ABSTRACT

Aim:

Liquid biopsy-based measurement of post-translational histone modifications (PTHMs) in bodily fluids of patients with cancer represents a novel area of biomarker research. Here, we tested the applicability of an approach combining enzyme-linked immunosorbent assays (ELISA)-like measurement of histone methylation and quantitative polymerase chain reaction to extract and quantify pericentric histone 4 lysine 20 trimethylation (H4K20me3)-related circulating DNA.

Materials and Methods:

After adding plasma into ELISA wells and letting H4K20me3-related nucleosomes bind to the antibody, nucleosomal DNA was detached from immune complexes using a buffer including NaHCO3, SDS, and proteinase K and purified. A single copy- and a multiple copy-sequence from pericentric regions of chromosomes 16 and 1 were amplified to quantify H4K20me3-related DNA from patients with colon cancer or colonic polyps.

Results:

Relative plasma level of H4K20me3-related nucleosomal DNA was lower in the patients with colon cancer than in controls for both target sequences, with a statistically significant difference at the chr16-specific target (relative values were 0.037 vs. 0.073, respectively; p=0.008).

Conclusion:

That H4K20me3-related nucleosomal DNA is present in decreased levels in colon cancer confirms our previous findings attained using other techniques. In conclusion, our findings indicate the applicability of direct extraction of protein-bound DNA from ELISA immune complexes and it could provide a surrogate means in the assessment of PTHMs or other protein-DNA interactions.

INTRODUCTION

In eukaryotic cells, nucleosomes are basic functional units of DNA packaging in chromatin. Each nucleosome is composed of an octamer of four core histones (H3, H4, H2A, and H2B), with wrapped DNA around them¹. The genetic information stored in the context of chromatin is subjected to two main types of epigenetic modifications, i.e. DNA methylation and various histone modifications. Post-translational modifications of N-terminal tails of histone proteins protruding from the nucleosomes affect the chromatin structure, thus their dysregulation impacts gene expression and represents a common mechanism across human cancers contributing to oncogenesis through the induction of epigenetic, transcriptomic, and phenotypic alterations².

Histone methylation, which is catalyzed by histone methyltransferases on three levels (mono-, di-, or trimethylation), mainly takes place on lysine residues of histone H3 and H4 and is a dynamic process with key roles in development and differentiation. The outcomes of this modification are highly context-dependent and can be associated with transcriptional repression or activation. Methylation of H4K20 is associated with both transcriptional activation and repression depending on methylation states. H4K20me1 catalyzed by PR-Set7 is associated with transcriptional activation, whereas H4K20me2/3 catalyzed by SUV4-20H1/2 is associated with the repression of transcription by maintaining pericentric and telomeric heterochromatin³. Loss of H4K20me3 has been described as a hallmark of cancer⁴.

Liquid biopsy-based detection of cancer-related alterations in PTHMs has been reported by our group and others^5-9. In the present study, our goal was to demonstrate the practicability of the extraction of circulating nucleosomal DNA from antigen-antibody complexes in enzyme-linked immunosorbent assays (ELISA) wells. This resembles chromatin immunoprecipitation (ChIP) assays where, however, there are relevant differences between the two approaches. In ChIP assays, protein A/G agarose beads are usually employed to bind antibodies of interest, and this is often associated with significant non-specific binding of DNA or non-target proteins to beads^10-12. In the approach used in the present study, we extracted H4K20me3-associated circulating DNA directly from H4K20me3-containing nucleosomes that bound to anti-H4K20me3 antibodies immobilized on assay strips. In this way, many background signals could be excluded. Following extraction, a single copy and a multiple copy sequence from pericentric regions of chromosomes 16 and 1 were amplified to quantify H4K20me3-related circulating DNA from patients with colon cancer or colonic polyps.

GİRİŞ

Ökaryotik hücrelerde, nükleozomlar, kromatin içindeki DNA paketlemesinin temel fonksiyonel birimleridir. Her nükleozom, etraflarına sarılmış DNA ile dört çekirdek histondan (H3, H4, H2A ve H2B) oluşan bir oktamerden oluşur¹. Kromatin bağlamında depolanan genetik bilgi, iki ana tip epigenetik modifikasyona, yani DNA metilasyonu ve çeşitli histon modifikasyonlarına tabidir. Nükleozomlardan çıkıntı yapan histon proteinlerinin N-terminal kuyruklarının post-translasyon modifikasyonları, kromatin yapısını etkiler, bu nedenle düzensizlikleri gen ekspresyonunu etkiler ve epigenetik, transkriptomik ve fenotipik değişikliklerin indüksiyonu yoluyla onkogeneze katkıda bulunan, insan kanserlerinde ortak bir mekanizmayı temsil eder².

