Nuclear organisation and

long-range regulation of gene

expression
F. Guesdon
GEM6410, October 2006
 Gene Expression is regulated at
several levels
 Control by transcription factors
Transcriptional control  Long-range control
 Epigenetic control

RNA processing control  Splicing

 Half-life
 RNA surveillance
Translation control

Protein activity control

Topics for this lecture
1. Chromatin structure and remodelling
2. Introduction to epigenetics
3. Nuclear architecture
4. Long-range regulation: locus control
region
The Nucleus
 Complex structure for complex functions
 Packs 1.8 m of DNA !
 Expression of active genes
 mRNA processing and export
 Assembly of ribosomes

 DNA is packaged in a way to allow;

 Differential gene transcription
 DNA replication
 Gene recombination and cell division
DNA packaging changes through time
2 nm – DNA double helix

11 nm – Nucleosome
(11 nm fiber)

30 nm – 30 nm Fiber Interphase
chromatin:

300 nm – Loops I

700 nm – Loops II Metaphase

chromosomes

1400 nm – chromosome Alberts,

Mol. Biol. Cell.
 …And space.

Euchromatin:
Euchromatin:
•• Uncoiled
UncoiledDNA
DNA
••Available
Availablefor
fortranscription
transcription

Heterochromatin:
Heterochromatin: coiled
coiled (compacted)
(compacted) DNA,
DNA,
not
not available
available for
for transcription
transcription
 Determination of euchromatin
and heterochromatin domains
1. To a large extent, determined by DNA sequence

2 3 4

X Y

Chromosome structure of Drosophila Heterochromatin

melanogaster chromosomes 2, 3, 4, and Euchromatin
the sex chromosomes X and Y .
Centromere

2. But shows variability: Can be regulated locally by

covalent modifications of nucleosomes
  165 base pairs (bp) of DNA
wrapped around 8 histones.
Nucleosome  (Histones H2A, H2B, H3 and
H4) x 2.
 The DNA wound around the
octamer is not easily
accessible to regulatory
proteins; the linker regions are
much more accessible
 Chemical modifications of the
histone 'tails' regulate
nucleosome movement and
unwinding
 This has profound local effects
on the chromatin complex and
accessibility of DNA to
transcription factors and RNA
polymerase.
Interphase
chromosomes:
Mosaics of
solenoids/compacted
solenoids and extended
chromatin loops
I. Amphibian Lampbrush
Chromosomes: consist of
elaborately folded 30 nm
fibers and extended loops
containing expressed genes
 Histone modifications
 Chemical modification of histone tails: addition of
 Acetyl
 Methyl
 Ubiquitin
 or phosphate groups

 Complex regulation of chromatin state and gene

expression is effected by means of combinatorial
modifications of histones - the histone code

 Histone acetylation favors gene expression

Principal types of N-terminal histone
modifications and effects on chromatin

1. Acetylation of lysines: Neutralizes lysine +

charges and “loosens” histone/DNA interactions -
promotes de-condensation
2. Methylation of lysines: Replaces lysine + charges
by hydrophobic group and promotes formation of
highly compacted chromatin e.g. heterochromatin
3. Phosphorylation of serines: Helps to pack
nucleosomes together and thus tends to promote
higher levels of chromatin compaction e.g.
formation of metaphase chromosomes.
Histone modifications
 DNA methylation

 Methyl groups are found in the C residue of 5’-C-G-3’

dinucleotides
 Usually, the cytosines on both strands are methylated

5’-N - N - MC - G - N - N-3’
| | I I I I
3’-N - N - G- MC - N - N-5’

 After DNA replication, methylation on the parent strand

can promotes methylation of the new strand
Chromatin modifications regulates gene
expression
Euchromatin
Unmethylated DNA

methylated
DNA

recruitment
of (methyl-DNA binding domain)MBD’s

recruitment of
deacetylase complex

Deacetylated condensed
chromatin
Heterochromatin
Different histone tail modifications are
epigenetic markers for active and inactive
chromatin domains:

Silenced region
H3K9 Methylation
H3K4 Methylation (also H3K9 acetylation)

Similar results are also seen at the chicken B-globin locus.

Zhang & Reinberg (2001) Genes & Dev. 2343-2360.

