L2: Architecture of the nucleus 2

Chromosomes and organisation of the nucleus

The nucleosome subunites of chromatin allow for

• hierarchies of folding chromatin fibres 

For gene expression of replication

• dynamic accessibility of certain regions of DNA in the chromatin has to be provided

When is the most condensed state of chromatin found

• during metaphase of mitosis

→ chromosomes

Key principles of higher-order packing

1. loop formation

2. attachment of chromatin fibres

→ to an underlying scaffold or matric strucutre

Note: this shows the chromosome in interphase→ the most condensed phase needed for mitosis

In normal conditions: DNA is more lose and jus exists in its territory

How is chromatin condensation mediated

• condensins

  • large proteins

  • ‘strucutural maintenance of chromosomes’ proteins (SMCs)

HOw was this experimentally found out

• mutagenesis

• continue with all proteins

• until find the proteins that are needed for the condensation

RESULT:

1. SMCs→ unclosed loop thing

2. Kleisin→ ‘to glue’ closes the loop together

How does this work?

• Complexes of SMC2 and SMC4 

• with kleisin proteins (CAPs)

• clamp chromatin fibres

→ Condensation mediated!

Visualisation of chromosome condensation by SMC proteins: condensin

But you don’t walays want it to be condensed

• need phosphate? to break the kleisins

• so can de-condense after replication

Nuclear matrix

• when the crhomosomal DNA

• from underlying residual protein strucuture

• is liberated

HOw is this made?

• depleteing entire interphase nuclei or mitotic chromosomes

• from histones

  • e.g by detergents and high salt

The DNA in this nuclear matrix is attached in…

• loops of -60kbp

• to the residual matrix or scafforld

→ big fibres come out of uncerlying structure→ some more condesned than others

This DNA that is attached is called…

MAR

• matrix-associated regions

or

SAR

• scafford-associated regions

But how to experimentally show that there iis a scaffold?

1. Dissociate metaphase chromosome DNA from scaffold

2. Functional identification of DNA sequences that attach loops to chromosomal matric/scaffold

1. Dissociated metaphase chromosome

• shows evidence that there is a scaffold

• but not completely confirms how the scaffold works

2. Functional identification of DNA sequences that attach loops to chromosomal matric/scaffold: i.e finding out which parts of the DNA are ‘sticky’ for the scaffold to attach to (The MARs)

1. Adding nuclease to the MAR an SAR

• can isolate the DNA loops

2. wash away the proteins

3. degrade all DNA with DNAase

4. label

5. ass DNA

6. extract and sequence the DNA

7. see if they are the same region all the time?

Might have to check this!

First way to do experiment: conceptual stickiness (right handside)

1. DNA on the scaffold (which is made of proetins, laminins etc

2. DNAase→ degrades DNA off→ into fragments

3. Now add random small fragments of DNA

4. See what sticks

THEREFORE:

• Can see which parts of DNA have ability to stick to scaffold

but

• Ignores the context of continuous long strand and loops

OVERALL: just conceptual

Second way to do experiment: conceptual stickiness (left handside)

1. DNA on scaffold

2. Cleave with resistriction nucleases

3. cuts at certain points (almost like shaving the histone ball)

4. Extrat this DNA

5. anaylse

THEREFORE:

• we have taken off the DNA off the scaffold and sequenced

• so show what is actually on the scaffold

Features of MARs found out from these experiments

1. very A/T rich

2. contain weak consensus sites for DNA topoisomerase II

Not specific: BUT some regions are more sticky than others

• more stoacastic distribution

• taking into account the loop and tension

• to see which parts will be more likely to stick

What does this suggest about DNA topoisomerase II?

Might be involved in:

• controlling coiling of (topologically closed) DNA loops of the matrix

Other features of the nuclear matrix or scafford?

consistuents are ill-defined but…

1. SAF-A

   • Scaffold attachment factor A

   • directly bind to SAR elements

How can principle chromones arcitecture in interphase be visualised?

• in special cases

• light microscopy

Example of this?

Lampbrush chromosomes of newt oocytes

• highlight essential features of interphase chromatin organisation

Features of amphibian oocyte chromosomes

1. condensed for several months

2. in early meiotic prophase

3. very active in transcription→ desnsely packed nascent RNP particles coat the loops

non condensed parts are actively transcipbed and condesned are not

What is helpful about the RNP particles coating the loops

• allows them to be seen in the light microscope

But how do we know the loops are chromatin?

• use a floresecent histone

What does hybridisation of DNA probes to chromosomes preparations confirm?

1. organisation is strictly sequence specific

2. individual decondensed loops can correspond to particular active genes

Can transmission EM help differentiat individual chromosomes in interphase nuclei

• no

What is seen in the TEM instead?

1. densely packed heterochromatin

   • (no active genes)

2. (relatively) decondensed euchromatin

   • (active genes present) (lighter in colour)

What is involved in the formation of euchromatin and heterochromatin: histone code

Post-translational modifications of histone tails

• produce altered binding surfaces 

• for effector proteins

  • → influence chromatin strucuture

Example of these modifiations → SILENCE

HIstone H3:

1. methylated at lysine residue 9 (H3K9me)

2. bound by the HP1 heterochromatin protein

3. this HP1 itself brings with it other proteins

   • → Act to SILENCE the DNA 

Example of these modifiations → ACTIVATION H3 example

Histone H3

1. acteylation on lysine (H3K9)

2. brings chromatin remodelling enzymes 

3. open the chromatin strucutre

4. enable access of the transcription machinery

→ ACTIVATED

But how do we experimentally know this?

