Nuclear Organization

1. Nuclear Organization Yaniv Loewenstein Computational Biology Seminar, HUJI November 2006

2. Discrete membrane bound compartment • transcription • RNA processing • splicing The nucleus - introduction The nucleus defines eukaryotes. • No unequivocal evolutionary origin

3. Single cyclic “chromosome” Multiple linear chromosomes Endomembrane compartments (ER) Transcription & translation physically separated Prokaryotes vs. eukaryotes.

4. Origin of Eukaryote nucleus Still under much debate…(and numerous papers) • Invagination of plasmatic membrane • Nucleus connected to ER • Probable endosymbiotic origin • Partially incompatible w. known cell biology • Cytoskeleton proteins based phylogeny • Orthopoxvirus ancestor suggested (DNA-pol) • Selective force • Splicing\translation decoupling • metabolic compartmentalization (anabolic/catabolic)

5. nucleus lost nuclei regained Mitotic nucleus (by the textbook) “chromatin spaghetti”  shaped chromosomes

6. Talk overview • The nucleus - compartments overview • Nuclear domains (D. Spector) • DNA loops and rosettes • Transcription factories? (P. Cook’s) • Nuclear pores & expression regulation (P. Silver) • Transcriptional regulation. • Review of experimental results & models (T. Cremer) • A taste of recently published work • 3D FISHing – Bolzer et al. Challenge oversimplified text book dogmas.

7. Nuclear compartments • Nuclear envelope & lamina • Nuclear Pore Complexes (NPCs) • Chromosomal Territories (CT) • DNA is not a random spaghetti • Interchromatin Granule Clusters (IGC) • Splicing factor compartments - Speckles (D. Spector) • Nucleolus and sub-compartments • Others • Cell-type or condition specific.

8. The nuclear lamina A scaffolding structure at the nuclear periphery. • Nuclear shape maintenance & NPC spacing. • Organization of heterochromatin. • Often anchors interphase heterochromatin. • DNA replication. • Regulation of transcription factors.

9. Nuclear lamina (II) • Made of lamins A/B/C intermediate filaments. • RNPs involved • RNase somehow disrupts nuclear matrix. • Lamins phosphorylated in mitosis - nucleus breaks. • Dephosphorylation promotes chromosome vesicles fusion.

10. cytosol nucleoplasma Nuclear Pore Complex “I’m an importer/exporter”* Exports: • mostly mRNA. Imports • Nuclear proteins & snRNPs • Viruses. • Interacts with importins (karyopherins) Lamina associated

11. Nuclear Pore Complex (II) Cytoplasm • Small molecules diffuse • Active transport of large macromolecules. • Extends 95 nm into the nucleoplasm Nucleoplasm Suntharalingam and Wente, Dev. Cell 2003.

12. Heterochromatin concentrates near lamina Heterochromatin excluded from pores Mammalian Nucleus - EM white – euchromatin (open) black – heterochromatin (condensed)

13. Integral membrane protein RFBP interacts with RUSH, a SWI/SNF chromatin remodeling TF. Lamin (+Chromatin) Binding Proteins Foisner, R. J Cell Sci 2001;114:3791-3792

14. Genome-Wide Localization of the Nuclear Transport Machinery Couples Transcriptional Status and Nuclear Organization Cosalry J, … Silver PA, Cell 2004 Genes relocate from the nucleoplasm to the nuclear pore upon transcriptional induction.

15. % Counts (120 cells) at nuclear periphery GAL genes localize to periphery upon induction Green – GAL loucs (FISH) Red – NPC (periphery) Casolari J et al (Silver PA) Cell 117 (2004) 427-439

16. Known NPC components(used by Casolari 2004) Arrows depict known physical interactions Casolari J et al (Silver PA) Cell 117 (2004) 427-439

17. Glucose Galactose GAL1,2,7,10 0% 0% 100% 100% NPC binds induced GAL genes Nuclear basket Myosin like (non pore) . .. … Casolari J et al (Silver PA) Cell 117 (2004) 427-439

18. Genomic localization (microarray)(in a nutshell) Nuclear transport subcomplexes show similar genome occupancy specificities

19. Correlated Expression & NPC binding

20. … RAP1 & NPC binding co-localizes • Rap1 - DNA binding protein associated with • Telomeres, • Silent mating-type loci • Many active genes • Boundary activity.

21. ? ? Levels of transcription regulation DNA sequence alone - can't explain orchestrated activity of thousands of genes. Epigenetics - DNA methylation, nucleosome modifications, insulators etc. Nucleus architecture - a higher topological level of regulation.

22. Chromatin Packing 2 nm 105mm Double helix 11nm “Beads-on-a-string” ~x7 30 nm fiber of packed nucleosomes ~x100 30 nm Chromosomal loops attached to nuclear scaffold 300 nm Condensed section of metaphase chromosome 700 nm ~x104 Entire metaphase chromosome 1400 nm 5-10 mm

23. One rosette

24. Nuclear lamina ? Chromosomal Territory (long term silencing) DNA Rosettes CT Heterchromain Euchromatin Labrador and Corces, 2002. Cell 111, 151 -154.

25. C | In a particular tissue, a chromatin domain becomes open after activation of the flanking insulators and vice versa. Loop regulation A | Linear layout of interphase chromatin. Yellow - open chromatin Blue – highly condensed chromatin Red –Domains with regulatable insulators under cell differentiation. B | During development, domains of higher-order chromatin structure are organized by active insulators (purple). Inactive insulators and the domain they flank (green) remain in the heterochromatin compartment. • Labrador and Corces, 2002. Cell 111, 151-154.

