Project 3: Mechanics of Nuclei, Chromosomes and Chromatin in Cancer

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ABSTRACT (RESEARCH PROJECT 3) Project 3 aims to understand organization and function of chromosomes and the nucleus in cancer cells, engaging the Center's focus on spatio-temporal dynamics of chromatin and information transfer in cancer. All functions of the cell are ultimately controlled by the nucleus, in which genes are expressed, under elaborate regulatory control. The molecular organization of chromosomes and the underlying chromatin is central to gene regulation. How many copies of genes are present, what patterns of transcription factors are bound to gene regulatory elements, which regions are heavily repressed by heterochromatinization, and which regions carry epigenetic marks all play a central role in controlling gene expression. In cancer, these factors are often – and usually in combination – dysregulated, with concomitant aberrant structure of the nucleus and chromosomes. Project 3 seeks to analyze the variation in chromatin structure – from the fiber level to chromosomes to the whole cell nucleus – using physical tools in combination with state-of-the-art cell biological approaches. Aim 1 of the project examines the hypothesis, supported by an array of preliminary data, that the physical organization and mechanics of chromosomes and nuclei are altered in cancer cells. This will be tested using micromechanical studies of nuclei and chromosomes, combined fluorescence imaging of major chromosome- and nucleus-organizing proteins, and partial-wave spectroscopy measurements of nuclear disorder (PWS, a technology supported by the proposed Nanocytometry Core). Experiments will be carried out in a range of tumorigenic and non-tumorigenic cell types being studied in the other Projects. Experiments using patient-derived xenografts (PDX) will test ideas developed in cell lines in a patient disease context (supplied by the proposed PDX Core). Aim 2 will test how removal of specific chromosome-organizing proteins (using siRNA and CRISPR methods) affects chromosome and nuclear structure, and then will use the results of both Aim 1 and Aim 2 in combination with Hi-C data to develop mathematical models of metaphase chromosome and interphase nucleus organization. Aim 3 will focus on how chromatin fibers themselves are physically altered in different cell types, in single-chromatin-fiber assembly and mechanics experiments using nuclear extracts from cells studied in Aims 1 and 2, a primary goal being to correlate “open” or “soft” chromatin states with specific histone modifications. The result of Project 3 will be a comprehensive study of how chromatin and chromosomes are remodeled in cancer cells relative to normal cells, and its research will be tightly linked to Project 1 and Project 2 via parallel studies of the same cell lines, use of the physical assays developed in Project 3, and through cooperation on development of siRNA and CRISPR methodologies.