Mitotic Chromosome Condensation Andrew Belmont

Transcription

1 Mitotic Chromosome Condensation Prof. Department of Cell and Developmental Biology University of Illinois At Urbana-Champaign 1 Mitotic chromosome condensation: Still a mystery Goals for this lecture: Perspective Phenomenology Functions What we thought we knew What we think we know What we d like to know 2 Perspective: million fold multiplier Human diploid nucleus: 2 x 3 x10 9 bp x.34 nm/bp ~ 2 meters DNA/diploid nucleus 2 meters 2000 km (1250 miles) ~ 10 μm nucleus 10 meter sailboat 2 nm DNA diameter 2 mm diameter DNA ~ 50 nm persistence length (150 bp) ~ 5 cm persistence length of DNA 3 1

2 Perspective: evolutionary and developmental comparisons 80,000 fold variation in eukaryotic genome sizes Large variations in chromosome sizes: 200 kb smallest yeast chromosome versus 240 Mbp largest human chromosome Linear compaction ratio ~160 fold in budding yeast versus ~20,000 fold in human Duration: several minutes to hours But molecular mechanisms underlying mitotic chromosome condensation thought to be conserved 4 Phenomenology: stages of mitosis Prophase Begins with first sign of chromosome condensation Ends with nuclear envelope breakdown Progressive chromosome condensation throughout G2 Early Middle Late 5 Stages of mitosis (1) Prometaphase From nuclear envelope breakdown to chromosome alignment in middle of mitotic spindle Ptk1 Prometaphase: Conly Rieder laboratory 6 2

3 Stages of mitosis (2) Metaphase From chromosome alignment to beginning of sister chromatid separation Anaphase Sister chromatid segregation to poles Telophase Nuclear envelope reformation, beginning of chromosome decondensation and cleavage of daughter cells Histone H2B- GFP Human HT1080 cell Rieder and Khodjakov, Science 300:91 (2003) 7 Primary functions of mitosis Topological separation, resolution of sister chromatids Thought to be driven by process of chromosome condensation Successful segregation of sister chromatids to daughter cells 8 Experimental demonstration of effect of chromosome length on chromosome segregation I. Schubert and J.L. Oud, Cell 88:515 (1997) % longest arm not separated: 0% wt, 39% FHE (sterile) % micronuclei (larger than 1 micron): 0% wt, 3.5% FHE 9 3

4 Lower levels of chromatin organization Sailboat analogy: 50 km of 3 cm diameter chromatin fiber in 10 meter diameter nucleus 10 Two common observations 30 nm chromatin fibers visible at periphery under conditions of low divalent Ca, Mg++ or polycations Evidence for larger, possibly helical higher level folding hierarchical folding 11 Early whole mount electron microscopy Honey Bee embryonic chromosomes Folded Fiber Model E.J. DuPraw, Nature 206:338 (1965) 12 4

5 Hints of helical folding at highest level of compaction Harrison et al., J. Cell. Sci. 56:409 (1982) Ris, H., Methods Cell Bio. 22:77 (1981) DuPraw, E.J., Nature 209:577 (1965) 13 What we thought we knew: Experimental foundation of textbook model 14 Key advance Structural analysis hard: Different isolation procedures, different structures High packing density Difficult size scale- ~ nm resolution over ~ microns Deliberately unfold chromosomes Inspiration- revealing cytoskeleton in whole mount EM by high salt, detergent extraction Use analogous approach on isolated chromosomes 15 5

6 Paulson and Laemmli, Cell 12:817 (1977) 16 Metaphase chromatid cross-sections- swelling in different buffers Marsden and Laemmli, Cell 17:849 (1979) 17 Short region of DNA double helix Beads-on-a-string form of chromatin 30-nm chromatin fiber of packed nucleosomes Section of chromosome in an extended form Condensed section of metaphase chromosome Entire metaphase chromosome From The Art of MBoC Garland Publishing, Inc. 18 6

7 Interpretation Nonhistone protein network scaffolding as organizer of metaphase chromosome structure Radial loop model for mitotic chromosome organization Obvious questions: Do specific DNA sequences form the base of the DNA loops? Do specific nonhistone proteins form the chromosome scaffold? 19 Do specific DNA sequences form the base of the DNA loops? SARs MARs = (Scaffold attachment regions) (Matrix attachment regions) AT rich same sequences identified by both assays High affinity binding sites for topoisomerase 2 BURs (base pair unwinding regions) located within SAR/MARs; these sequences preferentially become single stranded after DNA supercoiling Increase transcription of transgenes when placed nearby; further increases when transgene is flanked by SAR/MARs 20 Question 1: Are there specific DNA sequences at the base of chromatin loops? Yes, but Caveats: In vivo attachments require special preparationnuclei stabilization, LIS Can not rule out release and binding of in vivo fragments during procedure Questions related to possible protein precipitation during procedures contributing to matrix/scaffold 21 7

