DNA Information and Organization at Supranucleosomal Scales: Chromatin Folding and Higher Order Structure, Heterochromatin and Domain-Wide Repression

Project Leader
John F. Marko, Molecular Biosciences/Physics & Astronomy

Team Members
Vadim Backman, Biomedical Engineering
Vinayak Dravid, Materials Science and Engineering
Michelle Le Beau, Medicine (University of Chicago)
Jonathan Licht, Feinberg School of Medicine
Adilson Motter, Physics & Astronomy
Thomas O'Halloran, Chemistry
Teri Odom, Chemistry

Project 3 Mitotic Chromosone.jpg
A mitotic chromosome is extracted from its cell.

Project Summary
Project 3 seeks to uncover the connection between higher-order chromatin structure and gene regulation mechanisms relevant to cancer. Cellular differentiation is linked to silencing of broad gene groups, often with accompanying formation of "facultative heterochromatin,” a compact (more-folded) state of chromatin. Intrinsically preferred higher order structures may be explicitly encoded in genomic DNA sequence. An important effect of higher-order structure may be that it, rather than discrete DNA sequence elements, defines and regulates the origins of replication.  Changes in higher order chromatin structure may appear in precancerous tissue, suggesting a hypothesis that optical detection of the “field effect” used in cancer diagnosis may follow from measurement of amounts and distribution of heterochromatin. Resolving these uncertainties depends on the development of technologies for physical analyses of nuclear and chromatin structures and their application to cancer model systems. This will allow understanding of the basic chromosome organization changes associated with turning on malignancy, an advance that will, in turn, lead to the emergence of new diagnostic tools. Physical tools being used include chemically sensitive and high-resolution electron microscopy methods such as protein-specific nanoparticle labeling techniques to image chromatin domains; non-imaging light-scattering techniques sensitive to variations and textures in live cell chromatin domains at submicron length scales; single-molecule, single-chromosome, and single-genome micromanipulation studies of chromatin folding; and high-resolution mass spectroscopy for detection of post-translational modifications of chromatin proteins. Data is being used to construct quantitative models explaining how sequence features are correlated with and control higher-order chromatin structure; how that control is modified in cancerous cells; and how spatially correlated patterns of global repression impact cell-cycle dynamics. This work will ultimately establish the existence of and transfer modes for a high-level layer of information, partially genetic and partially epigenetic, involved in switching chromatin structure and gene expression during cancer development.

Project 3 Force Measurement.jpgForce-measurement experiment on newt chromosone: Left pipette is deflected by force generated from chromosone stretching caused by displacement of right pipette bar.

Objective 1
This aspect of Project 3 tests the hypothesis that cancer cells display distinctive patterns of higher-order chromatin organization. High-resolution electron microscopy is being used to analyze chromatin density and three-dimensional packing in nuclei, chromosomes, and defined chromatin segments. Experiments are based on use of new high-resolution electron-microscopy techniques and targeted metal-nanoparticle labels to decorate specific chromatin structures, including core histones, linker histones, non-histone proteins (including condensin and cohesin complexes and topoisomerase II), and sequence-specific DNA-binding factors (transcription factors). Specific extracted chromatin fragments are being used to observe how 3-D chromatin structures are related to measured nucleosome position distribution and how nucleosome positioning changes during onset of cancer. The question of how those changes impact 3-D chromatin structure will thus be resolved. Using chemically sensitive microscopy techniques we will further test the hypothesis that metal occupancy in chromatin constitutes an unanticipated level of epigenetic control of chromatin architecture and gene accessibility important in cancer.

Objective 2
Our objective here is to test the hypothesis that chromatin in cancer cells has distinctive strengths of molecular interactions associated with differences in packaging. We are directly probing nucleosome-nucleosome interactions in single-chromatin-fiber stretching experiments on euchromatin and heterochromatin segments reconstituted from nuclear extracts and from purified proteins. We are defining how nucleosome-nucleosome stacking (nucleosome adhesion) forces are modulated by chemical modifications of histones including those relevant to cancerous changes. We are also carrying out complementary mechanical stiffness of whole metaphase chromosomes, whole nuclei, and interphase chromosomes extracted from nuclei. RNAi techniques are being used to reduce cellular concentrations of specific chromatin-folding proteins; this will allow us to determine their differential roles in normal versus cancer cells.

