Transcript Nuclear Organization

Nuclear Organization
Yaniv Loewenstein
Computational Biology Seminar, HUJI
November 2006
The nucleus - introduction
The nucleus defines eukaryotes.
– No unequivocal evolutionary origin
Discrete membrane bound compartment
– transcription
– RNA processing
– splicing
Prokaryotes vs. eukaryotes.
Single cyclic
“chromosome”
Multiple linear
chromosomes
Endomembrane
compartments (ER)
Transcription & translation
physically separated
Origin of Eukaryote nucleus
under much
numerous papers)
•Still
Invagination
of debate…(and
plasmatic membrane
– Nucleus connected to ER
• Probable endosymbiotic origin
– Partially incompatible w. known cell biology
– Cytoskeleton proteins based phylogeny
• Orthopoxvirus ancestor suggested (DNA-pol)
• Selective force
– Splicing\translation decoupling
– metabolic compartmentalization (anabolic/catabolic)
Mitotic nucleus (by the textbook)
nucleus lost
“chromatin spaghetti”  shaped chromosomes
nuclei regained
Talk overview
• The nucleus - compartments overview
–
–
–
–
Nuclear domains (D. Spector)
DNA loops and rosettes
Transcription factories? (P. Cook’s)
Nuclear pores & expression regulation (P. Silver)
• Transcriptional regulation.
– Review of experimental results & models (T. Cremer)
– A taste of recently published work
• 3D FISHing – Bolzer et al.
Challenge oversimplified text book dogmas.
Nuclear compartments
1. Nuclear envelope & lamina
2. Nuclear Pore Complexes (NPCs)
3. Chromosomal Territories (CT)
–
DNA is not a random spaghetti
4. Interchromatin Granule Clusters (IGC)
–
Splicing factor compartments - Speckles (D. Spector)
5. Nucleolus and sub-compartments
6. Others
–
Cell-type or condition specific.
The nuclear lamina
A scaffolding structure
at the nuclear periphery.
• Nuclear shape maintenance & NPC spacing.
• Organization of heterochromatin.
– Often anchors interphase heterochromatin.
• DNA replication.
• Regulation of transcription factors.
Nuclear lamina (II)
• Made of lamins A/B/C
intermediate filaments.
• RNPs involved
– RNase somehow disrupts
nuclear matrix.
• Lamins phosphorylated in
mitosis - nucleus breaks.
– Dephosphorylation promotes
chromosome vesicles fusion.
Nuclear Pore Complex
“I’m an importer/exporter”*
Exports:
– mostly mRNA.
cytosol
Imports
– Nuclear proteins & snRNPs
– Viruses.
– Interacts with importins
(karyopherins)
nucleoplasma
Lamina associated
Nuclear Pore Complex (II)
Cytoplasm
Small molecules diffuse
Active transport of large
macromolecules.
Extends 95 nm into the
nucleoplasm
Nucleoplasm
Suntharalingam and Wente, Dev. Cell 2003.
Mammalian Nucleus - EM
Heterochromatin concentrates near lamina
Heterochromatin
excluded from
pores
white – euchromatin (open)
black – heterochromatin (condensed)
Lamin (+Chromatin)
Binding Proteins
Integral membrane protein RFBP interacts with
RUSH, a SWI/SNF chromatin remodeling TF.
Foisner, R. J Cell Sci 2001;114:3791-3792
Genome-Wide Localization of the
Nuclear Transport Machinery
Couples Transcriptional Status
and Nuclear Organization
Cosalry J, … Silver PA, Cell 2004
Genes relocate from the
nucleoplasm to the nuclear pore
upon transcriptional induction.
GAL genes localize to periphery
% Counts (120 cells) at
upon induction
nuclear periphery
Green – GAL loucs
(FISH)
Red – NPC
(periphery)
Casolari J et al (Silver PA) Cell 117 (2004) 427-439
Known NPC components
(used by Casolari 2004)
Arrows depict known
physical interactions
Casolari J et al (Silver PA) Cell 117 (2004) 427-439
NPC binds induced GAL genes
Glucose
Galactose
Nuclear basket
Myosin like (non pore)
GAL1,2,7,10
.
..
