc
Explain how ‘readers’ work
PTMs form epitopes that are “read” by proteins, which normally would not bind to non-modified histones (or would bind but with a much lower affinity)
proteins that bind specifically to modified histones are called “readers”.
Usually they bring a new activity to the vicinity of a modified histone or alter the structure of chromatin simply by binding to a histone mark.
e.g. PHD finger domain of a tumour suppressor ING2 binds only trimethyl groups of histone H3 (H3K4Me3
How does histone code work?
Translation of a modification mark into biological function involves
- Writers (enzymes) that are responsible for modifying histones
- Readers (binders) that recognise and interact with modified histones
- Sometimes Effectors are needed, usually enzymes that change the status of chromatin to ‘close’ or to ‘open’, but some Readers may also do this
- Erasers (enzymes) that remove the modification
For the histone code to work properly, all these groups of proteins must be present at appropriate levels
Examples of the contribution of altered histone variants and their chaperones to different stages of tumour development
initiation :H3.3.K27M (Oncohistone mutation)
H2A- Z – another H2A histone variant leads to tumour growth
H3.3 -metastasis
MacroH2A is a histone variant of H2A – inhibits tumour growth and metastesis
How does misregulated epigenetic control lead to cancer formation?
DNA methylation
Non-coding RNA
Histone methylation
Histone acetylation
Can we try to repair epigenetic component cancers?
yes, there are some drugs used to combat epigentic changes
DNMT inhibators - block DNA methylation
HDAC inhibators: Block Histone acetylation
HMT inhibators: Block Histone methylation
Example of oncohistone/onconucleosomes
Mutations in the histones themselves have recently been linked to cancers, e.g. the discovery that mutations in histone H3 occur with high genetic penetrance within rare paediatric gliomas and sarcomas
Histone H3 examples:
K27 – trimethylated, in the hostone variemt where K -> M, methylation cannot occur, meaning PRC2 cannot bind
K36 – trimethylated also, into methyonine, SETD2 unable to bind
What is the 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
Features of centromeric chromatin
Unusual
different from euchromatin and heterochromatin
(→ unique set of histone marks, presence of centromeric proteins)
E.g. of a centromeric marker
histone H3 variant called CENP-A (CenH3)
How are centromeres defined
Epigenetically
Difference in CENP-A containing arrays of nucleosomes compared to ‘canonical’ ones
CENP-A containing - generally more condensed
What does CCAN stand for and what is it
Constitutive centromere-associated network (CCAN)
It is a point within centromeric chromatin where centromeric protein (CENPs) form complexes
At what point in the cell cycle are CCAN components at centromeres?
Throughout the cell cycle
At what point of the cell cycle are kinetochore components present
Only during mitosis
What is the kinetochore?
A multi-protein complex that forms at a centromere
Specialised structure, which allows sister chromatids to segregate during cell division
Site on a chromosome where microtubules attach
Three major functions of the kinetochore
- Capturing microtubules to form a connection between chromosomes and mitotic spindles
- Identifying incorrect attachments and repairing them
- Harnessing the force to generate movement of chromosomes during anaphase
When are kinetochores assembled?
Assembled on centromeric chromatin in the beginning of mitosis
Name two mitotic kinases involved in the process of kinetochore assembly
CDK1 and Aurora B
What is the structural core of a kinetochore
The KMN network:
- KNL1/Spc105 complex
- Mis12 complex
- Ndc80 complex
forms a physical connection between centromeres and microtubules of the mitotic spindle
Human KMN network is connected to centromeres via 2 separate pathways
Subunits of the KMN network form a binding platform for many regulatory proteins (including surveillance and correction mechanism components)
The affinity of Ndc80 complex to microtubules is regulated by phosphorylation via Aurora B kinase
Relate Centromeres/kinetochores to cancer
Many Centromere/kinetochore genes are misregulated in many cancers
Gene misexpression predicts cancer patient survival in response to radio/chemotherapy (Zhang 2016)
e.g. CANP-A (centromere) 85% of datasets had misregulation
CENP-U (85%)/CENP-K (80%) - CCAN (inner kinetochore)
NDC80 (85%) - KMN (outer kinetochore)
how is formation of the mitotic spindle achieved? (simple)
using different pathways
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
Incorrect spindle attachments…
are not stable and do not last
Correct spindle attachments..
become “locked” in space
What complex is involved in correcting improper attachments?
