“The study of expression levels of DNA methylation regulators in patients affected with congenital heart defects (CHDs)”
Background: Congenital heart defects (CHDs) have multifactorial etiology with the complex interplay of genetic and environmental factors. Environmental impact can have an epigenetic mechanism of CHD development. Many studies have reported the causal association between CHD and distinct DNA methylation profile which is one of the key epigenetic events, which has a vital role in normal embryonic development.
The products of DNMT1, DNMT3A, DNMT3B, and MBD2 are important regulators of the DNA methylation process.
Changes in the expression of these genes are implicated in congenital structural cardiac defects. Hence, in this proof-of-concept study, we have compared the expression levels of these genes in the blood samples of healthy controls and CHD cases while investigating the etiology of CHD.
Methods: In this study with 48 CHD cases and 47 healthy controls, total RNA was isolated from the whole blood samples using TRI reagent. Quantitative RT PCR (qRT-PCR) was used to analyze the mRNA levels of DNMT1, DNMT3A, DNMT3B, and MBD2. The expression levels have been analyzed by relative quantification.
Results: We observed that DNMT3B (fold change = -2.563; p = .0018) and DNMT3A (fold change = -2.169; p = .05) were significantly downregulated in CHD patients, whereas the expression of DNMT1 and MBD2 was not significantly different between cases and controls.
Conclusions: Lower expression of de novo methyltransferases, namely, DNMT3B and DNMT3A in CHD cases, may be an important contributor to the mechanism of CHD pathogenesis. Further studies with age-matched controls and analysis of global DNA methylation profile are required to investigate the proposed causal association.
Keywords: DNA methylation; DNA methyltransferase; DNMT3B; congenital heart defect; de novo methyltransferase.
H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeler function
The Isw1b chromatin-remodeling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II.
Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain.
Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeler.
The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP.
The ability to bind H3K36me3 and DNA promotes the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo.
Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodeling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs.
Clinical and Biological Significance of DNA Methylation-Driven Differentially Expressed Genes in Biochemical Recurrence After Radical Prostatectomy
Background: Biochemical recurrence (BCR) after radical prostatectomy indicates poor prognosis in patients with prostate cancer (PCA). DNA methylation (DNAm) is a critical factor in tumorigenesis and has attracted attention as a biomarker for the diagnosis, treatment, and prognosis of PCA.
However, the predictive value of DNAm-derived differentially expressed genes (DMGs) in PCA with BCR remains elusive.
Methods: We filtered the methylated genes and the differentially expressed genes (DGEs) for more than 1,000 clinical samples from the TCGA cohort using the chAMP and DESeq2 packages of R language, respectively.
Next, we integrated the DNAm beta value and gene expression data with the Mithymix package of R language to obtain the DMGs.
Then, 1,000 times Cox LASSO regression with 10-fold cross-validation was performed to screen signature DMGs and establish a predictive classifier.
Univariate and multivariate cox regressive analyses were used to identify the prognostic factors to build a predictive model, and its performance was measured by receiver operating characteristics, calibration curves, and Harrell’s concordance index (C-index).
Additionally, a GEO dataset was used to validate the prognostic classifier.
Results: One hundred DMGs were mined using the chAMP and Methymix packages of R language.
Of these, seven DMGs (CCK, CD38, CYP27A1, EID3, HABP2, LRRC4, and LY6G6D) were identified to build the prognostic classifier (Classifier) through LASSO analysis. Moreover, univariate and multivariate Cox regression analysis determined that the Classifier and pathological T stage (pathological_T) were independent predictors of BCR (hazard ratio (HR 2.2), (95% CI 1.4-3.5), p < 0.0012, and (HR 1.8), (95% CI 1.0-3.2), p < 0.046).
A nomogram based on the Classifier was constructed, with high prediction accuracy for BCR-free survival in TCGA and GEO datasets.
GSEA enrichment analysis showed that the DMGs were mainly enriched in the metabolism pathways.
Conclusion: We identified and validated the nomogram of BCR-free survival for PCA patients, which has the potential to guide treatment decisions for patients at differing risks of BCR.
