22

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

We probe how genetic variability across the three major histocompatibility complex (MHC) class I genes (human leukocyte antigen [HLA] A, B, and C) may affect susceptibility to and severity of severe acute respiratory syndrome 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19). We execute a comprehensive in silico analysis of viral peptide-MHC class I binding affinity across all known HLA -A, -B, and -C genotypes for all SARS-CoV-2 peptides. We further explore the potential for cross-protective immunity conferred by prior exposure to four common human coronaviruses. The SARS-CoV-2 proteome is successfully sampled and presented by a diversity of HLA alleles. However, we found that HLA-B*46:01 had the fewest predicted binding peptides for SARS-CoV-2, suggesting individuals with this allele may be particularly vulnerable to COVID-19, as they were previously shown to be for SARS (1). Conversely, we found that HLA-B*15:03 showed the greatest capacity to present highly conserved SARS-CoV-2 peptides that are shared among common human coronaviruses, suggesting it could enable cross-protective T-cell based immunity. Finally, we report global distributions of HLA types with potential epidemiological ramifications in the setting of the current pandemic.

No abstract

No abstract

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.

SummaryThe normal aging process is a complex phenomenon associated with physiological alterations in the function of cells and organs over time. Although an attractive candidate for mediating transcriptional dysregulation, the contribution of epigenetic dysregulation to these progressive changes in cellular physiology remains unclear. In this study, we employed the genome-wide HpaII tiny fragment enrichment by ligation-mediated PCR assay to define patterns of cytosine methylation throughout the rat genome and the luminometric methylation analysis assay to measure global levels of DNA methylation in the same samples. We studied both liver and visceral adipose tissues and demonstrated significant differences in DNA methylation with age at > 5% of sites analyzed. Furthermore, we showed that epigenetic dysregulation with age is a highly tissue-dependent phenomenon. The most distinctive loci were located at intergenic sequences and conserved noncoding elements, and not at promoters nor at CG-dinucleotide-dense loci. Despite this, we found that there was a subset of genes at which cytosine methylation and gene expression changes were concordant. Finally, we demonstrated that changes in methylation occur consistently near genes that are involved in metabolism and metabolic regulation, implicating their potential role in the pathogenesis of age-related diseases. We conclude that different patterns of epigenetic dysregulation occur in each tissue over time and may cause some of the physiological changes associated with normal aging.

Intrauterine growth restriction (IUGR) increases susceptibility to age-related diseases, including type 2 diabetes (T2DM), and is associated with permanent and progressive changes in gene expression. Our study was designed to test whether epigenomic dysregulation mediates the cellular memory of this intrauterine event. To test this hypothesis, we isolated pancreatic islets from control and IUGR (induced by bilateral uterine artery ligation at day 18 of fetal life) animals at 7 weeks of age. Using the HELP (HpaII tiny fragment enrichment by ligationmediated PCR) assay, we generated the first DNA methylation map at almost 1 million unique sites throughout the rat genome in normal pancreatic islet cells, allowing us to identify the changes that occur as a consequence of IUGR. We validated candidate dysregulated loci with quantitative assays of cytosine methylation and gene expression. IUGR changes cytosine methylation at ϳ1,400 loci (false discovery rate of 4.2%) in male rats at 7 weeks of age, preceding the development of diabetes and thus representing candidate loci for mediating the pathogenesis of metabolic disease that occurs later in life. Epigenetic dysregulation occurred preferentially at conserved intergenic sequences, frequently near genes regulating processes known to be abnormal in IUGR islets, such as vascularization, ␤-cell proliferation, insulin secretion, and cell death, associated with concordant changes in mRNA expression. These results demonstrate that epigenetic dysregulation is a strong candidate for propagating the cellular memory of intrauterine events, causing changes in expression of nearby genes and long term susceptibility to type 2 diabetes.Perturbations in the intrauterine environment resulting in poor fetal growth increase the susceptibility for age-related diseases, particularly type 2 diabetes mellitus (T2DM) 4 and cardiovascular disease (1, 2). Moreover, intrauterine growth restriction (IUGR) can cause permanent and progressive changes in gene expression, affecting important metabolically active tissues such as pancreatic islets (3-6). These changes are often present at birth or during early life, and can precede the development of overt disease in rodents by months and in humans by decades. Although the mechanisms that may mediate the "fetal origin of adult disease" (7) remain unclear, dysregulation of the epigenome could play an important role and may explain the changes in gene expression that are heritably maintained through cell divisions in IUGR animals throughout life (8).We have developed an animal model of IUGR caused by uteroplacental insufficiency, which limits the supply of critical substrates (e.g. nutrients, oxygen, growth factors, and hormones) to the fetus (9). This deficient intrauterine environment affects fetal development through permanent and progressive dysregulation of gene expression and function of susceptible cells (e.g. pancreatic ␤-cells) and leads to the development of T2DM in adulthood (3, 6, 9). In particular, expression of pancreatic and duodenal home...