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1.
Mol Med Rep ; 25(3)2022 03.
Article in English | MEDLINE | ID: mdl-35059744

ABSTRACT

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart malformation accounting for ~10% of cases. Although the pathogenesis of TOF is complex and largely unknown, epigenetics plays a huge role, specifically DNA methylation. The protein δ like non­canonical Notch ligand 1 (DLK1) gene encodes a non­canonical ligand of the Notch signaling pathway, which is involved in heart development. However, the epigenetic mechanism of DLK1 in the pathogenesis of TOF is yet to be elucidated. Therefore, the present study aimed to clarify its specific mechanism. In this study, immunohistochemistry was used to detect the protein expression of DLK1 and the methylation status of the DLK1 promoter was measured via bisulfite sequencing PCR. Dual­luciferase reporter assays were performed to examine the influence of transcription factor ETS proto­oncogene 1 (ETS1) on DLK1 gene expression. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay, both in vivo and in vitro, were used to verify the binding of the ETS1 transcription factor to the DLK1 promoter as well as the influence of methylation status of DLK1 promoter on this binding affinity. The expression of DLK1 in the right ventricular outflow tract was significantly lower in patients with Tetralogy of Fallot (TOF) than that in controls (P<0.001). Moreover, the methylation level of CpG site 10 and CpG site 11 in the DLK1_R region was significantly decreased in TOF cases compared with controls (P<0.01). The integral methylation levels of DLK1_R and the methylation status of the CpG site 11 were both positively associated with DLK1 protein expression in TOF cases. ETS1 was found to inhibit DLK1 transcriptional activity by binding to the CpG site 11 and this affinity could be influenced by the methylation level of the DLK1 promoter. These findings demonstrated that the hypomethylation of the DLK1 promoter could increase the binding affinity of ETS1 transcription factor, which in turn inhibited DLK1 gene transcriptional activity and contributed to the development of TOF.


Subject(s)
Calcium-Binding Proteins/genetics , CpG Islands/genetics , DNA Methylation , Gene Expression Regulation , Membrane Proteins/genetics , Proto-Oncogene Protein c-ets-1/genetics , Tetralogy of Fallot/genetics , Asian People/genetics , Base Sequence , Binding Sites/genetics , Calcium-Binding Proteins/metabolism , Child, Preschool , China , Female , HEK293 Cells , Humans , Infant , Male , Membrane Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Protein c-ets-1/metabolism , Sequence Analysis, DNA/methods , Tetralogy of Fallot/ethnology , Tetralogy of Fallot/metabolism
2.
Mol Med Rep ; 22(5): 4412-4422, 2020 Nov.
Article in English | MEDLINE | ID: mdl-33000281

ABSTRACT

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease (CHD). Although a lower methylation level of whole genome has been demonstrated in TOF patients, little is known regarding the DNA methylation changes in specific gene and its associations with TOF development. NOTCH4 is a mediator of the Notch signalling pathway that plays an important role in normal cardiac development. However, the role of epigenetic regulation of the NOTCH4 gene in the pathogenesis of TOF remains unclear. Considering the NOTCH4 low mutation frequency and reduced expression in the TOF patients, we hypothesized that abnormal DNA methylation change of NOTCH4 gene may influence its expression and responsible for TOF development. In this study, we measured the promoter methylation status of NOTCH4 and was measured and its regulation mechanism was explored, which may be related to TOF disease. Additionally, the promoter methylation statuses of NOTCH4 was measured in order to further understand epigenetic mechanisms that may serve a role in the development of TOF. Immunohistochemical analysis was used to examine NOTCH4 expression in right ventricular outflow tract myocardial tissues in patients with TOF. Compared with healthy controls, patients with TOF displayed significantly reduced in NOTCH4 expression (P=0.0055). Moreover, bisulphite sequencing suggested that the methylation levels of CpG site 2 in the NOTCH4 promoter was significantly higher in the patients than in the controls (P=0.0459). NOTCH4 expression was negatively associated with CpG site 2 methylation levels (r=­0.51; P=0.01). ETS1 transcription factor can serve as transcriptional activators by binding to specific DNA sequences of target genes, such as DLL4 and NOTCH4, which serves an important role in normal heart development. Dual­luciferase reporter and electrophoretic mobility shift assays indicated that the ETS1 transcription factor could bind to the NOTCH4 promoter region. However, binding of ETS1 to the NOTCH4 promoter was abrogated by methylation at the putative ETS1 binding sites. These findings suggested that decreased NOTCH4 expression in patients with TOF may be associated with hypermethylation of CpG site 2 in the NOTCH4 promoter region, due to impaired binding of ETS1.