Chromosome passenger complex (CPC)
4 subunits of CPC
- Aurora B (kinase)
- INCENP (scaffold and activation)
- Survivin (centromere targeting)
- Borealin (activation and interactions in cytokinesis)
What is CPC involved in and name a key regulator of cell division that it crosstalks with
involved in mitosis and cytokinesis
Plk1/Polo
what makes up the inner centromere?
CPC and cohesin
How does Aurora B help to correct improper attachments?
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.
What does Aurora A kinase do? and where is it located?
localised primarily to centrosomes and it controls centrosomal activities, e.g. mitotic spindle formation
What does Aurora B kinase do? and where is it located?
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
What is Aurora C kinase involved in?
Meiosis
What happens to aurora kinases in cancers?
The expression of all 3 Auroras was found to be elevated in different cancers, which may be related to the incorrect number of chromosomes in cancer cells
Can we counteract Aurora kinase overexpression in cancer cells?
Yes - with aurora kinase inhibators
e.g. Hesperadin, ZM447439 (also targets MEK, Src and Lck)
Explain the expected phenotypes after aurora A inhibition
- Progression through mitosis
- Incorrect centriole separation
- chromosome misallignment
- abnormal spindle formation
-G2/M arrest
this results in cell death by apoptosis
Explain the expected phenotypes after aurora B inhibition
- Progression through mitosis
- defective chromosome spindle attachment
- cytokinesis failure
- Polyploidy (p53 dependant)
This results in cell death by apoptosis
Aurora B kinase (ABK) inhibators in development:
AT9283 - For ABK and AAK, Phase II completes (Hay 2016)
Chiauranib - For ABK, Phase I and II competes (Sun 2019)
Examples of targets for the anti-mitotic aurora kinase inhibitors
-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)
Examples of anti-mitotic drugs already used in cancer treatment
Target -Drug example
Aurora A/B - AT9283
Microtubules/spindle - Taxanes
Kinesin spindle protein - Ispinesib
Wee1 - MK-1775
^ SAC checkpoint
Cyclin D/CDK - Ribociclib
^ G1 checkpoint
Whats the cohesin complex made of?
- SMC proteins 1 and 3
- non-SMC subunits - Rad21/Scc1 and Scc3, which bind other proteins that regulate cohesin function
When is the cohesin complex used?
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 enzyme is important for cohesin activity?
ATPase - plays several different roles at different stages of the cell cycle
Function of cohesin in mitosis
- Sister chromatid cohesion (at centromeres)
- Holding together sister centrioles
Functions of cohesin in meiosis
- Pairing of homologous chromosomes
- Assembly of the axes of synaptonemal complex
- Coordination of sister kinetochores during first meiotic division
Functions of cohesin in 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
Explain the cohesin cycle
In vertebrates cohesin is loaded onto DNA just after mitosis (G1) in a “non-cohesive” form (it will bind only to one strand of DNA) - 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
explain the removal of cohesin that allows chromosomes to segregate
95% of cohesin removal happens in the prophase pathway by mitotic kinases
Some cohesin stays bound (usually at the centromere/centrmeric regions)
This cohesin gets removed in the metaphase pathway by an enzyme called separase, this cohesin cannot be recycled, once its removed, anaphase immediately starts
Protein Shugoshin and Protein Phosphatase 2A protect centromeric cohesin
How is the activity of seperase controlled?
Both the activation of seperase and inactivation of Cdk1 are triggered by the degradation of regulatory proteins
There are two major prerequisites of the metaphase to anaphase transition:
- inactivation of Cdk1
- activation of Separase
How does the degradation of regulatory proteins, allowing for activation of seperase and inactivation of Cdk1 occur?
via the proteasome pathway, which requires ubiquitylation of the targeted proteins. Anaphase-Promoting Complex / Cyclosome (APC/C), a E3-type ubiquitin ligase, modifies substrates destined for degradation by “tagging” them with a small protein ubiquitin.
What is the activity of the APC/C under control of?
The spindle assembly checkpoint
How does the spindle assembly checkpoint (SAC) work?
