Cancer Chromosome Biology- Lecture 4 to 8

What does spectral karyotyping identify?

Chromosomes by using special fluorescent dyes; chromosomes 1–23 dyed in different colours.

What can spectral karyotyping reveal in cancer chromosomes?

Fusion between different chromosomes and different karyotypes within the same tumour.

Define chromosomal territory.

Distinct nuclear regions where individual chromosomes are located, with different euchromatin/heterochromatin compartments.

What are TADs?

Topologically associating domains where sequences of DNA interact more frequently with each other than with sequences outside their TAD.

Which proteins mark TADs and form chromatin loops?

Cohesins and CTCF.

Does the TAD/looping organization apply during mitosis?

No, it applies for chromosome interphase only, NOT MITOSIS.

What can binding of a transcriptional activator to chromatin cause?

A change in the position of that chromatin domain, moving to an active compartment.

What happens when a repressor binds to chromatin?

The active compartment can move to the inactive position causing repression.

Why must chromatin be open for transcription?

Because condensed chromatin prevents transcription factors from binding to the DNA strand.

How does chromatin looping aid transcription?

Allows interactions between distant regions/enhancers/promoters forming protein complexes that aid transcription.

What can disruption of chromatin regions/domains lead to?

Cancer development.

Which factor is frequently mutated in cancer and can affect chromatin boundaries?

CTCF (or its binding sites).

Give an example of cancer where CTCF mutation contributes to disease.

Uterine cancer (CTCF mutation has a high contribution).

What is a deep deletion?

Both alleles are gone, no activity of that gene.

Where are CTCF and cohesin binding sites often mutated?

In the non-coding cancer genome; they are mutational hotspots.

How can methylation affect CTCF binding?

Methylation of the sequence that CTCF would bind to can prevent binding and disturb loop boundaries.

What was found in T-cell acute lymphoblastic leukemia regarding insulated neighborhoods?

Microdeletions eliminated boundary sites of important proto-oncogenes leading to elevated expression.

What are histones?

Small, highly conserved proteins forming the basic subunit of eukaryotic chromatin, the nucleosome.

What proteins form the histone octamer?

H2A, H2B, H3, H4 (two copies of each form the octamer).

What part of histones protrudes through the nucleosome and is heavily modified?

N-terminal tails.

Name four post-translational modifications of histone tails mentioned.

Phosphorylation, methylation, acetylation and ubiquitination.

What is an example of competitive modification on H3 K4?

K4 can be either acetylated or methylated on a competitive basis.

Which modification typically leads to gene activation?

Acetylation.

Which modification is associated with gene silencing?

Methylation (site dependent) leading to silencing of genes.

Who are the 'writers' in the histone code?

Methyltransferases, acetyltransferases, kinases and ubiquitin ligases that carry out PTMs.

Who are the 'readers' in the histone code?

Proteins with bromo, chromo and tudor domains that identify modifications.

Who are the 'erasers' in the histone code?

Demethylases and other enzymes that remove PTMs from histone tails.

Give three examples of epigenetic regulation.

DNA methylation, non-coding RNAs, histone modification, and histone variant exchange.

What is the role of histone variants in cancer?

Certain histone variants and their chaperones contribute to different stages of tumor development depending on tissue type.

Which histone variant can inhibit tumour growth?

MacroH2A (according to the notes).

Which histone variant can help tumour growth?

H2A.Z (according to the notes).

What effect does H3.3 have according to the notes?

H3.3 will contribute to invasion.

What effect does macroH2A have on metastasis?

MacroH2A will inhibit metastasis.

Why is the histone code important in cancer biology?

Mutations in writers, readers or erasers contribute to tumour development by altering epigenetic regulation.

What is histone acetylation stated to be responsible for in the notes?

Usually responsible for gene silencing and transcription repression of many genes but especially tumour suppressor genes. (Note: the notes also state acetylation at promoters/enhancers/gene body promotes transcription especially oncogenes.)

What is histone methylation's relevance in cancer?

Oncohistones are frequently mutated histone associated with cancers; methylation sites determine activation or silencing.

Give an example of an oncohistone mutation mentioned.

H3K27M in pediatric gliomas.

How can non-coding RNA function in cancer?

