Opitz syndrome (OS) is a genetically heterogeneous disorder characterized by defects of the ventral midline, including hypertelorism, cleft lip and palate, heart defects, and mental retardation. We recently identified the gene responsible for X-linked OS. The ubiquitously expressed gene product, MID1, is a member of the RING finger family. These proteins are characterized by an N-terminal tripartite protein-protein interaction domain and a conserved C terminus of unknown function. Unlike other RING finger proteins for which diverse cellular functions have been proposed, the function of MID1 is as yet undefined. By using the green f luorescent protein as a tag, we show here that MID1 is a microtubule-associated protein that inf luences microtubule dynamics in MID1-overexpressing cells. We confirm this observation by demonstrating a colocalization of MID1 and tubulin in subcellular fractions and the association of endogenous MID1 with microtubules after in vitro assembly. Furthermore, overexpressed MID1 proteins harboring mutations described in OS patients lack the capability to associate with microtubules, forming cytoplasmic clumps instead. These data give an idea of the possible molecular pathomechanism underlying the OS phenotype.
Recent research suggests that epigenetics, especially DNA methylation, plays a mechanistic role in aging. Epigenetic clocks, which measure changes in a few hundred specific CpG sites, can accurately predict chronological age in a variety of species, including humans. These clocks are currently the bestbiomarkers for predicting mortality in humans. Additionally, several studies have characterized the effects of aging across the methylome in a wide variety of tissues from humans and mice. A small fraction (~2%) of the CpG sites show age-related changes, either hypermethylation or hypomethylation with aging. Evaluation of non-CpG site methylation has only been examined in a few studies, with about ~0.5% of these sites showing achange with age. Therefore, while only a small fraction of cytosines in the genome show changes in DNA methylation with age, this represents 2 to 3 million cytosines in the genome. Importantly, the only study to compare the effect of aging on DNA methylation in male and female mice and humans found that N95% of the age-related changes in DNA methylation in the hippocampus were sexually divergent, i.e., the methylation did not differ between males and females atyoung age but age-related changes occurred in one sex but not the other. The age-related changes in DNA methylation tend to be enriched and under-represented in specific genomic contexts, with some commonalities between tissues and species that require further investigation. The strongest evidence that the age-related changes in DNA methylation play a role in aging comes from studies of anti-aging interventions (e.g., caloric restriction, dwarfism, and rapamycin treatment) in mice. These anti-aging interventions deaccelerate the epigenetic clocks and reverse/prevent 20 to 40% of the age-related changes in DNA methylation. It will be important in the future to demonstrate that at least some of the age-related changes in DNA methylation directly lead to alterations in the transcriptome of cells/tissues that could potentially contribute to aging.
Summary 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.
Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific central nervous system cell types. Analysis of gene expression and DNA modifications in astrocytes or microglia from the same animal demonstrates differential usage of DNA methylation and hydroxymethylation in CpG and non-CpG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any brain cell type for which a cell type-specific cre is available.
Apoptotic cell death is a potential driver of RD, and in order to understand the mechanism of degeneration and potential treatments, we studied rhodopsin mutant RD model, P23H-1 rats. Investigating this genetic model of human RD allows us to investigate the association of sphingolipid metabolites with the degeneration of the retina in P23H-1 rats and the effects of a specific modulator of sphingolipid metabolism, FTY720. We found that P23H-1 rat retinas had altered sphingolipid profiles that, when treated with FTY720, were rebalanced closer to normal levels. FTY720-treated rats also showed protection from RD compared with their vehicletreated littermates. Based on these data, we conclude that sphingolipid dysregulation plays a secondary role in retinal cell death, which may be common to many forms of RDs, and that the U.S. Food and Drug Administration-approved
Epigenetic alterations are a hallmark of aging and age‐related diseases. Computational models using DNA methylation data can create “epigenetic clocks” which are proposed to reflect “biological” aging. Thus, it is important to understand the relationship between predictive clock sites and aging biology. To do this, we examined over 450,000 methylation sites from 9,699 samples. We found ~20% of the measured genomic cytosines can be used to make many different epigenetic clocks whose age prediction performance surpasses that of telomere length. Of these predictive sites, the average methylation change over a lifetime was small (~1.5%) and these sites were under‐represented in canonical regions of epigenetic regulation. There was only a weak association between “accelerated” epigenetic aging and disease. We also compare tissue‐specific and pan‐tissue clock performance. This is critical to applying clocks both to new sample sets in basic research, as well as understanding if clinically available tissues will be feasible samples to evaluate “epigenetic aging” in unavailable tissues (e.g., brain). Despite the reproducible and accurate age predictions from DNA methylation data, these findings suggest they may have limited utility as currently designed in understanding the molecular biology of aging and may not be suitable as surrogate endpoints in studies of anti‐aging interventions. Purpose‐built clocks for specific tissues age ranges or phenotypes may perform better for their specific purpose. However, if purpose‐built clocks are necessary for meaningful predictions, then the utility of clocks and their application in the field needs to be considered in that context.
BackgroundNCBI’s Gene Expression Omnibus (GEO) is a rich community resource containing millions of gene expression experiments from human, mouse, rat, and other model organisms. However, information about each experiment (metadata) is in the format of an open-ended, non-standardized textual description provided by the depositor. Thus, classification of experiments for meta-analysis by factors such as gender, age of the sample donor, and tissue of origin is not feasible without assigning labels to the experiments. Automated approaches are preferable for this, primarily because of the size and volume of the data to be processed, but also because it ensures standardization and consistency. While some of these labels can be extracted directly from the textual metadata, many of the data available do not contain explicit text informing the researcher about the age and gender of the subjects with the study. To bridge this gap, machine-learning methods can be trained to use the gene expression patterns associated with the text-derived labels to refine label-prediction confidence.ResultsOur analysis shows only 26% of metadata text contains information about gender and 21% about age. In order to ameliorate the lack of available labels for these data sets, we first extract labels from the textual metadata for each GEO RNA dataset and evaluate the performance against a gold standard of manually curated labels. We then use machine-learning methods to predict labels, based upon gene expression of the samples and compare this to the text-based method.ConclusionHere we present an automated method to extract labels for age, gender, and tissue from textual metadata and GEO data using both a heuristic approach as well as machine learning. We show the two methods together improve accuracy of label assignment to GEO samples.
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 nextgeneration 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-theart for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.
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