Our study deepens the understanding of DMGs in the pathogenesis of PCA.
Global DNAmethylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system
Although DNA methylation epigenetically regulates development, data on global DNA methylation during the development of limb buds (LBs) are scarce.
We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum-supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture.
Fischer rat front- and hind-LBs at 13th and 14th gestation days (GD) were cultivated at the air-liquid interface in Eagle’s Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively.
The methylation of repetitive DNA sequences (SINE rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by Safranin O.
Upon isolation, weak Safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures towards day 14, except in the SF-cultivated GD13 hind-LBs, where Safranin O staining was almost absent on day 3.
That was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day three. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th day was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day three and then decreased.
In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive towards differentiation in vitro, which is of importance for regenerative medicine strategies.
Alterations in DNA methylation associate with fatty liver and metabolic abnormalities in a multi-ethnic cohort of pre-teenage children
Non-Alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in children.
Epigenetic alterations, such as through DNA methylation (DNAm), may link adverse childhood exposures and fatty liver and provide non-invasive methods for identifying children at high risk for NAFLD and associated metabolic dysfunction.
We investigated the association between differential DNAm and liver fat content (LFC) and liver injury in pre-adolescent children.
Leveraging data from the Newborn Epigenetics Study (NEST), we enrolled 90 mother-child dyads and used linear regression to identify CpG sites and differentially methylated regions (DMRs) in peripheral blood associated with LFC and alanine aminotransferase (ALT) levels in 7-12yo children.
DNAm was measured using Infinium HumanMethylationEPIC BeadChips (Illumina).
LFC and fibrosis were quantified by magnetic resonance imaging proton density fat fraction and elastography. Median LFC was 1.4% (range, 0.3-13.4%) and MRE was 2.5 kPa (range, 1.5-3.6kPa). Three children had LFC ≥ 5%, while six (7.6%) met our definition of NAFLD (LFC ≥ 3.7%). All children with NAFLD were obese and five were Black.
Epizen DNA Methylation Kit |
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| Z4201-050 | GenDepot | 50 Rxns | 392.4 EUR |
Epizen DNA Methylation Kit |
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| Z4201-200 | GenDepot | 4x50 rxns | 1161.6 EUR |
DNA Methylation Detection Kit |
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| K5082100 | Biochain | 1 kit | 337 EUR |
DNA methylation inhibitor, Dichlone |
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| 2860-1000 | Biovision | each | 170.4 EUR |
DNA methylation inhibitor, Dichlone |
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| 2860-500 | Biovision | each | 130.8 EUR |
Zebularine (DNA methylation inhibitor) |
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| SIH-560-25MG | Stressmarq | 25 mg | 373.2 EUR |
Zebularine (DNA methylation inhibitor) |
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| SIH-560-5MG | Stressmarq | 5 mg | 151.2 EUR |
Epizen DNA Methylation-Gold Kit |
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| Z4101-050 | GenDepot | 50 Rxns | 433.2 EUR |
Epizen DNA Methylation-Gold Kit |
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| Z4101-200 | GenDepot | 4x50 rxns | 1081.2 EUR |
EpiArt DNA Methylation Library Kit |
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| NE101-01 | Vazyme | 24 rxn | 1066.8 EUR |
EpiArt DNA Methylation Library Kit |
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| NE101-02 | Vazyme | 96 rxn | 3513.6 EUR |
Epizen DNA Methylation-Direct Kit |
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| Z4001-050 | GenDepot | 50 Rxns | 513.6 EUR |
Epizen DNA Methylation-Direct Kit |
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| Z4001-200 | GenDepot | 4x50 rxns | 1243.2 EUR |
LFC was associated with 88 DMRs and 106 CpGs (FDR<5%). The top two CpGs, cg25474373 and cg07264203, mapped to or near RFTN2 and PRICKLE2 genes. These two CpG sites were also significantly associated with a NAFLD diagnosis.
As higher LFC associates with an adverse cardiometabolic profile already in childhood, altered DNAm may identify these children early in disease course for targeted intervention. Larger, longitudinal studies are needed to validate these findings and determine mechanistic relevance.