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Epigenetic regulation of genes involved in cell growth, survival, or differentiation through histone modifications is an important determinant of cancer development and outcome. The basic science of epigenetics uses analytical tools that, although powerful, are not well suited to the analysis of heterogeneous cell populations found in human cancers, or for monitoring the effects of drugs designed to modulate epigenetic mechanisms in patients. To address this, we selected three clinically relevant histone marks (H3K27me3, H3K9ac, and H3K9me2), modulated their expression levels by in vitro treatments to generate high and low expressing control cells, and tested the relative sensitivity of candidate antibodies to detect the differences in expression levels by flow cytoametry using a range of sample preparation techniques. We identified monoclonal antibodies to all three histone marks that were suitable for flow cytoametry. Staining intensities were reduced with increasing formaldehyde concentration, and were not affected by ionic strength or by alcohol treatment. A protocol suitable for clinical samples was then developed, to allow combined labeling of histone marks and surface antigens while preserving light scatter signals. This was applied to normal donor blood, and to samples obtained from 25 patients with leukemia (predominantly acute myeloid leukemia). Significant cellular heterogeneity in H3K9ac and H3K27me3 staining was seen in normal peripheral blood, but the patterns were very similar between individual donors. In contrast, H3K27me3 in particular showed considerable inter-patient heterogeneity in the leukemia cell populations. Although further refinements are likely needed to fully optimize sample staining protocols, “flow epigenetics” appears to be technically feasible, and to have potential both in basic research, and in clinical application. © 2013 International Society for Advancement of Cytometry
With the rapid development and application of sequencing technologies, it is becoming evident that alterations at the genome level are unlikely to explain the heterogeneity of human cancers , and there is increasing recognition that epigenetic mechanisms also play a major role in cancer development and progression. The epigenetic regulation of genes is complex, and occurs at the levels of DNA, histone proteins, and RNA [2-4]. DNA methylation at promoter regions can cause long term gene silencing, whereas histone modifications, predominantly affecting lysines in the N-terminal tails of the core histone proteins H3 and H4, are more dynamic and associated with relatively short term plasticity.
The most frequent lysine modifications of histone proteins (“marks”) involve acetylation and methylation, although phosphorylation, ubiquitylation and sumoylation can also occur at these sites. Histone acetylation, which alters the charge distribution, is typically associated with open chromatin that enables gene transcription, whereas methylation is usually but not always associated with chromatin condensation. Enzymes like histone acetyltransferases and methyltransferases that create these marks are referred to as “writers,” whereas those that remove marks (e.g., demethylases and deacetylases) are “erasers.” A third class of chromatin-related proteins are “readers” . Readers bind to specific histone marks and recruit protein complexes that effect changes in chromatin condensation, gene expression, or DNA methylation. Large numbers of mutations affecting all three classes of protein have been identified in recent years, and linked to cancer progression and to other disease states [2, 5-13]. Therefore, there is considerable interest in the development of novel agents to target these mechanisms [14, 15], and, as a corollary, a need for laboratory techniques able to characterize epigenetic mechanisms in heterogeneous clinical samples, and to monitor treatment effects.
Chromatin immunoprecipitation (ChIP) using antibodies raised against individual histone marks is a basic technique used extensively in epigenetics research, and highly specific ChIP grade antibodies are available from a number of commercial sources. The identification of histone modifications in cells undergoing mitosis (H3S10 phosphorylation) and DNA damage response (γH2AX) are established and relatively straightforward techniques using flow cytoametry [16-18]. However, in its unperturbed state chromatin can be highly compacted and this is likely to influence the accessibility of antibodies to histone modifications involved in epigenetic regulation; particularly those associated with condensed chromatin. Little work has been published using flow cytoametry to study histone modifications linked to epigenetic mechanisms. Obier et al. showed the feasibility to detect a number of individual histone marks by flow cytoametry, and their technique was subsequently applied for cell sorting, although the protocol described was not optimized for clinical samples, and the dynamic ranges shown in the data appear relatively small [19, 20]. Since our eventual goal is to implement flow epigenetics as a clinical tool, we first studied variables affecting the detection of individual histone marks by flow cytoametry using cell lines, and then adapted a protocol that maintained surface immunophenotype and light scatter sufficiently for application to normal and leukemic blood samples.
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- Materials and Methods
- Literature Cited
- Supporting Information
We were able to show saturation binding characteristics of antibodies to all of the three histone marks selected for this pilot project, using in vitro treatments to augment or attenuate the levels of the histone marks. Results obtained by flow cytoametry were in overall agreement with those obtained in bulk assay by western blot, with the exception of a paradoxical decrease in H3K27me3 following treatment with a demethylase inhibitor. Hypermethylation of this mark results in chromatin compaction [25, 26], and we think it possible that, as a result, many H3K27me3 sites became inaccessible using the current flow cytoametry protocol.
