Development of a High Throughput Flow Cytometry Method to Analyze Changes in Epigenetic Histone Modifications in the Cell

Abstract

Epigenetic studies commonly include assays such as Chromatin Immunoprecipitation (ChIP), western blotting, and immunofluorescence (IF), which are used to measure changes in chromatin modifications and in DNA methylation patterns associated with diseased states and resulting from drug treatments (Lo et al. 2004). Clinically, changes in epigenetic modifications are determined by intensive western blotting assays. However, other methods such as IF and flow cytometry are starting to become more prevalent, as these techniques are crucial to show that the drug response in a cell is specific before the drug enters clinical trials. The purpose of this study was to develop a high throughput flow assay to analyze a large number of histone modifications and to compare this assay to western blotting methods for ease of use and quantitative analysis. First, I utilized antibodies against total Histone H3, H3K27Me2, H3K27Me3, H3K4Me3 and H3K9Me3 to develop a flow assay, to determine optimal drug treatment conditions, and compare to western blotting for assaying changes in histone modification in cells. Using this flow assay, I found that the EZH2-specific methyltransferase inhibitor, GSK343, most optimal drug treatment condition was 5μM for 96h in Jurkat cells, showing a 14–fold reduction in H3K27Me2 levels and a 21–fold reduction in H3K27Me3 levels. Additionally, I found that the general methyltransferase inhibitor, Adox, rapidly induced cell death and apoptosis without showing significant changes in histone methylation. Finally, my results demonstrated that flow is a faster, more quantitative and less strenuous method compared to western blotting. Second, I determined a medium-throughput flow protocol using a panel of thirteen monoclonal antibodies against key methylation and acetylation sites on total Histone H3. I determined one flow protocol, which used 0.3% Triton X-100 permeabilization and worked for all thirteen antibodies. Finally, I utilized this medium-throughput flow assay and antibody panel to interrogate the specificity of GSK343 and for that it specifically indicated methylation of H3K27, the known substrate of EZH2. For this project, I was able to develop a medium-throughput flow protocol using a panel of thirteen monoclonal antibodies. I found this flow assay easier, more efficient and more quantitative than western blotting. Using the medium-throughput flow assay, I analyzed the specificity of GSK343 for EZH2. It is important to know of any off-target effects of epigenetic drugs before they are used on humans for drug therapies. Results from this project lay the groundwork so that I can expand upon and develop a high-throughout flow assay by introducing a larger panel of antibodies, potentially to every H2A, H2B, H3 and H4 modification site, for screening other epigenetic drugs for specificity.