David R. Stanford

Maf1p, a Negative Effector of RNA Polymerase III in Saccharomyces cerevisiae

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

Targeted DNA methylation analysis by next-generation sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female mice

BackgroundChanges to the epigenome with aging, and DNA modifications in particular, have been proposed as a central regulator of the aging process, a predictor of mortality, and a contributor to the pathogenesis of age-related diseases. In the central nervous system, control of learning and memory, neurogenesis, and plasticity require changes in cytosine methylation and hydroxymethylation. Although genome-wide decreases in methylation with aging are often reported as scientific dogma, primary research reports describe decreases, increases, or lack of change in methylation and hydroxymethylation and their principle regulators, DNA methyltransferases and ten-eleven translocation dioxygenases in the hippocampus. Furthermore, existing data are limited to only male animals.ResultsThrough examination of the hippocampus in young, adult, and old male and female mice by antibody-based, pyrosequencing, and whole-genome oxidative bisulfite sequencing methods, we provide compelling evidence that contradicts the genomic hypomethylation theory of aging. We also demonstrate that expression of DNA methyltransferases and ten-eleven translocation dioxygenases is not differentially regulated with aging or between the sexes, including the proposed cognitive aging regulator DNMT3a2. Using oxidative bisulfite sequencing that discriminates methylation from hydroxymethylation and by cytosine (CG and non-CG) context, we observe sex differences in average CG methylation and hydroxymethylation of the X chromosome, and small age-related differences in hydroxymethylation of CG island shores and shelves, and methylation of promoter regions.ConclusionThese findings clarify a long-standing misconception of the epigenomic response to aging and demonstrate the need for studies of base-specific methylation and hydroxymethylation with aging in both sexes.

CNS-wide Sexually Dimorphic Induction of the Major Histocompatibility Complex 1 Pathway With Aging

The major histocompatibility complex I (MHCI) pathway, which canonically functions in innate immune viral antigen presentation and detection, is functionally pleiotropic in the central nervous system (CNS). Alternative roles include developmental synapse pruning, regulation of synaptic plasticity, and inhibition of neuronal insulin signaling; all processes altered during brain aging. Upregulation of MHCI components with aging has been reported; however, no systematic examination of MHCI cellular localization, expression, and regulation across CNS regions, life span, and sexes has been reported. In the mouse, MHCI is expressed by neurons and microglia, and MHCI components and receptors (H2-K1, H2-D1, &bgr;2M, Lilrb3, Klra2, CD247) display markedly different expression profiles across the hippocampus, cortex, cerebellum, brainstem, and retina. MHCI components, receptors, associated inflammatory transcripts (IL1&agr;, IL1&bgr;, IL6, TNF&agr;), and TAP (transporter associated with antigen processing) components are induced with aging and to a greater degree in female than male mice across CNS regions. H2-K1 and H2-D1 expression is associated with differential CG and non-CG promoter methylation across CNS regions, ages, and between sexes, and concomitant increased expression of proinflammatory genes. Meta-analysis of human brain aging data also demonstrates age-related increases in MHCI. Induction of MHCI signaling could contribute to altered synapse regulation and impaired synaptic plasticity with aging.

Sexually divergent DNA methylation patterns with hippocampal aging

DNA methylation is a central regulator of genome function, and altered methylation patterns are indicative of biological aging and mortality. Age‐related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome‐wide analyses of age‐related methylation changes have considered the factor of sex in a controlled animal model. High‐depth, genome‐wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non‐CG (CH) contexts demonstrated age‐related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic and intronic regions and under‐represented in promoters, CG islands, and specific enhancer regions in both sexes, suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex‐specific. Lifelong sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome‐wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites was confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.

