Abstract
Background: Alterations in DNA methylation and histone modifications have been implicated in carcinogenesis. Although tumor-specific alterations in DNA methylation can be detected in the serum and plasma of cancer patients, no data are available on the presence of histone modifications in circulating blood. We investigated whether histone methylation, as a model of histone modifications, is detectable in plasma. Because methylation at histone 3 lysine 9 (H3K9) has been demonstrated to be enriched at sites of repetitive ALU elements, we addressed the specificity of histone-methylation detection and hypothesized that if monomethylated H3K9 (H3K9me1) is detectable in plasma, the concentrations in mononucleosomes and oligonucleosomes would be different. We also analyzed a single-copy gene, CDKN2A.
Methods: We enrolled 21 multiple myeloma patients in the study. We used ELISA and real-time PCR analysis to evaluate nucleosomes and cell-free DNA, respectively, as evidence of the presence of histones and associated DNA in circulating blood. H3K9me1 was analyzed by chromatin immunoprecipitation.
Results: ELISA and real-time PCR assays indicated the presence of free nucleosomes and DNA in plasma, and the results were quantitatively correlated (P < 0.001). The detection of histone methylation on free nucleosomes was sequence dependent. Fragments representing mono- and oligonucleosomes differed with respect to H3K9me1 concentrations (P = 0.004), in accordance with our hypothesis. In addition, the detection rate and concentrations of H3K9me1 were significantly higher on the fragment covering both mononucleosomes and oligonucleosomes than on the CDKN2A promoter (P < 0.001).
Conclusions: If validated in further studies, our findings may be a basis for investigations of cancer-specific alterations in histone modifications in the circulation.
Epigenetic changes, such as DNA methylation and histone modifications, represent an additional mechanism for the non-Mendelian inheritance of phenotypic variation. Of the >50 known modes of modification, including acetylation, methylation, phosphorylation, and ubiquitination, alterations in the histone tail exhibit the greatest range of variation in epigenetic regulation (1)(2). These different types of histone modifications occur at multiple and specific sites, and different combinations may lead to distinct consequences with respect to such chromatin-dependent functions as gene expression (3)(4).
Histone methylation is involved in the regulation of fundamental processes, including heterochromatin formation, X chromosome inactivation, genomic imprinting, transcriptional regulation, and DNA repair (5)(6). Histones are methylated on either lysine or arginine. Lysine side chains can be mono-, di-, or trimethylated at several residues (7), and the simultaneous in vivo presence of mono-, di-, and trimethylated lysines including histone 3 lysine 9 (H3K9)1 has been shown (8)(9). Although the biological significance of these differences is unknown, variation in the extent of methylation may correlate with different degrees of gene regulation (10). H3K9 monomethylation (H3K9me1) and dimethylation are predominantly regulated by G9a methyltransferase, whereas trimethylation is achieved via the Suv39h enzymes (11)(12). Imbalance in this dynamic process has been linked to cancer, and Suv39h proteins are among the enzymes responsible for the epigenetic regulation important in neoplastic transformation (13).
The concentrations of circulating cell-free DNA (cf-DNA) increase in various benign and malignant pathologic conditions, including cancers (14)(15). The potential of the distinctive characteristics of cf-DNA as a tool in monitoring and managing cancer has been widely investigated (16). In serum and plasma, DNA is thought to exist predominantly as mononucleosomes and oligonucleosomes (17)(18). The nucleosome structure seems to protect DNA against rapid digestion by endonucleases (19). Circulating nucleosomes can be quantified by real-time PCR as well as via immunologic assays, which are particularly well suited for serial measurements (20).
Chromatin modifications are at least as widespread and important in cancer as alterations in DNA methylation (21); however, in contrast with DNA methylation, which has been widely investigated in serum and plasma, no evidence is yet available on the presence of histone modifications in circulating nucleosomes. We investigated whether histone methylation is detectable in the circulation. H3K9me1 has previously been shown to be enriched at sites of heterochromatic ALU repeats (22)(23). Given this finding, we chose H3K9me1 in ALU elements (24) as detected by chromatin immunoprecipitation (ChIP) as a model for detecting histone methylation in circulating nucleosomes.
