Chapter 4: The cell nucleus

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Chapter 4: The cell nucleus

Chapter 4: The cell nucleus

Birthe Fahrenkrog, 29.09.11


1. Chromatin organisation

2. Nuclear compartments

Chapter 4: The cell nucleus

3. The nuclear envelope and nucleocytoplasmic transport

http://cshperspectives.cshlp.org/cgi/collection/the_nucleus


Of evolutionary uncertain origin.

The cell nucleus

Defined by the nuclear envelope, a double-membrane continuous with

the endoplasmic reticulum.

Nuclear processes are often essential and include initial steps of gene

expression (transcription, processing and transport of RNA precursors),

as well as DNA replication, repair and recombination.

The nucleus is an optimised environment for performing regulated gene

expression at the levels of chromatin, transcription, RNA processing and

export of RNAs.

The nucleus allows regulation of cellular processes by determining the

nuclear versus cytoplasmic location of key molecules in a spatial and

temporarily controlled fashion.

Nucleus is not nucleus.


From DNA to chromosomes

adapted from: Ball, 2003 Nature 421: 421-22


“Naked” chromosome

The chromosomes

Mitotic chromosomes

adapted from: Pollard and Earnshaw, Cell Biology


Chromatin

1944: Avery, MacLeod and McCarthy showed that genes are made of

DNA.

1953: Watson and Crick discovered structure of DNA.

The revolutionary idea that genetic information is protein-free proved too

simple: DNA is coated with at least an equal amount of protein, forming

a complex called chromatin.

The principle protein components of chromatin are proteins called

histones.

Core histones are among the most highly conserved proteins, especially

histones H3 and H4. Pea and mammalian histone H4 differ only in 2 of

102 amino acids.


Heterochromatin

confined to small patches

Chromatin

mouse lymphocyte human cell

condensed throughout cell cycle

predominantly silent (fewer genes, repressed)

characterised by distinct epigenetic marks

Euchromatin

relative dispersed, net-like

less compact

most active part of the genome

accessible for the transcription machinery


Packaging DNA: the nucleosomes

adapted from: Felsenfeld and Groudine, 2003 Nature 421: 448-53

DNA wrapped around core histones

5- to 10-fold compaction

50-fold compaction

1000-fold compaction

10-fold compaction


Transmission EM

adapted from: Pollard and Earnshaw, Cell Biology

Nucleosomes: beads on a string

Dark-field EM


Nucleosome core particles are:

Nucleosomes

8 core histones (H2A, H2B, H3 and H4) with

approximately 147 bp of DNA wrapped

around them in 1.7 left-handed helical turns.

Linker histones H1/H5 binding organises an

additional 20 bp, provides further compaction

and determines the geometry of DNA

entering and existing the core particle.


Histones

Histones are the most abundant chromosomal proteins.

Evolutionary highly conserved.

They are characterised by highly charged N-terminal tail domains and a globular core

domain, known as histone fold domain.

The tail domains are relatively unstructured and extend out from the nucleosome through

DNA turns.

The tails are highly modified by posttranslational modifications, which is an important

mechanism to regulate DNA accessibility.

adapted from: Talbert and Henikoff, 2010, Nature Rev. Mol. Cell. Biol. 11: 264-75

The core domains have no sequence similarity, but a common fold, the histone fold structure.

Consists of three α-helices (α 1, α 2 and α 3) separated by two loops (L1 and L2).


Nucleosome assembly

adapted from: Wikipedia


Crystal structure of the nucleosome core particle

Luger et al.,1997, Nature 389: 251-260; Davey et al., 2002, J. Mol. Biol. 319: 1097-1113.


Crystal structure of the nucleosome core particle

Luger et al.,1997, Nature 389: 251-260; Davey et al., 2002, J. Mol. Biol. 319: 1097-1113.


Luger et al.,1997, Nature 389: 251-260;

Davey et al., 2002, J. Mol. Biol. 319: 1097-1113.

