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

• ddynamic 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

How is chromatin condensation mediated

• condensins

  • large proteins

  • ‘strucutural maintenance of chromosomes’ proteins (SMCs)

How does this work?

• Complexes of SMC2 and SMC4 

• with kleisin proteins (CAPs)

• clamp chromatin fibres

→ Condensation mediated!

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

This DNA that is attached is called…

MAR

• matrix-associated regions

or

SAR

• scafford-associated regions

Adding nuclease to the MAR an SAR

• can isolate the DNA loops

→ can then be sequenced

Features of MARs

1. very A/T rich

2. contain weak consensus sites for DNA topoisomerase II

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

What is helpful about the RNP particles coating the loops

• allows them to be seen in the light microscope

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)

What is involved in the formation of euchromatin and heterochromatin

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

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

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

What has high throughput DNA sequencing techniques enabled us?

• 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?

• ligation of proximal DNA segments

• and the sequencing of these junctions

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

Many Interphase activites have been shown to occur where?

• at discrete subnucleuar sites, or foci

  • NOT: dispersed or in solution

Nuclear compartmentalisation

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

WHat is the nucleolus

• large subnuclear compartment

• separated from the rest of the nucleus

  • by region of condensed heterochromatin

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

What does the nuclear envelope consist of

1. 2 membranes 

2. underlying lamina

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

What are these special kinds of proteins

• Lamins

  • A B and C

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