Objective 3
Here we are testing the hypothesis that the distinctive light-scattering properties of precancerous cells used in detection of the "field effect" are predominantly due to altered higher-order chromatin structure. Non-imaging techniques sensitive to sub-optical length scales including diffuse-wave light scattering, focused laser spot scattering, and dynamic light scattering will be used to analyze live cells in culture and in tissue.

Objective 4
This area examines the hypothesis that changes in higher-order chromatin structure associated with change from normal to cancer growth are associated with changes in usage patterns of origins of DNA replication. Data for nucleosome positions and 3D chromatin structure (Objective 1 above and Project 2) will be correlated with analysis of origin use patterns.

Objective 5
Our goal here is to test the hypotheses that (i)large-scale chromatin structure is encoded in DNA sequence information and (ii) that global gene repression associated with more tightly compacted chromatin domains can drastically alter cell-cycle dynamics. Data from Objectives1-4 is being analyzed to obtain sequence profiles of chromatin with different 3-D structure, mechanical, and optical properties, thus providing insight into a possible high-level "layer" of DNA information. Patterns of cancer-related heterochromatin and gene repression (combining expression data from Project 4 with results of Objectives 1-4) are being used to study effects of spatially-correlated global repression on dynamics in existing models of gene regulation.

Publications for Project 3

Alipour E, Marko JF. Self-organization of domain structures by DNA-loop-extruding enzymes. Nucleic Acids Res. 40, 11202-12 (2012).  PMCID:PMC3526278.

Backman, V. Role of cytoskeleton in controlling the disorder strength of cellular nanoscale architectur”. Biophys. J., 99, 989–996 (2010). PMCID:PMC2913198.  

Bai H, Kath JE, Zörgiebel FM, Sun M, Ghosh P, Hatfull GF, Grindley ND, Marko JF. Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end. Proc Natl Acad Sci U S A. (2012) 109:16546-51. PMCID: PMC3478594.  

Bai, H., Sun, M., Ghosh, P., Hatfull, G.F., Grindley, N.D.F., Marko, J.F. “Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase”. Proc. Natl. Acad. Sci. USA, 108, 7914-7924 (2011). PMID: 21502527 

Bhattacharyya S., Yu H., Mim C., Matouschek A. Regulated protein turnover: snapshots of the proteasome in action, Nat Rev Mol Cell Biol. 15:122-133, 2014.  

Boustany, N. N., Boppart, S. A., Backman, V. Microscopic imaging and spectroscopy with scattered light. Annual Review of Biomedical Engineering, 12, 285–314 (2010). PMID:20617940

Bruns AM, Pollpeter D, Hadizadeh N, Myong S, Marko JF, Horvath CM. ATP hydrolysis enhances RNA recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology 2 (LGP2). J Biol Chem. (2013)  Jan 11; 288 (2): 938-46. PMCID: PMC3543043

Cherkezyan L, Stypula-Cyrus Y, Subramanian H, White C, Dela Cruz M, Wali RK, Goldberg MJ, Bianchi LK, Roy HK, Backman V. Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study. BMC Cancer. 2014 Mar 14;14:189. PMCID:PMC3995586

Cornelius, SP, Lee JS, Motter AE. “Dispensability of Escherichia coli's latent pathways”. Proc. Natl. Acad. Sci. USA 108, 3124-3129. (2011). PMCID: PMC3044393

Damania D, Subramanian H, Backman V, Anderson EC, Wong MH, McCarty OJ, Phillips KG. Network signatures of nuclear and cytoplasmic density alterations in a model of pre and postmetastatic colorectal cancer. J Biomed Opt. 2014 Jan;19(1):16016. PMCID: PMC4019418

Fishbain, S., Prakash, S. Herrig, A., Elsasser, S., Matouschek, A. “Rad23 escapes degradation because it lacks a proteasome initiation region”. Nat. Commun. 2, 192 doi: 10.1038/ncomms1194, 2011. PMID: 21304521

Fu H, Chen H, Marko JF, Yan J. “Two distinct overstretched DNA states”. Nucl. Acids Res. 38, 5594-600 (2010). PMCID:PMC2938222.

