Download Bio 402/502 Section II, Lecture 1

Bio 402/502
Section II, Lecture 6
Chromosome territory and nuclear organization
Dr. Michael C. Yu
Experimental approaches studying nuclear trafficking
Immunofluorescent tags
•
Transfect cells with proteins tagged with GFP, RFP, YFP, etc. Assess nuclear vs. cytoplasmic location
by IF (immunofluorescence)
•
Or, you can transfect cells which are epitope tagged and use antibodies conjugated with
fluorescently-tag to perform IF.
Confocal microscopy
SRPK1: cytoplasm
SC35: nuclear
Combined image
(Ding et al, 2006)
Experimental approaches to study nuclear trafficking
Permeabilized cells/cell free assay:
1.
Use digitonin to permeabilized cells, releasing cytosol
2.
This allows nuclear memebrane, nucleus and other organelles to remain
intact
3.
Add back different cytosolic fractions or antibody blockade, or other
biochemical manipulations to determine the components needed for
nuclear trafficking
Functional relevance of nuclear trafficking
• Bring into nucleus transcription factors, proteins for ribosome and
spliceosome assembly, and other proteins needed for nuclear functions.
• Export RNAs and ribosomes out of nucleus in a regulated manner. Each
is exported via a specific pathway.
• Shuttling of cellular proteins that go back and forth between nucleus and
cytoplasm (nuclear transport receptors, HnRNPs, etc.).
• Pathogens (mainly viruses) usurp nuclear trafficking machinery:
Viral genome import into and export out of the nucleus
Virus entry into nucleus
Virus exit from nucleus
Shuttling proteins encoded by viruses
• Pathogens can also destroy cellular nuclear trafficking machinery.
Internal organization of the nucleus
•
Chromosomes are discrete nuclear
bodies separated by an interchromatin
compartment
•
High order chromatin structure;- hetero—
localized to periphery of the nucleus;
inner membrane; euchromatin--distributed throughout the nucleus
•
Each chromosome occupies a distinct
territory; centromere, telomeres
Chromosomes during the cell cycle
Mitosis
Interphase
DNA folding: a long-standing mystery
Interphase nucleus
30 nm
“higher order”
Mitosis
800 nm
(Alberts et al)
• Most “higher-order” structures can’t be resolved by light microscope
Predominant 3-D patterns in the nucleus
200 3-D reconstructions of NIH-3T3 chromosomes
500 nm
• Thick (~ 400 nm) fiber and higher-order structures
• Frequent associations between gene clusters
• Gene sequence based
• Intermediate states
Human chromosome territories in HeLa cells
500 nm
(Foster & Bridger, 2005)
Green: HSA3, blue: HSA5, red: HSA11
Experimental approach used to probe chromosome
structure in the nucleus
Fluorescence in situ hybridization (FISH)
dsDNA in
fixed cell
Fluorescence imaging
denature
Labeled
DNA
probe
*
(Lindsay Shopland ,Institute for Molecular Biophysics)
hybridize
*
Interphase chromosomes form “territories”, not rods
• Chromosomes occupy discrete territories & has distinct chromosomearm and chromosome-band domains
mitotic chromosomes
(Lindsay Shopland ,Institute for Molecular Biophysics)
interphase chromosomes
Discovery of chromosome territories
(Heard & Bickmore, 2007)
• Idea conceived a long time ago
(1900’s)
• Experiment in 1980’s defined CT:
use laser to first induce genome
damage
• CT model: predict damage only
localized to a small subset of
chromosomes
• Random model: predict damage
only distributed on many
chromosomes
Damages mostly localized to chromosome 1 & 2
Tids and bits about chromosome territories (CTs)
Human fibroblast
nucleus
CTs
plants
Higher eukaryotes
(Maeburn and Misteli, 2007)
Chromosome
painting
Nucleoplasmic
channels within CT
Models of chromatin
structure within CT
• All cells have them, except lower eukaryotes
• Interior of CT are permeated by interconnected networks of
channels
• DNA structure within CT is non-random
• Folding of chromosome to a specific form: mechanism??
Chromosome Territories: a unit of nuclear organization
• Chromosomes have preferred position with respect to the center or
periphery of the nucleus
• Variability between celltypes
• Non-random neighbors:
purpose is to facilitate
proper gene expression!