Materials and Methods
plasma samples
If H3K9me at heterochromatic ALU elements is detectable in plasma, such detection is not expected to indicate a disease-specific event. Therefore, our study group consisted of only 21 patients with multiple myeloma at diagnosis, and the patients’ clinical characteristics were not considered. We have previously demonstrated that patients with multiple myeloma display variable concentrations of nucleosomal DNA in their blood plasma (25). The study protocol was approved by the local ethics committee of Istanbul Medical Faculty (No. 2006/1029). Peripheral blood was collected into EDTA-containing tubes and centrifuged immediately at 3000g for 30 min. Plasma aliquots were stored at −80 °C until use.
nucleosome elisa
The commercially available Cell Death Detection ELISAPLUS Kit (Roche Diagnostics) was used according to the manufacturer’s instructions to test for the presence of histone-associated DNA fragments (mononucleosomes and oligonucleosomes) in plasma. This quantitative sandwich enzyme immunoassay contains 2 monoclonal mouse antibodies, the first of which is directed against histones (H1, H2A, H2B, H3, and H4) and binds to the streptavidin-coated microtiter plate via its biotin tail. The second is a peroxidase-labeled antibody that recognizes the DNA component of nucleosomes bound to the first antibody, and its binding is detected colorimetrically. A positive signal is evidence for the presence of both histones and associated DNA in the plasma. Samples were analyzed in triplicate, and mean absorbance values were converted to milliunits, the relative concentrations of the circulating nucleosomes.
quantification of alu-specific free dna in the plasma
We extracted circulating DNA from 200 μL plasma with the High Pure Viral Nucleic Acid Kit (Roche Diagnostics) and quantified ALU-specific cf-DNA by quantitative real-time PCR with the LightCycler (Roche Diagnostics) and the double-stranded DNA–binding dye SYBR Green I as the fluorescent molecule. The primer pair we used amplifies both apoptotic and nonapoptotic fragments representing total DNA and produces a 115-bp amplicon (26). Absolute concentrations (expressed in micrograms per liter) were obtained from a calibration curve generated from serial dilutions of genomic DNA (10 ng to 0.1 pg; r = −0.99). The PCR mix contained 2 μL SYBR Green mix (composed of Taq DNA polymerase, dNTP Mix, SYBR Green I dye, and 10 mmol/L MgCl2), 2 mmol/L MgCl2, 200 pg of each primer, and 3 μL of DNA in a total volume of 20 μL. A hot start of 10 min at 95 °C was followed by 45 PCR cycles of denaturation at 95 °C, annealing at 64 °C, and extension at 72 °C (10 s each). The mean concentration for 3 independent PCR amplifications was specified as the amount of free DNA in each patient.
chromatin immunoprecipitation
ChIP assays are used to evaluate the association of proteins with specific DNA regions and are the standard technique for analyzing histone methylation. The technique consists of cross-linking proteins with DNA in cells on Petri dishes, harvesting the cells, and sonicating the cells, first to isolate and break down the nuclei and then to fragment the DNA (desired size usually 200–1000 nucleotides). Finally, soluble chromatin is immunoprecipitated with an antibody that recognizes the protein of interest. Because positive ELISA signals indicate the presence of soluble and stable histone complexes and short stretches of associated DNA (mono- and oligonucleosomes) in the circulation and because there is no indication in the literature of a cross-linking requirement in immunologic assays (27), we omitted these steps and started directly with the preanalytical steps of immunoprecipitation. We modified the protocol of the Protein Agarose A Immunoprecipitation Kit (Roche Diagnostics) to immunoprecipitate nucleosomes from plasma. In brief, we mixed 200 μL of plasma with 800 μL of buffer 1 (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 10 mL/L Nonidet P40, 5 g/L sodium deoxycholate, and protease inhibitors), added 50 μL of a homogeneous protein agarose A suspension (25-μL bed volume) to the samples in a preclearing step designed to reduce the background signal that can occur because of nonspecific adsorption of irrelevant plasma proteins to protein agarose A, incubated the agarose bead suspension overnight at 4 °C on a rocking platform, and eventually pelleted the agarose beads by centrifugation at 12 000g for 20 s. We transferred 1 mL of the supernatants to fresh 1.5-mL plastic tubes, added 1 μg anti-H3K9me1 antibody (Upstate), and incubated at 4 °C for 1 h. We used an antihistone antibody that recognizes all histone proteins to identify the nucleosomal input, and a sample without antibody served as a negative control. To capture the immunocomplex, we added 50 μL of protein agarose A, pelleted the beads, and washed twice with buffer 1. Next, we washed the pelleted beads with 1 mL of high-salt buffer (50 mmol/L Tris-HCl, pH 7.5, 500 mmol/L NaCl, 1 mL/L Nonidet P40, and 0.5 g/L sodium deoxycholate) and then with 1 mL of low-salt buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mL/L Nonidet P40, and 0.5 g/L sodium deoxycholate). Finally, we suspended the pellets in a buffer containing SDS and proteinase K and incubated the suspension for 30 min at 56 °C. We extracted the DNA with the phenol/chloroform method, precipitated the DNA with 0.3 mol/L sodium acetate and 2.5 volumes of absolute ethanol at −20 °C for 4 h, and centrifuged at 13 000g for 30 min. The purified DNA was suspended in 20 μL PCR-grade water.
To address the specificity of histone-methylation detection, we investigated the differences in H3K9me1 concentration between 2 overlapping ALU fragments, ALU115 and ALU247 (Fig. 1⇓ ), which represent apoptotic mononucleosomes and larger oligonucleosomes, respectively. The single-copy, transcriptionally active CDKN2A gene [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)]2 was used as a control. We amplified and quantified ALU115 and ALU47 fragments from precipitated DNA as previously described (26). We amplified the CDKN2A gene with the primers previously reported (28) and quantified CDKN2A sequence as described for ALU fragments. We used the mean values of 3 experiments to indicate the H3K9me1 concentration for each sequence.
Map of ALU sequences.
To demonstrate the specificity of histone-methylation detection, we compared 2 overlapping fragments of 115 bp and 247 bp (ALU115 and ALU247) that represent mono- and oligonucleosomes, respectively, with respect to their enrichment in ChIP. Adapted from (35).
We considered as positive samples that had entered the logarithmic phase of the amplification curve during the LightCycler PCR before cycle 41. After amplification, melting-curve analyses indicated melting peaks at approximately 84 °C for ALU115, 86 °C for ALU247, and 85 °C for CDKN2A. Finally, we electrophoresed LightCycler PCR products on a 20-g/L agarose gel stained with ethidium bromide to confirm the presence of the 115-, 247-, and 150-bp amplicons for the ALU115, ALU247, and CDKN2A sequences, respectively.
statistical analyses
Quantitative differences in H3K9me1 concentration between ALU115, ALU247, and CDKN2A were evaluated with the Mann–Whitney U-test. We evaluated quantitative correlations between the variables (e.g., histone methylation, nucleosome ELISA, or free plasma DNA) with the Spearman rank correlation test. P values <0.05 were considered statistically significant.
