L8: chromatin structure and genome stability

Question 1

What are the possible histone modifications?

Answer 1

• H1
• H2A
• H2B
• H3
• H4
• Core histones are: H2A, H2B, H3, H4, most positively charged, need to be able to shield from negative charges from the DNA
• Linker histone – important for folding the chromatin fibre up, C terminus long tail

Question 2

What are chromatin readers and writers?

Answer 2

Chromatin writers
Enzymes that add chemical modifications to DNA and histones. For example, protein arginine methyltransferases (PRMTs) methylate arginine residues.
Chromatin readers
Proteins that identify and interpret the chemical modifications added by chromatin writers. Readers bind to post-translational modifications (PTMs) on histones and can have either activating or repressive functions.

Question 3

What is the role of H2AX and what is the mechanism?

Answer 3

• H2AX is present across the entire genome on low level
• If damage is recognised in the chromatin by protein complexes: ATM and ATR (which are kinases)
• So they are able to phosphorylate on H2AX in the damaged sequence turning it into gammaH2AX – marks that bit of chromatin as damaged
• This allows recruitment of other proteins that are actually responsible for recruitment of repair proteins

Question 4

What are chromatin remodellers?

Answer 4

Chromatin remodelers are multiprotein complexes that alter chromatin structure by restructuring nucleosomes. They regulate a variety of cellular processes, including DNA repair and transcription, by controlling access to genomic DNA.

Question 5

What is the mutator hypothesis regarding the genomic instability?

Answer 5

Mutator hypothesis – the proposal that genomic instability promotes tumorigenesis by increasing the rate at which mutations in oncogenes and tumour suppressor genes arise during the multistep development of cancer.

Question 6

What are the epigenetic effects of H3.3 mutation?

Answer 6

• glioblastoma
• H3.3 is a variant of a histone H3 that’s normally found around the promotor of genes
• Mutations are found on K27M or G34R/V
• These modified histones affect how chromatin interacts with other chromatin related machinery
• Although histone proteins are highly conserved, you do get mutations causing very severe cancers
• H3.3 mutations are mostly prevalent in childhood patients

Question 7

How is fluorescence in situ hybridization useful?

Answer 7

FISH (Fluorescence in situ hybridization)
- In situ hybridization
o Examine position of a gene on a chromosome
o Examine position of chromosome in nucleus
o Examine distribution of transcript in cytoplasm
- Basic hybridization technique
o Label probe
o Hybridize via base pairing target

Question 8

How is array CGH useful?

Answer 8

• For looking for a copy number of individual chromosomes, you can take probes in different colours and hybridize them to microarray
• If you have amplification or deletion you can see more of one colour than the other

Question 9

What are common fragile sites?

Answer 9

Common fragile sites
- Regions of genome prone to damage under replication stress, such as aphidicolin treatment, form breaks cytogenetically visible on metaphase spreads
- AT-rich conserved between mice and humans in syntenic regions, associated with large genes, replicate late in cell cycle, cancer involvement
- Monogenic disorders with increased CFS formation: Werner’s syndrome, Bloom’s syndrome, Seckel syndrome
- Differential expression in different cell types and different individuals – epigenetic contribution to fragility

Question 10

Which proteins are important for correcting these lesions in common fragile sites?

Answer 10

• RecQ helicases recognise these regions that have not properly replicated
• If these helicases are mutated, we can increase the number of common fragile sites
• when humans have mutations in BLM or WRN gene helicases, they have a very high chance of developing cancers, because these cells are no longer able to repair these lesions that occur after replication stress
• Bloom syndrome: rare autosomal recessive disease characterized by short stature, predisposition to the development of cancer and genomic instability
• Werner syndrome: WRN helicase mutated, known as adult progeria, is a rare autosomal recessive progeroid syndrome (PS), and is characterized by the appearance of premature aging

Question 11

What is Bloom syndrome?

Answer 11

• Bloom syndrome: rare autosomal recessive disease characterized by short stature, predisposition to the development of cancer and genomic instability

Question 12

What is Werner syndrome?

Answer 12

• Werner syndrome: WRN helicase mutated, known as adult progeria, is a rare autosomal recessive progeroid syndrome (PS), and is characterized by the appearance of premature aging

Question 13

What are the possible mechanisms for common fragile sites formation?

Answer 13

• Not known for sure
• Replication fork collapse (possible): if you stress replication inside cells, replication fork falls apart, and you are unable to replicate, and lesions persist while you go through mitosis
• Replication initiation paucity (possible): maybe there aren’t enough origins across the genome for actually firing replication, or some of them are even turned off
• Transcription-replication collisions model (most possible): long genes take more than one cell cycle to be transcribed, so you need to be simultaneously transcribing these genes, actually also replicating the DNA for the cells that undergo the cell cycle. So, replication and transcription machinery might clash with each other. When they clash they might fall off the chromating, leaving some regions of chromatin unreplicated