• Complex folded surface
with active genes(red)
extends (or loops) into the
interchromatin space
CTs have separate arm domains
• Actively transcribed genes (white) are remotely located from
centeromeric heterochromatin. Recruitment of the same genes can occur
(black) to the centeromeric heterochromatin; results in silencing
Variable chromatin density is observed for CTs
• Loose chromatin (light yellow) expands into the interchromatin
compartment
• Dense chromatin (dark brown) is remote from the interchromatin
compartment
Chromatin territories have varied domain for replication
• Early replicating domains (green) & mid-to-late-replicating domains (red)
• Gene poor domain (red) is located closer to the nuclear periphery
• Gene rich domain (green) is located between gene poor compartments,
closer to the interior of nucleus
Reason for genome organization as chromosome territories
Mmu14
Genes on a chromosome are distributed
in patterns
Low gene density - 20 genes/5 Mb
Genes organized into discrete clusters
separated by gene “deserts”
There’s gene “rich” and gene “poor” regions
(Peterson, et al., 2002)
Identify gene clusters/gene desert on a chromosome
using FISH
Mouse chromosome 14:
Gene clusters
NIH-3T3
Deserts
Tiled BACs
5 Mb
Gene
“Desert”
Gene
Cluster
Different
fluorescent
labels
DNA
NIH-3T3 fibroblast
Sequentially expressed genes and CTs
Chromosomal organization of genes
in the mouse Hoxb complex
Differential expression of Hoxb
cluster genes detected by RT-PCR
(Chambeyron and Bickmore 2004)
Model system: mouse Hoxb gene cluster
Decondensation of Hoxb throughout the development
(Chambeyron and Bickmore 2004)
Control probes:
Red:Hoxb1
Green:Hoxb9
FISH experiment determines the change in the location between Hoxb1 and
Hoxb9 as development progresses
Measurement of CT movement in & out of CT
Distance from
edge of CT
Outside CT
Hoxb1
0
Hoxb9
Control gene
Inside CT
(Chambeyron and Bickmore 2004)
0
2
4
6
8
10
12 days
• Mean position of Hoxb1 and Hoxb9 relative to territory edge
• Shows extrusion of the Hoxb genes out of CT
Model for Hoxb progressive looping out of CT
“looping out” of Hoxb cluster
Hoxb cluster
“reeling back” of Hoxb cluster
Chromosome
territory
(Chambeyron and Bickmore 2004)
RA=retinoic acid to induce the development of mouse ES cells
Open regions of a chromosome may likely be located on
the outside of CT
11p15.5 probes
(high gene density)
11p15.5
11p14
11p14 probes
(low gene density)
11p13
(Gilbert et al, 2004)
Chromosome 11p
Chromosome territory
Gene density
Openness
• Visualization of outside localization may due to the manifestation
of an open-structured chromatin “looping” of its long stretches of
chromatin out of its CT
• Advantage for a chromosome to “loop” out it’s gene rich region?
Localization of transcription machineries throughout the
nucleus
Erythroid cell
colocation
DNA-FISH:
locating
genes
Eraf
Hbb
5 mm
Colocalization:
association with the
same RNAPII focus
Genes on
Mouse Chr 7
RNA-FISH:
locating
transcribed
genes
RNA
Polymerase II
transcription
factory
(Osborne et al, 2004)
What is the most a more “efficient” way to get genes transcribed?
Model of dynamic association of genes with
transcription factories
Transcribed
genes
RNA
Polymerase II
transcription
factory
Potentiated
genes
Chromosome
territory
(Osborne et al, 2004)
Spatial organization of chromosomes affects gene expression
(O’Brien, et al, 2003)
• Association of gene loci with NPC, nuclear periphery, or specific
nuclear bodies can all affect gene gene expression
• Compactness of chromatin influence gene activity
• Movement of chromatin towards transcription machinery facilitates
gene transcription
Chromosome conformation capture (3C)
• Method used to determine genome organization in the nucleus
1.
Genes
Regulatory elements
2.
3.
4.
Crosslinking fixes chromatin fragments in
close proximity
Restriction enzyme digests fragments
chromatin
Ligation of chromatin fragment ends
Interaction between two designated genomic
loci is tested by PCR with specific primers
Can hybridize to microarray/large scale
sequenceing to get systems wide info (4C)
(Job Dekkar, Umass Medical School)
Colocalization of genes in the nucleus for expression or coregulation
Chromosome territory
Cis-interaction/trans
interaction
Cis and trans
co-association
Speckle
(Fraser & Bickmore, 2007)
Transcription factory
Chromatin loop
Correlation between chromosome location and gene expression
Models of the chromosome territory
(Heard & Bickmore, 2007)
Interchromosome domain
The lattice model
Interchromatin compartment
Models of the chromosome territory: interchromosome domain
Splicing-factor
enriched
speckles (red)
RNAPII
(light blue)
(Heard & Bickmore, 2007)
• Interchromosome domain:
-Boundary between the surface of a CT and gene expression machinery compartment
-Predict active genes are all located at the surface of CTs
Models of the chromosome territory: interchromatin compartment
Splicing-factor
enriched
speckles (red)
RNAPII
(light blue)
(Heard & Bickmore, 2007)
• Interchromatin compartment:
-Surface of a CT is invaginated to allow contact with gene expression machinery
-Loops of decondensed chromatin containing active genes may loop out into this
compartment
-Genes from different CTs can localize together with gene expression factories or
splicing-factor enriched speckles
Models of the chromosome territory: lattice model
Splicing-factor
enriched
speckles (red)
RNAPII
(light blue)
(Heard & Bickmore, 2007)
• Lattice Model:
-Extensive intermingling of chromatin fibres from periphery and adjacent CTs
-Genes from different CTs can localize together with gene expression factories or
splicing-factor enriched speckles
Events of nuclear reorganization during X-chromosome inactivation
chromosome
X-active
X-inactive
Upregulation of Xist transcription
Transcription factory
Xist RNA
(Fraser & Bickmore, 2007)
Coating of chromosome by Xist RNA excludes transcriptional machinery, thus
silences genes on the chromosome
CT re-organization during X chromosome inactivation
Coating of Xist
RNA on a
chromosome
Organization of two X
chromosomes
(Heard & Bickmore, 2007)
Coating of chromosome by Xist RNA excludes transcriptional machinery, thus
silences genes on the chromosome
Chromosome arrangements are probabilistic and have a
preferred average position
Human Chr 18
(gene poor)
Human Chr 19
(gene dense)
Homologous to
Human Chr 19
Homologous to
Human Chr 18
(Tanabe et al, 2002)
Topological conservation of CTs across the evolution