Results
the concentrations of nucleosomes and free alu-specific dna are correlated
Before H3K9me1 analysis, we demonstrated the presence of circulating histones and DNA in plasma. We quantified nucleosomes by ELISA and analyzed circulating DNA by real-time PCR to quantify ALU repeat–specific sequences. Circulating nucleosomes were present in all samples from the multiple myeloma patients, but there were substantial interpatient differences (Table 1⇓ and Fig. 2A⇓ ). The difference between the lowest and highest values was 43.6-fold, indicating variation in the concentrations of circulating apoptotic nucleosomes in plasma. Interindividual variation in the total free DNA in the plasma was much higher (101.3-fold), with a median of 44.3 μg/L and a mean of 259.3 μg/L (Table 1⇓ and Fig. 2B⇓ ). Samples with lower or higher nucleosome concentrations also displayed low or high concentrations, respectively, of ALU-specific DNA fragments, suggesting that these 2 variables were correlated. A statistical analysis revealed a highly significant positive correlation between the nucleosome amount and total DNA (P < 0.001, Spearman rank test).
Concentrations of nucleosomes, free DNA, and H3K9me1 in plasma.
Free nucleosome and DNA concentrations in plasma.
(A), The relative concentrations of histone-associated DNA fragments (mono- and oligonucleosomes) were measured with an ELISA kit for cell-death detection. A positive signal indicates the presence of both histones and associated DNA in the circulation. (B), The presence of free DNA in the plasma was analyzed by amplifying the ALU115 fragment via real-time PCR. Each patient value is the mean of 3 experiments. The graphs display the lowest and highest concentrations, the median (broken line), and 95% confidence intervals (boxes).
volume-dependent detection of histone methylation in plasma
After demonstrating that histones and intact DNA are present in the circulation, we investigated the presence of histone methylation in plasma. Because plasma samples may have different H3K9me1 concentrations, we used a 200-μL volume of a plasma mixture to demonstrate the characteristics of histone-methylation detection. In this primary analysis, we analyzed for the presence of H3K9me1 on the ALU115 fragment because this fragment covers both mono- and oligonucleosomes. The ALU115 fragment was amplified from ChIP-precipitated DNA by real-time PCR. With the use of positive and negative controls, we demonstrated the presence of H3K9me1-specific signals, which were validated by the amplification curves, the specific melting peaks at approximately 84 °C, and the presence of the 115-bp fragment on agarose gels. To demonstrate volume dependence and linearity of detection, we set up a plasma-dilution series. With decreasing amounts of plasma (200, 20, 2, 0.2, and 0 μL), H3K9me1 was linearly detectable to 2 μL (r = −0.98).
h3k9me1 detection is sequence dependent
In subsequent analyses, we addressed the issue of the specificity of histone-methylation detection in plasma by comparing 2 overlapping fragments from ALU elements for their enrichment by the anti-H3K9me1 antibody (Fig. 1⇑ ). The ALU115 signal represents DNA bound to mono- and oligonucleosomes, and the ALU247 signal indicates DNA only from oligonucleosomes. H3K9me1 was detected on ALU115 in 17 (81%) of 21 samples and on ALU247 in only 47.6% of the samples. More importantly, ALU115 showed significantly higher concentrations of antibody-precipitated DNA than ALU247 (Table 1⇑ and Fig. 3⇓ ; P = 0.004). This result is in accord with our observation that the concentration of free mononucleosomal DNA in plasma is substantially higher than that of oligonucleosomes (data not shown). Our findings show that the 2 overlapping multiple-copy sequences that differ only in nucleosomal size can have different detection rates and amounts of histone methylation, indicating that the detection of histone methylation is sequence specific.
Sequence-dependent detection of histone methylation.
The concentrations of H3K9me1 on ALU115, ALU247, and CDKN2A gene fragments were measured. Data represent the mean of 3 experiments. Statistically significant differences are indicated. Shown are lowest and highest concentrations, the median, and 95% CIs (boxes).
A comparison of H3K9me1 detection for ALU115 and the single-copy CDKN2A sequence provided further demonstration of the sequence dependence of histone-methylation detection. We detected H3K9me1 at the CDKN2A promoter at a rate of only 33.3% (Table 1⇑ ) and found, as expected, that ALU115 and CDKN2A showed highly significant differences in H3K9me1 concentrations among samples (P < 0.001, Mann–Whitney U-test; Fig. 3⇑ ). Again, these results indicate that the detection of H39me1 in plasma is sequence dependent.
