DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

SignificanceThe phenomenon of genetic balance is characterized by the finding that addition or subtraction of a single chromosome to the whole set is more detrimental than altering the dosage of the complete complement. The molecular basis of this principle has not been studied previously in a comprehensive manner. In this study, the set of all five trisomic chromosomes and a ploidy series of diploid, triploid, and tetraploid in Arabidopsis were examined for global gene expression modulations. The results indicate an impact of genomic stoichiometry on the landscape of gene expression, which has implications for how gene expression operates, the evolution of duplicate genes, and the underlying basis of quantitative traits.

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). An exception are human pre-implantation embryos, where dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities with naïve pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X-chromosome are active and express the long non-coding RNAs XACT and XIST , the master regulator of XCI, which are silenced upon entry into meiosis. These findings uncover XACT as hPGC-marker, describe XCD associated with XIST -expression in hPGCs, and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Additionally, we found a unique X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.

Inherent genetic programming and environmental factors affect fetal growth in utero. Epidemiologic data in growth-altered fetuses, either intrauterine growth restricted (IUGR) or large for gestational age (LGA), demonstrate that these newborns are at increased risk of cardiometabolic disease in adulthood. There is growing evidence that the in utero environment leads to epigenetic modification, contributing to eventual risk of developing heart disease or diabetes. In this study, we used reduced representation bisulfite sequencing to examine genome-wide DNA methylation variation in placental samples from offspring born IUGR, LGA, and appropriate for gestational age (AGA) and to identify differential methylation of genes important for conferring risk of cardiometabolic disease. We found that there were distinct methylation signatures for IUGR, LGA, and AGA groups and identified over 500 differentially methylated genes (DMGs) among these group comparisons. Functional and gene network analyses revealed expected relationships of DMGs to placental physiology and transport, but also identified novel pathways with biologic plausibility and potential clinical importance to cardiometabolic disease. Specific loci for DMGs of interest had methylation patterns that were strongly associated with anthropometric presentations. We further validated altered gene expression of these specific DMGs contributing to vascular and metabolic diseases (SLC36A1, PTPRN2, CASZ1, IL10), thereby establishing transcriptional effects toward assigning functional significance. Our results suggest that the gene expression and methylation state of the human placenta are related and sensitive to the intrauterine environment, as it affects fetal growth patterns. We speculate that these observed changes may affect risk for offspring in developing adult cardiometabolic disease.