Üç seviyede (mono-, di- veya trimetilasyon) histon metiltransferazlar tarafından katalize edilen histon metilasyonu, esas olarak histon H3 ve H4’ün lizin kalıntıları üzerinde gerçekleşir ve gelişim ve farklılaşmada kilit rolleri olan dinamik bir süreçtir. Bu modifikasyonun sonuçları büyük ölçüde kontekste bağlıdır ve transkripsiyonel represyon veya aktivasyon ile ilişkilendirilebilir. H4K20’nin metilasyonu, metilasyon durumlarına bağlı olarak hem transkripsiyonel aktivasyon hem de represyon ile ilişkilidir. PR-Set7 tarafından katalize edilen H4K20me1, transkripsiyonel aktivasyon ile ilişkiliyken, SUV4-20H1/2 tarafından katalize edilen H4K20me2/3, perisentrik ve telomerik heterokromatini sürdürerek transkripsiyonun represyonu ile ilişkilidir³. H4K20me3 kaybı, kanserin ayırt edici özelliği olarak tanımlanmıştır⁴.

PTHM’lerde kansere bağlı değişikliklerin sıvı biyopsiye dayalı tespiti, grubumuz ve diğerleri tarafından bildirilmiştir^5-9. Bu çalışmada amacımız, dolaşımdaki nükleozomal DNA’nın enzime bağlı immünosorbent testi (ELISA) kuyularında antijen-antikor komplekslerinden ekstraksiyonunun uygulanabilirliğini göstermekti. Bu, kromatin immünopresipitasyon (ChIP) tahlillerine benzer, ancak burada iki yaklaşım arasında göreceli farklılıklar vardır. ChIP tahlillerinde, protein A/G agaroz boncuklar genellikle ilgili antikorları bağlamak için kullanılır ve bu genellikle DNA’nın veya hedef olmayan proteinlerin boncuklara non-spesifik bağlanmasıyla ilişkilendirilir^10-12. Bu çalışmada kullanılan yaklaşımda, H4K20me3 ile ilişkili dolaşımdaki DNA’yı, test şeritleri üzerinde immobilize edilmiş anti-H4K20me3 antikorlarına bağlanan H4K20me3 içeren nükleozomlardan doğrudan çıkardık. Bu şekilde, birçok arka plan sinyali hariç tutulabilir. Ekstraksiyonun ardından, kolon kanseri veya kolon polipleri olan hastalardan alınan H4K20me3 ile ilgili dolaşımdaki DNA’yı ölçmek için kromozom 16 ve 1’in perisentrik bölgelerinden tek bir kopya ve çoklu kopya dizisi amplifiye edildi.

MATERIALS AND METHODS

Study Participants

The study cohort included individuals who underwent colonoscopy procedures at the Surgical Department of İstanbul University, İstanbul Faculty of Medicine Hospital. Individuals with pathologically confirmed polyps in the colon (control group, n=15) and those with pathologically confirmed colon cancer (cancer group, n=15) were enrolled in the study. The characteristics of the patients with colon cancer are shown in Table 1. The study was approved by the İstanbul University, İstanbul Faculty of Medicine Ethics Committee (approval no: 2018/1095) and informed consent was obtained from each participant. Written informed consent forms were read by each patient and signed approvals were obtained before their operation.

Quantification of Circulating Nucleosomes

Blood samples were taken into EDTA tubes before the colonoscopy procedure and immediately centrifuged (10 min at 717 × g) to obtain the plasma fraction. After re-centrifugation, plasma was stored in aliquots at -80 °C until assayed. Plasma levels of circulating nucleosomes were determined using the Cell-Death Detection ELISA kit (Roche Diagnostics, Mannheim, Germany), as previously reported⁶. We applied 20 μL of plasma twice and the mean signal values, measured in optical density, were considered to be relative plasma concentrations.

