Using Nonsense Suppression to Generate Pure Histone H3 with Homogenous Lysine Acetylation On Up to Four Select Residues for Use in Biochemical and Biophysical Assays

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2014-04-15
Authors
Young, Isaac
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Chromatin is an important regulator of how the genetic material of eukaryotes is transcribed, replicated, and repaired. A major method of this regulation is through the post-translational modifications of histone proteins that comprise nucleosomes, the fundamental building blocks of chromatin. To better understand how post-translational modifications function, we are using in vitro approaches to study how histone H3 lysine acetylation, a mark associated with actively transcribed genes, affects chromatin structure and function. As a first step, we generated H3 histones with multiple lysine acetylation marks at specific positions using nonsense suppression techniques. We were able to generate sufficient quantities of pure protein for biochemical and biophysical studies. Using pure histone components, we have reconstituted nucleosomes with acetylation marks. To determine what extent histone H3 acetylation affects chromatin structure, we have developed a single molecule stability assay using total internal reflection fluorescence microscopy. This assay allows us to follow the disassembly of individual fluorescently labeled nucleosomes in real time. To study to what extent histone H3 acetylation recruits enzymes to neighboring nucleosomes, we have developed an on bead dinucleosome acetylation kinetics assay. This assay allows us to incorporate previously acetylated histones in one position and monitor enzyme-mediated acetylation on neighboring nucleosomes.

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