Histone tails between DNA grooves

random coil conformation

adapted from: Hansen, 2002, Ann. Rev. Biophys. Biomol. Struct. 31: 361-92


adapted from: Felsenfeld and Groudine, 2003 Nature 421: 448-53

Histone modifications


Higher order chromatin structure


low ionic strength

(< 100 mM salt)

The 30-nm fiber

high salt

(200 - 300 mM)

adapted from: Olins and Olins, 2003 Nature Rev. Mol. Cell. Biol. 4: 809-14.

10-nm fiber

beads on a string

30-nm fiber

coil of nucleosomes, 6 per turn


one-start helix

solenoid

The 30-nm fiber

two-start helix

zigzag

adapted from: Robinson and Rhodes, 2006, Curr. Opin. Struct. Biol. 16: 336-43.


tetranucleosome, no linker histones,

167 bp repeat, 20 bp linker DNA,

X-ray crystallography, 9Å resolution

The 30-nm fiber

Two-start helix

Schalch et al., 2005, Nature 436: 138-41.

Robinson and Rhodes, 2006, Curr. Opin. Struct. Biol. 16: 336-43.

25 nm in diameter, 5-6 nucleosome per 11 nm


22 nucleosomes, linker histones,

varying repeat and linker DNA length,

negative-stain EM

Robinson et al., 2006, PNAS 103: 6506-11.

Robinson and Rhodes, 2006, Curr. Opin. Struct. Biol. 16: 336-43.

The 30-nm fiber

One-start helix

3D reconstitution with nucleosome repeat length of 177-197 bp

33 nm in diameter, 5.4 nucleosome per turn


The 30-nm fiber

depends on nucleosome repeat length and the linker histones

Routh et al., 2008, PNAS 105: 8872-8877.


Further reading

1. Zlatanova et al., 2009, Structure 17: 160-171

The nucleosome family: dynamic and growing.”

2. Segal and Widom, 2009, Trends Genetics 25: 335-343

“What controls nucleosome positions?”

3. Clapier and Cairns, 2009, Annu. Rev. Biochem. 78: 273-304

The biology of chromatin remodeling complexes.”

4. Postnikov and Bustin, 2010, BBA 1799: 62-68

“Regulation of chromatin structure and function by HMGN proteins.”

5. Bernstein and Hake, 2006, Biochem. Cell Biol. 84: 505-17

The nucleosome: a little variation goes a long way.”


Pathologies


Systemic lupus erythematosus

- Systemic autoimmune inflammatory disease characterised by the production of

autoantibodies against the native nucleosome, its DNA component

! and/or its histone components (H2A, H2B), other nuclear proteins and cytoplasmic

components.

- Autoantibodies may be present for many years before the clinical onset of the disease.

- No single cause for SLE, but complex genetic basis for the disease, sunlight and drugs may

precipitate the conditions.

- Affects primarily women in their reproductive years (female to male rate: 9:1).

- Prevalence: 1:2500 in European-derived populations, 1:800 in populations with African or

Asian ancestry.

- Early symptoms are rather non-specific: fatigue, malaise, joint pain, photosensitive skin

rashes, chest pain, headache, dry eyes and mouth, mild hair loss, ....

DʼCruz, 2006, BMJ 332: 890-894


Other nucleosome-associated diseases

Circulating nucleosomes can be observed in case of:

sepsis, infections, stroke, heart infarction, cancer and others.

Defects in DNA methylation are associated with various developmental disorders:

α-thalassemia/mental retardation syndrome (ATR-X syndrome), ICF syndrome, Rett

syndrome, Rubinstein-Taybi syndrome, Coffin-Lowry syndrome and others.

Mutations in chromatin remodeling machinery: cancer


Chromatin organisation

www.lumc.nl/con/1050/85468/812090046152537/812100117522537/


- 30.000-75.000 genes

The human genome

- 3.2 billion basepairs of DNA packed into higher-order chromatin structure.

- Arranged in the human interphase nucleus among 46 chromosome territories.

- Chromosomes need to be arranged such that they are readily accessible to

transcriptional regulators that mediate their expression or repression.

- All this regulation takes place within the confines of the cell nucleus having an

average volume of 600-1500 µm 3 .

- The organisation is disassembled and reassembled during each cell cycle.

- Determining how nuclear functions are organised and coordinated, both

spatially and temporally, is central to understanding the proper workings of

the cell and the alterations that are associated with various diseases.


