A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms

Leakey, Tatiana I.; Zieliński, Jerzy; Siegfried, Rachel N.; Siegel, Eric R.; Fan, Chun‐Yang; Cooney, Craig A.

doi:10.1093/nar/gkn210

Cited by 38 publications

(29 citation statements)

References 34 publications

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“…Sequencing chromatograms were analyzed using ab1 Peak Reporter (ThermoFisher, Waltham, MA) to generate numerical peak height data. Methylation at individual cytosines was calculated using Mquant as previously described (Leakey et al, 2008).…”

Section: Methodsmentioning

confidence: 99%

Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers

Vyhlidal

et al. 2016

Drug Metabolism and Disposition

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Members of the human CYP3A family of metabolizing enzymes exhibit developmental changes in expression whereby CYP3A7 is expressed in fetal tissues, followed by a transition to expression of CYP3A4 in the first months of life. Despite knowledge about the general pattern of CYP3A activity in human development, the mechanisms that regulate developmental expression remain poorly understood. Epigenetic changes, including cytosine methylation, have been suggested to play a role in the regulation of CYP3A expression. The objective of this study was to investigate changes in cytosine methylation of the CYP3A4 and CYP3A7 genes in human pediatric and prenatal livers. The methylation status of cytosinephospho-guanine dinucleotides was determined in 16 pediatric liver samples using methyl-seq and confirmed by bisulfite sequencing of 48 pediatric and 34 prenatal liver samples. Samples were separated by age into five groups (prenatal, < 1 year of age, 1.8-6 years, 7-11 years, and 12-17 years). Methyl-seq anaylsis revealed that cytosines in the proximal promoter of CYP3A7 are hypomethylated in neonates compared with adolescents (P < 0.001). In contrast, a cytosine 383 base pair upstream of CYP3A4 is hypermethylated in liver samples from neonates compared with adolescents (P = 0.00001). Developmental changes in methylation of cytosines in the proximal promoters of CYP3A4 and CYP3A7 in pediatric livers were confirmed by bisulfite sequencing. In addition, the methylation status of cytosine in the CYP3A4 and CYP3A7 proximal promoters correlated with changes in developmental expression of mRNA for the two enzymes.

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Section: Methodsmentioning

confidence: 99%

Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers

Vyhlidal

et al. 2016

Drug Metabolism and Disposition

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“…The PCR products were purified and sequenced at the DNA sequencing facility of Heinrich-Heine University. DNA methylation was quantified by the Mquant method as described (33). The height of the thymine peak at a CpG dinucleotide was subtracted from the average signal of 10 surrounding thymine peaks to quantify DNA methylation at this site.…”

Section: Subcellular Fractionation Of Hscs By Differential Centrifugamentioning

confidence: 99%

The Role of Embryonic Stem Cell-expressed RAS (ERAS) in the Maintenance of Quiescent Hepatic Stellate Cells

Nakhaei‐Rad¹,

Nakhaeizadeh²,

Götze³

et al. 2016

Journal of Biological Chemistry

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Hepatic stellate cells (HSCs) were recently identified as liverresident mesenchymal stem cells. HSCs are activated after liver injury and involved in pivotal processes, such as liver development, immunoregulation, regeneration, and also fibrogenesis. To date, several studies have reported candidate pathways that regulate the plasticity of HSCs during physiological and pathophysiological processes. Here we analyzed the expression changes and activity of the RAS family GTPases and thereby investigated the signaling networks of quiescent HSCs versus activated HSCs. For the first time, we report that embryonic stem cell-expressed RAS (ERAS) is specifically expressed in quiescent HSCs and down-regulated during HSC activation via promoter DNA methylation. Notably, in quiescent HSCs, the high level of ERAS protein correlates with the activation of AKT, STAT3, mTORC2, and HIPPO signaling pathways and inactivation of FOXO1 and YAP. Our data strongly indicate that in quiescent HSCs, ERAS targets AKT via two distinct pathways driven by PI3K␣/␦ and mTORC2, whereas in activated HSCs, RAS signaling shifts to RAF-MEK-ERK. Thus, in contrast to the reported role of ERAS in tumor cells associated with cell proliferation, our findings indicate that ERAS is important to maintain quiescence in HSCs.

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“…Band densities were determined using Labworks image acquisition and analysis software (Ultra-violet Products, Ltd., Cambridge, UK). Percent methylation was calculated as the density ratio of the two methylated bands to the three total bands (15). To confirm CO-BRA results, PCR products for samples of interest were cloned into the PGEM-T vector (Promega), and approximately 20 clones of each individual sample were sequenced.…”

mentioning

confidence: 99%

Aberrant DNA methylation of imprinted H19 gene in human preimplantation embryos

Chen

Shi

Zheng

et al. 2010

Fertility and Sterility

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A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms

Cited by 38 publications

References 34 publications

Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers

Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers

The Role of Embryonic Stem Cell-expressed RAS (ERAS) in the Maintenance of Quiescent Hepatic Stellate Cells

Aberrant DNA methylation of imprinted H19 gene in human preimplantation embryos

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