Lecture 5 - Nuclear organisation Flashcards
How can nuclei change in cancer?
- Shape and margin irregularity
- changes to chromatin compaction/texture
- altered nucleoli
- variations in size
When is heterochromatin replicated?
late in S phase
When is euchromatin replicated?
Usually earlier in S phase
Why is euchromatin replicated early in S phase?
So the cell has 2 copied of it for longer
What is a SARs or MARs?
Scaffold/Matrix Attachment Regions
What do some people believe about the nuclear matrix?
There is an interconnecting network extending from the nuclear matrix through the cytoskeleton to the extracellular matrix
Why is it difficult to see the ‘interconnecting network’?
Usually studied in cultured cells which are different from cells in tissues
How is chromatin organised?
In loop attachments formed by periodic attachment to the nuclear matrix MARs and SARs
What happens at the base of the loops of chromatin?
Site of assembly of molecular machines involved in txn, splicing and DNA replication
Why is it good that everything happens at the loops of chromatin?
Regulatory elements can all come together there
How can the loops of chromatin cause disease?
- SATB1 (special AT-rich binding protein) is normally at loop bases
- Loss deregulates expression oof 2% of genes
- In KO genes move from loop from base
- Overexpression linked with aggressive breast and prostate cancer
- Shows importance of spatial arrangement
What are nucleoli?
Sites of ribosome synthesis
What is in the nucleus?
Chromatin occupies 35% of volume
Matrix occupies 10%
How is chromatin organised in interphase?
Distinct chromosomal bands segregate into different regions giving rise to chromosome territories.
Resulting in establishment of distinct high order genome compartments with functionally distinct chromatin fractions
What can you see when you differentially fluorescently label 2 chromosomes?
They move to different parts of the nucleus
How do you perform chromatin conformation capture (3C)?
- Use a chemical cross linker to make interacts between chromatin and protein assembly
- Digest chromatin into fragments
- Elute anything not cross linked
- Ligate generating an artificial DNA strand
- Analyse and look at frequency of two regions occurring together
What is 4C?
3C but in different time frames
What are common in leukemias and lymphomas that can be cured by chemo?
Balanced translocations
What are unbalanced rearrangements?
Common in solid tumours and are associated with more disorganisation and are difficult to cure
What are the two models of order of chromatin?
Deterministic and self-organisation
What is the deterministic model of chromatin?
- Structural evidence and adapter proteins keep everything together
What is the self-organisation model of chromatin?
Active regions and silenced regions between chromosomes
What does a stem cell require in terms of chromatin?
Access to all genes
What does a differentiating cell require in terms of chromatin?
Establishing order, long range interactions, lineage specific gene expression
What does a terminally differentiated cell require in terms of chromatin?
Long term functional specialisation
What did Coverley do with stem cells?
- Took stem cells and differentiated cells that had been induced from same cell line
- Added enzymes that digest chromatin to see the proteins bound to (left behind) the nuclear matrix (NC bound)
- Did this with Cyclin E
What results did Coverley get?
- In stem cell cyclin E is easy to extract
- In differentiated cell cyclin E shifted onto the NC
What is the implication of Coverley’s result?
Cyclin E’s function transitioned from not on the matrix to the matrix
What else did Coverley do with cyclin E and what was the result?
Tested in cancer cells
Cyclin E was not recruited onto nuclear matrix (cancer cell has no underlying architecture)
How can you find how many anchor points there are?
- Solubise nuclear membrane
- Extract histones
- Relieve supercoils
- DNA spreads into a halo only anchored at loop bases (S/MARs)
What features of nuclear morphology in cancer be used as a basis for diagnosis?
- Measure of nuclear and tissue disorganisation
- Predictor of aggressiveness
What can destabilisation of nuclear order facilitate?
Accumulation of other changes (genetic/epigenetic)
