RESUMO
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.
Assuntos
Epigênese Genética , Desenho Universal , Metilação de DNA , DNA/genética , Luciferases/metabolismo , Transferência de EnergiaRESUMO
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.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Ilhas de CpG , DNA/genética , Oligonucleotídeos , Luciferases/genéticaRESUMO
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.
Assuntos
5-Metilcitosina , Metilação de DNA , 5-Metilcitosina/metabolismo , Citosina/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transferência de Energia , Luciferases/metabolismoRESUMO
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.
Assuntos
Ilhas de CpG , Citosina/química , Metilação de DNA , Fator A de Crescimento do Endotélio Vascular/química , Motivos de Aminoácidos , Soluções Tampão , Dicroísmo Circular , Quadruplex G , Humanos , Concentração de Íons de Hidrogênio , Substâncias Intercalantes , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Estrutura Secundária de Proteína , Temperatura , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
N6-methyladenine (m6A) is the most abundant RNA modification in eukaryotic RNA. Further, m6A has been identified in the genomic DNA of both eukaryotes and prokaryotes. The G-quadruplex (G4) structure is a non-canonical nucleic acid structure formed by the stacking of G:G:G:G tetrads. In this study, we evaluated the effect of m6A modifications on G4 structures formed by GGA repeat oligonucleotides, d(GGA)8, d(GGA)4, and r(GGA)4. The d(GGA)8 forms an intramolecular tetrad:heptad:heptad:tetrad G4 structure, while d(GGA)4 forms a dimerized intermolecular tetrad:heptad:heptad:tetrad G4 structure. r(GGA)4 forms a dimerized intermolecular tetrad:hexad:hexad:tetrad G4 structure. Circular dichroism melting analysis demonstrated that (1) m6A modifications destabilized the G4 structure formed by d(GGA)8, (2) m6A modification at A3 disrupted the G4 structure formed by d(GGA)4, and (3) m6A modification at A3 destabilized the G4 structure formed by r(GGA)4. m6A modifications may be involved in controlling G4 structure formation to regulate biological functions.
RESUMO
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.
Assuntos
Quadruplex G , Recombinases , Ilhas de CpG , DNA/genética , DNA/metabolismo , Metilação de DNA , Recombinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Hidrogéis , Fator A de Crescimento do Endotélio Vascular , Animais , Peptídeos , Ratos , Ratos Wistar , Microtomografia por Raio-XRESUMO
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.
Assuntos
Perda do Osso Alveolar , Regeneração Tecidual Guiada Periodontal , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/cirurgia , Animais , Bovinos , Seguimentos , Humanos , Minerais , Perda da Inserção Periodontal/tratamento farmacológico , Perda da Inserção Periodontal/cirurgia , Resultado do TratamentoRESUMO
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.
Assuntos
Adenosina/análogos & derivados , Quadruplex G , Proteínas Proto-Oncogênicas c-kit/genética , Adenosina/química , Adenosina/genética , Metilação de DNA , Epigênese Genética , Humanos , Modelos Moleculares , Regiões Promotoras GenéticasRESUMO
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.
Assuntos
5-Metilcitosina/metabolismo , Metilação de DNA , Dioxigenases/metabolismo , Cromatografia Líquida de Alta Pressão , Ilhas de CpG , Proteínas de Ligação a DNA/genética , Transferência de Energia , Ensaio de Imunoadsorção Enzimática , Células HeLa , Humanos , Proteínas Proto-Oncogênicas/genéticaRESUMO
Based on the valence configuration interaction (VCI) model and quantum chemical calculations, we theoretically investigate the potential of diazadibora-substituted phenanthrenes [(BN)2-phenanthrenes] as novel singlet fission (SF) chromophores. (BN)2-substitution to phenanthrene is performed to exhibit a captodative effect, which is found to enhance both diradical character and exchange integral. These enhanced parameters induced by (BN)2-substitution are shown to bring energetically favorable SF with high triplet excitation energies. In order to reveal the relationship between diradical character and positions replaced by (BN)2, analyses based on the VCI model, odd-electron density, and resonance structures are conducted. Accordingly, a concrete design principle, which is inherent in and is understandable from the topology of (BN)2-phenanthrene, is presented. Furthermore, design strategies to fine-tuning of the diradical character are newly demonstrated based on the additional introduction of π-donor and π-acceptor. The present results provide feasible candidate molecules and novel design strategies toward the discovery of bright SF chromophores for the application to efficient organic solar cells.
RESUMO
Theoretical molecular design of efficient nonlinear optical (NLO) systems using singlet fission (SF) is performed for butterfly-shaped acenes with/without nitrogen and sulfur substitutions using quantum chemical calculations, exciton dynamics simulations, and Marcus theory. It is found that these large systems meet the energy level matching conditions of efficient SF and exhibit superior third-order NLO properties (second hyperpolarizability γ at the molecular scale) to a typical SF molecule, pentacene. In addition, we investigate SF rates and γ in the correlated triplet pair [1(TT)] state generated by SF for various slip-stacked dimer models of these systems. For molecular packing with relatively large 1(TT) yields, a significant increase in γ/monomer in the 1(TT) state is observed, which is in good agreement with the electronic-coupling-based design guidelines obtained from our previous study. In particular, the butterfly-shaped acenes involving heteroatoms are found to exhibit a significant increase in γ/monomer as compared to the other systems. By analyzing the excitation properties in the 1(TT) state and intermolecular orbital interactions, we clarify the origin of such γ enhancement. The obtained results contribute to the construction of design guidelines for efficient SF-induced-NLO materials and demonstrate that butterfly-shaped acenes have the potential to surpass conventional NLO systems.
