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Bio 402/502 Section II, Lecture 6. Chromosome territory and nuclear organization Dr. Michael C. Yu . Experimental approaches studying nuclear trafficking. Immunofluorescent tags

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bio 402 502 section ii lecture 6

Bio 402/502Section II, Lecture 6

Chromosome territory and nuclear organization

Dr. Michael C. Yu

slide2

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

Combined image

SC35: nuclear

SRPK1: cytoplasm

(Ding et al, 2006)

slide3

Experimental approaches to study nuclear trafficking

Permeabilized cells/cell free assay:

  • Use digitonin to permeabilized cells, releasing cytosol
  • This allows nuclear memebrane, nucleus and other organelles to remain intact
  • Add back different cytosolic fractions or antibody blockade, or other biochemical manipulations to determine the components needed for nuclear trafficking
slide4

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.
slide5

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
DNA folding: a long-standing mystery

Interphase nucleus

Mitosis

“higher order”

30 nm

800 nm

(Alberts et al)

  • Most “higher-order” structures can’t be resolved by light microscope
predominant 3 d patterns in the nucleus
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
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

*

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

*

hybridize

(Lindsay Shopland ,Institute for Molecular Biophysics)

interphase chromosomes form territories not rods
Interphase chromosomes form “territories”, not rods
  • Chromosomes occupy discrete territories & has distinct chromosome-arm and chromosome-band domains

mitotic chromosomes

interphase chromosomes

(Lindsay Shopland ,Institute for Molecular Biophysics)

slide12

Damages mostly localized to chromosome 1 & 2

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
slide13

Tids and bits about chromosome territories (CTs)

Human fibroblast

nucleus

plants

Higher eukaryotes

CTs

(Maeburn and Misteli, 2007)

Chromosome

painting

Models of chromatin

structure within CT

Nucleoplasmic

channels 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??
slide14

Complex folded surface with active genes(red) extends (or loops) into the interchromatin space

Chromosome Territories: a unit of nuclear organization

  • Chromosomes have preferred position with respect to the center or periphery of the nucleus
  • Variability between cell-types
  • Non-random neighbors: purpose is to facilitate proper gene expression!
slide15

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
slide16

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
slide17

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
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
Identify gene clusters/gene desert on a chromosome using FISH

Tiled BACs

Different

fluorescent

labels

Gene clusters

Mouse chromosome 14:

NIH-3T3

Deserts

Gene

“Desert”

Gene

Cluster

5 Mb

NIH-3T3 fibroblast

DNA

slide20

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

slide21

Decondensation of Hoxb throughout the development

(Chambeyron and Bickmore 2004)

Red:Hoxb1

Green:Hoxb9

Control probes:

FISH experiment determines the change in the location between Hoxb1 and Hoxb9 as development progresses

slide22

Measurement of CT movement in & out of CT

Distance from

edge of CT

Outside CT

Hoxb1

Hoxb9

0

Control gene

Inside CT

days

0

2

12

4

6

8

10

(Chambeyron and Bickmore 2004)

  • Mean position of Hoxb1 and Hoxb9 relative to territory edge
  • Shows extrusion of the Hoxb genes out of CT
slide23

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

slide24

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?
slide25

Localization of transcription machineries throughout the nucleus

Erythroid cell

colocation

DNA-FISH:

locating

genes

Eraf

Hbb

RNA

Polymerase II

transcription

factory

5 mm

Colocalization: association with the same RNAPII focus

RNA-FISH:

locating

transcribed

genes

Genes on

Mouse Chr 7

(Osborne et al, 2004)

What is the most a more “efficient” way to get genes transcribed?

slide26

Model of dynamic association of genes with transcription factories

Transcribed

genes

RNA

Polymerase II

transcription

factory

Potentiated

genes

Chromosome

territory

(Osborne et al, 2004)

slide27

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
Chromosome conformation capture (3C)
  • Method used to determine genome organization in the nucleus
  • 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)

Genes

Regulatory elements

(Job Dekkar, Umass Medical School)

slide29

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

slide30

Models of the chromosome territory

(Heard & Bickmore, 2007)

Interchromosome domain

The lattice model

Interchromatin compartment

slide31

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
slide32

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
slide33

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
slide34

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

slide35

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

slide36

Chromosome arrangements are probabilistic and have a preferred average position

Human Chr 18

(gene poor)

Homologous to

Human Chr 19

Human Chr 19

(gene dense)

Homologous to

Human Chr 18

(Tanabe et al, 2002)

Topological conservation of CTs across the evolution

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