Germline status and micronutrient availability regulate a somatic mitochondrial quality control pathway via short-chain fatty acid metabolism.

Reproductive status, such as pregnancy and menopause in women, profoundly influences metabolism of the body. Mitochondria likely orchestrate many of these metabolic changes. However, the influence of reproductive status on somatic mitochondria and the underlying mechanisms remain largely unexplored. We demonstrate that reproductive signals modulate mitochondria in the Caenorhabditis elegans soma. We show that the germline acts via an RNA endonuclease, HOE-1, which despite its housekeeping role in tRNA maturation, selectively regulates the mitochondrial unfolded protein response (UPRmt). Mechanistically, we uncover a fatty acid metabolism pathway acting upstream of HOE-1 to convey germline status. Furthermore, we link vitamin B12's dietary intake to the germline's regulatory impact on HOE-1-driven UPRmt. Combined, our study uncovers a germline-somatic mitochondrial connection, reveals the underlying mechanism, and highlights the importance of micronutrients in modulating this connection. Our findings provide insights into the interplay between reproductive biology and metabolic regulation.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Distinct impacts of alpha-synuclein overexpression on the hippocampal epigenome of mice in standard and enriched environments.

Elevated alpha-synuclein (SNCA) gene expression is associated with transcriptional deregulation and increased risk of Parkinson's disease, which may be partially ameliorated by environmental enrichment. At the molecular level, there is emerging evidence that excess alpha-synuclein protein (aSyn) impacts the epigenome through direct and/or indirect mechanisms. However, the extents to which the effects of both aSyn and the environment converge at the epigenome and whether epigenetic alterations underpin the preventive effects of environmental factors on transcription remain to be elucidated. Here, we profiled five DNA and histone modifications in the hippocampus of wild-type and transgenic mice overexpressing human SNCA. Mice of each genotype were housed under either standard conditions or in an enriched environment (EE) for 12 months. SNCA overexpression induced hippocampal CpG hydroxymethylation and histone H3K27 acetylation changes that associated with genotype more than environment. Excess aSyn was also associated with genotype- and environment-dependent changes in non-CpG (CpH) DNA methylation and H3K4 methylation. These H3K4 methylation changes included loci where the EE ameliorated the impacts of the transgene as well as loci resistant to the effects of environmental enrichment in transgenic mice. In addition, select H3K4 monomethylation alterations were associated with changes in mRNA expression. Our results suggested an environment-dependent impact of excess aSyn on some functionally relevant parts of the epigenome, and will ultimately enhance our understanding of the molecular etiology of Parkinson's disease and other synucleinopathies.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3, a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased in H3K4me1 levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels, but also causes a decrease in H3K4me3 levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific demethylase 5 (KDM5/Lid), an enzyme that removes tri- and di- methyl marks from H3K4. Moreover, analysis of ChIP-seq data revealed that BRWD3 and KDM5 are significantly co- localized throughout the genome and that sites of H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

A role for N6-methyldeoxyadenosine in C. elegans mitochondrial genome regulation.

Epigenetic modifications provide powerful means for transmitting information from parent to progeny. As a maternally inherited genome that encodes essential components of the electron transport chain, the mitochondrial genome (mtDNA) is ideally positioned to serve as a conduit for the transgenerational transmission of metabolic information. Here, we provide evidence that mtDNA of C. elegans contains the epigenetic mark N6-methyldeoxyadenosine (6mA). Bioinformatic analysis of SMRT sequencing data and methylated DNA IP sequencing data reveal that C. elegans mtDNA is methylated at high levels in a site-specific manner. We further confirmed that mtDNA contains 6mA by leveraging highly specific anti-6mA antibodies. Additionally, we find that mtDNA methylation is dynamically regulated in response to antimycin, a mitochondrial stressor. Further, 6mA is increased in nmad-1 mutants and is accompanied by a significant decrease in mtDNA copy number. Our discovery paves the way for future studies to investigate the regulation and inheritance of mitochondrial epigenetics.

DNA methylation as a mediator of genetic and environmental influences on Parkinson's disease susceptibility: Impacts of alpha-Synuclein, physical activity, and pesticide exposure on the epigenome.

Parkinson's disease (PD) is a neurodegenerative disorder with a complex etiology and increasing prevalence worldwide. As PD is influenced by a combination of genetic and environment/lifestyle factors in approximately 90% of cases, there is increasing interest in identification of the interindividual mechanisms underlying the development of PD as well as actionable lifestyle factors that can influence risk. This narrative review presents an outline of the genetic and environmental factors contributing to PD risk and explores the possible roles of cytosine methylation and hydroxymethylation in the etiology and/or as early-stage biomarkers of PD, with an emphasis on epigenome-wide association studies (EWAS) of PD conducted over the past decade. Specifically, we focused on variants in the SNCA gene, exposure to pesticides, and physical activity as key contributors to PD risk. Current research indicates that these factors individually impact the epigenome, particularly at the level of CpG methylation. There is also emerging evidence for interaction effects between genetic and environmental contributions to PD risk, possibly acting across multiple omics layers. We speculated that this may be one reason for the poor replicability of the results of EWAS for PD reported to date. Our goal is to provide direction for future epigenetics studies of PD to build upon existing foundations and leverage large datasets, new technologies, and relevant statistical approaches to further elucidate the etiology of this disease.

