Quantification of Acetylated Histones (Acetyl-H3) (Z’ > 0.5)
Introduction: Histones are small proteins that bind to DNA and are involved in chromosome packing and the regulation of gene transcription. In the basal state, most chromatin is wrapped around histone complexes to form a “beads on a string” packing structure, such that 146 base pairs of DNA are typically associated with each histone core. In the condensed state, overall transcription of the chromatin is reduced, due to restricted availability of the DNA to transcription-promoting proteins and RNA polymerase. Addition of acetyl groups to the N-terminal tail region of histones H2A, H2B, H3, and H4 by histone acetyltransferases (HATs) leads to unwinding of the chromatin, which promotes transcription. Histone deacetylases (HDACs) have the opposite effect, removing acetyl groups, which promotes condensation of the chromatin and inactivation of transcription. One of the earliest identified and most widely known HDAC inhibitors is sodium butyrate which increases the levels of acetyl-histones when used at millimolar concentrations (Sealy and Chalkley 1978; Davie 2003) as diagrammed in Fig. 1. It is becoming widely appreciated that HDAC inhibitors represent a class of potential anti-cancer therapeutics, as inhibition of HDACs often inhibits tumor cell proliferation, stimulates apoptosis, and modifies differentiation and angiogenesis (Liu et al., 2006; Ouasissi and Ouaissi 2006). Accordingly, there is much active research to discover novel HDACs and considerable screening efforts are being directed towards ascertaining the optimal structure-activity relationships for HDACs inhibition and cellular efficacy (Su et al., 2000, Curtin and Glaser 2003; Monneret 2005)
Figure 1. The relationship between histone acetylation, chromatin condensation and gene transcription.
The levels of histones vs. acetylated-histones are controlled by HAT (producing acetyl-histone) and HDAC (which remove acetyl groups) enzymes. Addition of sodium butyrate to cultured cells inhibits HDACs leading to an accumulation of acetyl-histones within the nuclei.
Assay of acetylated histones via high content screening techniques
While high throughput assays have been performed to identify potential HDAC inhibitors such assays have often been indirect, detecting changes in transcriptional activity rather than acetyl-histone levels within the intact cellular environment (Su et al., 2000; Curtin and Glaser 2003). An alternative approach is to utilize high content screening techniques to directly measure the presence of acetyl histones within the nuclei as shown in Fig. 2. To enable this strategy, Vala Sciences has developed a reagent and software kit for the visualization and quantification of drug effects on nuclear acetyl-H3 levels that will optimally work for confluent cultured cells plated on 96-well (or higher) format plates. To validate the kit reagents and quantification methodology, HeLa cells were plated on glass-bottomed 96-well dishes and exposed to various concentrations of sodium butyrate for 48 hours. The cells were then stained for acetyl-H3 and for nuclei (with DAPI) and imaged in a high throughput manner with a Beckman IC 100 digital microscopy work station.
Figure 2. Exposing HeLa cells to sodium butyrate leads to increased nuclear acetyl-histone expression.
HeLa cells were exposed to either DMSO (controls) or 10 mM sodium butyrate (NaB) for 48 hours then visualized for DAPI (A and C) or acetyl histones (Acetyl-His).
The scanned images were then analyzed automatically by Vala Sciences CyteSeerTM image cytometry software platform, which quantified nuclear acetyl-H3. For each cell in each field of view, values were calculated for the cytoplasmic and nuclear levels of acetyl-H3. As shown in Fig. 3, the ratio of median nuclear to median cytoplasmic (MNI/MCI) acetyl-H3 (left panel) and the percentage of cells with nuclear acetyl-H3 above a threshold (right panel) increased with sodium butyrate concentrations. The % of cells expressing acetyl-H3 above a threshold increased 36-fold. To characterize the robustness of the assay, Z’ calculations were performed as a measure of the dynamic range of the assay (Z’ = 1-((3*SDmin)+(3*SDmax))/|MeanMax-MeanMin|). Z’ values were 0.50 for MNI/MCI and 0.61 for the % of cells above a threshold level of acetyl-H3. Z’ values of 0.5-1.0 are considered optimal, but screens with 0.2 < Z < 0.5 can be used with 2-3 replicates per compound (Zhang et al., 1999; Granas et al. 2005). Thus, these Z’ values both achieved the optimal range for primary screens.
Figure 3. Quantification of acetyl-H3 in HeLa cells exposed to the indicated concentrations of sodium butyrate for 48 hrs.
Left: Acetyl-H3 is quantified as the ratio of median nuclear intensity to median cytoplasmic intensity (MNI/MCI). Right: The percentages of cells with acetyl-H3 levels above a threshold are shown. Each bar represents the mean ± SD for n=8 wells.
Discussion
The combination of Vala’s reagent kit for staining acetyl-H3 in cultured cells and Vala’s CyteSeer image cytometry software is well suited to drug discoveryand chemical genomic studies designed to identify or characterize the ability of compounds to inhibit HDAC activity. The technique has several advantages over previously utilized strategies to identify modulators of HDAC activity. For example, it is likely that the Vala reagent kit and CyteSeer can be applied to numerous cell types (validated here with HeLa cells), which may be important for particular applications. In contrast to strategies in which cells stably transfected with luciferase reporter genes where alterations in HDAC activity are inferred from differences in luciferase expression, Vala’s reagent kit directly measures acetyl-H3. Also, it is very likely that additional high content information can be obtained in parallel to the assay of acetyl-H3 by simultaneously staining other proteins with dyes emitting different fluorescent wavelengths and imaging them. Additionally, ploidy information can also be obtained from DAPI-stained nuclei through use of Vala reagents and CyteSeer (e.g., see Vala Sciences_CulturedCellploidyApplicationnote). In addition to kits and software, Vala Sciences provides technical support, assay optimization and screening services and is committed to enabling customers to achieve the highest performance automation for assay measurements and high content screening.
References
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Davie, J. R. 2003. Inhibition of histone deacetylase activity by butyrate. J. Nutr. 133:2485S-2493S.
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Liu, T., Kuljaca, S., Tee, A., and Marshall, G. M. 2006. Histone deacetylase inhibitors: multifunctional anticancer agents. Cancer Treatment Reviews 32:157-165.
Monneret, C. 2005. histone deacetylase inhibitors. Eur. J. Medicinal Chem. 40:1-13.
Ouaissi, M., and Ouaissi, A. 2006. Histone deacetylase enzymes as potential drug targets in cancer and parasitic diseases. J. Biomedicine and Biotechnology , Article ID 13474: 1-10.
Sealy, L., and Chalkley, R. 1978. The effect of sodium butyrate on histone modification. Cell 14:115-121.
Su, G. H., Sohn, T. A., Ryu, B., and Kern, S. E. 2000. A novel histone deacetylase inhibitor identified by high-throughput transcriptional screening of a compound library. Cancer Res. 60:3137-3142.
Zhang et al., 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomolecular Scr. 4:67-73
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