Increased circulating progranulin is not sufficient to induce cardiac dysfunction or supraventricular arrhythmia.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, and the incidence of new-onset AF has been increasing over the past two decades. Several factors contribute to the risk of developing AF including age, preexisting cardiovascular disease, chronic kidney disease, and obesity. Concurrent with the rise in AF, obesity has followed the same two-decade trend. The contribution of circulating proteins to obesity-related AF is of particular interest in the field. In this study, we investigated the effects of increased circulating levels of the glycoprotein progranulin on the development of supraventricular arrhythmias and changes to cardiac function. AAV8-mediated overexpression of full-length mouse progranulin was used to increase plasma protein levels and determine susceptibility to supraventricular arrhythmias and changes in cardiac structure and function. C57Bl/6N mice were subjected to increased circulating levels of progranulin for 20 weeks. Cardiac conduction was evaluated by surface ECG with and without isoproterenol challenge, and cardiac structure and function were measured by echocardiography after 20 weeks of circulating progranulin overexpression. Increased circulating levels of progranulin were maintained throughout the 20-week study. The cardiac structure and function remained unchanged in mice with increased circulating progranulin. ECG indices (P wave duration, P amplitude, QRS interval) were unaffected by increased progranulin levels and no arrhythmogenic events were observed following the isoproterenol challenge. In our model, increased levels of circulating progranulin were not sufficient to induce changes in cardiac structure and function or elicit ECG abnormalities suggestive of susceptibility to supraventricular arrhythmias.

Epigenomic regulation by labile iron.

Iron is an essential micronutrient metal for cellular functions but can generate highly reactive oxygen species resulting in oxidative damage. For these reasons its uptake and metabolism is highly regulated. A small but dynamic fraction of ferrous iron inside the cell, termed intracellular labile iron, is redox-reactive and ready to participate multiples reactions of intracellular enzymes. Due to its nature its determination and precise quantification has been a roadblock. However, recent progress in the development of intracellular labile iron probes are allowing the reevaluation of our current understanding and unmasking new functions. The role of intracellular labile iron in regulating the epigenome was recently discovered. This chapter examine how intracellular labile iron can modulate histone and DNA demethylation and how its pool can mediate a signaling pathway from cAMP serving as a sensor of the metabolic needs of the cells.

Vitamin C regulates Schwann cell myelination by promoting DNA demethylation of pro-myelinating genes.

Ascorbic acid (vitamin C) is critical for Schwann cells to myelinate peripheral nerve axons during development and remyelination after injury. However, its exact mechanism remains elusive. Vitamin C is a dietary nutrient that was recently discovered to promote active DNA demethylation. Schwann cell myelination is characterized by global DNA demethylation in vivo and may therefore be regulated by vitamin C. We found that vitamin C induces a massive transcriptomic shift (n = 3,848 genes) in primary cultured Schwann cells while simultaneously producing a global increase in genomic 5-hydroxymethylcytosine (5hmC), a DNA demethylation intermediate which regulates transcription. Vitamin C up-regulates 10 pro-myelinating genes which exhibit elevated 5hmC content in both the promoter and gene body regions of these loci following treatment. Using a mouse model of human vitamin C metabolism, we found that maternal dietary vitamin C deficiency causes peripheral nerve hypomyelination throughout early development in resulting offspring. Additionally, dietary vitamin C intake regulates the expression of myelin-related proteins such as periaxin (PRX) and myelin basic protein (MBP) during development and remyelination after injury in mice. Taken together, these results suggest that vitamin C cooperatively promotes myelination through 1) increased DNA demethylation and transcription of pro-myelinating genes, and 2) its known role in stabilizing collagen helices to form the basal lamina that is necessary for myelination.

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD.

BACKGROUND: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. METHODS AND RESULTS: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. CONCLUSIONS: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

Oscillatory cAMP signaling rapidly alters H3K4 methylation.

Epigenetic variation reflects the impact of a dynamic environment on chromatin. However, it remains elusive how environmental factors influence epigenetic events. Here, we show that G protein-coupled receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target-Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II).

Vitamin C supplementation expands the therapeutic window of BETi for triple negative breast cancer.