Visualisation of histone modes by immunofloresence microscopy

• antibodies

How this works: finding out if methylation is associated with heterochromatin

1. Label nucleus with methH3-K9 antibody → red

2. Label with antibody for heterochromatin

3. Merge to see yellow colour if they are in the same place

4. Double check with DAPI that there is more DNA (e.g the unlabbeled euchromatin)

What have hybridisation experiments help show about chromosomes

• Experiements with whole nucleus preparations

• with probes specific for individual chromosomes 

  • → chromosome painting

RESULT:

1. evidence for concept that individual chromsomes occupy discrete territories  

   • within the interphase nucleus

however

2. Locations of individual chromosomes and individual genetic loci within the nucleus are dynamic

But how do we visualise indivisual chromsomes to find their territoires

1. need DNA sequence

2. abel part of it that is spcific to the chromosome

Map of the chromosome territores

→ bt can these move around? Are they dynamic?

note that HeLa cells are not like this→ once mutated, cancer cells lose their territorial organisation

How to find synamic behaviour of chromosome

• tag over time:

RESULT:

• dynamic

• but does stay in rrough territory

Next question to ask about the territories

• Which parts of the DNA touch eachother

• within and between the territories

• (within same chromosome or between different chromosomes)

HOw do we find this out

• Hi-C→ high throughput DNA sequencing

What has high throughput DNA sequencing techniques enabled us? (Hi-C)

• Enabled the mapping of 3D DNA-DNA interactions

• within the nucleus

What is this technique called?

Hi-C

→ High-resolution Chromosome conformation capture

Principles of this technique?

1. Cross link the DNA→ this will stick together anything that is close to eachother

2. Cut crosslink of DNA with restriction enzyme

3. sticky ends

4. fill with biotin mark

5. ligation of proximal DNA segments

6. purify and shear DNA→ pull down biotin (around 100bp long)

7. sequence of these junctions, using paired ends

Bulk experiment: get a better understanding of the loops of DNA→ looking at ALL the different interactions and closeness of all chromosomes in the genome at once

Results of this e.g interactions within the Chr 14

• show zones of high interction topologically associated domains (TADs)

I think the shape of this graph is trivial and is a coincidence that it is kinda a straight line

Evidence obtained by Hi-C has been used for

1. modelling interactions within chromosome territories 

   • at chromosomal resolution

2. dynamic spatial segregation of chromatin fibres

   • into open and closed domains at megabase resolution

really good to get an almost gene level view of what genes could influence eachtoother due to the proximity of their terrioties

Many Interphase activites have been shown to occur where?

• at discrete subnucleuar sites, or foci

  • NOT: dispersed or in solution

Nuclear compartmentalisation

Next question to ask

• Are there specific places for:

  • DNA replication foci

  • transciption foci

  • nucleoli

• or are they just in random places

How to find this out

• tag with fluorescent 

green= sites of replication

red=nuclear DNA

Examples of this nuclear compartmentalisation

1. clusters of DNA replication forks (replication foci)

2. clusters of RNA synthesis and processing machinery (transciption foci)

Features of the intracellular locations and patterns of these foci?

1. highly dynamic

2. seem to be associated with the nuclear matrix

Tagging the parts of DNA which are replicating also gives infor for the timing of replication during interphase:

1. EARLY: euchromatin

2. LATE: heterochromatin

DNA replication foci and trasciption foci

WHat is the nucleolus

• large subnuclear compartment

• separated from the rest of the nucleus

  • by region of condensed heterochromatin

How are nucleoli dynamic

1. Early G1: separately made from each chromsome territory

2. aggregate mid G1

3. finaally one nucleous late G1/S phasse

What happens with in the nucleolus

1. rDNA is transciptbed by RNA polymerase 1

2. generatres pre rRNA

3. these RNAs are processed to generate mature rRNAs

4. assembled with imported ribosomal proteins

5. to generate ribosome subunits

6. THEN exported out of the nucleus

7. complete ribosomes are assembled in the cytoplasm

What surrounds the chromatin in interphase nucleus

• nuclear envelope (the nuclear frontier

What does the nuclear envelope consist of

1. 2 membranes 

2. underlying lamina

   • 2 dimensional meshworkd made of a spcieal kind of intermediate filament proteins

lamina itself is used to help repair DNA after damage e.g UV light

What are these special kinds of proteins

• Lamins

  • A B and C

• cross linked protein fibres

• solid stuff the keep chromatin in

  • the membrane is only like a bubble

HOw does the nucleaur envelope interact with the nucleus

Direct contact to

1. chromatin

2. inner nuclear membrane

How do lamin fibres contribute to architecture of nuclear matrix (as some evidence suggests)

• Lamin fibres extend into the lumen of the nuclei

What does the inner membrane contain

• receptors that bind the nuclear lamina

Features of the outermembrane

1. continuous with the rough endoplasmic reticulum

2. contains ribosomes

Both the inner nad outer membrane …

• encapsulate the perinuclear space

  • which is continuous with the lumen of the ER

Both nuclear membranes are perforated by…

• nuclear pore complexes

What are nuclear pore complexes

• huge macromoleular complexes

• aorund 150MDa

• made of 50-100 different proteins

  • nucleoporins

What do Nucleuar pore complexes form

• aqueous channels across the nuclear envelope

• allows diffusion of small molecules into and out of nucleus

But what is their main function?

• regulated transport of mactomolecules

• into and out of the nucleus