Melanie Ehrlich, PhD

We have been studying epigenetics since the 1970’s. My interest in naturally modified DNAs derived from my postdoc and early PI studies of one of the weirdest known DNAs, SP-15 DNA. This DNA has 62% of its T replaced by 5-(4',5'-dihydroxypentyl)uracil which has a phosphoglucuronate moiety attached by a phosphodiester linkage to one of the hydroxyl groups of the pentyl side chain. Decades later, in collaboration with Andrew Koprinski and Kenneth Ehrlich (my husband), SP-15 DNA was sequenced. Recently, Ken and I were honored by the naming of a new bacteriophage family as Ehrlichviridae.

The prelude to our work on DNA methylation in vertebrates was our research on a bacteriophage DNA (XP12 DNA) with all of its cytosine residues methylated. In collaboration with Charles Gehrke, we published the first study demonstrating tissue-specific differences in the levels of DNA methylation in humans. In 1983, our lab and, independently, the lab of Bert Vogelstein were the first to publish that there are abnormalities in DNA methylation in human cancers. Later, we were the first to report very frequent hypomethylation of highly repeated DNA sequences in human cancers. Our findings on genome-wide, cancer-associated DNA hypomethylation have been confirmed by numerous laboratories.

Our interest in how certain transcription factors might distinguish between methylated and nonmethylated DNA recognition sites led to the first report of a vertebrate DNA-binding protein with specificity for DNA methylation (MDBP/RFX). Alongside our many studies of this protein, we continued analyzing aberrant DNA hypermethylation vs. hypomethylation in human cancer, e.g., and the relationships between cancer-associated DNA hypomethylation and pericentromeric chromosome rearrangements. We also investigated this relationship in normal chorionic villus cultures. As a follow-up to studying the associations between DNA hypomethylation and chromosomal rearrangements, we studied a rare DNA methyltransferase deficiency (immunodeficiency, centromeric region instability, facial anomalies, ICF) in which there are bizarre diagnostic rearrangements of hypomethylated satellite DNA in the pericentromic regions of chromosomes 1 and 16.

Results from our research on additional targets of abnormal DNA hypomethylation in the ICF syndrome converged with our earlier findings of sequences displaying gamete-associated DNA hypomethylation. One of the most interesting sequences that we found to display hypomethylation in ICF cells was a macrosatellite repeat sequence (D4Z4) that is associated with a type of dominantly inherited muscular dystrophy (facioscaulohumeral muscular dystrophy, FSHD). D4Z4 is very similar to a sequence with sperm-specific DNA hypomethylation that we had previously cloned.

To better understand normal and aberrant muscle formation, we profiled genome-wide expression in myoblasts (skeletal muscle progenitor cells) and myotubes (multinucleated differentiation products of myoblasts) from controls and FSHD patients. We uncovered the first convincing evidence for generalized dysregulation of gene expression in this disease. To better understand transcription control in FSHD and other muscular dystrophies as well as muscle aging and cachexia, we characterized genome-wide epigenetic changes associated with myogenesis and postnatal skeletal muscle using ENCODE and RoadMap whole-genome profiles of human DNA methylation and chromatin epigenetic marks in myogenic progenitor cells, skeletal muscle tissue, and many different types of samples. Among the genes we studied in detail for their relationships between skeletal muscle lineage-specific epigenomics (including DNA hydroxymethylation) and transcriptomics were the early differentiation-linked HOX genes, ubiquination-associated ASB family genes and KLHL family genes, and Notch-signaling genes. These studies provided new insights into the variety of transcription modulatory roles played by differential DNA methylation, intragenic enhancers, and distant intergenic enhancers. For example, we described evidence for dual-purpose enhancers that upregulate expression of the gene in which they reside in one cell type and upregulate an adjacent gene in another cell type.

In collaboration with Sriharsa Pradhan at New England Biolabs, we recently generated the first methylomes (whole-genome bisulfite sequencing, WGBS, and enzymatic methylation sequencing, EM-seq) from primary cultures of myoblasts that used quality-controlled cell cultures. This quality control is especially important for DNA methylation analysis of myoblasts. The cell cultures we used were >90% myoblasts. Our previous studies of genome-wide DNA methylation in myoblasts involved reduced representation bisulfite sequencing (RRBS) profiles that had been generated by ENCODE from our myoblast cell cultures. With WGBS and EM-seq profiles, there was virtually 100% coverage of CpGs in the genome instead <5% from RRBS. The WGBS and EM-seq profiles of myoblasts as well as WGBS profiles of six heterogenous cell cultures were used to determine myoblast differentially methylated regions (DMRs). Myoblast and skeletal muscle DMRs were compared to chromatin states, open chromatin, CTCF binding, and MYOD binding both globally and in specific gene neighborhoods. Our results highlight unusual relationships between epigenetics and gene expression that illustrate the interplay between DNA methylation and chromatin epigenetics in the regulation of transcription (submitted). These studies also revealed unexpected similarities in transcription and epigenetics between two highly dissimilar cell populations, myoblasts and cerebellum.  

In a series of reporter gene studies, we targeted in vitro DNA methylation to myoblast-hypermethylated enhancer or promoter elements and demonstrated that methylation of as little as three clustered CpGs could repress a potent MYOD1 enhancer element. From other reporter gene assays coupled with bioinformatics of primary cell cultures and tissues, we provided evidence that tissue-specific DNA methylation at some 5’ gene regions can down-modulate cell type-specific transcription rather than silence it. 

Our research on developmental epigenetic changes expanded to include heart and aorta and associated diseases. We discovered tissue-specific expression-linked super-enhancers at five genes that are critical for cardiac fibrosis . From another bioinformatic study of atherosclerotic vs. control aorta methylomes, we provided evidence for atherosclerosis-linked DNA methylation changes in aorta that might help minimize or reverse the standard contractile character of many of these cells by abnormally down-modulating smooth muscle-related enhancers. We also used in-depth tissue-comparative bioinformatics, including epigenomics and transcriptomics, to inform analysis of genetic variants identified in genome-wide association studies (GWAS) of osteoporosis and obesity traits in collaboration with Hong-Wen Deng.

Our studies of the effects of DNA hypomethylation and hypermethylation on gene expression include our ongoing interest in cancer-associated DNA hypermethylation. We are currently examining the intersection of differentiation-linked changes in DNA methylation with cancer-associated changes in DNA methylation. We are also collaborating with Prescott Deininger on a study of methylation changes at AluY repeats in cancer cells and the promoter and enhancer elements that can drive their independent expression. In addition, with Giovani Piedimonte and Michael Moore, we are studying changes in DNA methylation of peripheral neurons upon infection with Respiratory Syncytial Virus.