BACKGROUND: Bromodomain and extra-terminal inhibitors (BETi) have shown efficacy for the treatment of aggressive triple negative breast cancer (TNBC). However, BETi are plagued by a narrow therapeutic window as manifested by severe toxicities at effective doses. Therefore, it is a limitation to their clinical implementation in patient care. METHODS: The impact of vitamin C on the efficacy of small compounds including BETi was assessed by high-throughput screening. Co-treatment of TNBC by BETi especially JQ1 and vitamin C was evaluated in vitro and in vivo. FINDINGS: High-throughput screening revealed that vitamin C improves the efficacy of a number of structurally-unrelated BETi including JQ1, I-BET762, I-BET151, and CPI-203 in treating TNBC cells. The synergy between BETi and vitamin C is due to suppressed histone acetylation (H3ac and H4ac), which is in turn caused by upregulated histone deacetylase 1 (HDAC1) expression upon vitamin C addition. Treatment with JQ1 at lower doses together with vitamin C induces apoptosis and inhibits the clonogenic ability of cultured TNBC cells. Oral vitamin C supplementation renders a sub-therapeutic dose of JQ1 able to inhibit human TNBC xenograft growth and metastasis in mice. INTERPRETATION: Vitamin C expands the therapeutic window of BETi by sensitizing TNBC to BETi. Using vitamin C as a co-treatment, lower doses of BETi could be used to achieve an increased therapeutic index in patients, which will translate to a reduced side effect profile. FUND: University of Miami Sylvester Comprehensive Cancer Center, Bankhead Coley Cancer Research program (7BC10), Flight Attendant Medical Research Institute, and NIH R21CA191668 (to GW) and 1R56AG061911 (to CW and CHV).

SCO2 mutations cause early-onset axonal Charcot-Marie-Tooth disease associated with cellular copper deficiency.

Recessive mutations in the mitochondrial copper-binding protein SCO2, cytochrome c oxidase (COX) assembly protein, have been reported in several cases with fatal infantile cardioencephalomyopathy with COX deficiency. Significantly expanding the known phenotypic spectrum, we identified compound heterozygous variants in SCO2 in two unrelated patients with axonal polyneuropathy, also known as Charcot-Marie-Tooth disease type 4. Different from previously described cases, our patients developed predominantly motor neuropathy, they survived infancy, and they have not yet developed the cardiomyopathy that causes death in early infancy in reported patients. Both of our patients harbour missense mutations near the conserved copper-binding motif (CXXXC), including the common pathogenic variant E140K and a novel change D135G. In addition, each patient carries a second mutation located at the same loop region, resulting in compound heterozygote changes E140K/P169T and D135G/R171Q. Patient fibroblasts showed reduced levels of SCO2, decreased copper levels and COX deficiency. Given that another Charcot-Marie-Tooth disease gene, ATP7A, is a known copper transporter, our findings further underline the relevance of copper metabolism in Charcot-Marie-Tooth disease.

Vitamin C Sensitizes Melanoma to BET Inhibitors.

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo-/- mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment.Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572-83. ©2017 AACR.

cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool.

It is widely accepted that cAMP regulates gene transcription principally by activating the protein kinase A (PKA)-targeted transcription factors. Here, we show that cAMP enhances the generation of 5-hydroxymethylcytosine (5hmC) in multiple cell types. 5hmC is converted from 5-methylcytosine (5mC) by Tet methylcytosine dioxygenases, for which Fe(II) is an essential cofactor. The promotion of 5hmC was mediated by a prompt increase of the intracellular labile Fe(II) pool (LIP). cAMP enhanced the acidification of endosomes for Fe(II) release to the LIP likely through RapGEF2. The effect of cAMP on Fe(II) and 5hmC was confirmed by adenylate cyclase activators, phosphodiesterase inhibitors, and most notably by stimulation of G protein-coupled receptors (GPCR). The transcriptomic changes caused by cAMP occurred in concert with 5hmC elevation in differentially transcribed genes. Collectively, these data show a previously unrecognized regulation of gene transcription by GPCR-cAMP signaling through augmentation of the intracellular labile Fe(II) pool and DNA hydroxymethylation.