The discovery of chromosome territories

Carl Rabl (1885) and Theodor Boveri (1887) proposed that each chromosome

maintains its individuality during the cell cycle and Boveri explained this

behaviour in terms of chromosome territories.

The individuality of chromosomes was the basis for Boveriʼs “chromosome

theory of heredity” (1904) suggesting that individual chromosomes carry the

heritable genetic information.

Early 1980ʼs Thomas and Christoph Cremer demonstrated the existence of

chromosome territories experimentally in groundbreaking microlaser

experiments.


The discovery of chromosome territories

Meaburn and Misteli (2008) Nature 445: 379-381


The discovery of chromosome territories

CHO cells

Zorn et al. (1979) Exp. Cell Res. 124: 111-119; Meaburn and Misteli (2008) Nature 445: 379-381


Chromosomes are organised into territories

Cremer and Cremer (2001) Nature Rev. Genetics 2:292


Localisation of chromosomes in the human nucleus

human lymphocyte

18

chromosome position depends on gene content, protein modifications (acetylation),

and RNA/RNPs

19

Spector (2003) Annu. Rev. Biochem. 72: 573-608;

Croft et al. (1999) J. Cell Biol. 145: 1119-1131

chromosome 18:

85 Mb of DNA

2.6% of the physical length of the genome

low density of CpG islands

replicates most of its DNA in late S phase

gene-poor

chromosome 19:

67 Mb of DNA

2% of the physical length of the genome

high density of CpG islands

replicates most of its DNA in early S phase

gene-rich

but no correlation to chromosome size, centromeres or nuclear periphery (emerin)


Chromosomes are organised into territories

gene-poor territories

(Chromosome 18)

gene-rich territories

(Chromosome 19)

Cremer and Cremer (2001) Nature Rev. Genetics 2:292


Chromosome position is maintained throughout

interphase

Croft et al. (1999) J. Cell Biol. 145: 1119-1131

human lymphocytes


DNA

Chromosome position reversibly

changes in quiescent cells

Proliferating Quiescent Senescent

early G1 midG1 late G1

Bridger et al. (2000) Curr. Biol. 10: 149-152

human fibroblasts


Positioning of genes in chromosome territories

Folle (2008) Mut. Res. 658: 172-183

Interchromosome domain (ICD) model

CTs are separated by interchromosome domains (ICDs)

active genes are located at the periphery of CTs, inactive ones

in the interior

supported by the findings that RNA transcripts predominantly

accumulate in the space surrounding chromosomes and

demonstration of functional space between CTs

however: transcription sites have been found deep within CTs

by BrdU incorporation


Positioning of genes in chromosome territories

Branco and Pombo (2007) Trends Cell Biol. 17: 127-134

CT-IC model

CTs are subdivided into ~1 Mbp chromatin

domains, constituting a level of chromatin

organisation above the 30 nm fibre

ICD is a network of gaps between chromatin

domains that enable proteins access into the CT

each 1 Mbp domain is built up as a rosette of

small loops, termed ~100 kbp chromatin

domains

100 kbp domains are in contact with the ICD:

active genes locate at the periphery, inactive

genes at the interior of the 100 kbp domains


Positioning of genes in chromosome territories

Lattice model

Electron spectroscopic imaging (ESI)

Branco and Pombo (2007) Trends Cell Biol. 17: 127-134

chromatin is organised into 10 nm and 30 nm fibres with varying local concentrations

within the nucleus, no other higher order structures such as 1Mbp domain

irresolvable by lower-resolution methods

proteins (N) DNA (P) net DNA (P)


Positioning of genes in chromosome territories

Branco and Pombo (2007)

Trends Cell Biol. 17: 127-134

Lattice model

No large spaces other than those occupied by bodies such as nucleolus or PML bodies.

Fibres from different chromosomes are able to intermingle to a certain extent at the edges of CTs.

Fibres form a nearly-continuous lattice across the whole nucleoplasm.

Folding into a 30 nm fibre is sufficient to regulate gene activity by limiting accessibility to its interior

without the need to invoke a more complex structure such as 100 kbp domain.

pol II found at the surface of chromatin fibres throughout the whole nucleus.