Summary: levels of chromatin
organization
Nucleosomes: Package DNA into the 10 nm chromatin fiber.
Fundamental unit of chromatin (“beads on a string”);
Nuclesomes consist of histones.
30 nm chromatin fiber or solenoid: Created by coiled 11 nm
fiber.
Interphase chromatin exists as highly condensed solenoid
with interspersed extended loops containing transcribed
genes.
Metaphase chromosomes: looping/coiling of solenoid DNA
into highly compacted, transcriptionally silent, form of
chromatin- occurs during mitosis.
Importance of DNA methylation
 It acts as a signal for histone de-
acetylation and chromosome compaction
 This can inhibit binding of transcription
factors to DNA
 Often inhibitory - compaction of promoters
and enhancers
 Methylation can be heritable - it is an
epigenetic mechanism of regulation
Methyl-cytosine: the 5th base
Role of chromatin modifications
in cell differentiation:
 Exemples: Haematopoietic cell differentiation

1. Embryonic stem cell -------> B lymphocyte

2. Precursor T cell -------> TH1 or TH2
phenotype

 Two possible mechanims:

 Sequential gene activation
 Progressive gene silencing
Epigenetic hypothesis for B cell
differentiation
 Silencing may be reflected by
change in chromatin structure rather than
by the presence of a repressor.
Experimental approach
 Does the spatial position of a locus in the
nucleus correlate with its expression?

 Two techniques:
1. FISH - fluorescent labelling of genes in nuclei.
2. Replication timing assay - Measures whether a
gene replicates early or late during mitosis.
Epigenetic and chromosome organisation
changes occur during B lymphocytes
differentiation
Amanda Fischer, MRC clinical Sciences Centre, London:

 Nonequivalent nuclear location of immunoglobulin alleles in B

lymphocytes. Skok et al., Nat Immunol. 2:848-854 (2001)

 Nuclear organisation and gene expression. Baxter,

Merkenschlager and Fisher, Curr. Opin. Cell Biol. 14:372-376
(2002)
What is “epigenetic” information?
 Information “added” to the genome
 Usually without change to DNA sequence
 Transmissible to daughter cells through
mitosis
 Reflects history of the cell lineage
Examples of normal epigenetic
control
 X chromosome silencing, mosaicism
 Parental imprinting
 Cellular differentiation
 T cell maturation
 siRNA
Single cell assays of gene activation
of plasminogen activator inhibitor 2
Low LPS High LPS

Stimulus: bacterial lipopolysccharide (LPS)

 PAI-2 mRNA detection

Low LPS High LPS

As stimulus gets stronger (more LPS), more cells

start expressing PAI-2
The level of expression in individual positive cells is
the same regardless of the dose of LPS
The PAI-2 gene is regulated by LPS in an on/off
manner - there are no intermediate states
 Transcription is stochastic
 Transcription is not regulated continuously
 Stochastic behaviour
 E.g; Plaminogen activator inhibitor II. (PAI-2)
 On/off model, probability, frequency…
 It is NOT regulated by continuous variation of rate of transcription.
 Stimulus affects the probability of expression, not its rate

David A Hume Blood vol 96 Number 7

Patterns of gene activity in
space and time
 Long term regulation (e.g. through
differentiation) is achieved by stable
modifications of chromatin
 Short-term regulation can display on/off
behaviour rather than gradual variations
 What are the mechanisms that explain
this behaviour?
Analysing the spatial organisation
of chromosomes and transcription
Three techniques to analyse spatial
organisation of chromosomes:

1. FISH (labelling of DNA markers or pre-

mRNA)
2. Immunofluorescence microscopy (detection
of DNA-binding proteins, enzymes)
3. CCC - analysis of chromosome
conformation
 Fluorescence in situ hybridisation
(FISH)
Hybridization of fluorescent probes to DNAin fixed cells (in
situ) shows spatial position of chromosomal markers or gene
Satellite DNA is used to mark
locations of heterochromatin
 Tandem Repeats
 Organized in long mega base sized arrays
 Pericentromeric and telomeric
 Important for heterochromatin formation
 Proper chromosomal behaviour in mitosis and meiosis.
 Reserved function, but not sequence.
 Examples:
 Humans: Alpha Satellite: 171bp repeat
 Mouse: Gamma Satellite: 234bp repeat
 Example

Su et al. Nat Gen; May 2004

 Th2 Cells Th1 Cells

Grogan JL et al. Immunity. 2001 Mar;14(3):205-15

Loacting active genes by DNA-FISH and
RNA-FISH
RNA FISH DNA FISH
RNAP II Immuno

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Detecting inactive and active regions
of a chromosome
 Colocalisation of expressed
genes In relation to Hbb-b1
IgF2 & Kcnq1ot1

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Eraf Uros IgF2 Kcnq1ot1 Hba

Same chromosome Other
 Transcription factories
 The expected frequency of random co-
localisation of two genes in trans(on different
chromosomes) is estimated to be less than 1%.
 The observed frequency(Hbb and Hba) is 7% -
Much higher than random
 This indicates that active genes from different
chromosomes can co-localise at sites of
transcription
 RNA Polymerase II factories; Limiting
factor in gene expression

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Differentiated or committed cell have limited number of

available RNAP II factories. (erythroid cells have around
4000 active genes)
 Transcription factories
 Discrete nuclear sites of nascent RNA production
and concentrated transcriptional componnents
e.g. RNA polymerase II (RNAP II).