Fu, H., Chen, H., Zhang. X., Qu,Y., Marko, J.F., Yan, J.. “Transition dynamics and selection of the distinct SDNA and strand unpeeling modes of double helix overstretching”. Nucl. Acids Res. 39, 3473-3481 (2011). PMCID: PMC3082884

Graham, J.S., Johnson, R.C., Marko, J.F. “Concentration-dependent exchange accelerates protein dissociation from double-stranded DNA”. Nucl. Acids Res. 39, 2249-2259 (2011). PMCID: PMC3064784

Graham, J.S., Johnson, R.C., Marko, J.F. “Counting proteins bound to a single DNA molecule”. Biochem. Biophys. Res.Commun. 415,131-4 (2011). PMID 22020072. PMC3215857

Hadizadeh Yazdi N, Guet CC, Johnson RC, Marko JF. Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions. Mol Microbiol. 86, 1318-33 (2012).  PMCID: PMC3524407

Hurst, S. J., Hill, H., MacFarlane, R.J., Wu, J., Dravid, V. P., Mirkin, C.A. “Synthetically Programmable DNA Binding Domains in Aggregates of DNA-Functionalized Gold Nanoparticles”. Small, Vol. 5 (19): pp: 2156-61 (2009), PMID 19618429

Inobe, T., Fishbain, S., Prakash, S., Matouschek, A. Defining the geometry of the two-component proteasome degron. Nat. Chem. Biol. 7, 161-167, 2011. PMID: 21278740. PMCID:PMC3129032

Inobe T., Matouschek A. Paradigms of protein degradation by the proteasome, Curr Opin Struct Biol. 24:156-164, 2014. PMCID: PMC4010099

Kawamura R. , Pope L., Christensen M., Anderson A., Meilke C., Boege F., Terekhova, K. Sun M., Marko J.F. “Mitotic chromosomes are constrained by topoisomerase-II-sensitive DNA entanglements”. J. Cell Biol. 188, 656-63 (2010). PMCID:PMC2835934  

Kim JS, Backman V, Szleifer I. Crowding-induced structural alterations of random-loop chromosome model, Phys. Rev. Lett. 106, 168102, 1-4 (2011). PMID 21599416

Kim, J.S., Pradhan, P., Backman,V., Szleifer, I. Influence of chromosome density variations on the increase in nuclear disorder strength in carcinogenesis. Physical Biology, 8(1), 015004, 1-6 (2011). PMID: 21301058, PMCID in process. 

Kraut DA, Israeli E, Schrader EK, Patil A, Nakai K, Nanavati D, Inobe T, Matouschek A. Sequence- and species-dependence of proteasomal processivity. ACS Chem Biol. 2012 Aug 17;7(8):1444-53. PMCID: PMC3423507

Kraut DA, Matouschek A. Proteasomal degradation from internal sites favors partial proteolysis via remote domain stabilization. ACS Chem Biol. 2011 Oct 21;6(10):1087-95. PMCID: PMC3199294

Lee, J.S., Nishikawa, T., Motter, A.E. Why optimal states recruit fewer reactions in metabolic network”. Discret. Contin. Dyn. Syst. A. 32(8), 2937-2950. (2012). PMCID in process. 

Lee, SM, Chen, H, O'Halloran, TV, Nguyen, SBT. Clickable' Polymer-Caged Nanobins as a Modular drug Delivery Platform. J Am Chem Soc. 131(26):9311-20. (2009). PMID:19527027

Marko JF Linking topology of large DNA molecules. Physica A 389, 2997-3001 (2010). PMCID: PMC2896270.

Marko JF. Scaling of linking and writhing numbers for spherically confined and topologically equilibrated flexible polymers. J. Stat. Phys. 142, 1353-1370 (2011), PMCID:PMC3115200

Marko, J.F., Neukirch, S. Competition between curls and plectonemes near the buckling transition of stretched supercoiled DNA, Phys. Rev. E 85, 011908 (2011). PMID:22400592

 Marko J.F., Neukirch S. Global force-torque phase diagram for the DNA double helix: structural transitions, triple points, and collapsed plectonemes, Phys Rev E Stat Nonlin Soft Matter Phys. 88:062722, 2013. PMCID:PMC3936674.  

