Freeman, D. M. | Search bioRxiv

bioRxiv2019

The conserved DNMT1 dependent methylation regions in human cells are vulnerable to environmental rotenone.

Allele-specific DNA methylation (ASM) describes genomic loci that maintain CpG methylation at only one inherited allele rather than having coordinated methylation across both alleles. The most prominent of these regions are germline ASMs (gASMs) that control the expression of imprinted genes in a parent of origin-dependent manner and are associated with disease. However, our recent report reveals numerous ASMs at non-imprinted genes. These non-germline ASMs are dependent on DNA methyltransferase 1 (DNMT1) and strikingly show the feature of random, switchable monoallelic methylation patterns in the mouse genome. The significance of these ASMs to human health has not been explored. Due to their shared allelicity with gASMs, herein, we propose that non-traditional ASMs are sensitive to exposures in association with human disease. We first explore their conservancy in the human genome. Our data show that our putative non-germline ASMs were in conserved regions of the human genome and located adjacent to genes vital for neuronal development and maturation. We next tested the hypothesized vulnerability of these regions by exposing human embryonic kidney cell HEK293 with the neurotoxicant rotenone for 24h. Indeed,14 genes adjacent to our identified regions were differentially expressed from RNA-sequencing. We analyzed the base-resolution methylation patterns of the predicted non-germline ASMs at two neurological genes, HCN2 and NEFM, with potential to increase the risk of neurodegeneration. Both regions were significantly hypomethylated in response to rotenone. Our data indicate that non-germline ASMs seem conserved between mouse and human genomes, overlap important regulatory factor binding motifs, and regulate the expression of genes vital to neuronal function. These results support the notion that ASMs are sensitive to environmental factors and may alter the risk of neurological disease later in life by disrupting neuronal development.

molecular biology↗

bioRxiv2019

Manganese-induced Parkinsonism in mice is reduced using a novel contaminated water sediment exposure model.

The effects of heavy metals on human health have become an important area of study. For instance, acute manganese toxicity is known to induce Parkinsonism. Heavy metals including manganese enter the aquatic environment from both anthropogenic and natural processes. These metals accumulate within water sediments and their behavior is then dependent upon the sediment composition and phase. These metal-sediment interactions remain to be explored within in-vivo animal studies. To study the effect of these interactions, herein we successfully developed an exposure model in mice that encapsulates the aquatic microenvironment of heavy metals before exposure. Male and female C57/BL6 mice were exposed to manganese contaminated sediment via their drinking water (Sed_Mn) or to manganese placed directly into their drinking water with no prior sediment interaction (Mn) for six weeks. Sediment interaction did not alter total manganese in drinking water (mg/L) or weekly manganese consumption (mg) in males (54.9{+/-}1.5 mg) or females (44.6{+/-}1.0 mg) over the six-week exposure period. We analyzed motor impairment, a common feature in Parkinsons disease, using the beam traversal, cylinder, and accelerating rotarod behavioral tests. We observed Parkinsons like deficits in motor control in both treatment groups as early as four weeks of exposure in males but not in females. Intriguingly, mice given water incubated with manganese spiked sediment (Sed_Mn) performed better overall compared to mice given manganese directly in water (Mn) despite having similar exposure in males and females. Male Sed_Mn mice compared to Mn mice had a 146% reduction in time to cross the beam traversal test (p<0.05), a 10% increase in rearing activity in the cylinder test (p<0.05), and a 14% increase in time remaining on the rotarod (not significant). Female Sed_Mn mice compared to Mn mice had no change in the time to cross the beam traversal test, a 36% increase in rearing activity in the cylinder test (p<0.05), and a 35% increase in time on the rotarod (p<0.05). Our study indicates that metal-sediment interactions may alter metal toxicity in mammals and introduces a new exposure model to test the toxicity of metal contaminants of drinking water.

pharmacology and toxicology↗