Development and Implementation of an Automated Multiplexed Method to Study Genome-Wide DNA Methylation

DNA methylation (DNAm) is an epigenetic mark with a critical role in regulating gene expression. Altered clinical states, including toxin exposure and viral infections, can cause aberrant DNA methylation in cells, which may persist during cell division. Current methods to study genome-wide methylome profiles of the cells require a long processing time and are expensive. Here, a novel technique called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which is amenable to automation. Up to 15 different samples can be combined into the same run of Mx-MeDIP-Seq, using only 25 ng of DNA per sample. Mx-MeDIP-Seq was used to study DNAm profiles of peripheral blood mononuclear cells (PBMCs) in two biologically distinct RNA viral infections with different modes of transmission, symptoms, and interaction with the host immune system: human immunodeficiency virus1 (HIV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Analysis of 90 hospitalized patients with SARS-CoV-2 and 57 healthy controls revealed that SARS-CoV-2 infection led to alterations in 920 methylated regions in PBMCs, resulting in a change in transcription that affects host immune response and cell survival. Analysis of publicly available RNA-Sequencing data in COVID-19 correlated with DNAm in several key pathways. These findings provide a mechanistic view toward further understanding of viral infections. Genome-wide DNAm changes post HIV-1-infection from 37 chronically ill patients compared to 17 controls revealed dysregulation of the actin cytoskeleton, which could contribute to the establishment of latency in HIV-1 infections. Longitudinal DNAm analysis identified several potentially protective and harmful genes that could contribute to disease suppression or progression.