As tumour promoting or suppressing; can act by RNA–RNA, RNA–DNA, RNA–protein interactions and roles vary by cancer.

How can DNA methylation affect drug resistance?

DNA methylation is associated with drug resistance; hypermethylation at gene body can activate oncogenes and promoter methylation can repress genes like p16, p53, BRCA1.

Can the epigenetic component of cancer be targeted?

Yes; specific inhibitors are being developed to interfere with enzymes carrying out methylation, acetylation, etc.

What are oncohistones/onconucleosome?

Mutated histones in oncogenes that can lead to cancer (mutations in histones can have high genetic penetrance in rare pediatric gliomas and sarcomas).

Name two enzyme complexes involved in epigenetics

PRC2 and SETD2.

How can mutation of Glycine at position 34 affect epigenetic regulation?

It can sterically hinder binding of SETD2 to the histone, affecting methylation and chromatin interactions.

Which lysine methylation affects PRC2 binding?

Methylation of Lysine 27 affects PRC2 binding.

Which lysine methylation affects SETD2 binding?

Methylation of Lysine 36 affects SETD2 binding.

What is prometaphase described as?

When microtubules are attached to kinetochores; one of the longest phases.

Define centromere

A constricted region on a chromosome that joins sister chromatids and where the kinetochore forms; specialized DNA fragment allowing segregation.

What forms at the centromere?

The kinetochore, a protein+RNA complex that allows sister chromatids to segregate and where microtubules attach.

What histone variant marks centromeric chromatin?

CENP-A (CenH3).

Are human centromeres defined by DNA sequence according to the notes?

No; human centromeres are defined by presence of CENP-A rather than DNA sequence.

What is CCAN?

Constitutive Centromere Associated Network: multiprotein complexes recruited to centromeres, important for functioning centromeres and kinetochores.

Are CCAN components present throughout the cell cycle?

Yes, centromeric (CCAN) components are at centromeres throughout the entire cell cycle.

When are kinetochore components present?

During mitosis only; they are disassembled after mitosis.

What are the main functions of the kinetochore?

Capturing microtubules, identifying incorrect attachments and repairing them, producing force to move chromosomes during anaphase.

Name the structural core of the kinetochore / KMN network components.

KNL1/Spc105 complex, Mis12 complex, Ndc80 complex.

What does KNL1/Spc105 bind?

Binds Mis12 and Ndc80.

What is the role of the Mis12 complex?

Bridge between CENP-C protein and NDC80 complex.

What does the Ndc80 complex do?

Binds microtubules to the kinetochore.

How does the kinetochore connect to microtubules via two pathways?

First uses CENP-C protein; second uses CENP-T, CENP-W, CENP-S, CENP-X.

Which kinase regulates Ndc80 affinity to microtubules?

Aurora B kinase via phosphorylation.

What does the spindle assembly checkpoint (SAC) bind to?

Kinetochore components that are not attached properly to microtubules.

What happens to centromere/kinetochore genes in many cancers?

They are misregulated or overexpressed (e.g., SKA3, NUF2 in CENP-C complex).

What correlation was found with overexpression of some centromere/kinetochore genes?

Correlates with increased genomic instability, adverse tumour properties, and predicts poor survival in breast and lung cancer patients.

Name three categories of centromere/kinetochore genes used in the study.

CENP-A (centromere), CCAN (inner kinetochore), KMN (outer kinetochore).

What are the different types of microtubules mentioned?

Astral microtubules, interpolar microtubules, kinetochore microtubules.

What is required for correct segregation (bi-polarity)?

Kinetochores attaching to opposing centrosomes to create bipolarity.

How are incorrect attachments handled?

They are unstable and repaired by the chromosome passenger complex/Aurora B complex.

What are the four subunits of the CPC?

Aurora B kinase, INCENP, survivin, borealin.

What is INCENP's role?

Scaffold and activation; long protein.

What is survivin's role?

Centromere targeting and secures INCENP at the anchor; binds chromatin at inner centromere.

What is borealin's role?

Activation and interactions in cytokinesis.

Where is the Aurora B complex localized during mitosis?

