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.