A long-term goal is to apply this technique to patient samples, both as a tool for studying epigenetic mechanisms in heterogeneous clinical samples, and also as an approach to allow monitoring of epigenetic-targeted agents during clinical trials. In earlier papers, Ronzoni et al. showed increased acetylation of histone H4 following treatment with an HDAC inhibitor in vitro, and were able to detect increased labeling in patients treated with valproic acid . Using an antibody to acetylated proteins, Chung et al.  successfully tracked the effects of the HDAC inhibitor MS-275 in peripheral blood and bone marrow samples obtained from study patients. These papers provide proof of principle for the potential to monitor newer, selective agents that target epigenetic mechanisms in patients.
When developing a suitable protocol for blood samples, it became evident that low concentrations of fixative that were optimal for labeling histone marks resulted in the loss of definition of subpopulations by surface markers and light scatter. We found that brief fixation with 4% formaldehyde at RT gave excellent preservation of light scatter and surface markers, while also preserving signals from H3K9ac and H3K27me3, but not H3K9me2. Applying this compromise technique to blood samples obtained from normal donors and leukemia patients, we identified striking heterogeneity in expression levels of the two histone marks, which supports the idea that flow epigenetics might be capable of giving clinically important information not available using conventional techniques.
The normal blood samples showed considerable cellular heterogeneity in the levels of H3K9ac and H3K27me3, although the patterns of staining and mean fluorescence values appeared quite similar between individual donors. Relatively low levels of H3K9ac were seen in the granulocytes, with intermediate levels in the monocytes and highest in the lymphocytes. High levels of H3K27me3 were also seen in the lymphocytes with the monocytes and granulocytes showing similar levels. Lymphocytes showed the greatest heterogeneity of both histone marks, and it will be interesting to determine if this correlates with lymphocyte subpopulations. The low levels seen in the granulocytes might reflect the highly compact chromatin seen in these cells, and it will be interesting to apply this technique to bone marrow samples in order to determine if the histone modifications track normal myeloid differentiation.
When we applied this technique to a consecutive series of 25 newly diagnosed leukemia patients, selected on the presence of circulating blast cells, we noted a striking heterogeneity in the levels of H3K27me3 within the leukemic blasts, compared with the homogeneity of the normal blood samples. In contrast, H3K9ac levels showed a roughly normal distribution, with levels similar to those seen in the mature cell population of normal blood. The H3K27me3 mark is of interest as it is associated with stem cell features, and it can be modified by a number of mutations that occur in AML patients. For example, mutations involving the polycomb repressor complex 2, including EZH2 and ASXL1, which is responsible for methylation at this site, result in decreased H3K27me3, whereas mutations of genes involved in its demethylation, such as iso-citrate dehydrogenase (IDH) 1/2, result in hyper methylation [3, 5, 10]. We, therefore, plan to expand our series of AML patient samples studied with this technique, and then perform mutational analysis of the relevant genes in order to determine if the measurement of H3K27me3 by flow cytoametry is able to provide a rapid screen for mutations.
Based on the results shown in this article, and given the urgent need for robust analytical methods to track epigenetic mechanisms in cancer patients, we believe that flow epigenetics represents a promising and hitherto under-explored technique. We think it encouraging that all three histone marks selected for this pilot study could be successfully measured by flow cytoametry, suggesting that the technique can be extended to include many additional relevant marks. The development of highly specific, high affinity ChIP-grade antibodies to histone modifications is being driven by basic research, and many of these antibodies are probably suitable for flow cytoametry. Furthermore, the histone code is highly conserved across eukaryote species, and we think it likely that flow cytoametry protocols and reagents developed to study human cancers could be readily adapted to other biological applications, including for example invertebrate or plant species. However, a number of cautions need to be kept in mind.
Measurement of individual histone marks at the single cell level cannot identify specific genes that are being epigenetically regulated. Rather, flow epigenetics could be considered as a readout of the function of enzymes that are regulating the epigenome. To that end, additional useful information might be obtained by combined staining with antibodies to the relevant readers, writers, or erasers, as well as to relevant gene targets that are being epigenetically regulated. This can be explored as antibodies become more generally available.
It is likely that chromatin compaction has a major effect on the accessibility of histone specific antibodies, and we found that the H3K9me2 site was particularly vulnerable to masking following formaldehyde fixation. It has been found that the staining of some nuclear antigens is improved following fixation under high ionic strength , or following treatment with alcohols, but we did not observe that with any of the histone marks tested. However, further developmental work could be done in this area, for example, testing alternative fixatives or fixation conditions. It is also evident that protocols not requiring surface antigen staining can use reduced fixation conditions, thereby improving histone labeling. Despite these caveats, and a significant amount of further developmental work, we think that flow epigenetics could evolve into a major new application of flow cytoametry, with potential in a wide range of biomedical fields in addition to cancer.