8 Question 2: Are there specific nonhistone proteins making up the chromosome scaffold? Improved mitotic chromosome scaffold preparations showed two major proteins Sc1-170 kd Sc2-135 kd 22 Sc1 = Topoisomerase 2 Topoisomerase 2 localizes to chromosome and chromosome scaffold axis Chromosome spread DAPI Topo 2 Ab IF Earnshaw and Heck, JCB 100:1716 (1985) 23 Three independent experimental paths led to identification of condensin complex containing Sc2 (1) 1. Mitotic Scaffold Biochemistry Chicken Human Saitoh, Goldberg, Wood, Earnshaw, JCB 127: 303 (1994) 24 8

9 Three independent experimental paths led to identification of condensin complex containing Sc2 (2) 2. In vitro assembly of mitotic chromosomes using frog egg extracts 25 Hirano and Mitchison, JCB 120:601 (1993) Hirano, Kobayashi, Hirano, Cell 89:511 (1997) Three independent experimental paths led to identification of condensin complex containing Sc2 (3) 2. In vitro assembly of mitotic chromosomes using frog egg extracts XCAP-C and XCAP-E in complex XCAP-E homologous to Sc2 Hirano and Mitchison, Cell 79:449 (1994) 26 Three independent experimental paths led to identification of condensin complex containing Sc2 (4) 3. Yeast Molecular Genetics 1. Screen for loss of mini-chromosomes Secondary screen for nondysjunction of minichromosomes SMC1 SMC= stability of minichromosomes 2. Sequence homology SMC2 (Sc2, XCAP-E) Gene knockout Defects in segregation, reduced mitotic condensation Strunnikov, Hogan, Koshland, Genes Dev. 9:587 (1995) 27 9

10 SMC family of proteins hinge ATP binding coiled-coil arm ATP hydrolysis engagement head SMC3 SMC1 SMC2 SMC4 SMC2 SMC4 SMC5 SMC6 SMC SMC Scc2 Scc1 H H2 Nse ScpA Pds5 Scc3 cohesin D2 G condensin I D3 G2 condensin II SMC5-6 ScpB bacterial SMC complex kleisin HEAT Losada and Hirano, Genes Dev. 19:1269 (2005) 28 Vindication of radial loop/scaffold model Axial localization of Sc1/Topoisomerase 2 and Sc2/SMC2/XCAP-E confirmed: In chromosome halo/scaffold preparations In unextracted, native chromosomes In live cells Sc1 and Sc2 appeared to be functionally important for chromosome condensation: Topoisomerase inhibitors inhibited chromosome condensation Condensin SMC2/SMC4 complex depletion blocks later steps of in vitro chromosome condensation 29 Textbook model: condensation by condensins Molecular Biology of the Cell Garland Science

11 What we thought we knew: Challenges to textbook model Chromosome diameter determined by radial loop size Radial loop size determined by spacing of SAR/MAR sequences In vitro chromosome assembly after histone H1 depletion produced longer, thinner chromosomes Engineered chromosome regions with variable SAR/MAR frequency show normal chromosome diameter 31 Challenges to textbook model: progressive condensation during prophase Radial loop/scaffold model suggests chromosome condensation driven by formation of scaffold Constant loop size implies constant chromosome diameter BUT: Prophase condensation suggests hierarchical folding with several layers of folding, change in chromosome diameter during prophase Kireeva et al., JCB 166:775 (2004) 32 Evidence for nm subunit in metaphase chromosomes Strukov, Wang, Belmont, JCB 162:23 (2003) 33 11

12 Alternative model: Radial loop, helical coil model Prophase chromatid organized as radial loop structure Prophase chromatid coils to form metaphase chromatid Prediction: Linear scaffold would form at center of prophase chromatid, scaffold would be helical in metaphase chromatid 34 AND: SMC2 staining in metaphase chromatid appears right down chromosome center, no evidence for coiling at center of prophase chromatid Kireeva et al., JCB 166:775 (2004) 35 Challenges to textbook model BUT: Linear condensed prophase chromosomes appear prior to well defined axial staining of SMC2, topoisomerase 2 DAPI SMC2 Middle prophase Metaphase Kireeva et al., JCB 166:775 (2004) 36 12