Subject(s)
DNA Methylation , Down-Regulation , Receptor, Notch4/genetics , Receptor, Notch4/metabolism , Tetralogy of Fallot/genetics , Adolescent , Case-Control Studies , Child , Child, Preschool , CpG Islands , Female , Humans , Infant , Infant, Newborn , Male , Promoter Regions, Genetic , Proto-Oncogene Protein c-ets-1/metabolism , Sequence Analysis, DNA , Tetralogy of Fallot/metabolism
3.
Sci Rep ; 8(1): 12386, 2018 08 17.
Article in English | MEDLINE | ID: mdl-30120289

ABSTRACT

Heterotaxy syndrome (HTX) is characterized by left-right (LR) asymmetry disturbances associated with severe heart malformations. However, the exact genetic cause of HTX pathogenesis remains unclear. The aim of this study was to investigate the pathogenic mechanism underlying heterotaxy syndrome. Targeted next-generation sequencing (NGS) was performed for twenty-two candidate genes correlated with LR axis development in sixty-six HTX patients from unrelated families. Variants were filtered from databases and predicted in silico using prediction programs. A total of twenty-one potential disease-causing variants were identified in seven genes. Next, we used Sanger sequencing to confirm the identified variants in the family pedigree and found a novel hemizygous mutation (c.890G > T, p.C297F) in the ZIC3 gene in a male patient that was inherited from his mother, who was a carrier. The results of functional indicated that this ZIC3 mutation decreases transcriptional activity, affects the affinity of the GLI-binding site and results in aberrant cellular localization in transfected cells. Moreover, morpholino-knockdown experiments in zebrafish demonstrated that zic3 mutant mRNA failed to rescue the abnormal phenotype, suggesting a role for the novel ZIC3 mutation in heterotaxy syndrome.


Subject(s)
Heart Defects, Congenital/diagnosis , Heart Defects, Congenital/genetics , Heterotaxy Syndrome/diagnosis , Heterotaxy Syndrome/genetics , Homeodomain Proteins/genetics , Mutation , Transcription Factors/genetics , Adolescent , Adult , Aged , Animals , Child , DNA Mutational Analysis , Female , Genetic Association Studies , Genetic Predisposition to Disease , Genotype , Homeodomain Proteins/metabolism , Humans , Male , Middle Aged , Pedigree , Phenotype , Radiography, Thoracic , Tomography, X-Ray Computed , Transcription Factors/metabolism , Transcriptional Activation , Young Adult , Zebrafish
4.
Cardiol Young ; 28(3): 397-402, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29198211

ABSTRACT

Atrial septal defect is one of the most common CHD. The pathogenesis of atrial septal defect still remains unknown. Cx43 is the most prevalent connexin in the mammalian heart during development. Its genetic variants can cause several CHD. The aim of our study was to investigate the association of genetic variations of the Cx43 with sporadic atrial septal defect. A total of 450 paediatric patients were recruited, including 150 cases with atrial septal defect and 300 healthy controls. The promoter region of Cx43 was analysed by sequencing after polymerase chain reaction. All data were analysed by using the Statistic Package for Social Science 19.0 software. The frequency of the single nucleotide polymorphism rs2071166 was significantly higher in atrial septal defect cases than in healthy controls. The CC genotype at rs2071166 site in Cx43 was correlated with an increased risk for atrial septal defect (p<0.0001, odds ratio=3.891, 95% confidence interval 1.948-7.772) and the C allele was positively correlated with atrial septal defect (p=0.007, odds ratio=1.567, 95% confidence interval 1.129-2.175). In conclusion, our results confirmed that rs2071166 in Cx43 may be relevant with an increased atrial septal defect risk.


Subject(s)
Connexin 43/genetics , Heart Septal Defects, Atrial/genetics , Polymorphism, Single Nucleotide , Adolescent , Alleles , Case-Control Studies , Child , Child, Preschool , China , Female , Genetic Predisposition to Disease , Humans , Infant , Male , Risk Factors , Young Adult
5.
Sci Rep ; 7(1): 10435, 2017 09 05.
Article in English | MEDLINE | ID: mdl-28874875

ABSTRACT

Abnormal level of Cx43 expression could result in CHD. Epigenetic modification and disease-associated, non-coding SNPs might influence gene transcription and expression. Our study aimed to determine the role of histone modification and an rSNP (rs2071166) in the Cx43 promoter in patients with TOF. Our results indicate that H3K18ac bind to Cx43 promoter and that their levels are reduced in TOF patients relative to controls. The relationship between the non-coding SNP in the Cx43 gene and TOF patients was evaluated in 158 patients and 300 controls. The C allele of rs2071166 was confirmed to result in an increased risk of TOF (OR = 1.586, 95%CI 1.149-2.189). Individuals with the CC genotype at rs2071166 also showed a significant susceptibility to TOF (OR = 2.961, 95%CI 1.452-6.038). The mRNA level in TOF who were CC genotype was lower than that in patients with the AA/AC genotype. Functional analysis in cells and transgenic zebrafish models showed that rs2071166 decreased the activity of the promoter and could block the interaction between RXRα and RARE. This is the first study to illustrate that epigenetic modification and an rSNP in the Cx43 promoter region play a critical role in TOF by impacting the transcriptional activity and expression level of Cx43.


Subject(s)
Connexin 43/genetics , Histone Code , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Tetralogy of Fallot/genetics , Acetylation , Alleles , Asian People/genetics , Biomarkers , Genetic Predisposition to Disease , Genotype , Histones/metabolism , Humans , Immunohistochemistry , Lysine/metabolism , Phenotype , Protein Binding , RNA, Messenger/genetics , Retinoid X Receptor alpha/metabolism , Risk Factors , Sequence Analysis, DNA , Tetralogy of Fallot/metabolism
6.
Pediatr Res ; 80(1): 159-68, 2016 07.
Article in English | MEDLINE | ID: mdl-26991262

ABSTRACT

INTRODUCTION: Retinoic acid X receptor alpha (RXRα) and Connexin 43 (Cx43) both play a crucial role in cardiogenesis. However, little is known about the interplay mechanism between the RXRα and Cx43. METHODS: The activations of retinoic acid response element (RARE) in Cx43 were measured by luciferase transfection assay. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) was performed to prove that RXRα can directly bind to the RARE sequence. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to analyze the RXRα and Cx43 mRNA level and protein level in cells. RESULTS: In this study, we confirmed the negative association of the gene expression between the RXRα and Cx43 in the cell level. Interestingly, a functional RARE was detected in the region from -1,426 to -314 base pairs upstream from the transcriptional start site of Cx43. Moreover, we also prove that RXRα can directly bind to this RARE sequence in vitro and in vivo. CONCLUSIONS: RXRα negatively regulates the transcription and expression by directly binding to the RARE in the promoter of Cx43. The RARE-like sequence harbored in the Cx43 promoter region may serve as a functional RARE in the retinoic acid (RA) signaling pathway.


Subject(s)
Connexin 43/genetics , Promoter Regions, Genetic , Retinoic Acid Receptor alpha/genetics , Animals , Chromatin Immunoprecipitation , Connexin 43/metabolism , Gene Expression Regulation , HeLa Cells , Heart/embryology , Heart/growth & development , Humans , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Real-Time Polymerase Chain Reaction , Response Elements , Retinoic Acid Receptor alpha/metabolism , Signal Transduction
7.
Zhongguo Dang Dai Er Ke Za Zhi ; 15(10): 817-21, 2013 Oct.
Article in Chinese | MEDLINE | ID: mdl-24131830

ABSTRACT

OBJECTIVE: To study the expression of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in children with tetralogy of Fallot (TOF), and to investigate the role of histone acetylation and acetylation-related enzymes in the pathogenesis of TOF. METHODS: Myocardial tissue samples in the TOF group were obtained from 46 children with TOF who underwent radical operation, and myocardial tissue samples in the control group were obtained from 16 children who suffered accidental deaths and had no cardiac anomalies as shown by autopsy. The acetylation of H3K9, H3K18 and H3K27 was evaluated by immunohistochemistry. The mRNA expression of HATs and HDACs in the myocardium was measured by real-time PCR. The correlation between mRNA expression of HATs and HDACs and histone acetylation was analyzed. RESULTS: Compared with the control group, the TOF group showed significantly increased acetylation of H3K9 (P=0.0165) and significantly decreased acetylation of H3K18 (P=0.0048) and H3K27 (P=0.0084). As to 4 HATs and 6 HDACs, the mRNA expression of EP300 and CBP was significantly higher in the TOF group than in the control group (P=0.025; P=0.017), and there was no significant difference in the mRNA expression of other HATs and HDACs between the two groups. The correlation analysis revealed a positive correlation between H3K9 acetylation and mRNA expression of EP300 (r=0.71, P<0.01) and CBP (r=0.72, P<0.01). CONCLUSIONS: Upregulated mRNA expression of EP300 and CBP may be associated with increased H3K9 acetylation, suggesting that EP300 and CBP might affect cardiac development by regulating H3K9 acetylation.


Subject(s)
Histone Acetyltransferases/genetics , Histone Deacetylases/genetics , Histones/metabolism , Tetralogy of Fallot/metabolism , Acetylation , E1A-Associated p300 Protein/genetics , Female , Humans , Infant , Male , Myocardium/metabolism , Peptide Fragments/genetics , RNA, Messenger/analysis , Sialoglycoproteins/genetics
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