…
0%
100%
0%
100%
Casolari J et al (Silver PA) Cell 117 (2004) 427-439
Genomic localization (microarray)
(in a nutshell)
Nuclear transport
subcomplexes show
similar genome
occupancy
specificities
Correlated Expression & NPC binding
RAP1 & NPC binding co-localizes
Rap1 - DNA binding protein associated with
•Telomeres,
•Silent mating-type loci
•Many active genes
•Boundary activity.
…
Levels of transcription regulation
DNA sequence alone - can't explain
orchestrated activity of thousands of genes.
Epigenetics - DNA methylation, nucleosome
modifications, insulators etc.
?
?
Nucleus architecture - a higher
topological level of regulation.
Chromatin Packing
105 mm Double helix
2 nm
“Beads-on-a-string”
11nm
30 nm fiber of
packed nucleosomes
30 nm
Chromosomal loops
attached to nuclear
scaffold
300 nm
~x7
~x100
Condensed section
of metaphase
chromosome
700 nm
~x104
5-10 mm
Entire metaphase
chromosome
1400 nm
One rosette
DNA Rosettes
Nuclear lamina
?
CT
Chromosomal Territory
(long term silencing)
Heterchromain
Euchromatin
Labrador and Corces, 2002. Cell 111, 151 -154.
Loop regulation
A | Linear layout of interphase chromatin.
Yellow - open chromatin
Blue – highly condensed chromatin
Red –Domains with regulatable insulators
under cell differentiation.
B | During development, domains of
higher-order chromatin structure are
organized by active insulators
(purple). Inactive insulators and the
domain they flank (green) remain in the
heterochromatin compartment.
C | In a particular tissue, a chromatin
domain becomes open after activation
of the flanking insulators and vice
versa.
Labrador and Corces, 2002. Cell 111, 151-154.
Looped
domains
-splicing-proteins
green staining
(light micrography)
Granules may
represent
splicing
machineries.
DNA-loop regulation
•Back to the text book…
•Scaffold rich in topoisomerases
•RNase sensitive lamina.
The nucleolus
Production and assembly of
ribosome components
– various small RNA
– telomerase function modulation
– oncogene regulation
A non membranous compartment.
Do tRNA genes affect chromosomes positions?
Thompson et al, (2003) Science 302 1399-1401
tRNAs on yeast chromosomes
Nucleolar tRNA localization
tRNA genes
nucleolar
colocalization
Thompson et al, (2003) Science 302 1399-1401
The nucleolus
How hundreds of tRNA genes found in
many chromosomes are arranged and
clustered in the nuclear space?
tRNA gene localization depends on
Pol III complex formation
(52% of 440 cells)
(87% of 715 cells)
Legend: SUP3 - tRNA gene, URA3 (red) - adjacently inserted
gene (non-RNA probe). U14 (green) - nucleolar probe
Lessons from the nucleolus
• Inactivation of the promoter at a single locus
removes its nucleolar association.
=> Nucleolar localization requires tRNA gene
transcription-complex formation.
• Organization of tRNA genes profoundly affects the
spatial genome packaging.
• Are gene types coordinated in 3D to regulate
transcription?
– Nuclear structure prediction from gene activity?
Spector, D. L. J Cell Sci 2001;114:2891-2893
Splicing factor granules - speckles
Differential distribution of factors involved in pre-mRNA
processing in the yeast cell nucleus. Potashkin, J.A., …, Spector,
D.L. 1990. MCB. 10: 3524-3534.
Associations between distinct pre-mRNA splicing components
and the cell nucleus. Spector, D.L., …, Maniatis, T. 1991. EMBO J.
10: 3467-3481.
Nascent pre-mRNA transcripts are associated with nuclear
regions enriched in splicing factors. Huang, S. and Spector, D.L.
1991. Genes & Dev., 5: 2288-2302.
U1 and U2 snRNAs are present in nuclear speckles. Huang, S. and
Spector, D.L. 1992. PNAS 89: 305-308.
…
…..
……..
2006
Speckles in the IC space
Green – splicing factors
snRNPs ABs
Speckled
pattern
Blue (DAPI) DNA
Cajal bodies
Speckles occur in
nuclear specific regions
containing little or no
DNA.
diffused in the nucleoplasm
Lamond & Spector. 2003. Nature
Rev. Mol. Cell Biol. 4, 605-612
Nuclear sub-compartments
Nucleolus - rRNA synthesis (various subcomp.)
Cajal bodies
– snRNP biogenesis (e.g. U1,2, 4-6).
– Trafficking to speckles (snRNPs) or nucleoli (snoRNPs).
