Packing and Unpacking the Genome: Single-Molecule Manipulation of Chromatin

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In this project, the PIs will use single-molecule optical tweezers to elucidate the structural, mechanical and thermodynamic consequences of biologically-relevant post-translational modifications (PTMs) on the behavior of individual nucleosomes and on chromatin condensation. The fundamental biological significance of the proposed research is that the packaging of DNA into nucleosomes and chromatin, and its modulation by histone modifications and histone H1, are essential aspects of gene regulation in all eukaryotes. Specific objectives of this project include: (1) assemble nucleosomes and chromatin, using histone H4 with prescribed single and multiple site-specific acetylation; (2) compare the behavior of chromatin in the presence of magnesium ions or magnesium ions and linker histone H1, which are key factors regulating condensation, to the behavior of chromatin without these factors; (3) determine accurate free energy differences between the different molecular states encountered, using an intelligent force-stepping protocol; (4) compare the behavior of nucleosomes containing histone H4 with acetylated tails to those containing H4 with mutated tails, that mimic acetylation, revealing the role of charge. Thus, this project will provide observations and measurements needed to create and validate physical models of eukaryotic gene expression. The broader impacts of this project include: (1) these studies have potential human health benefits, because several diseases, including asthma and a number of cancers, are linked to histone PTMs; (2) by following Yale's Integrated Graduate Program in Physical and Engineering Biology (IGPPEB) and serving as teaching fellows in a novel introductory physics sequence, that seeks to engage life-science undergraduates, the graduate students involved in this project will join a new generation of researcher/teachers, who excel at quantitative approaches, have the biological sophistication to identify and apply their skills to cutting-edge biological problems, and are excited to educate future generations; (3) to encourage an interest in science in the next generation, the PIs will work with Breakthrough New Haven, a high school preparation program for socio-economically disadvantaged students in Grades 7 and 8, to implement hands-on science modules for Breakthrough's summer program, and will host high-school students for summer research projects in their laboratories.This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences.