Detection of CpG methylation level using methyl-CpG-binding domain-fused fluorescent protein.

Methylation of cytosine to 5-methylcytosine on CpG dinucleotides is the most frequently studied epigenetic modification involved in the regulation of gene expression. In normal tissues, tissue-specific CpG methylation patterns are established during development. In contrast, alterations in methylation patterns have been observed in abnormal cells, such as cancer cells. Cancer type-specific CpG methylation patterns have been identified and used as biomarkers for cancer diagnosis. In this study, we developed a hybridization-based CpG methylation level sensing system using a methyl-CpG-binding domain (MBD)-fused fluorescent protein. In this system, the target DNA is captured by a complementary methylated probe DNA. When the target DNA is methylated, a symmetrically methylated CpG is formed in the double-stranded DNA. MBD specifically recognizes symmetrical methyl-CpG on double-stranded DNA; therefore, the methylation level is quantified by measuring the fluorescence intensity of the bound MBD-fused fluorescent protein. We prepared MBD-fused AcGFP1 and quantified the CpG methylation levels of the target DNA against SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) using MBD-AcGFP1. This detection principle can be applied to the simultaneous and genome-wide modified base detection systems using microarrays coupled with modified base binding proteins fused to fluorescent proteins.

Hybridization-based CpG methylation level detection using methyl-CpG-binding domain-fused luciferase.

Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.

Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer.

Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused Oplophorus luciferase (Oluc). Moreover, BRET-based hydroxymethylation and hemi-methylation level sensing systems have been developed using hydroxymethyl-CpG and hemi-methyl-CpG binding domain-fused Fluc. These studies suggest that target epigenetic modifications can be simultaneously quantified using target-modification-binding protein-fused luciferases. In this study, we focused on the SnoopTag (SnT)/SnoopCatcher (SnC) protein ligation system to establish a universal design for fusion protein construction for any combination. SnT spontaneously forms an isopeptide bond with SnC; therefore, any kind of fusion protein would be constructed by the SnT/SnC system. To establish the proof of concept, MBD-SnT, CXXC-SnT, and SnC-Oluc were prepared and ligated MBD-SnT or CXXC-SnT to SnC-Oluc. The ligation products of MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc showed luciferase activity and specific binding activity to methyl-CpG and unmethyl-CpG, respectively. The BRET signal using MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc increased the amount of methyl-CpG and unmethyl-CpG in genomic DNA, respectively. There was a significant negative correlation between the BRET signals; therefore, the global DNA methylation level was quantified using the BRET signals (R2 = 0.99, and R.S.D. <3.5%). These results indicate that the SnT/SnC protein ligation system can be utilized to construct target modification-binding protein-fused luciferases in any combination that detects target modifications in genomic DNA based on BRET.

Quantitative detection of CpG methylation level on G-quadruplex and i-motif-forming DNA by recombinase polymerase amplification.

Detection of CpG methylation levels holds immense potential for application in medical diagnosis of various diseases. In this study, we report the development of a recombinase polymerase amplification (RPA)-based CpG methylation level sensing system on G-quadruplex (G4) and intercalated motif (i-motif)-forming regions, which are stabilized by CpG methylation. This detection system is based on the principle that DNA polymerase is stalled at the methylated G4 and i-motif-forming region, which results in a decrease in the initial elongation efficiency of RPA. This reduction in turn affects the onset of amplification depending on the extent of CpG methylation; therefore, the methylation level is quantified by RPA. We demonstrate that the onset of amplification was delayed by CpG methylation when PCR products containing the vascular endothelial growth factor (VEGF) G4 and i-motif-forming region were used as the template. Furthermore, onset of amplification was delayed with the increase in CpG methylation of the VEGF region on genomic DNA. These results demonstrate that the sensing system is capable of directly detecting the methylation level at a constant temperature (39 °C) within 30 min without performing bisulfite conversion or affinity capture of methylated DNA.

Bioluminescence Resonance Energy Transfer for Global DNA Methylation Quantification.

Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several cancers; therefore, global methylation levels have been considered as biomarkers for cancer diagnosis. Bisulfite conversion analysis has been widely used as the gold standard method for quantification of DNA methylation levels. However, this method requires cumbersome and time-consuming steps. To quantify global DNA methylation levels in homogeneous solutions, we exemplify a sensing system based on bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (MBD-FLuc) and unmethyl-CpG binding domain (CXXC)-fused firefly luciferase (CXXC-FLuc). MBD-FLuc and CXXC-FLuc bind to methylated and unmethylated CpGs, respectively, in the genomic DNA to excite BOBO-3, an intercalating dye on genomic DNA. These BOBO-3 emission intensities depend on the methylated and unmethylated CpG content. The global DNA methylation levels can be quantified from the BOBO-3 emission intensities. Moreover, we introduce a multicolor BRET assay using MBD-FLuc and CXXC-fused Oplophorus luciferase (CXXC-OLuc) for the simultaneous quantification of methylated and unmethylated CpG content in genomic DNA. CXXC-OLuc excites the BOBO-1 DNA-intercalating dye depending on the unmethylated CpG content. Thus, the emission intensities of BOBO-1 and BOBO-3 excited by CXXC-OLuc and MBD-FLuc, respectively, can be simultaneously measured, thereby enabling the determination of global DNA methylation level in a single step. Here, we describe the detailed protocols for the expression of MBD-FLuc, CXXC-FLuc, and CXXC-OLuc in Escherichia coli and determine the global DNA methylation levels using these BRET assays.

Quantification of Global DNA Hydroxymethylation Level Using UHRF2 SRA-Luciferase Based on Bioluminescence Resonance Energy Transfer.

5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD (R2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng µL-1. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.

Investigating the Effects of Dehydrated Human Amnion-Chorion Membrane on Periodontal Healing.

Each growth factor (GF) has different effects and targets, and plays a critical role in periodontal healing. Dehydrated human amnion-chorion membrane (dHACM) contains various GFs and has been used to enhance wound healing. The purpose of this study was to evaluate the effects of dHACM on periodontal healing, using in vitro and in vivo experimental approaches. Standardized periodontal defects were created in rats. The defects were randomly divided into three groups: Unfilled, filled with hydroxypropyl cellulose (HPC), and dHACM+HPC. At 2 and 4 weeks postoperatively, periodontal healing was analyzed by microcomputed tomography (micro-CT), and histological and immunohistochemical analyses. In vitro, periodontal ligament-derived cells (PDLCs) isolated from rat incisors were incubated with dHACM extract. Cell proliferation and migration were evaluated by WST-1 and wound healing assay. In vivo, micro-CT examination at 2 weeks revealed enhanced formation of new bone in the dHACM+HPC group. At 4 weeks, the proportions of vascular endothelial growth factor (VEGF)-positive cells and α-smooth muscle actin (α-SMA)-positive blood vessels in the dHACM+HPC group were significantly greater than those in the Unfilled group. In vitro, dHACM extracts at 100 µg/mL significantly increased cell proliferation and migration compared with control. These findings suggest that GFs contained in dHACM promote proliferation and migration of PDLCs and angiogenesis, which lead to enhanced periodontal healing.

Stabilization of VEGF i-motif structure by CpG methylation.

The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemi-protonated cytosine base pairs (C-C+) under acidic conditions. The i-motif structure formation is involved in biological processes such as transcription regulation. Therefore, the identification of factors controlling i-motif formation is important in elucidating the cellular functions it controls. We previously reported that the VEGF G-quadruplex structure is stabilized by CpG methylation. In this study, the effect of CpG methylation on the stability of the VEGF i-motif structure was investigated. The VEGF i-motif-forming oligonucleotide contains four cytosines on CpG sites, and three of the four cytosines (C4, C15, and C20) are involved in C-C+ formation in the i-motif structure. Circular dichroism (CD) spectra analysis demonstrated that full CpG methylation increased the pH of mid transition (pHT) of the i-motif structure by 0.1, and the melting temperature (Tm) by 5.1 °C in 25 mM sodium cacodylate buffer at pH 5.0. Moreover, single methylation at C4, C15, and C20 increased Tm by 0.5, 1.7, and 2.0 °C in the buffer, respectively. These results demonstrated that CpG methylation stabilized the VEGF i-motif structure.

Healing of Experimental Periodontal Defects Following Treatment with Fibroblast Growth Factor-2 and Deproteinized Bovine Bone Mineral.

The aim of this study was to investigate the effects of fibroblast growth factor (FGF)-2 used in combination with deproteinized bovine bone mineral (DBBM) on the healing of experimental periodontal defects. Periodontal defects created in rats were treated by FGF-2, DBBM, FGF-2 + DBBM, or left unfilled. Microcomputed tomography, histological, and immunohistochemical examinations were used to evaluate healing. In vitro cell viability/proliferation on DBBM with/without FGF-2 was assessed by WST-1. Cell behavior was analyzed using scanning electron and confocal laser scanning microscopy. Osteogenic differentiation was evaluated by staining with alkaline phosphatase and alizarin red. Bone volume fraction was significantly greater in FGF-2 and FGF-2 + DBBM groups than in other groups at 2 and 4 weeks postoperatively. In histological assessment, newly formed bone in FGF-2 and FGF-2 + DBBM groups appeared to be greater than other groups. Significantly greater levels of proliferating cell nuclear antigen-, vascular endothelial growth factor-, and osterix-positive cells were observed in FGF-2 and FGF-2 + DBBM groups compared to Unfilled group. In vitro, addition of FGF-2 to DBBM promoted cell viability/proliferation, attachment/spreading, and osteogenic differentiation. The combination therapy using FGF-2 and DBBM was similarly effective as FGF-2 alone in the healing of experimental periodontal defects. In certain bone defect configurations, the combined use of FGF-2 and DBBM may enhance healing via promotion of cell proliferation, angiogenesis, and osteogenic differentiation.

Periodontal surgery using rhFGF-2 with deproteinized bovine bone mineral or rhFGF-2 alone: 2-year follow-up of a randomized controlled trial.

AIM: To compare outcomes of rhFGF-2 + DBBM therapy with rhFGF-2 alone in the treatment of intrabony defects. This study provides 2-year follow-up results from the previous randomized controlled trial. MATERIALS AND METHODS: Defects were randomly allocated to receive rhFGF-2 + DBBM (test) or rhFGF-2 (control). Treated sites were re-evaluated at 2 years postoperatively, using original clinical and patient-centred measures. RESULTS: Thirty-eight sites were available for re-evaluation. At 2 years, both groups showed a significant improvement in clinical attachment level (CAL) from baseline. A gain in CAL of 3.4 ± 1.3 mm in the test group and 3.1 ± 1.5 mm in the control group was found. No significant inter-group difference was noted. Both groups showed a progressive increase in radiographic bone fill (RBF). The test treatment yielded greater RBF (56%) compared with the control group (41%). The control treatment performed better in contained defects in terms of CAL and RBF. There was no significant difference in patient-reported outcomes between groups. CONCLUSIONS: At 2-year follow-up, the test and cotrol treatments were similarly effective in improving CAL, whereas the test treatment achieved a significantly greater RBF. In both treatments, favourable clinical, radiographic, and patient-reported outcomes can be sustained for at least 2 years. TRIAL REGISTRATION: The University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) 000025257.

Treatment with functionalized designer self-assembling peptide hydrogels promotes healing of experimental periodontal defects.

BACKGROUND/OBJECTIVES: It has been reported that self-assembling peptide (SAP) hydrogels with functionalized motifs enhance proliferation and migration of host cells. How these designer SAP hydrogels perform in the treatment of periodontal defects remains unknown. This study aimed to test the potential of local application of designer SAP hydrogels with two different functionalized motifs in the treatment of experimental periodontal defects. MATERIAL AND METHODS: In vitro, viability/proliferation of rat periodontal ligament-derived cells (PDLCs) cultured on an SAP hydrogel RADA16 and RADA16 with functionalized motifs, PRG (integrin binding sequence) and PDS (laminin cell adhesion motif), was assessed. Cell morphology was analyzed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). In vivo, standardized periodontal defects were made mesially in the maxillary first molars of Wistar rats. Defects received RADA16, PRG, PDS or left unfilled. At 2 or 4 weeks postoperatively, healing was assessed by microcomputed tomography, histological and immunohistochemical methods. RESULTS: Viability/proliferation of PDLCs was significantly greater on PRG than on RADA16 or PDS at 72 hours. rPDLCs in the PRG group showed enhanced elongations and cell protrusions. In vivo, at 4 weeks, bone volume fractions in the PRG and PDS groups were significantly greater than the RADA16 group. Histologically, bone formation was more clearly observed in the PRG and PDS groups compared with the RADA16 group. At 4 weeks, epithelial downgrowth in the hydrogel groups was significantly reduced compared to the Unfilled group. In Azan-Mallory staining, PDL-like bundles ran in oblique direction in the hydrogel groups. At 2 weeks, in the area near the root, proliferating cell nuclear antigen (PCNA)-positive cells were detected significantly more in the PRG group than other groups. At 4 weeks, in the middle part of the defect, a significantly greater level of vascular endothelial growth factor (VEGF)-positive cells and α-smooth muscle actin (SMA)-positive blood vessels were observed in the PRG group than in other groups. CONCLUSION: The results indicate that local application of the functionalized designer SAP hydrogels, especially PRG, promotes periodontal healing by increasing cell proliferation and angiogenesis.

Quantification of global DNA methylation level using 5-methylcytosine dioxygenase.

DNA methylation is one of the best studied epigenetic modifications. Alteration of the global DNA methylation level occurs in abnormal cells, such as those associated with cancers and Alzheimer's disease. Several assays are used to determine the global DNA methylation level, including the bisulfite-based assay, high-performance liquid chromatography (HPLC)-based assay, enzyme-linked immunosorbent assay (ELISA), and methyl acceptance assay. However, these assays require several cumbersome steps to detect methylation levels. We developed a simpler enzymatic assay for the quantification of the global DNA methylation level using the Ten-eleven translocation (TET) protein. TET proteins mediate DNA demethylation through the oxidation of 5-methylcytosine (5mC) in CpG in mammalian cells. Succinate is produced during this oxidation reaction, and the amount of succinate produced correlates to the global DNA methylation level. The catalytic domain of the TET2 was expressed in Escherichia coli (E. coli), and the purified TET2 catalytic domain was reacted with human genomic DNA. The reaction solution was used for enzymatic succinate quantification with no purification step. The results showed that the succinate produced through TET-mediated oxidation increased with increasing global DNA methylation levels in human genomic DNA, which was determined using the bisulfite method. These results show that the global DNA methylation level is quantifiable by measuring the amount of succinate produced by the TET2-mediated 5mC oxidation reaction. Graphical abstract.

Destabilisation of the c-kit1 G-quadruplex structure by N6-methyladenosine modification.

N6-methyladenine (m6dA) has been recently discovered in eukaryotic genomic DNA. However, there have been few reports on its biological roles. G-quadruplex (G4) is a non-canonical nucleic acid structure formed by the stacking of G-tetrads. G4-forming sequences are enriched with cis-regulatory elements in genomic DNA and the G4 structures have important roles in various cellular functions. We previously reported that CpG methylation stabilized vascular endothelial growth factor (VEGF) G4 structure. Here we report that m6dA modification destabilizes the human c-kit1 G4 structure. These results suggest that epigenetic modifications may affect G4 formation in order to regulate the biological functions.

Combined effects of systemic parathyroid hormone (1-34) and locally delivered neutral self-assembling peptide hydrogel in the treatment of periodontal defects: An experimental in vivo investigation.