Gems – snRNP maturation.
Cleavage bodies – cleavage & poly-A proteins foci.
Perinucleolar compartment (PNC)
– small RNAs
– Predominantly found in cancer cells.
PML bodies – associated with various cancers.
Spector DL. J Cell Sci. 2001, 114(Pt 16):2891-3.
Cell type specific domains
GATA-1 nuclear bodies (GATA TF)
– cell type specific to murine haemopoietic cells
– not active in transcription
Heat Shock Factor 1 (HSF1 TF) foci
– physiological state specific for HS cells
– not in HSP70/90 or HSP90 transcription sites
Additional levels of transcriptional regulation?
Spector DL. J Cell Sci. 2001, 114(Pt 16):2891-3.
Transcription factories & fixed pol?
Permeabilized human nucleus of HeLa cells
Red - TOTO-3 stains DNA, Green - bromo-UTP nascent RNA transcripts.
Cook P. 2002. Nature Genetics 32, 347–52
Transcription factories
Foci concentrated transcripts.
# foci << # active genes
# foci << # polymerases
==> “Transcription factories”
• Similar to bacteria nucleoids.
• Pol aggregates + RNA interactions
(inhibited by RNase).
Cook P. 2002. Nature Genetics 32, 347–52
A fixed polymerase?
The “untwining
problem” – no known
mechanism.
Kinetics consistent
with the existence of
loops of 7.5−175 kb
Experimental result
Cook P. 2002. Nature Genetics 32, 347–52
Regulated-exchange model .
•Speckles created by PPI of pre-mRNA
splicing factors.
•Basal level of factor exchange with
nucleoplasmic pool, regulated by
phosphorilation.
•Cell-type-specific (de)phosphorylation.
•Phosphorylation level modulation of
speckle proteins results in an increased
release and recruitment to transcription
sites.
Lamond & Spector. 2003. Nature
Rev. Mol. Cell Biol. 4, 605-612
• Gathers a large-body of previous experimental
work.
• Understanding gene reg. at the topological level:
– Reviews several testable models.
– Offers the CT-IC model.
Chromosome Territories (CT)
Chromosomes occupy discrete territories in the
cell nucleus (evidence since the 70s).
Methods:
• FISH detects specific DNA seqs in single cells.
– 3D positioning of individual (in)active genes
– Using various fluorochromes in conjuction.
– Secondary coloring (antibodies etc.).
• S-phase DNA labeling persists in daughter cells
– Can be analyzed in EM.
– One patch per chromatid
• Sponge-like CT structure.
• Accessible interchromatin invaginations.
Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.
CT–IC model – supporting
a | A giant chromatin loop with
several
active genes (red)
structural
features
expands from the CT surface into
the IC space.
Short (p) arm
long (q) arm
b | Top, actively transcribed
genes (white) are located on a
chromatin loop that is remote
from centromeric
heterochromatin. Bottom,
recruitment of the same genes
(black) to the centromeric
heterochromatin leads to their
silencing
A living HeLa cell nucleus.
Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.
CT–IC model – supporting
structural features
c | CTs have variable chromatin density (dark brown, high
density; light yellow, low density). Loose chromatin expands
into the IC, whereas the most dense chromatin is remote
from the IC.
A living HeLa cell nucleus.
Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.
d | CT showing early-replicating chromatin domains
(green) and mid-to-late-replicating chromatin domains
(red). Each domain comprises 1 Mb. Gene-poor
chromatin (red), is preferentially located at the nuclear
periphery and in close contact with the nuclear lamina
(yellow), as well as with infoldings of the lamina and
around the nucleolus (nu). Gene-rich chromatin (green)
is located between the gene-poor compartments.
CT–IC model – supporting
structural features
A living HeLa cell nucleus.
Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.
CT–IC model – supporting
structural features
f | The CT–IC model predicts
that the IC (green) contains
complexes (orange dots) and
larger non-chromatin
domains (aggregations of
orange dots) for transcription,
splicing, DNA replication and
repair.
e | Higher-order chromatin structures
built up from a hierarchy of chromatin
fibres. Inset: this topological view of
gene regulation indicates that active
genes (white dots) are at the surface of
convoluted chromatin fibres. Silenced
genes (black dots) may be located
towards the interior of the chromatin
structure.
A living HeLa cell nucleus.
Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.