On the other hand, we found no correlation between H3K9me1 concentration and either nucleosome concentration (P = 0.17, Spearman rank test) or total DNA (P = 0.15), suggesting that the detection of H3K9me1 is independent of the concentration of circulating nucleosomes.
Discussion
To our knowledge, this study is the first to show that histone methylation is detectable in circulating blood. We based this analysis on an investigation of H3K9me1, a modification that is generally associated with gene silencing (29). We modified the ChIP protocol by not cross-linking DNA with chromatin proteins and not fragmenting the DNA to smaller fragments. The fact that the ELISA analysis of circulating nucleosomes requires the presence of histone-DNA complexes makes cross-linking and DNA fragmentation unnecessary. Accordingly, circulating nucleosomes have been used in immunologic assays (20) without any indication of a requirement for cross-linking (27).
A previous demonstration that H3K9 methylation is higher at ALU repeats (22)(23) prompted our decision to investigate the specificity of the analysis. Our comparison of 2 overlapping fragments from ALU repeats that differ in size and represent mono- or oligonucleosomes showed quantitative differences in the detectable amounts of histone methylation. In addition, a comparison of multiple copies of the ALU115 fragment with the promoter of the single-copy CDKN2A gene demonstrated that the detection of histone methylation was sequence specific. H3K9 in the ALU element was methylated at a higher rate and in significantly higher amounts than in the CDKN2A promoter. As further evidence supporting this specificity, we found that the H3K9me1 concentration decreased with decreasing plasma volume.
Of interest was the correlation between histone methylation and either nucleosomes or amplifiable DNA. We observed no quantitative association, although the nucleosome and amplifiable DNA concentrations are correlated, as has previously been reported (30). In addition, the amount of H3K9me1 varied appreciably among samples. These findings suggest that the amount of methylated histone residues in plasma is independent of the nucleosome concentration. Increased concentrations of circulating nucleosomes have been reported not to be disease specific (20)(31). Also worth noting is that some samples contained no methylated H3K9 residues, although they did have detectable concentrations of circulating nucleosomes. The reason for this difference is not clear.
In this preliminary study, we used only an antibody against the monomethylated H3K9. Also of interest is how higher degrees of methylation (di- and trimethylation) differ in the circulation. Different degrees of methylation may represent different levels of gene regulation, depending on the physiological role in the regulation of gene expression or their localization in the genome. Several studies have used antibodies specific for different methylation states to investigate the localization of these residues. Rice et al. (12) reported that mono- and dimethylated residues were specifically localized at silent domains within the euchromatin and that H3K9 trimethylation was more frequent in pericentric heterochromatin. Our finding that H3K9me1 was detectable in the CDKN2A promoter region in 33.3% of the plasma samples may indicate the presence of a repressive histone modification at transcriptionally active genes; however, the extent to which methylation of histone lysine residues is involved in mammalian gene regulation has not yet been completely defined.
Methylated DNA is thought to preferentially occur in nucleosomes in plasma and serum (32). Our findings indicate that circulating nucleosomes may be useful for investigating the relationships between disease and the various types and degrees of histone modifications or variations in their quantities. As with the detection of tumor-related molecular and epigenetic changes in circulating nucleic acids, it should be possible to trace tumor-related alterations in histone modifications in the circulation. For example, the antitumor effects of histone deacetylase or histone methyltransferase inhibitors (33) could be evaluated by monitoring changes in the quantities of the corresponding modification in the circulation. Further studies are warranted to determine whether and to what extent detection of histone modifications in the circulation is relevant for diseases such as cancer. Methodologic advances that enable analysis of the global pattern of histone modifications on a genome-wide scale (34) may help to assess the relevance of the pattern of plasma histone modifications in cancer.
Acknowledgments
Grant/Funding Support: This work was supported by the Istanbul University Research Fund (Project No. 564/14082006).
Financial Disclosures: None declared.
Footnotes
- © 2008 The American Association for Clinical Chemistry