Project 3: Mechanics of Nuclei, Chromosomes and Chromatin in Cancer
Vadim Backman, Biomedical Engineering, Northwestern University
Robert Goldman, Cell and Molecular Biology, Northwestern University
Leonid Mirny, Health Sciences Technology/Physics, MIT
Adilson Motter, Physics and Astronomy, Northwestern University
Igal Szleifer, Biomedical Engineering, Northwestern University
The large-scale structure of chromosomes is being increasingly appreciated as crucial to the regulation of genes, and dysregulation of higher-order chromosome organization is a hallmark of most cancers. The overarching hypothesis driving this Project is that there are profound changes in organization of nuclei and chromosomes with cell type, particularly for rapidly growing cancer cells. Preliminary data show strong effects of cell cycle timing on the structure and mechanics of metaphase chromosomes, and strong changes in nuclear organization in cancer cells, including changes driven by post-translational modifications of histones.
Aim 1: How does the distribution of chromosome-organizing proteins in mitotic and interphase chromosomes vary with cell type and differ between normal and cancer cells? Here we will test the hypothesis that cell cycle timing and cancer cell state plays a major role in organization of Structural Maintenance of Chromosome (SMC) complexes on metaphase chromosomes, and that there are aberrant metaphase chromosome and nuclear organization in rapidly dividing cancer cells. “Pipette-spray” immunofluorescence imaging of protein distributions in nuclei and metaphase chromosomes, and pipette-based nanoelasticity measurements, wide-field and single-molecule fluorescence imaging (STORM) will be used, in conjunction with partial wave spectroscopy (PWS) and electron microscopy (EM) to quantify variation in nuclear structure. Methods to label specific chromosomal loci using CRISPR-based methods, to study changes in positioning of specific loci in different cell types, and to study prophase chromosomes will be developed.
Aim 2: What is the role of chromatin-organizing proteins in the folding of metaphase chromosomes and in establishing interphase nuclear structure, in normal and cancer cells? How do these results correlate with mathematical models of chromosomes? We will use siRNA and parallel CRISPR techniques to carry out targeted knockdowns of SMC complex to test the hypotheses that (a) cohesin plays a role in stabilizing metaphase chromosomes, (b) that metaphase chromosomes have mechanical stiffness correlated with mitosis duration and SMC distributions, and (c) that knockdowns of cohesin and SMC5/6 lead to chromosomes and nuclei with aberrant mechanical properties, using optical imaging and micromechanical analysis in conjunction with PWS and EM studies. Parallel experiments using knockdowns of nuclear-envelope-organizing factors and chromatin-NE connection factors will be used to determine the relative roles of nuclear envelope and chromatin in controlling nuclear envelope structure and mechanics. We will integrate the results of Specific Aim 1 and 2 to develop polymer simulation and statistical mechanics based models for chromosome folding, addressing the questions of how SMC complexes organize metaphase chromosomes, the origin of the “fractal globule” statistics seen in interphase nuclei, the link between chromosome structure and gene expression, and the role of protein “crowding” in controlling chromatin structure and dynamics.
Aim 3: How does variation of the varied post-translational histone modifications characteristic of cancer affect the dynamics of chromatin fibers? We will use a newly developed human-cell-extract single-chromatin-fiber assembly method to study the variation in assembly, mechanics and dynamics of chromatin fibers. We will test the hypothesis that heavily acetylated chromatin is “open” and “softer”, while heavily deacetylated chromatin is “closed” and “stiffer”, using highly quantitative piconewton-scale “magnetic tweezers” micromechanical assays. To test the hypothesis that cancer chromatin is itself physically distinct from normal chromatin extracts prepared from cells studied in Aims 1 and 2 (varied normal and cancer phenotypes), and cells with oncogenic histone modification mutations (e.g., MMSET mutations) will be analyzed, and results will be correlated with the results of Aims 1 and 2.
These studies, integrating parallel efforts of cancer cell biology (Licht, Crispino, Goldman), physics and engineering (Backman, Marko), and molecular modeling (Motter, Mirny, Szleifer) researchers will provide a unified analysis of how higher-order chromatin structure varies across a variety of cell types ranging from non-tumorigenic to highly tumorigenic, and how those changes feedback to drive cancer progression. The aims are focused on questions tightly integrated with the other Projects of the proposed PSOC, namely physio-chemical dysregulation of chromatin organization in cancer by metal ion imbalances (Project 1) and structural and regulatory roles of chromatin looping interactions in cancer (Project 2). Methods and resources used in Project 3 will be involved in the research of Projects 1 and 2 (e.g., relevant cell lines, knockdown and labeling reagents). Project 3 will employ both cores, for PWS/ STORM analysis (Nanocytometry core) and to provide patient-derived cancer xenografts (PDX core).