Transcript ppt - Chair of Computational Biology

V3 Nuclear architecture affects gene regulation
Eukaryotic genomes are regulated on 3 different levels:
(1) On the sequence level (V2). This includes the 1D organization of functional
sequence elements in the genome:
- coding regions,
- regulatory sequences that bind sequence-specific transcription factors (TFs)
- sequence elements that determine the 3D folding of the chromatin
(2) On the chromatin level (V1, V5-V8)
- different histone compositions
- the ¨histone code¨ (see lectures on epigenetics)
(3) On the nuclear level (today)
- the 3D structure and functional compartimentalization of the genome inside
the interphase nucleus.
Driel et al. J. Cell. Sci. 116, 4067 (2003)
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Nuclear architecture
A striking feature of nuclear architecture is the existence of distinct structural and
functional compartments.
Well characterized nuclear substructures include the nuclear lamina, nucleoli,
PML and Cajal bodies, and nuclear speckles.
Also, a growing number of components of the machinery that is required for
transcription or its repression are known to have a non-homogeneous
distribution in the nucleoplasm.
At the level of the genome itself, the genetic material is folded and packaged
in the nucleus into higher-order structures that are likely to contribute to the
regulation of gene expression.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Organization of the mammalian cell nucleus
Major goal: identify the principles
that govern the spatial organization
of the genome.
The nucleus is characterized by a
compartmentalized distribution of
functional components. The nuclear
envelope contains pores and rests on
a meshwork of intermediate filaments,
the nuclear lamina. Nucleolar
organizer regions cluster to form
nucleoli.
In the chromosome territory–
interchromatin compartment (CT–IC)
model, chromatin is organized in
distinct CTs.
Also depicted are nuclear speckles,
PML bodies and Cajal bodies located in
wider IC lacunas.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Model of functional nuclear architecture
(a) CTs have complex folded surfaces. Inset: topological model of gene
regulation. A giant chromatin loop with several active genes (red) expands from
the CT surface into the IC space.
Cremer, Cremer Nat. Rev. Gen. 2, 292 (2001)
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Molecular model of nuclear pore complex
Localization volumes of all 456 proteins in the NPC (excluding the FG-repeat
regions) in 4 different views.
The proteins are colour-coded according to their assignment to the 6 NPC
modules.
Alber et al. Nature (2007)
Chromatin movement
In vivo studies showed that the positions of labelled chromatin are constrained
during interphase within a radius of ca. 0.5 – 1 m.
This is less than 1% of the volume of a typical spherical mammalian nucleus that
has a diameter of 10 m.
Only during early G1, long-range movements of  2 m are observed.
In Drosophila, labelled topoisomerase II that binds to a heterochromatic repeat
block on chromosome X could explore about half of the radius of a Drosphophila
nucleus (2 m) indicating constrained diffusion.
Current work addresses whether the chromatin movements are correlated with
changes in gene expression.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Nuclear architecture affects gene regulation
One classical example of organization within the nucleus is the distinction
between decondensed, transcriptionally active euchromatin and more condensed,
generally inactive heterochromatin.
Individual chromosomes occupy distinct positions in the nucleus, referred to as
chromosome territories.
As a result of different compaction levels, different chromosome segments
adopt a complex organization and topography within their chromosome territory.
Gene-rich regions tend to be oriented towards the nuclear interior, whereas genepoor regions tend to be oriented towards the periphery. This principle of
nuclear organization is evolutionarily conserved.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Chromosome territories in the chicken
(a-c) staining of different chromosomes.
(d) optical section through a chicken fibroblast nucleus showing various mutually
exclusive CTs.
Cremer, Cremer Nat. Rev. Gen. 2, 292 (2001)
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The interchromatin compartment
LASER CONFOCAL sections through a HeLa
cell nucleus with GFP-tagged H2B, and staining
of speckles
a Section showing GFP-tagged chromatin (high
density, white; low density, grey), two nucleoli
(nu) and the interchromatin compartment (IC)
space (black). Note the variability in the width of
this space with examples of IC lacunas
(asterisks). Inset: expansions of less condensed
chromatin into the IC space at higher
magnification.
b Speckles visualized in the same section using
antibodies to the non-snRNP splicing factor SC35.
c Overlay of sections (chromatin, green;
speckles, red) shows that speckles form clusters
in IC lacunas. These lacunas are only partially
filled by the speckles, leaving space for other
non-chromatin domains.
…
Cremer, Cremer Nat. Rev. Gen. 2, 292 (2001)
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Regulation on the nuclear level
(1) A classical example is the nucleolus,
in which the rRNA-coding gene clusters of several chromosomes are brought
together to create a subnuclear domain that is dedicated to rRNA synthesis and
processing and pre-ribosome synthesis.
(2) Clustering of heterochromatin e.g. near the nuclear envelope.
Difficulty: we lack experimental tools to manipulate nuclear structure!
So far, researchers have mostly analyzed correlations.