Extraction of H4k20me3-related Nucleosomal DNA from Antibody-bound Nuclesosomes in ELISA

We used the commercially available EpiQuik Global Tri-Methyl Histone Quantification Kit (Epigentek, Farmingdale, NY, USA), which enables ELISA-like measurements of H4K20me3. Nuclesomal DNA was, however, extracted from antibody-bound nucleosomes before the H4K20me3 measurement was completed. For this, 50 µL plasma was added into ELISA wells and H4K20me3-related nucleosomes were allowed to bind to the antibody for 1 hour. After several washing steps, bound DNA was extracted as follows: The extraction buffer [100 mM NaHCO₃, 1% SDS and proteinase K (250 µg/mL)] was added on H4K20me3-antibody complexes and incubated at 65 °C for 15 min leading to the disassociation of nucleosomal DNA from immune complexes. Detached DNA was transferred into fresh tubes, diluted to 100 µL, and purified using spin columns. DNA was eluted in 50 µL elution buffer and stored at -20 °C after checking the purity.

Quantitation of Nucleosomal DNA by Quantitative Polymerase Chain Reaction (PCR)

We amplified two genomic sequences from the pericentric regions of chromosomes 1 and 16 because H4K20me3 is enriched in pericentric heterochromatin. The primers for chr16 allow the amplification of a single copy sequence and had the following sequences: 5’AATCCAATGGAATCATCGAGT-3’ and 5’-GGTGATTTCATTCAAGTCCATTC-3’.

The primers for chr1 amplify at least 40-copies and were 5’-GGTTCCATTTGATGATGATTCC-3’ and 5’-ATCGAGTGGAATCGAATGG-3’. LINE1 element, which is found in all genomic regions of the human genome and enriched near centromeres, was used as the reference sequence to normalize the target sequences. Primer sequences of LINE1 resulting in 148-bp amplicon were 5’-AAAGCCGCTCAACTACATGG-3’ and 5’-TGCTTTGAATGCGTCCCAGAG-3’. Real-time PCR was conducted in a light-cycler 480 with SYBR green as the fluorescence molecule, according to the instructions. Reactions were run in double and relative concentrations of pericentric sequences were calculated using the ∆∆Ct method.

Statistical Analysis

The study findings are presented as median, quartiles, and total ranges. Discrimination between the study groups was performed using the Mann-Whitney U test. P-values of <0.05 were considered statistically significant. Calculations were performed using the SPSS 21.0 statistical software (IBM Corporation, Armonk, NY, USA).

RESULTS AND DISCUSSION

All plasma samples taken from patients with colon cancer and controls had detectable levels of circulating nucleosomes with slightly higher levels in the cancer group (Figure 1A). Following the extraction of nucleosomal DNA from H4K20me3-ELISA immune complexes, DNA was present in all samples and used as a template for the amplification of pericentric sequences from Chr 1 and 16. The relative plasma level of H4K20me3-related nucleosomal DNA was lower in the patients with colon cancer than in the controls for both target sequences (Figure 1B and 1C). At the chr1-specific target, relative values of H4K20me3-related nucleosomal DNA were 2.74 and 3.2 (p=0.16) in the patients with colon cancer and those with polyps, respectively. At the chr16-specific pericentric region, the difference between the patients with colon cancer and the controls was statistically significant (relative values: 0.037 vs. 0.073, respectively; p=0.008). These findings show that H4K20me3-related circulating nucleosomal DNA is present in decreased levels in colon cancer compared to the premalignant state and confirm our previous findings when ChIP- or ELISA-like approaches were used^5,6. Even if the number of patients in the study is very small, the patients with more advanced disease (n=7) had lower levels of both target sequences than those with localized disease (n=8) (2.26 vs. 3.4 for Chr1-specific target and 0.036 vs. 0.049 for Chr16-specific target, respectively). Overall, decreased plasma levels of H4K20me3-related nucleosomal DNA are likely to be a reflection of H4K20me3 loss in colonic tumors, which has been described as a hallmark of cancer^4,13.

To our knowledge, this is the first report to describe the applicability of extracting circulating nucleosomal DNA from ELISA immune complexes. Even though our study does not include a direct comparison of this approach with ChIP, this method may be superior to ChIP in preventing background noise signals because protein A/G agarose beads used in ChIP assays non-specifically bind DNA and non-target proteins^10-12.

The study of PTHM alterations in bodily fluids of individuals with disease represents a relatively new research field. Different techniques such as ChIP, ELISA-like measurement or more recently mass spectrometry have been used for the measurement of PTHMs in circulating nucleosomes^5-9, where cancer has been the main topic. The research conducted up to now has revealed the potential of liquid biopsy-based detection of PTHMs as surrogate cancer biomarkers. However, given the complexity in the number and the regulation of PTHMs, the lack of standardization of pre-analytics, and the method of their measurement, considerable research is required before validated markers become available for clinical use.