Alpha-synuclein overexpression induces epigenomic dysregulation of glutamate signaling and locomotor pathways.

Parkinson's disease (PD) is a neurological disorder with complex interindividual etiology that is becoming increasingly prevalent worldwide. Elevated alpha-synuclein levels can increase risk of PD and may influence epigenetic regulation of PD pathways. Here, we report genome-wide DNA methylation and hydroxymethylation alterations associated with overexpression of two PD-linked alpha-synuclein variants (wild-type and A30P) in LUHMES cells differentiated to dopaminergic neurons. Alpha-synuclein altered DNA methylation at thousands of CpGs and DNA hydroxymethylation at hundreds of CpGs in both genotypes, primarily in locomotor behavior and glutamate signaling pathway genes. In some cases, epigenetic changes were associated with transcription. SMITE network analysis incorporating H3K4me1 ChIP-seq to score DNA methylation and hydroxymethylation changes across promoters, enhancers, and gene bodies confirmed epigenetic and transcriptional deregulation of glutamate signaling modules in both genotypes. Our results identify distinct and shared impacts of alpha-synuclein variants on the epigenome, and associate alpha-synuclein with the epigenetic etiology of PD.

Diverse mating phenotypes impact the spread of wtf meiotic drivers in Schizosaccharomyces pombe.

Meiotic drivers are genetic elements that break Mendel's law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing (mating between individuals from distinct lineages) because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity for cells from distinct clonal lineages to mate varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed levels of preferential mating between cells from the same clonal lineage can slow, but not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs linked to the driver. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

The fission yeast, Schizosaccharomyces pombe, is a haploid organism, meaning it has a single copy of each of its genes. S. pombe cells generally carry one copy of each chromosome and can reproduce clonally by duplicating these chromosomes and then dividing into two cells. However, when the yeast are starving, they can reproduce sexually. This involves two cells mating by fusing together to create a 'diploid zygote', which contains two copies of each gene. The zygote then undergoes 'meiosis', a special type of cell division in which the zygote first duplicates its genome and then divides twice. This results in four haploid spores which are analogous to sperm and eggs in humans that each contain one copy of the genome. The spores will grow and divide normally when conditions improve. The genes carried by each of the haploid spores depend on the cells that formed the zygote. If the two 'parent' yeast had the same version or 'allele' of a gene, all four spores will have it in their genome. However, if the two parents have different alleles, only 50% of the offspring will carry each version. Although this is usually the case, there are certain alleles, called meiotic drivers, that are transmitted to all offspring even in situations where it is only carried by one parent. Meiotic drivers can be found in many organisms, including mammals, but their behavior is easiest to study in yeast. Meiotic drivers known as killers achieve this by disposing of any 'sister' spores that do not inherit the same allele of this gene. This 'killing' can only happen when only one of the 'parents' carries the driver. This scenario is thought to rarely occur in species that inbreed, as inbreeding leads to both gene copies being the same. However, this does not appear to be the case for S. pombe, which contain a whole family of killer meiotic drivers, the wtf genes, despite also being reported to mainly inbreed. To investigate this contradiction, López Hernández et al. isolated several genetically distinct populations of S.pombe. These isolates were grown together to determine how often the each one would outcross (mate with an individual from a different population) or inbreed. The results found that levels of inbreeding varied between isolates. Next, López Hernández et al. used mathematical modelling and experimental evolution analyses to study how wtf drivers spread amongst these populations. This revealed that wtf genes spread faster in populations with more outcrossing. In some instances, the wtf driver was linked to a gene that could harm the population. In these cases, López Hernández et al. found than inbreeding could purge these drivers and stop them from spreading the dangerous alleles through the population. López Hernández et al. establish a simple experimental system to model driver evolution and experimentally demonstrate how key parameters, such as outcrossing rates, affect the spread of these genes. Understanding how meiotic drivers spread is important, as these systems could potentially be used to modify populations important to humans, such as crops or disease vectors.

Extreme Acid Modulates Fitness Trade-Offs of Multidrug Efflux Pumps MdtEF-TolC and AcrAB-TolC in Escherichia coli K-12.