Driel et al. J. Cell. Sci. 116, 4067 (2003)
Biological Sequence Analysis
SS 2009 – lecture 3
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Model of structural constraints on chromatin mobility
A model of structural constraints
on chromatin mobility and gene–
gene interactions.
a Three hypothetical chromosome
territories — green, blue and red —
are shown.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Model of structural constraints on chromatin mobility
Filled circles: Subchromosomal ~1 Mb
domains.
For the green territory, the lighter-green
circles indicate gene-poor chromatin; for
all territories, dark-coloured circles
indicate gene-dense chromatin.
Dark background shading: areas that
contain transcriptionally active genes.
b | The three panels show how the
chromatin that is located where the
same three territories are adjacent to
each other (shown as a shaded
region in panel a) might become
repositioned over time; the panels
indicate 3 consecutive time points
during interphase.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
As the chromatin moves over time,
there are concomitant changes in the
positioning of genes that are involved in
interchromosomal interactions (the
positions of 3 such genes, one from
each territory, are indicated by black
circles around the ~1 Mb domains in
which they are located).
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The multiloop subcompartment model
b, c Two 3D models of the
internal ultrastructure of a
~1-Mb chromatin domain.
b The nucleosome chain is compacted into a 30-nm chromatin fibre and folded
into ten 100-kb-sized loop domains according to the multiloop
subcompartment model. Occasionally, 30-nm fibres are interrupted by short
regions of individual nucleosomes (small white dots). The arrow points to a
red sphere, with a diameter of 30 nm, that represents a TF complex.
Cremer, Cremer Nat. Rev. Gen. 2, 292 (2001)
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The multiloop subcompartment model
b, c Two 3D models of the
internal ultrastructure of a
~1-Mb chromatin domain.
c | Each of the ten 100-kb chromatin domains was modelled under the
assumption of a restricted random walk (zig-zag) nucleosome chain. Each
dot represents an individual nucleosome. Nine 100-kb chromatin domains
are shown in a closed configuration and one in an open chromatin
configuration with a relaxed chain structure that expands at the periphery of
the 1-Mb domain.
The open domain will have enhanced accessibility to partial transcription
complexes preformed in the interchromatin compartment. By contrast, most
of the chromatin in the nine closed domains remains inaccessible to larger
factor
Cremer,
Cremer Nat. Rev. Gen. 2, 292 (2001)
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complexes
(arrows).
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Localization at the nuclear envelope
Eukaryotic genomes contain 3 classes of chromatin. The establishment and
maintenance of chromatin states is related to their spatial distribution with the
interphase nucleus.
(1) Open or actively transcribed chromatin, which contains genes with
engaged RNA polymerases.
(2) Potentially active chromatin, which contains promoters that are poised to
respond to activating signals, but from which stable transcripts are rare or
non-existent.
In yeast, these two states account for the vast majority of chromatin.
(3) In mammals, they only comprise a small fraction of the genome. In
differentiated somatic cells, most DNA is in a transcriptionally silent
heterochromatic state. Here, genes are generally repressed, gene
promoters are inaccessible to TFs.
Akhtar, Gasser, Nat. Rev. Genet. 8, 507 (2007)
SS 2009 – lecture 3
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Transcriptionally silent heterochromatic states
Positioning of chromatin at the nuclear envelope can contribute to gene regulation
in both a positive and negative manner.
Sites that anchor silent chromatin are mechanistically distinct from those for active
genes (budding yeast experiments).
In budding yeast, heterochromatin
binds the nuclear envelope through
Esc1 (green) which forms distinct
foci with nuclear pores (Nup49
labelled red).
Akhtar, Gasser, Nat. Rev. Genet. 8, 507 (2007)
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Localization to nuclear envelope
In yeast nuclei, envelopeassociated proteins such as
Esc1 (enhancer of silent
chromatin 1) are present in
foci at the peripheri. However,
they do not coincide with the
pores (Immuno-EM).
Esc1 binds Sir4 (silent information regulator 4) which is an integral component of
repressed heterochromatin in yeast.
This interaction is necessary and sufficient to anchor silent chromatin at the
nuclear envelope.
Akhtar, Gasser, Nat. Rev. Genet. 8, 507 (2007)
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Localization to nuclear envelope
In metazoan nuclei, the nuclear envelope is underlaid by a continuous meshwork
of lamins and lamin-associated proteins (LAPs) which preferentially associate
with inactive chromatin regions.
Akhtar, Gasser, Nat. Rev. Genet. 8, 507 (2007)
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Putative role of NPC in coupling transcription and
mRNA processing by gene looping in yeast.
Although not all transcriptional
activity in the nucleus will be
subject to this mode of regulation,
the budding yeast NPC seems to
work together with transcriptional
activation mechanisms to finetune gene activity.