Analysis of DNA modifications in aging research

As geroscience research extends into the role of epigenetics in aging and age-related disease, researchers are being confronted with unfamiliar molecular techniques and data analysis methods that can be difficult to integrate into their work. In this review, we focus on the analysis of DNA modifications, namely cytosine methylation and hydroxymethylation, through next-generation sequencing methods. While older techniques for modification analysis performed relative quantitation across regions of the genome or examined average genome levels, these analyses lack the desired specificity, rigor, and genomic coverage to firmly establish the nature of genomic methylation patterns and their response to aging. With recent methodological advances, such as whole genome bisulfite sequencing (WGBS), bisulfite oligonucleotide capture sequencing (BOCS), and bisulfite amplicon sequencing (BSAS), cytosine modifications can now be readily analyzed with base-specific, absolute quantitation at both cytosine-guanine dinucleotide (CG) and non-CG sites throughout the genome or within specific regions of interest by next-generation sequencing. Additional advances, such as oxidative bisulfite conversion to differentiate methylation from hydroxymethylation and analysis of limited input/single-cells, have great promise for continuing to expand epigenomic capabilities. This review provides a background on DNA modifications, the current state-of-the-art for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.

Caloric-restriction induced alterations in CG and non-CG methylation attenuate age-associated changes in the old brain.

Brain aging is marked by cognitive decline and susceptibility to neurodegeneration. Caloric-restriction (CR) increases neurogenesis, improves memory function, and protects from age-associated neurological disorders. Epigenetic mechanisms, including DNA methylation, are vital to normal CNS cellular and memory functions, and are dysregulated with aging. The beneficial effects of CR have been proposed to work through epigenetic processes, but this is largely unexplored. We therefore tested whether life-long CR prevents age-related DNA methylation changes in the brain. Hippocampal DNA from young (3 months) and old (24 months) male mice fed ad libitum and 24 month old mice fed a 40% calorierestricted diet from 3 months of age were examined by genome-wide bisulfite sequencing to measure methylation with base-specificity. Over 27 million CG and CH (non-CG) sites were examined. Of the ~40,000 differentially methylated CGs (dmCGs) and ~80,000 CHs (dmCHs) with aging, >1/3 were prevented by CR and were found across genomic regulatory regions and gene pathways. CR also caused alterations to CG and CH methylation at sites not differentially methylated with aging, and these CR-specific changes demonstrated a different pattern of regulatory element and gene pathway enrichment than those affected by aging. CR-specific DNMT1 and TET3 promoter hypermethylation corresponded to reduced gene expression. These findings demonstrate that CR attenuates age-related CG and CH hippocampal methylation changes, in combination with CR-specific methylation that may also contribute to the neuroprotective effects of CR. The prevention of age-related methylation alterations is also consistent with the pro-longevity effects of CR working through an epigenetic mechanism.Brain aging is marked by cognitive decline and increased susceptibility to neurodegeneration. Epigenetic mechanisms, including DNA methylation, are vital to CNS cellular function and memory formation, and are dysregulated with aging and age-related neurodegenerative disease. Caloric-restriction (CR), an established pro-longevity intervention, increases neurogenesis, improves memory function, and protects from age-associated pathologies. However, the molecular mechanisms promoting the neuroprotective effect of CR remain largely unknown. We tested the role of DNA methylation as a mechanism for CR-induced neuroprotection in the old hippocampus. Hippocampal DNA from young (3M) and old (24M) male mice fed ad libitum and 24M old mice fed 40% calorie-restricted diet from 3M of age were examined by genome-wide bisulfite sequencing to measure methylation levels at base-specific resolution. Over 22 million CG and CH (non-CG) sites were examined. Of the ~40,000 differentially methylated CGs (dmCGs) and ~80,000 CHs (dmCHs) observed with aging, 35% and 38%, respectively, were prevented by CR. Unique to dmCHs, CR preferentially prevented age-related hypermethylation. A diet-specific methylation response was observed in both CG and CH contexts. Diet-induced dmCHs were enriched in unexpected genomic locations including promoters and CG islands including hypermethylation of DNMT1 and Tet3 promoters corresponding to reduced gene expression. These findings demonstrate for the first time that caloric-restriction prevents age-induced cytosine methylation changes in the old brain in combination with diet-specific methylation changes that may function to maintain epigenetic homeostasis through epigenetic auto-regulation. The prevention of age-dmCGs/CHs by CR emphasizes the prominent role of DNA methylation as a driver of the aging process.Brain aging is marked by cognitive decline and increased susceptibility to neurodegeneration. Epigenetic mechanisms, including DNA methylation, are vital to CNS cellular function and memory formation, and are dysregulated with aging and age-related neurodegenerative disease. Caloric-restriction (CR), an established pro-longevity intervention, increases neurogenesis, improves memory function, and protects from age-associated pathologies. However, the molecular mechanisms promoting the neuroprotective effect of CR remain largely unknown. We tested the role of DNA methylation as a mechanism for CR-induced neuroprotection in the old hippocampus. Hippocampal DNA from young (3M) and old (24M) male mice fed ad libitum and 24M old mice fed 40% calorie-restricted diet from 3M of age were examined by genome-wide bisulfite sequencing to measure methylation levels at base-specific resolution. Over 22 million CG and CH (non-CG) sites were examined. Of the ~40,000 differentially methylated CGs (dmCGs) and ~80,000 CHs (dmCHs) observed with aging, 35% and 38%, respectively, were prevented by CR. Unique to dmCHs, CR preferentially prevented age-related hypermethylation. A diet-specific methylation response was observed in both CG and CH contexts. Diet-induced dmCHs were enriched in unexpected genomic locations including promoters and CG islands including hypermethylation of DNMT1 and Tet3 promoters corresponding to reduced gene expression. These findings demonstrate for the first time that caloric-restriction prevents age-induced cytosine methylation changes in the old brain in combination with diet-specific methylation changes that may function to maintain epigenetic homeostasis through epigenetic auto-regulation. The prevention of age-dmCGs/CHs by CR emphasizes the prominent role of DNA methylation as a driver of the aging process.