Marko JF. Biophysics of protein-DNA interactions and chromosome organization. Physica A. 2015 Jan 15;418:126-153. PMCID:PMC4235750

Michor, F., Liphardt, J., Ferrari, M., Widom, J. What does physics have to do with cancer? Nat Rev Cancer. Aug 18;11(9):657-70. doi: 10.1038/nrc3092. Review. (2011). PMID: 21850037

Neukirch S, Marko JF., Analytical description of extension, torque, and supercoiling radius of a stretched twisted DNA. Phys. Rev. Lett. 106, 138104 (2011).PMID: 21517425.  PMCID:PMC3120040

Padinhateeri, R., Marko, J.F. Nucleosome positioning in a model of active chromatin remodeling enzymes. Proc. Natl. Acad. Sci. USA 108, 7799-7803 (2011). PMID:21518900, PMCID:PMC3093463

Parmar JJ, Marko JF, Padinhateeri R. Nucleosome positioning and kinetics near transcription-start-site barriers are controlled by interplay between active remodeling and DNA sequence. Nucleic Acids Res. 2014 Jan 7;42(1):128-36. PubMed Central PMCID: PMC3874171

Park, H., Pontius, W., Guet, C.C., Marko, J.F., Emonet, T., Cluzel, P. Interdependence of behavioural variability and response to small stimuli in bacteria.Nature 468, 819-823. Epub 2010 Nov 14. (2010). PMCID: PMC3230254

Parsaeian A., de la Cruz M.O., Marko J.F. Binding-rebinding dynamics of proteins interacting nonspecifically with a long DNA molecule, Phys Rev E Stat Nonlin Soft Matter Phys. 88:040703, 2013. PMCID: PMC3894571

Pederson T, Marko JF. Nuclear physics (of the cell, not the atom). Mol Biol Cell. 2014 Nov 5;25(22):3466-9. PMCID: PMC4230604

Physical Sciences - Oncology Centers Network, Agus DB, Alexander JF, Arap W, Ashili S, Aslan JE, Austin RH, Backman V, Bethel KJ, Bonneau R, Chen WC, Chen-Tanyolac C, Choi NC, Curley SA, Dallas M, Damania D, Davies PC, Decuzzi P, Dickinson L, Estevez-Salmeron L, Estrella V, Ferrari M, Fischbach C, Foo J, Fraley SI, Frantz C, Fuhrmann A, Gascard P, Gatenby RA, Geng Y, Gerecht S, Gillies RJ, Godin B, Grady WM, Greenfield A, Hemphill C, Hempstead BL, Hielscher A, Hillis WD, Holland EC, Ibrahim-Hashim A, Jacks T, Johnson RH, Joo A, Katz JE, Kelbauskas L, Kesselman C, King MR, Konstantopoulos K, Kraning-Rush CM, Kuhn P, Kung K, Kwee B, Lakins JN, Lambert G, Liao D, Licht JD, Liphardt JT, Liu L, Lloyd MC, Lyubimova A, Mallick P, Marko J, McCarty OJ, Meldrum DR, Michor F,Mumenthaler SM, Nandakumar V, O'Halloran TV, Oh S, Pasqualini R, Paszek MJ, Philips KG, Poultney CS, Rana K, Reinhart-King CA, Ros R, Semenza GL, Senechal P, Shuler ML, Srinivasan S, Staunton JR, Stypula Y, Subramanian H, Tlsty TD, Tormoen GW, Tseng Y, van Oudenaarden A, Verbridge SS, Wan JC, Weaver VM, Widom J, Will C, Wirtz D, Wojtkowiak J, Wu PH. A physical sciences network characterization of non-tumorigenic and metastatic cells. Sci Rep. 2013 Apr 25; 3:1449. PMCID: PMC3636513.

Pradhan, P., Damania, D., Joshi, H., Turzhitsky, V., Subramanian, H., Roy, H.K., Taflove, A., Dravid, V., Backman, V. Quantification of Nanoscale Density Fluctuations by Electron Microscopy: probing cellularalterations in early carcinogenesis. Physical Biology, 8, 026012, 1-9. (2011). PMID: 21441647, PMCID:PMC3332100

Pradhan, P., Damania, D., Turzhitsky, V., Backman, V. Quantification of nanoscale density fluctuations using electron microscopy: Light-localization properties of biological cells. Applied Physics Lett. 97, 243704, 1-3 (2010) PMID: 21221251, PMCID: PMC3017571

Schrader EK, Harstad KG, Holmgren RA, Matouschek A. A three-part signal governs differential processing of Gli1 and Gli3 proteins by the proteasome. J Biol Chem. 2011 Nov 11;286(45):39051-8. PCID: PMC3234730

Schrader, E.K., Wilmington, S.R., Matouschek, A.Making it easier to regulate protein stability. Chem. Biol. 17, 917-918, (2010). PMCID:PMC2990474