26. Looped domains -splicing-proteins green staining (light micrography) Granules may represent splicing machineries.

27. DNA-loop regulation • Back to the text book… • Scaffold rich in topoisomerases • RNase sensitive lamina.

29. The nucleolus Production and assembly of ribosome components • various small RNA • telomerase function modulation • oncogene regulation A non membranous compartment. Do tRNA genes affect chromosomes positions? Thompson et al, (2003) Science 302 1399-1401

30. tRNAs on yeast chromosomes

31. Nucleolar tRNA localization

32. tRNA genes nucleolar colocalization Thompson et al, (2003) Science 302 1399-1401

33. The nucleolus How hundreds of tRNA genes found in many chromosomes are arranged and clustered in the nuclear space?

34. tRNA gene localization depends on Pol III complex formation (52% of 440 cells) (87% of 715 cells) Legend: SUP3 - tRNA gene, URA3 (red) - adjacently inserted gene (non-RNA probe). U14 (green) - nucleolar probe

35. Lessons from the nucleolus • Inactivation of the promoter at a single locus removes its nucleolar association. => Nucleolar localization requires tRNA gene transcription-complex formation. • Organization of tRNA genes profoundly affects the spatial genome packaging. • Are gene types coordinated in 3D to regulate transcription? • Nuclear structure prediction from gene activity?

36. Spector, D. L. J Cell Sci 2001;114:2891-2893

37. Splicing factor granules - speckles Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus. Potashkin, J.A., …, Spector, D.L. 1990. MCB. 10: 3524-3534.  Associations between distinct pre-mRNA splicing components and the cell nucleus. Spector, D.L., …, Maniatis, T. 1991. EMBO J. 10: 3467-3481. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Huang, S. and Spector, D.L. 1991. Genes & Dev., 5: 2288-2302. U1 and U2 snRNAs are present in nuclear speckles. Huang, S. and Spector, D.L. 1992. PNAS 89: 305-308. … ….. …….. 2006

38. Speckles in the IC space Green – splicing factors snRNPs ABs Blue (DAPI) DNA Speckled pattern Speckles occur in nuclear specific regions containing little or no DNA. Cajal bodies diffused in the nucleoplasm Lamond & Spector. 2003. Nature Rev. Mol. Cell Biol. 4, 605-612

39. Nuclear sub-compartments Nucleolus - rRNA synthesis (various subcomp.) Cajal bodies • snRNP biogenesis (e.g. U1,2, 4-6). • Trafficking to speckles (snRNPs) or nucleoli (snoRNPs). Gems – snRNP maturation. Cleavage bodies – cleavage & poly-A proteins foci. Perinucleolar compartment (PNC) • small RNAs • Predominantly found in cancer cells. PML bodies – associated with various cancers. Spector DL. J Cell Sci. 2001, 114(Pt 16):2891-3.

40. Cell type specific domains GATA-1 nuclear bodies (GATA TF) • cell type specific to murine haemopoietic cells • not active in transcription Heat Shock Factor 1 (HSF1 TF) foci • physiological state specific for HS cells • not in HSP70/90 or HSP90 transcription sites Additional levels of transcriptional regulation? Spector DL. J Cell Sci. 2001, 114(Pt 16):2891-3.

41. Transcription factories & fixed pol? Permeabilized human nucleus of HeLa cells Red - TOTO-3 stains DNA, Green - bromo-UTP nascent RNA transcripts. Cook P. 2002. Nature Genetics 32, 347–52

42. Transcription factories Foci concentrated transcripts. # foci << # active genes # foci << # polymerases ==> “Transcription factories” • Similar to bacteria nucleoids. • Pol aggregates + RNA interactions (inhibited by RNase). Cook P. 2002. Nature Genetics 32, 347–52

43. The “untwining problem” – no known mechanism. Kinetics consistent with the existence of loops of 7.5−175 kb Experimental result A fixed polymerase? Cook P. 2002. Nature Genetics 32, 347–52

44. Regulated-exchange model . • Speckles created by PPI of pre-mRNA splicing factors. • Basal level of factor exchange with nucleoplasmic pool, regulated by phosphorilation. • Cell-type-specific (de)phosphorylation. • Phosphorylation level modulation of speckle proteins results in an increased release and recruitment to transcription sites. Lamond & Spector. 2003. Nature Rev. Mol. Cell Biol. 4, 605-612

45. Gathers a large-body of previous experimental work. • Understanding gene reg. at the topological level: • Reviews several testable models. • Offers the CT-IC model.

46. Chromosome Territories (CT) Chromosomes occupy discrete territories in the cell nucleus (evidence since the 70s). Methods: • FISH detects specific DNA seqs in single cells. • 3D positioning of individual (in)active genes • Using various fluorochromes in conjuction. • Secondary coloring (antibodies etc.). • S-phase DNA labeling persists in daughter cells • Can be analyzed in EM. • One patch per chromatid

47. Sponge-like CT structure. • Accessible interchromatin invaginations. Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.

49. CT–IC model – supporting structural features a | A giant chromatin loop with several active genes (red) expands from the CT surface into the IC space. b | Top, actively transcribed genes (white) are located on a chromatin loop that is remote from centromeric heterochromatin. Bottom, recruitment of the same genes (black) to the centromeric heterochromatin leads to their silencing Short (p) arm long (q) arm A living HeLa cell nucleus. Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.

50. c | CTs have variable chromatin density (dark brown, high density; light yellow, low density). Loose chromatin expands into the IC, whereas the most dense chromatin is remote from the IC. CT–IC model – supporting structural features A living HeLa cell nucleus. Cremer T et al. Nat Rev Genet. 2001 Apr;2(4):292-301.