Abstract
ABSTRACTPost-translational modifications on histones can be stable epigenetic marks or transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here, we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. In extracts of marine organisms, we also screened chemical compounds that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.
Highlights
In eukaryotes, DNA is wrapped around eight histone proteins to form nucleosomes, which are the basic units of chromatin
Multicolor immunofluorescence-based single-cell analysis To analyze the global levels of multiple modifications in single cells, HeLa cells were grown on coverslips, fixed and immunolabeled with various antibodies directly conjugated to fluorescent dyes
Evaluating the robustness of the assay system To evaluate the robustness of the assay, we examined the effects of antibody staining conditions using objective lenses with different numerical apertures (NAs)
Summary
DNA is wrapped around eight histone proteins to form nucleosomes, which are the basic units of chromatin. Posttranslational modifications of histones play a crucial role in gene regulation by altering chromatin structure and/or recruiting reader proteins (Jenuwein and Allis, 2001; Kouzarides, 2007). Sitespecific acetylation and methylation of histone H3 lysine (H3K) residues are associated with gene activation and silencing. Some modifications are stable epigenetic marks, whereas others turnover rapidly and/or exhibit dynamic changes in response to external and internal stimuli (McBrian et al, 2013; Niu et al, 2015) and during the cell cycle (Black et al, 2012). H4 acetylation and H3 phosphorylation drastically increase during the S and M phases of the cell cycle, respectively (Chahal et al, 1980; Gurley et al, 1975). Recent analyses have revealed that histone methylation states fluctuate during the cell cycle (Bar-Ziv et al, 2016; Petruk et al, 2013; Xu et al, 2012), possibly reflecting the balance between methylation and demethylation enzymes (Greer and Shi, 2012; Shmakova et al, 2014)
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