Bio 402/502 Section II, Lecture 6 Chromosome territory and nuclear organization Dr. Michael C. Yu.

Bio 402/502Section 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.

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

Chromosomes during the cell cycle

Mitosis

Interphase

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


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


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)


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


Cis and transco-association

Cis-interaction/transinteraction

Speckle

Chromatin loopTranscription factory

Models of the chromosome territory


Interchromosome domain

Interchromatin compartment

The lattice model

Models of the chromosome territory: interchromosome domain


• 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


• 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


RNAPII (light blue)

Models of the chromosome territory: lattice model


• 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


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


Organization of two X chromosomes

Coating of Xist RNA on a chromosome

Chromosome arrangements are probabilistic and have a preferred average position

(Tanabe et al, 2002)

Homologous to Human Chr 18

Homologous to Human Chr 19

Human Chr 18(gene poor)

Human Chr 19(gene dense)

Topological conservation of CTs across the evolution

Bio 402/502 Section II, Lecture 6 Chromosome territory and nuclear organization Dr. Michael C. Yu.

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