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.
SRPK1: cytoplasm SC35: nuclear Combined image
(Ding et al, 2006)
Confocal microscopy
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 nucleusVirus entry into nucleusVirus exit from nucleusShuttling 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
DNA folding: a long-standing mystery
(Alberts et al)30 nm 800 nm“higher order”
• Most “higher-order” structures can’t be resolved by light microscope
Interphase nucleus Mitosis
Predominant 3-D patterns in the nucleus
500 nm
• Thick (~ 400 nm) fiber and higher-order structures• Frequent associations between gene clusters• Gene sequence based• Intermediate states
200 3-D reconstructions of NIH-3T3 chromosomes
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
Labeled DNA probe
denature
hybridize* *
Fluorescence imaging
(Lindsay Shopland ,Institute for Molecular Biophysics)
Interphase chromosomes form “territories”, not rods
mitotic chromosomes interphase chromosomes
(Lindsay Shopland ,Institute for Molecular Biophysics)
• Chromosomes occupy discrete territories & has distinct chromosome-arm and chromosome-band domains
Discovery of chromosome territories
• Idea conceived a long time ago (1900’s)
• Experiment in 1980’s defined CT: use laser to first induce genome damage
• Random model: predict damage only distributed on many chromosomes
• CT model: predict damage only localized to a small subset of chromosomes
Damages mostly localized to chromosome 1 & 2
(Heard & Bickmore, 2007)
Tids and bits about chromosome territories (CTs)
(Maeburn and Misteli, 2007)
Chromosome painting
Nucleoplasmic channels within CT
plants Higher eukaryotes
Models of chromatin structure within CT
Human fibroblast nucleus
CTs
• 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
• Non-random neighbors: purpose is to facilitate proper gene expression!
• Variability between cell-types
• 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
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)
Genes on a chromosome are distributed in patterns
GeneCluster
Gene “Desert”
Mouse chromosome 14:
5 M
bIdentify gene clusters/gene desert on a chromosome
using FISH
NIH-3T3Gene clustersDeserts
NIH-3T3 fibroblastDNA
Different fluorescent labels
Tiled BACs
Sequentially expressed genes and CTs
Model system: mouse Hoxb gene cluster
Chromosomal organization of genes in the mouse Hoxb complex
Differential expression of Hoxb cluster genes detected by RT-PCR
(Chambeyron and Bickmore 2004)
Decondensation of Hoxb throughout the development
FISH experiment determines the change in the location between Hoxb1 and Hoxb9 as development progresses
(Chambeyron and Bickmore 2004)
Red:Hoxb1
Green:Hoxb9Control probes:
Measurement of CT movement in & out of CT
Distance from edge of CT
Outside CT
Inside CT
0
days0
Hoxb1
Hoxb9
Control gene
(Chambeyron and Bickmore 2004) 122 4 6 8 10
• Shows extrusion of the Hoxb genes out of CT
• Mean position of Hoxb1 and Hoxb9 relative to territory edge
Model for Hoxb progressive looping out of CT
Chromosometerritory
Hoxb cluster
(Chambeyron and Bickmore 2004)
RA=retinoic acid to induce the development of mouse ES cells
“looping out” of Hoxb cluster
“reeling back” of Hoxb cluster
Open regions of a chromosome may likely be located on the outside of CT
(Gilbert et al, 2004)
Chromosome 11p
Gene density
Openness
Chromosome territory
11p15.5
11p14
11p13
11p15.5 probes(high gene density)
11p14 probes(low gene density)
• 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
(Osborne et al, 2004)Genes on Mouse Chr 7
DNA-FISH:locating genes
RNA-FISH:locating transcribed genes
Hbb
Eraf
RNAPolymerase IItranscriptionfactory
colocation
5 m
Erythroid cell
What is the most a more “efficient” way to get genes transcribed?
Colocalization: association with the same RNAPII focus
Model of dynamic association of genes with transcription factories
(Osborne et al, 2004)
Chromosome territory
RNA Polymerase IItranscription factory
Transcribed genes
Potentiatedgenes
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
(Job Dekkar, Umass Medical School)
1. Crosslinking fixes chromatin fragments in close proximity
2. Restriction enzyme digests fragments chromatin
3. Ligation of chromatin fragment ends
4. 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)
Genes Regulatory elements
Colocalization of genes in the nucleus for expression or coregulation
(Fraser & Bickmore, 2007)
Correlation between chromosome location and gene expression
Chromosome territory
Cis and transco-association
Cis-interaction/transinteraction
Speckle
Chromatin loopTranscription factory
Models of the chromosome territory
(Heard & Bickmore, 2007)
Interchromosome domain
Interchromatin compartment
The lattice model
Models of the chromosome territory: interchromosome domain
(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
Splicing-factor enriched speckles (red)
RNAPII (light blue)
Models of the chromosome territory: interchromatin compartment
(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
Splicing-factor enriched speckles (red)
RNAPII (light blue)
Models of the chromosome territory: lattice model
(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
Splicing-factor enriched speckles (red)
RNAPII (light blue)
Events of nuclear reorganization during X-chromosome inactivation
chromosome
X-active
X-inactive
Transcription factory
Xist RNA
Upregulation of Xist transcription
Coating of chromosome by Xist RNA excludes transcriptional machinery, thus silences genes on the chromosome
(Fraser & Bickmore, 2007)
CT re-organization during X chromosome inactivation
Coating of chromosome by Xist RNA excludes transcriptional machinery, thus silences genes on the chromosome
(Heard & Bickmore, 2007)
Organization of two X chromosomes
Coating of Xist RNA on a chromosome
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