RESUMO
We theoretically investigate microscopic origins of vibronic coupling (VC) contributing to singlet fission (SF) dynamics in pentacene and its halogenated derivatives. The features of VCs related to diabatic exciton states and interstate electronic couplings (Holstein and Peierls couplings, respectively) are interpreted by the VC density (VCD) analysis, which allows one to clarify the relationship between the chemical structure and VC as spatial contribution. It is found for the pentacene dimer face-to-edge configuration in a herringbone crystal that characteristic intermolecular vibrations with low frequencies exhibit strong Holstein couplings for the intermediate charge-transfer (CT) exciton states as well as Peierls couplings. From VCD analysis, the comprising density of the intermolecular CT and that of the intermolecular vibration are found to be constructively mixed in the intermolecular space, leading to the enhancement of VC. Moreover, in order to assess the chemical modification manner for controlling VC, we design several halogenated pentacene derivatives with slip-stack configurations. Our strategy to enhance VCD by halogenation is found to be rational, whereas the peaks of VC spectra for the CT states in the slip-stack packings are observed in high frequency regions. We compare their SF dynamics based on the quantum master equation explicitly including the exciton state-dependent VCs. From the analysis on relative relaxation factors between the adiabatic exciton states, their difference in the SF rate is highlighted by exciton configurations in addition to VCs. The present study is expected to be a first step toward efficient SF based on the design of VC in terms of both the chemical structure and intermolecular packing.
RESUMO
Clinical use of 0.3% recombinant human fibroblast growth factor (rhFGF)-2 for periodontal regeneration received formal approval in Japan in 2016. The combination of growth factor and bone graft material is used to enhance periodontal healing in regenerative therapy. The exact effects of combination therapy on periodontal healing remain unknown, however. Here, we report three cases of chronic periodontitis treated with the combination of rhFGF-2 and deproteinized bovine bone mineral (DBBM). Following initial periodontal therapy, periodontal regenerative therapy using rhFGF-2 in combination with DBBM was performed to treat wide intrabony defects. Periodontal parameters and radiographic bone fill were reevaluated at 3 months, 6 months, and 1 year postoperatively. Oral health-related quality of life (OHRQL) was assessed as a patient-reported measure of outcome. At 1 year postoperatively, probing pocket depth and clinical attachment level showed a significant improvement in comparison with at baseline. An improvement was also noted in radiographic evidence of bone fill and total OHRQL scores. Combination therapy yielded clinically favorable results in the present cases.
Assuntos
Perda do Osso Alveolar , Periodontite Crônica , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/cirurgia , Animais , Bovinos , Periodontite Crônica/tratamento farmacológico , Periodontite Crônica/cirurgia , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Seguimentos , Regeneração Tecidual Guiada Periodontal , Humanos , Japão , Minerais , Perda da Inserção Periodontal , Qualidade de Vida , Resultado do TratamentoRESUMO
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.
Assuntos
Quadruplex G , Oxazóis/química , Sequência de Bases , Ciclização , Transferência Ressonante de Energia de Fluorescência , Transição de Fase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Telômero/química , Telômero/metabolismoRESUMO
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 á .
Assuntos
Metilação de DNA , Ilhas de CpG , Transferência de Energia , Células HeLa , Humanos , Medições Luminescentes/métodos , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Hidrogéis , Hormônio Paratireóideo , Animais , Peptídeos , Ratos , Ratos Wistar , Microtomografia por Raio-XRESUMO
An enhancer located upstream of the transcriptional start site of Ccnb1ip1 containing two GGA-rich regions and a 14-GGA repeat (GGA)14 region has been previously identified. Three copies of four GGA repeats in the c-myb promoter that form a tetrad:heptad:heptad:tetrad (T:H:H:T) dimerized G-quadruplex (G4) structure reportedly functions as both a transcriptional repressor and activator. Here, the secondary structures of the two GGA-rich and (GGA)14 regions were analyzed using circular dichroism spectral analysis, which indicated that the two GGA-rich DNAs formed parallel-type G4 structures, whereas (GGA)14 DNA formed the T:H:H:T dimerized G4 structure. Reporter assays demonstrated that individual regions did not show enhancer activity; however, the deletion of the (GGA)14 region resulted in 1.5-fold higher enhancer activity than that of the whole enhancer. These results indicate that the (GGA)14 region that forms the T:H:H:T dimerized G4 structure functions as a negative regulator of the Ccnb1ip1 enhancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Elementos Facilitadores Genéticos , Quadruplex G , Sequências Repetitivas de Ácido Nucleico , Animais , Dicroísmo Circular , Humanos , Camundongos , Células NIH 3T3 , Ubiquitina-Proteína LigasesRESUMO
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.
Assuntos
Ilhas de CpG , Metilação de DNA , Quadruplex G , Regiões Promotoras Genéticas , Fator A de Crescimento do Endotélio Vascular/genética , Cátions/química , Cátions/metabolismo , Dicroísmo Circular , Humanos , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.