Development of Methods to Study Synthetic Histones In Cellulo
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Date
2015-05
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The Ohio State University
Abstract
Post-translational modifications of histone proteins are essential to the regulation of chromatin structure and DNA accessibility in the eukaryotic cell. The ability to prepare synthetic, chemically-modified histone proteins has enabled fundamental insights into the functions of histone post-translational modifications. However, these powerful chemical approaches inherently limit studies to an in vitro context which lacks the complexity of native biological systems. Here we develop tools to introduce exogenous, chemically-synthesized histones into different eukaryotic cell lines for assembly into chromatin in the cell nucleus.
Previous studies have demonstrated successful uptake of recombinant H2A/H2B dimer and H32/H42 tetramer into the nuclei of Physarum polycephalum, a myxomycete slime mold. We repeat these results and extend them to show uptake of histones into a common air-borne mold of the Mucor genus, rat IEC cells, and human breast cancer MDA-231 cells. We demonstrate nuclear import of exogenous fluorescently-labeled histones into multiple cell lines through gel electrophoresis of isolated nuclei. Preliminary fluorescent microscopy experiments support the colocalization of DNA and fluorescently-labeled exogenous histones in live and in fixed cells. Avidin pull-downs of intact nucleosomes after uptake of biotin-labeled histone dimer indicate incorporation of these histones into chromatin. Our results suggest that certain histone proteins display cell penetrating characteristics, which may provide a method for the study of homogenous semi- or fully-synthetic chemically-modified histones within the complex systems of eukaryotic cells.
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Keywords
Physarum, Uptake, Histone, Synthetic