Bacterial genomes encode various multidrug efflux pumps (MDR) whose specific conditions for fitness advantage are unknown. We show that the efflux pump MdtEF-TolC, in Escherichia coli, confers a fitness advantage during exposure to extreme acid (pH 2). Our flow cytometry method revealed pH-dependent fitness trade-offs between bile acids (a major pump substrate) and salicylic acid, a membrane-permeant aromatic acid that induces a drug resistance regulon but depletes proton motive force (PMF). The PMF drives MdtEF-TolC and related pumps such as AcrAB-TolC. Deletion of mdtE (with loss of the pump MdtEF-TolC) increased the strain's relative fitness during growth with or without salicylate or bile acids. However, when the growth cycle included a 2-h incubation at pH 2 (below the pH growth range), MdtEF-TolC conferred a fitness advantage. The fitness advantage required bile salts but was decreased by the presence of salicylate, whose uptake is amplified by acid. For comparison, AcrAB-TolC, the primary efflux pump for bile acids, conferred a PMF-dependent fitness advantage with or without acid exposure in the growth cycle. A different MDR pump, EmrAB-TolC, conferred no selective benefit during growth in the presence of bile acids. Without bile acids, all three MDR pumps incurred a large fitness cost with salicylate when exposed at pH 2. These results are consistent with the increased uptake of salicylate at low pH. Overall, we showed that MdtEF-TolC is an MDR pump adapted for transient extreme-acid exposure and that low pH amplifies the salicylate-dependent fitness cost for drug pumps. IMPORTANCE Antibiotics and other drugs that reach the gut must pass through stomach acid. However, little is known of how extreme acid modulates the effect of drugs on gut bacteria. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump that otherwise incurs a fitness cost. At the same time, extreme acid amplifies the effect of salicylate selection against multidrug pumps. Thus, organic acids and stomach acid could play important roles in regulating multidrug resistance in the gut microbiome. Our flow cytometry assay provides a way to measure the fitness effects of extreme-acid exposure to various membrane-soluble organic acids, including plant-derived nutrients and pharmaceutical agents. Therapeutic acids might be devised to control the prevalence of multidrug pumps in environmental and host-associated habitats.

Neonatal Alcohol Exposure in Mice Induces Select Differentiation- and Apoptosis-Related Chromatin Changes Both Independent of and Dependent on Sex.

Prenatal alcohol exposure (PAE) affects many aspects of physiology and behavior, including brain development. Specifically, ethanol can influence expression of genes important for brain growth, including chromatin modifiers. Ethanol can also increase apoptotic cell death in the brain and alter epigenetic profiles such as modifications to histones and DNA methylation. Although differential sex outcomes and disruptions to the function of multiple brain regions have been reported in fetal alcohol spectrum disorder (FASD), the majority of our knowledge on molecular epigenetic and apoptotic dysregulation in PAE is based on data from males and is sometimes limited to assessments of the whole brain or one brain region. Here, we examined histone modifications, DNA methylation, and expression of genes involved in differentiation and proliferation related-chromatin modifications and apoptosis in the cerebral cortex and cerebellum of C57BL/6J mice exposed to an acute alcohol challenge on postnatal day 7, with a focus on differential outcomes between sexes and brain regions. We found that neonatal alcohol exposure altered histone modifications, and impacted expression of a select few chromatin modifier and apoptotic genes in both the cortex and cerebellum. The results were observed primarily in a sex-independent manner, although some additional trends toward sexual dimorphisms were observed. Alcohol exposure induced trends toward increased bulk H3K4me3 levels, increased Kmt2e expression, and elevated levels of Casp6 mRNA and bulk γH2A.X. Additional trends indicated that ethanol may impact Kdm4a promoter DNA methylation levels and bulk levels of the histone variant H2A.Z, although further studies are needed. We comprehensively examined effects of ethanol exposure across different sexes and brain regions, and our results suggest that major impacts of ethanol on bulk chromatin modifications underlying differentiation and apoptosis may be broadly applicable across the rodent cortex and cerebellum in both sexes.

NK cell detachment from target cells is regulated by successful cytotoxicity and influences cytokine production.

Natural killer (NK) cells participate in early immune defenses against pathogens and tumors and play a major role as immune effector and regulatory cells. The NK cell-mediated elimination of an infected or cancerous cell is a highly regulated process that requires the formation of a cell contact, the establishment of an immunological synapse and the polarization and release of lytic granules. Additionally, the detachment of NK cells from target cells is important for NK cells to bind and kill other cells in a process called serial killing. However, very little is known about this detachment process. Here, we show that NK detachment is directly connected to the successful killing of a target cell. The inhibition of killing due to reduced NK cell cytotoxicity or increased target cell resistance results in defective detachment and prolonged contact times. This effect leads to sustained Ca2+ flux in NK cells and the hypersecretion of proinflammatory cytokines. Linking defective cytotoxicity with enhanced cytokine secretion via reduced detachment may explain inflammatory pathologies in several diseases.