Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Histone acetylation at the sulfotransferase 1a1 gene is associated with its hepatic expression in normal aging.

OBJECTIVES: Phase II drug metabolism is poorly studied in advanced age and older adults may exhibit significant variability in their expression of phase II enzymes. We hypothesized that age-related changes to epigenetic regulation of genes involved in phase II drug metabolism may contribute to these effects. METHODS: We examined published epigenome-wide studies of human blood and identified the SULT1A1 and UGT1A6 genes as the top loci showing epigenetic changes with age. To assess possible functional alterations with age in the liver, we assayed DNA methylation (5mC) and histone acetylation changes around the mouse homologs Sult1a1 and Ugt1a6 in liver tissue from mice aged 4-32 months. RESULTS: Our sample shows a significant loss of 5mC at Sult1a1 (ß = -1.08, 95% CI [-1.8, -0.2], SE = 0.38, P = 0.011), mirroring the loss of 5mC with age observed in human blood DNA at the same locus. We also detected increased histone 3 lysine 9 acetylation (H3K9ac) with age at Sult1a1 (ß = 0.11, 95% CI [0.002, 0.22], SE = 0.05, P = 0.04), but no change to histone 3 lysine 27 acetylation (H3K27ac). Sult1a1 gene expression is significantly positively associated with H3K9ac levels, accounting for 23% of the variation in expression. We did not detect any significant effects at Ugt1a6. CONCLUSIONS: Sult1a1 expression is under epigenetic influence in normal aging and this influence is more pronounced for H3K9ac than DNA methylation or H3K27ac in this study. More generally, our findings support the relevance of epigenetics in regulating key drug-metabolizing pathways. In the future, epigenetic biomarkers could prove useful to inform dosing in older adults.

Epigenetic histone acetylation and Bdnf dysregulation in the hippocampus of rats exposed to repeated, low-dose diisopropylfluorophosphate.

AIMS: Deployment-related exposures to organophosphate (OP) compounds are implicated for Gulf War Illness (GWI) development in First GW veterans. However, reasons for the persistence of GWI are not fully understood. Epigenetic modifications to chromatin are regulatory mechanisms that can adaptively or maladaptively respond to external stimuli. These include DNA methylation and histone acetylation. DNA methylation changes have been reported in GWI but the role of histone acetylation in GWI has been less explored, despite its importance as an epigenetic mechanism for neurological disorders. MAIN METHODS: Male Sprague-Dawley rats were exposed to OP diisopropyl fluorophosphate (DFP, 0.5 mg/kg s.c., 5-d) and 6-m later brains were dissected for hippocampus. Western blotting, activity assays and chromatin immunoprecipitation (ChIP) were utilized for epigenetic analyses. Behavior was assessed using the Forced Swim Test (FST) and the Elevated Plus Maze (EPM). KEY FINDINGS: We observed a significant upregulation in HDAC1 protein along with a significant increase in HDAC enzyme activity in the hippocampus of DFP rats. A locus-specific ChIP study revealed decreases in H3K9ac at the brain derived neurotrophic factor (Bdnf) promoter IV coupled with a significant decrease in BDNF protein in DFP rat hippocampus. Treatment with HDAC inhibitor valproic acid reduced HDAC activity and decreased the FST immobility time in DFP rats. SIGNIFICANCE: Our research suggests that epigenetic alterations to histone acetylation pathways and decreased BDNF expression could represent novel mechanisms for GWI symptomatology and may provide new targets for developing effective drugs for GWI treatment.

Molecular mechanisms for the antidepressant-like effects of a low-dose ketamine treatment in a DFP-based rat model for Gulf War Illness.

Exposure to organophosphates (OP) during the First Gulf War is among one of the factors for Gulf War Illness (GWI) development in veterans and it has been challenging to treat GWI symptoms with existing therapies. Ketamine produces a rapid-onset and sustained antidepressant response, but there is no evidence whether ketamine treatment is effective for GWI depression. Repeated, low-dose exposure to diisopropyl fluorophosphate (DFP) mimic Gulf War related OP exposures and produces a chronic depressive state in rats. In this study, DFP-exposed rats treated with ketamine (10 mg/kg, i.p.) exhibited antidepressant-like effect on the Forced Swim Test at 1-h. This effect persisted at 24-h post ketamine, a time-point by which it is eliminated from the brain suggesting involvement of mechanisms that affect long-term synaptic plasticity. Western blot analysis showed significantly lower Brain-Derived Neurotrophic Factor (BDNF) levels in DFP rat brains. Ketamine produced a nonsignificant increase in BDNF expression at 1-h but produced a larger, significant (2.2-fold) increase at 24-h in DFP rats. We previously reported chronic hippocampal calcium elevations ([Ca2+]i) in DFP rats. Ketamine-treated DFP rats exhibited significantly lower [Ca2+]i at 1-h but not at 24-h. Interestingly, treatment with ANA-12, a TrkB-BDNF receptor antagonist, in DFP rats blunted ketamine's antidepressant-like effect at 24-h but not at 1-h. These experiments suggest that in a rat model of DFP-induced depression, inhibition of the NMDAR-Ca2+ contributes to the rapid-onset antidepressant effects of ketamine while the antidepressant actions that persisted at 24-h post ketamine administration involve upregulation of BDNF signaling.

DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver.

Aging is associated with reduced liver function that may increase the risk for adverse drug reactions in older adults. We hypothesized that age-related changes to epigenetic regulation of genes involved in drug metabolism may contribute to this effect. We reviewed published epigenome-wide studies of human blood and identified the cytochrome P450 2E1 (CYP2E1) gene as a top locus exhibiting epigenetic changes with age. To investigate potential functional changes with age in the liver, the primary organ of drug metabolism, we obtained liver tissue from mice aged 4-32 months from the National Institute on Aging. We assayed global DNA methylation (5-methylcytosine, 5mC), hydroxymethylation (5-hydroxymethylcytosine, 5hmC), and locus-specific 5mC and histone acetylation changes around mouse Cyp2e1. The mouse livers exhibit significant global decreases in 5mC and 5hmC with age. Furthermore, 5mC significantly increased with age at two regulatory regions of Cyp2e1 in tandem with decreases in its gene and protein expressions. H3K9ac levels also changed with age at both regulatory regions of Cyp2e1 investigated, while H3K27ac did not. To test if these epigenetic changes are associated with varying rates of drug metabolism, we assayed clearance of the CYP2E1-specific probe drug chlorzoxazone in microsome extracts from the same livers. CYP2E1 intrinsic clearance is associated with DNA methylation and H3K9ac levels at the Cyp2e1 locus but not with chronological age. This suggests that age-related epigenetic changes may influence rates of hepatic drug metabolism. In the future, epigenetic biomarkers could prove useful to guide dosing regimens in older adults.

Building a schizophrenia genetic network: transcription factor 4 regulates genes involved in neuronal development and schizophrenia risk.

The transcription factor 4 (TCF4) locus is a robust association finding with schizophrenia (SCZ), but little is known about the genes regulated by the encoded transcription factor. Therefore, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) of TCF4 in neural-derived (SH-SY5Y) cells to identify genome-wide TCF4 binding sites, followed by data integration with SCZ association findings. We identified 11 322 TCF4 binding sites overlapping in two ChIP-seq experiments. These sites are significantly enriched for the TCF4 Ebox binding motif (>85% having ≥1 Ebox) and implicate a gene set enriched for genes downregulated in TCF4 small-interfering RNA (siRNA) knockdown experiments, indicating the validity of our findings. The TCF4 gene set was also enriched among (1) gene ontology categories such as axon/neuronal development, (2) genes preferentially expressed in brain, in particular pyramidal neurons of the somatosensory cortex and (3) genes downregulated in postmortem brain tissue from SCZ patients (odds ratio, OR = 2.8, permutation P < 4x10-5). Considering genomic alignments, TCF4 binding sites significantly overlapped those for neural DNA-binding proteins such as FOXP2 and the SCZ-associated EP300. TCF4 binding sites were modestly enriched among SCZ risk loci from the Psychiatric Genomic Consortium (OR = 1.56, P = 0.03). In total, 130 TCF4 binding sites occurred in 39 of the 108 regions published in 2014. Thirteen genes within the 108 loci had both a TCF4 binding site ±10kb and were differentially expressed in siRNA knockdown experiments of TCF4, suggesting direct TCF4 regulation. These findings confirm TCF4 as an important regulator of neural genes and point toward functional interactions with potential relevance for SCZ.

The role of epigenomics in personalized medicine.

INTRODUCTION: Epigenetics is the study of reversible modifications to chromatin and their extensive and profound effects on gene regulation. To date, the role of epigenetics in personalized medicine has been under-explored. Therefore, this review aims to highlight the vast potential that epigenetics holds. AREAS COVERED: We first review the cell-specific nature of epigenetic states and how these can vary with developmental stage and in response to environmental factors. We then summarize epigenetic biomarkers of disease, with a focus on diagnostic tests, followed by a detailed description of current and pipeline drugs with epigenetic modes of action. Finally, we discuss epigenetic biomarkers of drug response. EXPERT COMMENTARY: Epigenetic variation can yield information on cellular states and developmental histories in ways that genotype information cannot. Furthermore, in contrast to fixed genome sequence, epigenetic patterns are plastic, so correcting aberrant, disease-causing epigenetic marks holds considerable therapeutic promise. While just six epigenetic drugs are currently approved for use in the United States, a larger number is being developed. However, a drawback to current therapeutics is their non-specific effects. Development of locus-specific epigenetic modifiers, used in conjunction with epigenetic biomarkers of response, will enable truly precision interventions.