Study Limitations

The limitation of our study is the small number of patients. Further studies with higher number of patients are needed.

BULGULAR VE TARTIŞMA

Kolon kanserli hastalardan ve kontrollerden alınan tüm plazma numuneleri, kanser grubunda biraz daha yüksek seviyelerle birlikte dolaşımdaki nükleozom seviyelerine sahipti (Şekil 1A). H4K20me3-ELISA immün komplekslerinden nükleozomal DNA’nın ekstraksiyonunun ardından, DNA tüm numunelerde mevcuttu ve Chr 1 ve 16’dan perisentrik dizilerin amplifikasyonu için bir şablon olarak kullanıldı. H4K20me3 ile ilişkili nükleozomal DNA’nın rölatif plazma seviyesi, her iki hedef dizi için de kolon kanserli hastalarda kontrollere göre daha düşüktü (Şekil 1B ve 1C). Chr1’e özgü hedefte, H4K20me3 ile ilişkili nükleozomal DNA’nın rölatif değerleri kolon kanserli hastalarda ve polipli hastalarda sırasıyla 2,74 ve 3,2 (p=0,16) idi. chr16’ya özgü perisentrik bölgede, kolon kanserli hastalar ile kontroller arasındaki fark istatistiksel olarak anlamlıydı (rölatif değerler: Sırasıyla 0,037 ve 0,073; p=0,008). Bu bulgular, H4K20me3 ile ilişkili dolaşımdaki nükleozomal DNA’nın, premalign duruma kıyasla kolon kanserinde düşük seviyelerde bulunduğunu göstermektedir ve ChIP veya ELISA benzeri yaklaşımlar kullanıldığında önceki bulgularımızı doğrulamaktadır^5,6. Çalışmadaki hasta sayısı çok az olsa bile, daha ileri hastalığı olan hastalarda (n=7) lokalize hastalığı olanlara göre her iki hedef sekans düzeyi daha düşüktü (n=8) (Chr1’e özgü hedef için 2,26’ya karşı 3,4 ve Chr16’ya özgü hedef için 0,036’ya karşı 0,049). Genel olarak, H4K20me3 ile ilişkili nükleozomal DNA’nın azalmış plazma seviyelerinin, kanserin ayırt edici özelliği olarak tanımlanan kolon tümörlerindeki H4K20me3 kaybının bir yansıması olması muhtemeldir^4,13.

Bildiğimiz kadarıyla bu, dolaşımdaki nükleozomal DNA’nın ELISA immün komplekslerinden çıkarılmasının uygulanabilirliğini tanımlayan ilk rapordur. Çalışmamız bu yaklaşımın ChIP ile doğrudan bir karşılaştırmasını içermese de, ChIP deneylerinde kullanılan protein A/G agaroz boncukları DNA ve hedef olmayan proteinleri non-spesifik bir şekilde bağladığı için bu yöntem arka plan gürültü sinyallerini önlemede ChIP’den daha üstün olabilir^10-12.

Hastalığı olan bireylerin vücut sıvılarındaki PTHM değişikliklerinin incelenmesi, nispeten yeni bir araştırma alanını temsil etmektedir. Kanserin ana konu olduğu, dolaşımdaki nükleozomlarda PTHM’lerin ölçümü için, ChIP, ELISA benzeri ölçüm veya daha yakın zamanda kütle spektrometrisi gibi farklı teknikler kullanılmıştır^5-9. Şimdiye kadar yapılan araştırmalar, PTHM’lerin vekil kanser biyobelirteçleri olarak sıvı biyopsiye dayalı tespit potansiyelini ortaya çıkardı. Bununla birlikte, PTHM’lerin sayısı ve düzenlenmesindeki karmaşıklık, pre-analitiklerin standardizasyonunun olmaması ve bunların ölçüm yöntemi göz önüne alındığında, valide edilmiş belirteçlerin klinik kullanım için uygun hale gelmesinden önce önemli araştırmalar gereklidir.

Çalışmanın Kısıtlılıkları

Çalışmamızın kısıtlılığı hasta sayımızın az olmasıdır. Daha fazla hasta sayısı ile yapılacak başka çalışmalara ihtiyaç vardır.

CONCLUSION

The detection of biomarkers in blood or other biologic fluids offers many advantages, including being minimally invasive and easily accessible. Here, we demonstrate the applicability of direct extraction of nucleosomal DNA from ELISA immune complexes, which represents a simplified method for the analysis of protein-DNA interactions and could provide an additional means in the assessment of PTHMs in liquid biopsy measurements.