At inner centromeres and then moves to the mid-zone of the central spindle for cytokinesis the inner centromere is where the Aurora B complex and cohesin is found.

Explain the kinetochore spring-like mechanism.

Outer kinetochore moves away or closer to inner centromere under higher or lower tension; low tension keeps outer kinetochore close to CPC where Aurora B phosphorylates Ndc80 destabilizing attachments.

What does Aurora B phosphorylation of Ndc80 do?

Regulates the stability of Ndc80 binding to microtubules; phosphorylation modulates affinity.

What effect does high tension have on Ndc80 and Aurora B reach?

High tension removes Ndc80 from reach of Aurora B kinase making the attachment stable.

What are the roles of Aurora A, B, and C as described?

Aurora A: centrosomes and mitotic spindle formation. Aurora B: component of CPC, involved in condensation, segregation and cytokinesis. Aurora C: in meiosis.

What happens if Aurora B amount is decreased?

Chromosomes are not properly arranged at the metaphase plate and are more scattered.

What effects result from Aurora A inhibition?

G2/M arrest, abnormal spindle formation, chromosome misalignment, incorrect centriole separation, leading to apoptosis.

What effects result from Aurora B inhibition?

Defective chromosome-spindle attachment, cytokinesis failure, polyploidy (p53 dependent) due to premature mitotic exit, leading to apoptosis.

What are common anti-mitotic drug targets listed?

Microtubules, kinesins, mitotic kinases (e.g., CDKs).

Name the most commonly used anti-mitotic drugs mentioned.

Taxanes.

What is the cohesin complex composed of?

SMC proteins 1 and 3 together with non-SMC subunits (Rad21/Scc1 and Scc3) form the cohesin complex.

What is the folding behavior of an SMC monomer?

SMC monomer folds in half to reach folded state and binds another SMC to form a dimer.

When is cohesin loaded on chromosomes?

During G1, after DNA replication it holds sister chromatids together.

With which protein does cohesin define chromatin borders during interphase?

With CTCF.

When is cohesin released from chromosomes?

Its release in prophase happens at the same time with axial compression of chromosomes during mitosis.

List cohesin functions during mitosis.

Sister chromatid cohesion and holding together sister centrioles.

List cohesin functions during meiosis.

Pairing homologous chromosomes, assembly of synaptonemal complex axes, coordination of sister kinetochores during first division.

List cohesin functions during interphase.

Sister chromatid cohesion, repair DNA breaks, assembly of DNA replication factories, regulation of transcription, organisation of chromatin loops+TADs.

What is Scc2 required for in the cohesin cycle?

Loading non-cohesive/unfolded cohesin onto DNA after mitosis.

What is Eco1's role during S phase?

Acetylates cohesin to establish cohesive (folded) cohesin to hold sister chromatids together.

How is mitotic cohesin removal regulated? (two stages)

First, Prophase pathway: mitotic kinases (Plk1) and Wapl phosphorylate cohesin in arms causing removal; second, Separase degrades cohesin from centromere so it cannot be reused.

What protects centromeric cohesin in prophase?

Shugoshin and protein prophase 2A protect centromeric cohesin by removing phosphorylation.

What triggers activation of separase?

Degradation of regulatory proteins by ubiquitination via APC/C leading to inactivation of Cdk1 and activation of separase.

What is APC/C?

Anaphase-promoting Complex, an E3 ubiquitin ligase that tags substrates for degradation by proteasome.

What complex creates the STOP signal that blocks APC/C?

Mitotic Checkpoint Complex (MCC) composed of BubR1, Bub3, Mad2 and Cdc20.

What happens when Bub3, BubR1 and Mad2 dissociate from APC/C?

Degradation of cyclin and securin happens leading to activation of separase and mitotic exit.

What are two outcomes of APC/C activation?

Degradation of securin activating separase leading to chromosome segregation; degradation of Cyclin B inactivating CDK1 leading to mitotic exit.

What does the Spindle Assembly Checkpoint (SAC) do?

SAC creates a STOP signal from unattached kinetochores to block APC/C, giving the cell time to establish proper attachments.

How does Aurora B activity affect SAC?

Aurora B activity is needed for proper SAC function; inhibition prevents recruitment of Mps1 to kinetochores to stop anaphase.