Positioning of genes in chromosome territories

The ICN model

Incorporates recent evidence obtained by cryo-FISH for intra- and interchromosomal associations.

ICN model predicts that functional interaction occurring within a CT and between CTs are mediated

by pol II or other nuclear components, form a network that contributes to the organisation of

chromosomes within the nucleus.

This cell type-specific network influences CT position and accessibility as well as chromosomal

translocations.

Branco and Pombo (2007)

Trends Cell Biol. 17: 127-134


Yeast interphase chromosomes are highly dynamic

Taddei and Gasser (2004) BBA 1677:120-128.

Heun et al. (2001), Trends Cell Biol. 11:519-525

Taddei and Gasser (2004) BBA 1677:120-128


Chromatin motion is constrained by associations

with nuclear compartments in human cells

lacO arrays

5p14

Chubb et al. (2002) Curr. Biol. 12: 439-445

nucleoplasmic

peripheral

nucleolar

nucleolar


Summary I

Interphase chromosomes are arranged non-randomly.

Chromosomes occupy a finite, mutually exclusive fraction of the nuclear volume and represent

structural units known as chromosome territories.

In mammals, CTs are arranged in a radial manner according to gene density.

CTs are distinct entities with no gross intermingling, but loop regions mediate intra- and

interchromosomal associations.

CTs have constrained mobility, but individual genomic loci randomly diffuse within confined regions

with a radius of ~0.5 µm.

CTs are permeable to proteins and other nuclear compartments (bodies) up to a size of ~600 kDa.

Active transcription sites are scattered throughout CTs and are not concentrated on their surface.

Genes can be found anywhere in a CT, regardless of their transcriptional activity


Are chromosome arrangements passed on from

one cell generation to the next?


Chromosome territories in dividing cells

7 10

Walter et al. (2003) J. Cell Biol. 160: 685-697.

HeLa nuclei of two-cell clones


7 10

Chromosome territories in dividing cells

Walter et al. (2003) J. Cell Biol. 160: 685-697.

HeLa nuclei of four-cell clones

Similarity between sister cells is not perfect.

Differences between clonally descendent cells increases with successive cell

divisions.


paternal

maternal

Chromosome territories in dividing cells

Differential demethylation of parental chromatin in the early mouse embryo

Mayer et al. (2000) Nature 403: 501-502; Gerlich and Ellenberg (2003) Curr. Opin. Cell Biol. 15: 664-671

Chromosome positions are not randomised during early embryonic mitoses.


Chromosome positioning in live cells

NRK cells

Gerlich et al. (2003) J. Cell Biol. 112: 751-764


Chromosome positioning in live cells

positions are heritable

NRK cells bleached in early G1

Gerlich et al. (2003) J. Cell Biol. 112: 751-764


Chromosome positioning in live cells

positions change during mitosis

HeLa cells / H2B-GFP arrested in G2 before bleaching

Walter et al. (2003) J. Cell Biol. 160: 685-697


It is a matter of degree.

Differences in cell type.

How to resolve?

Mother nuclei are analysed at different periods before mitosis, daughter G1

nuclei at different periods after metaphase.

Both studies suggest that a significant level of global chromosomal order is

established at the time of chromatid separation. Chromosome positioning

inherited during metaphase-telophase transition, some mobility in

prometaphase and early G1.

To look at individual loci.


How is chromosome positioning maintained?

Gerlich and Ellenberg (2003) Curr. Opin. Cell Biol. 15: 664-671


Summary II

Global patterns of chromosome arrangements are transmitted to subsequent

cell generations: direct descendents resemble each other more than unrelated

cells.

Some positional changes are observed (probabilistic order).

In live cells, chromosomes move preferentially to positions in postmitotic

daughter cells that are similar, but not identical, to the mother nucleus.

How this is established and maintained is far from being understood and still at

a highly speculative stage.


Chromosome translocations:

Pathologies

cancer, especially in leukaemia and sarcoma

miscarriage


Mitotic chromosomes

The chromosomes

“Naked” interphase chromosome

Pollard and Earnshaw, Cell Biology


Histone variants

adapted from: Bernstein and Hake, 2006 Biochem. Cell Biol. 84: 505-17

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