Bio 402/502 Section II, Lecture 6

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

2. 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)

3. 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

4. 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.

5. 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

6. Mitosis Interphase Chromosomes during the cell cycle

7. 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

8. 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

9. Human chromosome territories in HeLa cells 500 nm (Foster & Bridger, 2005) Green: HSA3, blue: HSA5, red: HSA11

10. * 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)

11. 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)

12. 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

13. 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??

14. 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!

15. 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

16. 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

17. 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

18. 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)

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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?

25. 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?

26. Model of dynamic association of genes with transcription factories Transcribed genes RNA Polymerase II transcription factory Potentiated genes Chromosome territory (Osborne et al, 2004)

27. 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

28. 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)

29. 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

30. Models of the chromosome territory (Heard & Bickmore, 2007) Interchromosome domain The lattice model Interchromatin compartment

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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