The SAGA chromatin-remodelling complex in yeast contains Sus1; Sus1 is also
present in the mRNA-export complex TREX, which interacts with Nup1. Nup2 also
interacts with the promoters of active genes, and the NPC-associated protein Mlp1
(myosin-like protein 1) accumulates at the 3′ end of active genes, where it contributes
to an RNA surveillance mechanism. Optimal activation can require both localization of
the induced gene at the NPC as well as at the 3′ UTR. Our model suggests that gene
looping, which results from the coincident NPC-tethering of an initiation complex and
mRNA-processing complexes that are associated with the 3′ UTR, will help to finetune the expression of certain genes. Finally, the pore protein Nup2 was found to
Akhtar, Gasser, Nat. Rev. Genet. 8, 507 (2007)
tether genes through a histone variant H2A.Z (Htz1) in yeast. This could reflect a
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heritable
that contributes
to forms of epigenetic control.
SS 2009 localization
– lecture 3
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3C method: analyze gene interactions in 3D
3C stands for chromosome conformation capture method.
Method measures the formation of crosslinks between chromatin segments after
formaldehyde fixation of whole cells or isolated nuclei.
A frequency of crosslinking above control levels indicates spatial proximity
4C method: combine 3C with microarrays.
3C and 4C methods indicate that long-range chromatin interactions (gene
kissing) are involved in the epigenetic regulation of gene expression.
One important example: H19 and Igf2 genes.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Regulatory models at imprinted loci
(A) The enhancer–blocker model (also known
as the boundary model) is well studied at the
Igf2/H19 locus and consists of an imprinting
control region (ICR) located between a pair of
reciprocally expressed genes that controls
access to shared enhancer elements.
On the paternal allele, the differentially
methylated domain (DMD) acquires
methylation (black circles) during
spermatogenesis, which leads to repression of
the H19 promoter. The hypomethylated
maternal DMD acts as an insulator element,
mediated through binding sites for the
methylation-sensitive boundary factor CTCF
(shaded ellipse). When CTCF is bound, Igf2
promoter access to the enhancers (E) distal to
H19 is blocked.
Blue boxes : paternally expressed
alleles,
red boxes : maternally expressed
alleles,
black boxes : silenced alleles, grey
boxes : nonimprinted genes.
Arrows on boxes indicate
transcriptional orientation.
PLOS Genet. 2, e147
(2006)
Protein Interactions and Chromatin Loops
•
•
reading the imprint:
candidate "imprinting
transcription factors"
CTCF, YY1
chromatin loop model
– DMRs interact via
proteins
– mediates
interaction with
the enhancers
H19
p
m
Igf2
p
m
Murrell et al. (2004) Nature Genet. 36: 889
• maternal chromosome: DMR1 and DMR unmethylated, CTFC bound  H19 is
expressed (interaction with the enhancers), Igf2 is silenced
• paternal chromosome: DMR and DMR2 methylated, no CTCF binding  Igf2 in
contact with enhancers, active; H19 silenced
Gene kissing
In this example of gene kissing, copies of the Drosophila melanogaster Fab7
regulatory element that are present on two different chromosomes co-localize in
the cell nucleus.
DAPI (4′,6-diamidino-2-phenylindole) is the DNA counterstain, sd shows the
position of a transgenic Fab7 copy that is inserted in the X chromosome at the
scalloped (sd) locus. Abd-B indicates the locus that is regulated by the
endogenous copy of the Fab7 element. The two loci ‘kiss’ each other in a
significant fraction of the nuclei, as seen in the merged panel.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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How do compartments form?
Two non-exclusive models have been proposed to explain how compartments
form and are maintained.
Compartments could correspond to pre-existing structures,
or they could be formed as a result of self-assembly.
It was reported in 1999 that maintenance of chromosome-territories requires
RNA.
For the case of the clustering of Fab7 transgenes, the presence of small 21-23 nt
long RNA generated by the RNAi machinery was correlated with spatial colocalization. Mutations in the RNAi machinery disrupted the long-range Fab7
interactions.
So also RNAs seem to have structural and regulatory roles in nuclear
architecture.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Chromatin mobility in nucleus
Chromatin mobility allows dynamic interactions
between genomic loci and between loci and other
nuclear structures. a and b show two chromosome
territories. Within each territory a gene locus is
indicated in red. Movement of chromatin is
depicted by arrows. Two possible configurations
are represented for each territory, with the dotted
outline of one superimposed on the other. The
transition involves repositioning of the two loci
within the three-dimensional space of the nucleus.
In this hypothetical example, the bottom
configuration in each case is favored on
transcriptional activation of the loci.
Two alternative models have been proposed to
account for the compartmentalization of nuclear
functions that are involved in gene expression.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Chromatin mobility in nucleus
a, compartments are pre-existing structures
containing molecular machineries that are
dedicated to specific nuclear functions.
Movement of chromatin from one compartment to
another leads to changes in expression of the
corresponding genomic regions.
Activation is triggered by repositioning of the
gene loci to an activating compartment, away from
silencing compartments.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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Chromatin mobility in nucleus
In b, compartments are transient self-organizing
entities.
In this case, gene activation leads to dissolution
of the silencing compartments, changes in gene
positioning and de novo assembly of an
activating compartment.
Once initiated, this state can be maintained by
the self-assembly of components that are
involved in gene regulation, as well as the
clustering of chromatin regions that contain
actively expressed genes.
Lanctot et al. Nat. Rev. Genet. 8, 104 (2007)
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