Unattached kinetochores generate “STOP” signal, which blocks activity of APC/C.
This signal is thought to be a complex of 4 proteins that is called Mitotic Checkpoint Complex (MCC).
It consists of BubR1, Bub3, Mad2 and Cdc20, and binds directly APC/C and inhibits it.
When all kinetochores become attached to microtubules of the mitotic spindle the MCC is no longer produced and APC/C becomes active
APC/C can then degrade cyclin B1 and securin and allow for progression to anaphase
When APC/C becomes active…
…the cascade of events leads to the inactivation of Cdk1 and activation of Separase, which removes cohesin from centromeric regions of chromosomes
What does securin do?
Securin keeps seperase inactive when an MCC (STOP) signal is present
When there is no MCC signal…
APC/C is active, and securin is inhibited so that seperase can be active and cut the cohesin
Whats a common feature of all SAC components and name two more proteins imprtant for SAC activity
they are recruited to unattached kinetochores, but not to properly attached ones.
- Plf1
- Aurora Kinase B
What leads to the generation of the MCC signal
Recruitment of SAC components tounattached kinetochores
What is the current model for sensing Kinetochore to mictorubule (MT-KT) attachemnets
mitotic kinase Mps1 is responsible for sensing the attachment of MTs to KTs
What does SAC activity allow for?
gives a cell more time to establish proper MT-KT attachments. Without this extra time cells may proceed into anaphase before these attachments are made
Explain how Aurora B has a dual role
- By participating in the error correction mechanism it generates unattached kinetochores that are recognised by SAC
- It directly participates in SAC
Are cohesin components muatated in cancer?
Yes, many mutations were found in cohesin components’ genes, most frequently within the STAG2 (SA2, Scc3-type) subunit
BUT! The number of mutations in STAG2 does not translate into the changes in the chromosome number in daughter cells. The same is true about other cohesin subunits.
Mutations in genes for cohesin components or changes in the expression level of those genes may affect cancer formation using many different pathways
What are some other roles of cohesin
Chromosome biorientation, Genome compartmentalisation, transcription regulation
Explain an extra function of cohesin not related to DNA
Cohesin complexes are present at centrosomes
A specialised variant of Sgo (Shugoshin) is also localised at centrosomes
Centrosomal Sgo protects centrosomal cohesin
Loss of function of centrosomal Sgo leads to mitotic aberrations, e.g. multipolar spindle formation
Protection of centrosomal cohesin requires PP2A (against prophase pathway)
Dissociation of cohesin from centrosomes requires activity of Separase
What are cancer cells characterised by?
High levels of aneuploidy and chromosmal instability
What is aneuploidy?
a number of chromosomes different from the usual 46 (in case of human cells) e.g. in Down’s syndrome, also called Trisomy 21, the total number of chromosomes in a cell is 47
What % of tumours is aneuploidy found in?
90% of solid tumours and ̴60% of haematological malignancies
How does aneuploidy arise?
missegregation of whole chromosomes during cell division. In many cancer cells the rate of the missegregation is increased, resulting in high frequency of chromosome gain or loss
Define chromosomal instability (CIN)
Lack of capacity to maintain the same number of chromosomes from one generation of cells to the next
in cancer cells, who is first, CIN or aneuploidy?
still a lot to learn about the ‘cause and effect’ problem in respect to appearance of aneuploidy and the role of CIN in this process.
But one study (Jason 2012) saw it turns out that aneuploidy may indeed drive chromosomal instability (this may not be true but it does show that they are linked
What is the difference between aneuploidy and CIN?
→ Aneuploidy is an acquired state of a cell
→ CIN is a process that may lead to aneuploidy and that may be driven by aneuploidy
This means that not all aneuploid cells must show chromosomal instability (e.g. many cell lines derived from tumours are aneuploid but do not show CIN)
And not always cell that are characterised by CIN must be aneuploid (e.g. in case when all cells with the wrong number of chromosomes die immediately, aneuploidy is not going to develop)
What effect does aneuploidy have on gene expression and protein level?
causes upregulation of genes carried by the additional chromosomes and misregulation of genes on other chromosomes
What effect does aneuploidy have on cell fitness and proliferation?