CT–IC model – supporting
structural features
g | CT with 1-Mb chromatin domains (red) and
IC (green) expanding between these domains.
Inset: the topological relationships between the
IC, and active and inactive genes. The finest
branches of the IC end between 100-kb
chromatin domains. Top: active genes (white
dots) are located at the surface of these
domains, whereas silenced genes (black dots)
are located in the interior. Bottom: alternatively,
closed 100-kb chromatin domains with silenced
genes are transformed into an open configuration
before transcriptional
activation.
A living
HeLa cell nucleus.
Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.
FISHing in 3D – Bolzer et al
A probabilistic 3D order of chromosomes & CTs
in quiescent and cycling human fibroblast
nuclei.
Methods
• Differential coloring of all 24 chromosome types.
– FISH with combinatorial labeling.
– Confocal microscopy.
– Conditions preserve 3D nucleus shape.
• 54 nuclei analyzed by “goldFISH”.
– A lot of technical details.
Bolzer A et al. (Cremer T) PLoS Biol. 2005 May;3(5):e157
Combinatorial coloring (2001)
DAPI
karyotype
color
combinations
Mutually exclusive CTs (chicken fibroblast)
Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.
Pseudo-coloring (Human – 2005)
Bolzer A et al. PLoS Biol. 2005 May;3(5):e157
3D image processing & simulations
Bolzer A et al. PLoS Biol. 2005 May;3(5):e157
Multi FISH analysis scheme
Promethaphase
chromosome “flowers”
Bolzer A et al. PLoS Biol. 2005 May;3(5):e157
3D reconstruction
Bolzer A et al. (Cremer T) PLoS Biol. 2005 May;3(5):e157
Radial positions for 54 nuclei
•Nuclei shape and size normalizions
•North/South etc. can’t be distinguished.
Results:
1. short chromosomes prefer the
nucleus center.
CN
2.periphery
No gene-richness correlation.
Bolzer A et al. PLoS Biol. 2005 May;3(5):e157
Significantly probabilistic positioning
• “Chromosome positioning patterns are statistical
representations … positions can be described, but
contain significant uncertainty”. [Parada, TCB 2003]
– Bolzer’s findings agree.
– Disagree w. reported fibroblasts precise positioning.
• Does’nt exclude some extent of determinism
– Gene-poor chromatin located beneath the nuclear
envelope of all reported cell types.
– Nucleoli positioning combines probabilistic and
deterministic patterns.
Bolzer A et al. PLoS Biol. 2005 May;3(5):e157
Bolzer PLoS 2005 - discussion
Findings in fibroblasts with flat ellipsoidal nuclei contradict
previous studies in cells with rounder nuclei.
• Central positioning of gene-rich CTs in lymphocytes.
• Cell-type-specific can’t be explained solely by
geometrical constrains of nucleus shape.
– 3D Simulations.
• A common rule relating GE to nuclear position:
– Gene-poor chromosomes located on nuclear envelope.
• Questions for future experiments
– Does nucleus shape enforce CT arrangements or vice versa?
– Nuclear shape causally connected with expression changes?
Bolzer A et al. PLoS Biol. 2005 May;3(5):e157
Nat Genet. 2006 Aug;38(8):936-41.
RNAi & chromatin insulators
• RNAi required for establishing centromeric chromatin
in S. pombe and D. melanogaster.
– Nuclear positioning of silent chromatin.
• Defects in telomere clustering in S. pombe
• Insulator proteins physical interaction lost when
RNase added.
• RNAi mutants show large phenotypic loss.
– “Insulator bodies” lost.
• Chromatin condensation affected.
– Transgenes introduction => phenotypic rescue.
CP190
RM62
Transcription regulation - summary
Chromosomal territories:
• Probabilistic but non-random chromosome location.
• chromatin condensation regulated by nuclear location.
– Sequence based?
– Regulatory insulators
• Sequence and factor dependent?
• RNA interactions (and RNAi)
• Exposure to nucleic factors in interchromatin
– DNA topology - Rosettes and loops
– Differential factor concentrations.
– Transcription factories.
• Can they be predicted from sequence?
• mRNA nuclear export regulation (not covered)
transcription regulation (revisited)
DNA sequence alone - can't explain
orchestrated activity of thousands of genes.
Epigenetics - DNA methylation, nucleosome
modifications, insulators etc.
?
?
Nucleus architecture - a higher
topological level of regulation.