Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model

Spencer, Netanya Y.; Yan, Ziying; Cong, Le; Engelhardt, John F.; Stanton, Robert C.

doi:10.1016/j.ab.2015.11.002

Cited by 8 publications

(4 citation statements)

References 28 publications

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“…Additionally, as G6PD is associated with nuclear enzymes that modify histones (acetylation and methylation) and DNA/RNA polymerases (Supplemental Table S5), we suspect that G6PD potentially regulates gene expression through chromatin modification, which has been suggested to persistently activate cells in the wall of PAs (18), followed by polymerase-dependent transcription. G6PD is present in the nucleus of hepatic cells (37), and its nuclear localization was recently confirmed by CRISPR-Cas9 editing (38). These studies support our idea that G6PD contributes to the epigenetic modification and regulation of the expression of genes that orchestrate the accumulation of persistently activated cell types involved in remodeling of hypertensive PAs.…”

Section: Discussionsupporting

confidence: 77%

Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation

Joshi

Kitagawa

Jacob

et al. 2020

American Journal of Physiology-Lung Cellular and Molecular Phys

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Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PDDef), and further show that established severe PH in Cyp2c44−/− mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PDDef, knockdown and inhibition in lungs: 1) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, 2) increased expression of Tet methylcytosine dioxygenase 2 ( Tet2) gene, and 3) upregulated expression of the coding genes and long noncoding (lnc) RNA Pint, which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated TET2 and lncPINT, and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.

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Section: Discussionsupporting

confidence: 77%

Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation

Joshi

Kitagawa

Jacob

et al. 2020

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…G2/M phase transition: Within cells, G6PD is localized in several subcellular compartments, including the plasma membrane, nucleus and cytoplasm 23,24 . Within the nucleus of hepatic cells, G6PD fuels NADPH oxidase-dependent superoxide anion production 25 , which in turn regulates gene expression in endothelial cells 26 .…”

Section: The Loss-of-function G6pd S188f Variant Led To Augmented Exp...mentioning

confidence: 99%

Studies in CRISPR-generated Mediterranean G6PD variant rats reveal G6PD orchestrates genome-wide DNA methylation and gene expression in vascular wall

Signoretti

Gupte

2023

Preprint

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Background: Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of DNMTs and TETs, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. Methods: In aorta of CRISPR-edited rats with the Mediterranean G6PD variant we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. Results: Here, we documented higher expression of Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PDS188F variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5,787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aorta from G6PDS188F as compared to wild-type rats. Further, we observed less large artery (aorta) stiffness in G6PDS188F as compared to wild-type rats. Conclusions: These results establish a noncanonical function of the wild-type G6PD and G6PDS188F variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveals G6PDS188F variant contributes to reduce large artery stiffness.

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“…Within cells, G6PD is localized in several subcellular compartments, including the plasma membrane, nucleus, and cytoplasm [17,18]. Within the nucleus of hepatic cells, G6PD fuels NADPH oxidase-dependent superoxide anion production [19], which in turn regulates gene expression in endothelial cells [20].…”

Section: The Loss-of-function G6pd S188f Variant and Differential Gen...mentioning

confidence: 99%

G6PD Orchestrates Genome-Wide DNA Methylation and Gene Expression in the Vascular Wall

Signoretti,

Gupte

2023

IJMS

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Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and the de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of epigenetic writers and erasers, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. In the aorta of CRISPR-edited rats with the Mediterranean G6PD variant, we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. Here, we documented higher expression of Dnmt1, Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PDS188F variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aortas from G6PDS188F as compared to wild-type rats. Our results demonstrated that nitric oxide, which is generated in a G6PD-derived NADPH-dependent manner, increases TET and decreases DNMT activity. Further, we observed less large artery (aorta) stiffness in G6PDS188F as compared to wild-type rats. These results establish a noncanonical function of the wild-type G6PD and G6PDS188F variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveal that the G6PDS188F variant contributes to reducing large artery stiffness.

show abstract

Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model

Cited by 8 publications

References 28 publications

Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation

Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation

Studies in CRISPR-generated Mediterranean G6PD variant rats reveal G6PD orchestrates genome-wide DNA methylation and gene expression in vascular wall

G6PD Orchestrates Genome-Wide DNA Methylation and Gene Expression in the Vascular Wall

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