Aneuploidy brings impaired proliferation and metabolism
BUT!
Aneuploidy is normal in certain cell types and its suppression leads to defects (e.g. certain cell types during development)
And, as a hallmark of cancer, most likely is beneficial to tumour cells because it:
Induces chromosomal instability
and
Adaptability
Explain how aneuploidy leads to the evolution of cancer
by causing chromosome missegregation and CIN, produces genetic heterogeneity in a cell population. This heterogeneity would make the population adaptable to a broader spectrum of environmental challenges/conditions. (similar to how a virus mutates) → EVOLUTION OF CANCER
Explain the different selections of aneuploid cells in tumour growth
Negative selection: aneupolidy will die out
Neutral selection: aneuploidy doesnt die out but is not especially selected for
Positive selection: aneupolidy is beneficial so spreasa, this usual;y leads to cancer
Give an example of how aneuploidy may participate in cancer evolution
Genome instability driven by aneuploidy can facilitate chemoresistance:
Resistance to chemotherapy is dictated by changes in gene copy number
Chemoresistance is achieved through altered expression of specific proteins
Due to heterogeneity it allows cells to get through different environmental constraints and eventually cause tumour development and cancer
What may be the origin of aneuploidy?
- Errors in kinetochore-microtubule attachments (e.g. merotely)
- Supernumerary centrosomes
- Weak Spindle Assembly Checkpoint fails to delay anaphase
- Impaired sister chromatin cohesion
- Cytokinesis failure
What are the 4 major forms of attachment of microtubules to chomosomes?
Amphitelic - correct model, both chromosomes attached correct therefore, the splindle checkpoint (SAC) inactive
merotelic - extra attachment, although wrong, centromeric tension means SAC turned off, but somehow these merotelic attachmenys are repaired before anaphase
Monotelic - only one chromosome attached, SAC active
Syntelic - two attachements from same chromosome, SAC active
When can failure of microtubule attachment occur, and what does this lead to?
when cells are overwhelmed with the number of erroneous attachments or when the repair system is impaired.
This leads to the missagregation of chromosomes
What can be said about merotelic attachments?
difficult to repair and may lead to chromosome missegregation that in turn will give rise to aneuploidy
Only a small fraction of aggregated chromsomes survive cell death
How do superneumarary centrosomes lead to aneuploidy
- Supernumerary centrosomes very rarely lead to multipolar divisions. Even if they do, typically cells die after such divisions.
Therefore it must be a different mechanism that leads to aneuploidy:
- 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 extra centrosomes and what is this a common characteristic of?
Common in cancer cells
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
Supernumerary centrosomes cause..
chromosome missegregation by increasing the rates of formation of merotelic attachments
Explain cytokinesis as a possible reason for aneuploidy
Cytokinesis defects affect the ploidy of a cell, and 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
All these pathways lead to aneuploidy, which is a numerical chromosomal aberration.
Features of structural chromosomal aberrations
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
Give an example of simple chromosome rearrangements, a.k.a. structural variations (SVs)
e.g. chromosomal translocation involved in cancer formation – Philadelphia chromosome (chronic myelogenous leukemia - CML)
conversion of the Abl proto-oncogene into an oncogene in individuals with chronic myelogenous leukemia. 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
Explain how some chromosome rearrangments are more complex, give an example
involving many points of break and fusions along the chromosomes
But there are also examples of extreme rearrangements that change chromosomes beyond recognition.
e.g. Chromothripsis:
Massive genomic rearrangement by pulverising chromosome and stitching it together is one of drivers of cancer genome evolution
Chromosome segregation defects can give rise to:
aneuploidy and/or chromothripsis
Chromosome missegregation often leads to formation of what?
Micronuclei -> DNA replication in micronuclei is defective -> This leads to extensive damage of DNA in micronuclei -> micronuclear chromosomes undergo chromothripsis -> some of the rearranged DNA is incorperated back into the genome
How may aneuploidy be used to fight cancer?
Targeting the adaptibility of heterogeneous aneuploids (Find reference)
Molecular targeting of defective functions in cancer cells must provide a wide therapeutic window to preferentially eliminate cancer cells.
It is based on the observation that certain levels of aneuploidy cannot be tolerated in cells