NOTE:
 Do not confuse “transcription factories” with
“transcription factors”
 Two similar names for very different things
 Chromosome territories and
transcription factories
 Chromosomes reside in specific sub-nuclear locations
(territories)
 Active genes tend to be found towards the interior of the
nucleus.
 Silent genes often associated with nuclear periphery/
heterochromatin
 Large loops extend outside the chromosome territory to
position active genes in transcription factories.
 Silent genes reposition away from the factories and close to
their chromosomal territory.
Eukaryotic gene regulatory structures

Single gene regulation

 Basal promoter (core promoter)
 TATA box (positions -40 to -25)
 ”Upstream" promoter
 Upstream regulatory Elements (UREs), positions -25 to as far as -200 bp
 Enhancer
 Silencer

Cluster regulation (Co-ordination of several genes)

 Insulator
 Locus Control Region
 Insulators
 Stretches of DNA (as few as 42 base pairs).
 Prevent a gene from being influenced by the

activation (or repression) of its neighbors.

T-cell antigen receptor ( TCR )

gene segments
Locus Control Regions (LCRs)
 DNA regulatory regions
 Not normally part of any gene
 Conserved between organisms
 Needed for expression of a group of linked
genes (cluster):
 Physiological levels
 Determines tissue specific expression
 Function in a copy number-dependent manner.
 Location independent
 Affects timing of replication of genes during mitosis
Currently known LCRs
 Locus Control Region (LCR)

Example: hemoglobin cluster

 Transgenic mice without LCR
 ß Thalassemy, but intact ß globin locus.
 Closed chromatin conformation.
 Properties of LCR
1. Transcriptional enhancer activity:
• Tissue specific
• E.g. Expression of globin genes confined to
erythroid cells when linked to ß globin LCR.
• ß globin LCR enhances linked heterogeneous
gene in erythroid cells.
• The gene is also expressed outside the erythroid
compartment.
 Properties of LCR

2. Copy Number dependent gene expression

 Properties of LCR
3. Timing and origin of DNA replication.

 ß-globin locus replicates late in most cells, but

early in erythroid cells.
 LCR directs early replication.
 Histone deacetylation occurs in late S phase.
• LCR participate in recruiting different histone
acetyltransferases.
A: LCR-bound factors direct
migration of DNA to a
transcription factory, or the
assembly of the factory

B: The chromatin fibre is reeled

in (arrows) to scan for
promoters

C: A gene is found; interactions

between promoter-, enhancer-
and LCR-bound factors stabilise
the gene in the factory and
cause high transcription levels

D: An enhancer-blocking
element, or silencer, might act
by preventing chromatin fibre
reeling and access of other
genes to factory
Example of study of an LCR:
T helper type 2 Th2 Cytokine Locus

Location:
•Human: Chromosome 5.
•Mouse: Chromosome 11.
CCC:
Chromosome Conformation Capture

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
* * * * * * * *

* = EcoRI = 20 Kb
Th2 Cytokine Locus

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CCC- Th2

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CCC- Th2

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Probing Th2 intrachromosomal
interactions with a reporter gene

Construct for BAC Transgenic Mice.

Th2 intrachromosomal
interactions
Locating the LCR:
BAC deletion analysis - step 1
BAC deletion analysis - step 2
BAC deletion analysis - step 3
 LCRs and Disease
Beta Globin- -Thalasemia

Deletion of LCR

Heavy Chain Immunoglobulin- Coeliac

Disease

Polymorphism in HS 1,2
Chromosomal Translocation
Importance of transcription
factories
Gene expression
 Gene regulation by LCR
 Increased efficiency of transcription:
 concentration of transcription machinery components at
specialised areas
 sharing enhancers

Sites of genetic changes

 Sites of chromosomal translocation
 Formation of gene cluster
 Conclusion
Regulation of gene expression involves:
 Transcription factors
 Signalling pathways

 Changes in the chromatin states

 Changes in spatial arrangements of active genes
 Folding and interaction between different regions
of DNA
 Concentration of regulatory and transcriptional
activities at specialised locations - Transcription
factories
 What you need to know
 You should understand the functional links between:
 DNA methylation
 Histone modifications
 Chromatin structure
 Gene expression / silencing
 Nuclear architecture
 Transcription factories
 Locus control regions

 You should know the principle and the aims of the two
following techniques:
 FISH and RNA-FISH
 CCC
End of lecture