13 Radial loop/scaffold model predicts chromosome condensation dependent on topoisomerase 2 and/or condensins Experimental test: Make conditional knockdown cell line Delete first wt allele Add transgene under control of an inducible promoter Delete second wt allele Now have cell line expressing protein only under control of inducible promoter Turn off promoter- protein levels drop over time What is the effect on chromosome condensation? 37 Chromosome condensation with topoisomerase 2 knockdown wt topo 2 off Merge topo 2 DAPI Carpenter and Porter, MBC 15:5700 (2004) 38 Chromosome condensation with SMC2 knockdown Hudson et al., Dev. Cell 5: 323 (2003) 39 13

14 What we think we know Continuum of prophase condensation Some type of higher order folding which may represent hierarchical folding of prophase chromatid Scaffold proteins localize axially relatively late in chromosome condensation and after linear chromosome axis apparent 40 What is the predominant phenotype of topoisomerase 2 and condensin knockdowns? Chromosome segregation defects for both topoisomerase 2 and condensins ScII ON wt CENP-C, DNA microtubules ScII OFF Topo 2 off Merge Topo 2 DAPI Carpenter and Porter, MBC 15:5700 (2004) Hudson et al., Dev. Cell 5: 323 (2003) 41 Condensins also required for chromosome stability ScII ON ScII OFF Hudson et al., Dev. Cell 5: 323 (2003) Hirato et al., J. Cell Sci. 117: 6435 (2004) 42 14

15 Two condensin complexes present: Condensin I: Excluded from nucleus Condensin II: Present within nucleus 43 Condensin 1 versus 2 Condensin 1: Cytoplasmic Stiffens centromeric chromatin Required to avoid delay in metaphase Condensin 2 Intranuclear Required for prophase condensation Stable (minutes) Required for chromosome hypercondensation Dynamic (minutes) Either condensin complex: Chromosome stability, segregation during anaphase 44 What we d like to know 45 15

16 Chromosome scaffold versus glue models Condensin Chromatin ATP hydrolysis ATP binding Prophase Prometaphase Metaphase Hirano, Nat Rev Mol Cell Biol. 7:311 (2006) Kireeva et al., JCB 166:775 (2004) 46 How do we incorporate experimental results which demonstrate primary condensin knockdown phenotype is chromosome stability not condensation? Two possibilities: Remaining condensins sufficient for condensation Difficult to fit into axial scaffold model Focuses attention on alternative actions of condensins Additional/alternative mechanism for chromosome condensation 47 How to connect condensin biochemical activities with chromosome structure? Enzymatic activities: ssdna to dsdna annealing Positive supercoiling/knotting? Chromosome compaction Separating effects of condensins on chromosome axis formation versus chromosome structure/compaction: Condensin subunit CAP-H2 overexpression drives separation of chromatids in salivary gland polytene nuclei Regulating cis and trans long-range chromosome interactions? Local compaction through supercoiling? Progressive condensation during prophase? Hartl et al., Science 322:1384 (2008) 48 16

17 Alternative mechanism for chromosome condensation? Knockdown of both condensin 1 and 2: Still see robust condensation after NEBD Defect in anaphase chromosome segregation, anaphase chromosome condensation Rescued by cyclin B3 expression SMC OFF SMC OFF +cyclin 3B mcherry-cyclinb3 Lac I-GFP DNA Vagnarelli et al., Nature Cell Biology 8:1133 (2006) 49 Model Regulator of Chromosome Architecture (RCA) acts as alternative condensation mechanism RCA-P active Anaphase chromosomes Repo-Man binds in anaphase, recruits PP1 phosphatase RCA inactive wt SMC2 off SMC2 off, high cyclin B3, or dom neg Repo-man 50 How do we integrate structural studies of chromosome structure with chromosome biochemistry? 51 17

18 We need a better understanding of chromosome structure (1) How is a linear chromosome axis formed? What is the role of condensins- particularly at early stages? Is chromatin reorganized within the mitotic chromosome or is mitotic chromosome condensation part of a continuum of interphase chromosome folding? Is there a hierarchical folding of chromatin in chromosomes? If so, how does this allow a conserved biochemical mechanism for chromosome condensation across different size scales of chromosomes? 52 We need a better understanding of chromosome structure (2) Is chromatin condensation separate from forming a linear axis and establishment of chromosome architecture? Is chromosome stability/elasticity separate from chromatin condensation and architecture? 53 The End Recommended reviews: A.S. Belmont, Mitotic chromosome structure, Current Opinion Cell Biology 18: (2006) Losada, A. and T. Hirano, Dynamics molecular linkers of the genome: the first decade of SMC proteins, Genes Dev. 19: (2005) Hirano, T., At the heart of the chromosome: SMC proteins in action, Nature Reviews Molecular and Cell Biology 7: (2006) Hudson, D.F., Marshall, K.M., and W.C. Earnshaw, Condensin: Architect of mitotic chromosomes, Chromosome Research 17: (2009) 54 18

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