How does higher-order chromatin structure differ in normal and cancer cells?

1. NU-PS-OC Project 3DNA information and organization at supranucleosomal scales: chromatin folding and higher-order structure,patterns of DNA replication initiationJohn F. MarkoNorthwestern University How does higher-order chromatin structure differ in normal and cancer cells?

2. Paulson & Laemmli Cell 12, 817 1977

3. Ono, Hirano, Cell 2003 SMC protein complexes Visualizatio of condensin I and condensin II on HeLa chromosomes. (A) Chromosome selected from a metaphase chromosome spread costained with DAPI and biotinylated anti-hCAP-G (condensin I, green) and antihCAP-G2 (condensin II, red). Merged images are shown in the right two panels. Bar, 2 mm. (B) Closeups of the chromosomal regions indicated by red boxes in (A). Bar, 2 mm. Reproduced from T. Ono et al, Cell 115, 109-121 (2003).

4. Maeshima and Laemlli Chromosoma 2005 Chromatin loops attached to SMC-rich axis Fixed, immuno-gold stained topo IIand SMC3 (hBar)

5. (PNAS 2008) Unresolved fundamental questions – chromatin is soft!

6. Cancer vs Normal Cells (Crispino, Licht, Lebeau also NCI) DNA Replication (Lebeau) Nanoparticle Labels (Odom, O’Halloran) EM and opticalvisualization + analysis (Backman, Dravid, O’H.) Cancer and cell disorder (Backman) Micromechanics (Marko) Network Models (Motter) NU-PS-OC Project 3

7. John Crispino cancer cell biology – What cells should we be looking at? Jon Licht Cancer-related chromatin modifications? Michelle Lebeau cancer cell biology – DNA replication and modification in cancer? (U Chicago) Accompanying large-scale chromosome architecture changes (“fragile” sites)? Teri Odom nanotechnology – 10 to 100 nm metal particles for labeling for optical and EM imaging Tom O’Halloran cell biology - antibody functionalization of nanoparticles and use of them for labeling cell structures Vinayak Dravid materials science – cryo EM visualization of cell structures Vadim Backman chem biol eng – light scattering and EM roughness analysis of cells John Marko biophysics – use of force-measurement techniques to analyze chromosome structure, mechanics Adilson Motter biophysics – What are suitable mathematical models for cell control systems (genetic, protein networks)

8. Cancer vs normal cells Prof. Jon Crispino Prof. John Licht Christine Will (cell handler) Dr. Laure Gilles • iPS cells • Weinberg cells (telomerase+Ras+SV40 oncoprotein) • Cells with specific mutations (MMSET histone methylase and JAK kinase) • 4. NCI cell lines (MCF-10A, MDA-MB231) (controls?) • What cancer/normal cell partners will show differing • higher-order chromatin structure?

9. NCI cells (60x phase) MCF-10A, passage 3 MDA-MB231, passage 3

10. NCI MDA-MB231 cells (60x phase) MDA-MB231, passage 3 MDA-MB231, passage 3

11. Electron Microscopy Visualization • (Cryo and non-cryo) • Prof. Tom O’Halloran (BMBCB) • Prof. Vinayak Dravid (Mat Sci Eng) • Dr. Jinsong Wu (NUANCE) • Dr. Shuyou Li (NUANCE) • Dhwanil Damania (Ph.D. student) • Ultrastructure of Stained and Unstained Chromatin • Nanoparticle labels for localization of specific proteins • Chemically sensitive EM to visualize metal distributions in chromatin • Instrumentation – highest resolution STEM system (Hitachi 2300), set up for TEM and back-scattering, chemical sensitivity, flash-frozen cryo and freeze-drying

12. Intrinsic High Contrast in ADF (Z-contrast) (E. Coli, unstained) ADF BF

13. STEM Imaging of Negatively Stained Chaperonin Complexes

14. STEM images of HT29 Cells collected by different detectors – freeze-dried cells Dark Field Detector Bright Field Detector Secondary electron Detector • Flash freezing followed by freeze-drying • No staining • Minimal distortion of cell • Can see subcellular structures (nucleus)

15. Typical EDS spectrum of the HT-29 HT29 cell – Zoomed in to nucleus STEM collected by dark-field detector X-ray element mapping of HT-29 Cell: Bright-field STEM image, P and K maps

16. Nanoparticles Prof. Teri Odom (Chem) Eunah You (Ph.D. student) Au particles, < 10 nm diam Comparable to nucleosomeor topo II size Smaller than SMCs Currently functionalizingwith antibodies to specifictargets

17. Micromechanical Study ofChromosomes and Chromatin • John Marko (BMBCB/P&A) • Mina Sun (Ph.D. student) • Houqing Yu (Ph.D. student) • PS-OC effort starting 2011 • Extract whole (metaphase) chromosomes for micromanipulation • Study mechanics +/- enzyme, biochemical treatments • Study mechanics +/- changes in cell properties (RNAi, cell type) • Infer properties of and differences in higher-order chromatin organization • Extend approach to interphase nuclei

18. Micromanipulation of individual mitotic chromosomes Inter- chromo- somal linker newt, Xenopus laevis, mouse, human

19. stretch

20. Chromosome stretching experiment. Pipettes are used to hold a mitotic chromosome, with left pipette fabricated with a deflection force constant ~1 nN/m to allow chromosome tension to be measured. Top image shows relaxed chromosome. As the right pipette is moved, the left pipette is observed to deflect from its zero-force position (thin white line). Digital image analysis allows pipette deflections to be measured to about 10 nm accuracy, translating to about 10 pN force resolution. Black bar, 10 m. Marko (Chromosome Res 2007)

21. 0 s 300 s 110 s 330 s 390 s 250 s 275 s 1100 s Digestion of mitotic chromosome by 1 nm AluI (AG^CT). Cutting DNA alone sufficiently often eliminates chromosome elasticity, then completely dissolves chromosome. Conclusion is that chromatin (DNA) connects non-DNA components of the chromosome together, i.e., protein “scaffold” is disconnected. (Poirier et al PNAS 2002)

22. The proteins that organize chromatin into a mitotic chromosome are not connected (bonded) to one anotherMitotic scaffold is a disconnected structure linkers (proteins) approximately 15 kb spacing (4 base RE sufficient to dissolve) 30 nm (or possibly thicker) chromatin fiber Mitotic Chromosome Are the linkers primarily proteins? SMCs (condensin I/II)? Not RNA. Linkers must be distributed throughout diameter, not just axially (bending)

23. Partial Wave Spectroscopic Microscopy Yolanda Stypula, PhD Student Dhwanil Damania, PhD student Vadim Backman, BME Vinayak P. Dravid, MSE (poster) Use light scattering and direct space correlation analysis to gauge degree of disorder in cells Systematic differences between “normal” and “cancer” cell cell models Possibilities for use as diagnostic tool

24. Analysis of Replication Dynamics in normal and cancer cells Dr. Isabelle Lucas Prof. Michelle Le Beau (U Chicago) Microarray (and deep sequencing)study of DNA replication origin usage (poster)

25. Objectives 2010/2011 • Establish techniques for (ST)EM of nuclei and chromosomes • Demonstrate targeting of antibody-labeled gold particles in nuclei and chromosomes (histones, SMCs, topo II) • Define “normal/cancer” cell partners for controlled differential studies • Begin micromanipulation experiments • Major question for cell line group: does it make sense to carry out “differential” studies on the “normal” MCF-10A vs MDA-MB231 “cancer” cells?