AIM: To evaluate in vivo combination therapy of systemic parathyroid hormone (PTH) and locally delivered neutral self-assembling peptide (SAP) hydrogel for periodontal treatment. MATERIALS AND METHODS: Viability/proliferation of rat periodontal ligament cells in a neutral SAP nanofibre hydrogel (SPG-178) was evaluated using WST-1 assay. Periodontal defects were created mesially to the maxillary first molars in 40 Wistar rats. Defects were filled with 1.5% SPG-178 or left unfilled. Animals received PTH (1-34) or saline injections every 2 days. Microcomputed tomography, histological, and immunohistochemical examinations were used to evaluate healing at 2 or 4 weeks postoperative. RESULTS: At 72 hr, cells in 1.5% SPG-178 showed increased viability/proliferation compared to cells in 0.8% SPG-178 or untreated controls. In vivo, systemic PTH resulted in significantly greater bone volume in the Unfilled group at 2 weeks (p = .01) and 4 weeks (p < .0001) than in the saline control. At 4 weeks, a significantly greater bone volume was observed in the PTH/SPG-178 (p = .0003) and PTH/Unfilled (p = .004) groups than in Saline/SPG-178 group. Histologically, greater bone formation was observed in PTH/SPG-178 at 4 weeks than in other groups. In the PTH/SPG-178 group, increased proportions of PCNA-, VEGF-, and Osterix-positive cells were observed in the treated sites. CONCLUSIONS: These findings suggest that intermittent systemic PTH and locally delivered neutral SAP hydrogel enhance periodontal healing.

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole.

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.

Multicolor bioluminescence resonance energy transfer assay for quantification of global DNA methylation.

Abnormal DNA methylations such as hypermethylation on tumor suppressor genes and global hypomethylation have been recognized as hallmarks of cancer. Previously, we reported a bioluminescence resonance energy transfer (BRET)-based global DNA methylation level assay using a methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc) and unmethylated CpG-binding domain-fused firefly luciferase (CXXC-Fluc). The BRET signal between MBD-Fluc and BOBO-3 DNA intercalating dye depends on the methylated CpG contents, whereas the BRET signal between CXXC-Fluc and BOBO-3 depends on the unmethylated CpG contents. Therefore, the global DNA methylation level can be quantified using the BRET assay. However, these assays must be performed separately, because the same luciferase fuses to both MBD and CXXC. In this study, we developed a one-step quantification assay of global DNA methylation based on a multicolor BRET assay using MBD-Fluc and CXXC-fused Oplophorus luciferase (CXXC-Oluc). We demonstrated that MBD-Fluc and CXXC-Oluc simultaneously excite BOBO-3 and BOBO-1 DNA intercalating dyes on genomic DNA, respectively. Moreover, the BRET signals produced from MBD-Fluc and CXXC-Oluc depended on the methylation status of the CpG contents. These results demonstrate that global DNA methylation can be quantified by this multicolor BRET assay in a single tube. Graphical abstract ᅟ.

Esterification of PQQ Enhances Blood-Brain Barrier Permeability and Inhibitory Activity against Amyloidogenic Protein Fibril Formation.

Several neurodegenerative diseases have a common pathophysiology where selective damage to neurons results from the accumulation of amyloid oligomer proteins formed via fibrilization. Considering that the formation of amyloid oligomers leads to cytotoxicity, the development of chemical compounds that are able to effectively cross the blood-brain barrier (BBB) and inhibit this conversion to oligomers and/or fibrils is essential for neurodegenerative disease therapy. We previously reported that pyrroloquinoline quinone (PQQ) prevented aggregation and fibrillation of α-synuclein, amyloid ß1-42 (Aß1-42), and mouse prion protein. To develop a novel drug against neurodegenerative diseases based on PQQ, it is necessary to improve the insufficient BBB permeability of PQQ. Here, we show that an esterified compound of PQQ, PQQ-trimethylester (PQQ-TME), has twice the BBB permeability than PQQ in vitro. Moreover, PQQ-TME exhibited greater inhibitory activity against fibrillation of α-synuclein, Aß1-42, and prion protein. These results indicated that esterification of PQQ could be a useful approach in developing a novel PQQ-based amyloid inhibitor.