Schutz-Sikma, E. A., Joshi, H. M., Ma, Q., MacRenaris, K. W., Eckermann, A. L., Dravid, V. P., Meade, T. J. “Probing the Chemical Stability of Mixed Ferrites: Implications for Magnetic Resonance Contrast Agent Design”. Chemistry of Materials. 23 (10), 2657-2664, (2011). PMCID:PMC3097046

Sheinin, M.Y., Forth, S., Marko, J.F., Wang, M.D.  Underwound DNA under tension: structure, elasticity, and sequence-dependent behaviors. Phys. Rev. Lett. 107,108102 (2011). PMID 2198153. PMCID PMC3201814  

Sing C.E., Olvera de la Cruz M., Marko J.F. Multiple-binding-site mechanism explains concentration-dependent unbinding rates of DNA-binding proteins, Nucleic Acids Res. 42:3783-3791, 2014. PMCID: PMC3973338.  

Strasser SD; Shekhawat G; Rogers J., Dravid VP, Taflove A, Backman V. Near-field penetrating optical microscopy: a live cell nanoscale refractive index measurement technique for quantification of internal macromolecular density. Optics Letters. 37(4):506-508 (2012). PMCID:PMC3357211

Stypula-Cyrus Y, Damania D, Kunte DP, Cruz MD, Subramanian H, Roy HK, Backman V. HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure. PLoS One. 2013 May 28;8(5):e64600. PMCID: PMC3665824

Stypula-Cyrus Y, Mutyal NN, Dela Cruz M, Kunte DP, Radosevich AJ, Wali R, Roy HK, Backman V. End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis. FEBS Lett. 2014 Mar 3;588(5):829-35. Epub 2014 Feb 1. PMCID: PMC4103177

Sun M, Nishino T, Marko JF. The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation. Nucleic Acids Res., Jul 2013; 41(12):6149-6160e-published (2013). PMID: 23620281PMCID: PMC3695518.  

Sun, M., Kawamura, R., Marko, J.F. Micromechanics of human mitotic chromosomes.  Phys. Biol. 8, 015003 (2011). PMID: 21301072, PMCID:PMC3150456

Terekhova K, Gunn KH, Marko JF, Mondragón A. Bacterial topoisomerase I and topoisomerase III relax supercoiled DNA via distinct pathways. Nucleic Acids Res. (2012) 40:10432-40. PMCID:PMC3488232.  

Terekhova K, Marko JF, Mondragón A. Studies of bacterial topoisomerases I and III at the single-molecule level. Biochem Soc Trans. 41, 571-5 (2013). PMCID: PMC3767994

Terekhova K, Marko JF, Mondragón A. Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III. Nucleic Acids Res. 2015;42(18):11657-67.. Epub 2014 Sep 17. PMID: 25232096.

Whichard, ZL, Motter AE, Stein PJ, Corey SJ. “Slowly produced microRNAs control protein levels”. J. Biol. Chem. 286, 4742-4748 (2011). PMCID: PMC3039369,  

Wu J., Kim AM, Bleher R, Woodruff TK, O'Halloran TV and Dravid VP, Imaging and elemental mapping of biological specimens with the Hitachi HD-2300A dual-EDS dedicated scanning transmission electron microscope. Ultramicroscopy.  128, 24-31 (2013). PMID: 3500508

Xiao B, Freedman BS, Miller KE, Heald R, Marko JF. Histone H1 compacts DNA under force and during chromatin assembly. Mol Biol Cell. 23, 4864-71 (2012).  PMCID: PMC3521692

Xiao B, Johnson, R., Marko JF  “Modulation of HU-DNA i interactions by salt concentration and applied force”.  Nucl. Acids Res. 38, 6176-85 (2010). PMCID:PMC2952867

Xiao, B., Zhang, H., Johnson, R.C., Marko, J.F. Force-driven unbinding of proteins HU and Fis from DNA quantified using a thermodynamic Maxwell relation. Nucl. Acids Res. 39, 5568-77 (2011). PMID 21427084. PMCID: PMC3141252

You, EA, Ahn, RW, Lee, MH, Raja, MR, O'Halloran, TV, Odom, TW. Size Control of Arsenic Trioxide Nanocrystals Grown in Nanowells. J Am Chem Soc. 131 (31):10863-5. (2009) PMCID:PMC3086295

Zhang H, Marko JF. Range of interaction between DNA-bending proteins is controlled by the second-longest correlation length for bending fluctuations. Phys Rev Lett. 109, 248301 (2012). PMID: 23368394.  PMCID in process.