Exposure to environmental enrichment attenuates addiction-like behavior and alters molecular effects of heroin self-administration in rats

ABSTRACT Environmental factors profoundly affect the addictive potential of drugs of abuse and may also modulate the neuro‐anatomical/neuro‐chemical impacts of uncontrolled drug use and relapse propensity. This study examined the impact of environmental enrichment on heroin self‐administration, addiction‐related behaviors, and molecular processes proposed to underlie these behaviors. Male Sprague‐Dawley rats in standard and enriched housing conditions intravenously self‐administered similar amounts of heroin over 14 days. However, environmental enrichment attenuated progressive ratio, extinction, and reinstatement session responding after 14 days of enforced abstinence. Molecular mechanisms, namely DNA methylation and gene expression, are proposed to underlie abstinence‐persistent behaviors. A global reduction in methylation is reported to coincide with addiction, but no differences in total genomic methylation or repeat element methylation were observed in CpG or non‐CpG (CH) contexts across the mesolimbic circuitry as assessed by multiple methods including whole genome bisulfite sequencing. Immediate early gene expression associated with drug seeking, taking, and abstinence also were examined. EGR1 and EGR2 were suppressed in mesolimbic regions with heroin‐taking and environmental enrichment. Site‐specific methylation analysis of EGR1 and EGR2 promoter regions using bisulfite amplicon sequencing (BSAS) revealed hypo‐methylation in the EGR2 promoter region and EGR1 intragenic CpG sites with heroin‐taking and environmental enrichment that was associated with decreased mRNA expression. Taken together, these findings illuminate the impact of drug taking and environment on the epigenome in a locus and gene‐specific manner and highlight the need for positive, alternative rewards in the treatment and prevention of drug addiction. HIGHLIGHTSEnvironmental enrichment decreases the reinforcement efficacy of heroin.Genome‐wide methylation is not altered in mesolimbic structures with heroin self‐administration or environmental enrichment.Gene expression and base‐specific methylation responses to heroin are modified by environmental enrichment.