Stabilization of G-quadruplex structure on vascular endothelial growth factor gene promoter depends on CpG methylation site and cation type.

BACKGROUND: DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure. METHODS: The thermostability of a methylated, 21-mer G4 structure located on the vascular endothelial growth factor (VEGF) gene promoter containing four CpG sites (C1, C6, C11, and C17) were investigated using circular dichroism (CD) spectral analysis. RESULTS: CD melting analysis revealed that VEGF G4 was stabilized by a single CpG methylation on C11 in the presence of Na+ and Mg2+. However, either C1 or C11 methylation enhanced VEGF G4 thermal stability in the presence of K+. CONCLUSIONS: Single CpG methylation appears to enhance VEGF G4 thermostability in a manner dependent on both the CpG methylation site and cation type. GENERAL SIGNIFICANCE: These results are expected to contribute to the elucidation of the roles of CpG methylation-stabilized G4 structures in biological processes.

CpG Methylation Changes G-Quadruplex Structures Derived from Gene Promoters and Interaction with VEGF and SP1.

G-quadruplex (G4) is a DNA/RNA conformation that consists of two or more G-tetrads resulting from four-guanine bases connected by Hoogsteen-type hydrogen bonds, which is often found in the telomeres of chromatin, as well as in the promoter regions of genes. The function of G4 in the genomic DNA is being elucidated and some G4-protein interactions have been reported; these are believed to play a role in vital cellular functions. In this study, we focused on CpG methylation, a well-known epigenetic modification of the genomic DNA, especially found in the promoter regions. Although many G4-forming sequences within the genomic DNA harbor CpG sites, the relationship between CpG methylation and the binding properties of associated proteins remains unclear. We demonstrated that the binding ability of vascular endothelial growth factor (VEGF) G4 DNA to VEGF165 protein was significantly decreased by CpG methylation. We identified the binding activity of G4 DNA oligonucleotides derived from gene promoter regions to SP1, a transcription factor that interacts with a G4-forming DNA and is also altered by CpG methylation. The effect of methylation on binding affinity was accompanied by changes in G4 structure and/or topology. Therefore, this study suggested that CpG methylation might be involved in protein binding to G4-forming DNA segments for purposes of transcriptional regulation.

A quantitative homogeneous assay for global DNA methylation levels using CpG-binding domain- and methyl-CpG-binding domain-fused luciferase.

Global DNA methylation levels have been considered as biomarkers for cancer diagnostics because transposable elements that constitute approximately 45% of the human genome are hypomethylated in cancer cells. We have previously reported a homogeneous assay for measuring methylated CpG content of genomic DNA based on bioluminescence resonance energy transfer (BRET) using methyl-CpG-binding domain (MBD)-fused luciferase (MBD-luciferase). In this study, a homogeneous assay for measuring unmethylated CpG content of genomic DNA in the same platform was developed using CXXC domain-fused luciferase (CXXC-luciferase) that specifically recognizes unmethylated CpG. In this assay, CXXC-luciferase recognizes unmethylated CpG on genomic DNA, whereby BRET between luciferase and the fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the genomic DNA concentration (R2 = 0.99) and unmethylated CpG content determined by the bisulfite method (R2 = 0.97). There was a significant negative correlation between the BRET signal of the CXXC-luciferase-based assay and that of the MBD-luciferase-based assay (R2 = 0.92). Moreover, we demonstrated that the global DNA methylation level determined using the bisulfite method was dependent on the ratio of the BRET signal in the MBD-luciferase-based assay to the total BRET signal in the MBD-luciferase- and CXXC-luciferase-based assays (R2 = 0.99, relative standard deviation < 2.2%, and analysis speed < 35 min). These results demonstrated that global DNA methylation levels can be quantified by calculating the BRET signal ratio without any calibration curve.