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Development-linked changes in DNA methylation and hydroxymethylation in humans: interview with Dr Melanie Ehrlich
Melanie Ehrlich, PhD, is Professor in the Human Genetics Program, the Tulane Cancer Center, and the Program in Bioinformatics and Genomics at Tulane University Health Sciences Center, New Orleans, LA, USA. She obtained her PhD in molecular biology in 1971 from the State University of New York at Stony Brook and completed postdoctoral research at Albert Einstein College of Medicine in 1972. She has been working on various aspects of epigenetics, starting with DNA methylation, since 1972. Her laboratory is currently studying the interrelationships of tissue-specific changes in DNA methylation and hydroxymethylation with alterations in chromatin structure and gene expression in differentiation and disease.
QQ Dr Ehrlich, you are currently Professor in the Tulane Cancer Center, the Human Genetics Program, & the Center for Bioinformatics & Genomics, at Tulane University Health Sciences Center (LA, USA). Can you tell our readers a little about your career to date? What have been your greatest achievements? In 1975, we were the first to report that the 5-methyl group of 5-methylcytosine (5mC) residues stabilizes the DNA double helix, just as it does for T residues. The 1970s and 1980s were a time when only a small percentage of molecular biologists considered vertebrate DNA methylation to be of major functional importance and much before ‘epigenetics’ became almost a household word. It was gratifying to have worked on 5mC-rich XP12 phage DNA, the DNA with the highest known melting temperature and phage SP-15 DNA, which has the lowest known melting temperature and which we found to have bizarre, naturally programed DNA base modifications. Continuing our studies on the biochemistry of 5mC in DNA, we showed that the 5-methyl group of 5mC residues in DNA conferred resistance to reaction with bisulfite, but susceptibility to heat-induced deamination. We were the first to demonstrate that cytosine methylation increases DNA recognition by certain verte-
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brate DNA-binding proteins (MDBP, later called RFX1–5). In 1982, my laboratory together with Charles Gehrke’s was the first to report tissuespecific differences in human genomic 5mC levels. These findings prompted us to identify, clone and sequence a class of sperm-specific hypomethylated DNA sequences in the 1980s. Our studies of tissue-specific DNA methylation also led us to demonstrate that in cancer, a kind of development gone awry, there are major changes in DNA methylation compared with normal tissues, as was also reported the same year, 1983, independently by Andy Feinberg with Bert Vogelstein. To bring balance to cancer epigenetics after an overly strong shift of attention from cancer DNA hypomethylation to the subsequently discovered cancer DNA hypermethylation [1] , we showed in collaborative studies that hypomethylation, like hypermethylation, is ubiquitous in human malignancies, can occur very early in oncogenesis, tends to increase with tumor progression and is often an excellent prognostic marker. These findings have been abundantly confirmed by others. We also discovered the extremely frequent hypomethylation of tandem DNA repeats in human cancers, including of a repeat that can undergo either predominantly demethyl-
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Melanie Ehrlich Hayward Genetics Program, Tulane Cancer Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA and Center for Bioinformatics & Genomics, Tulane Medical School, 1430 Tulane Avenue, New Orleans, LA 70112, USA
[email protected]
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ISSN 1750-1911
Interview Ehrlich ation or de novo methylation in cancer depending on the specimen. Subsequently, we studied tandem DNA repeats involved in the immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome and those in facioscapulohumeral muscular dystrophy (FSHD). The transcriptome analyses, DNA methylation profiling and histone chromatin immunoprecipitation studies of disease versus normal cell cultures in which we were engaged helped to define the nature of these genetic and epigenetic diseases, and, in the case of FSHD, to argue against several oversimplified hypotheses about disease etiology. In 1994, I founded the DNA Methylation Society (now the Epigenetics Society [2]) largely to counter misunderstandings about DNA methylation [1] promulgated by several prominent molecular biologists and by some reviewers of NIH grant proposals. Lately, it has been deeply satisfying to return to our earlier investigation of tissue-specific DNA methylation in human samples using powerful new tools. We participated with our collaborator Michelle Lacey in developing improved methods for statistically calling individual hypo- or hypermethylated CpGs and differentially methylated regions (DMRs) in methyomes from reduced representation bisulfite sequencing. Among our novel findings about human development-related changes in DNA
methylation is the discovery of early de novo methylation and prolonged demethylation in the skeletal muscle lineage with strong overrepresentation of hypermethylation near or at genes encoding development-associated DNA-binding proteins. In addition, our study of muscle lineage-specific hypomethylation and hydroxymethylation in the vicinity of genes-specifying Notch signaling proteins gives insights into the functioning of Notch signaling in skeletal muscle. We have also found much myogenesis-associated differential methylation in unexpected gene regions, which we are studying to elucidate their functionality.
QQ Today, we are focusing our interview on development-linked changes in DNA methylation & hydroxymethylation in humans. You have described a little about tissue-specific DNA methylation above. Can you provide our readers with some background information on the new topic of DNA hydroxymethylation? 5mC was long considered to be the only genetically programed base modification in vertebrate genomes. The presence of a genetically programed sixth base in mammalian DNA was proven only 6 years ago by Tahiliani et al. and Kriaucionis and Heintz. Subsequent work demonstrated that 5-hydroxymethylcytosine (5hmC), an enzymatic oxidation product of 5mC, could be an intermediate in tissue-specific loss of methylation. In addition, 5hmC often functions as a relatively stable component of DNA, which has very different functional correlates from those of 5mC. It is now being studied intensely for its effects on gene expression and chromatin organization.
QQ What have been the most exciting discoveries in the area of DNA methylation & hydroxymethylation recently?
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There are so many that I can give only a brief sample. The results of recent studies suggest that DNA methylation and hydroxymethylation act concertedly with chromatin modification to ‘fine-tune’ gene expression and to help shape the epigenome locally and regionally. In the area of disease and DNA epigenetics, there are very many recent articles supporting the importance of DNA epigenetic changes to the development of neoplasia, two of which are mentioned below in answer to the question about DNA modification in health and disease. Over 2000 articles on DNA methylation and cancer were published just in 2014. In addition, DNA methylation changes are being increasingly associated with other diseases. For example, in lupus there is often aberrant global and gene-specific demethylation in T cells, sometimes linked to environmental exposures and aging [3] . DNA hypomethylation and hypermethylation have been doc-
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DNA methylation & hydroxymethylation in development: Dr Melanie Ehrlich interview
umented to be altered during aging. In a recent study of the methylomes of mononuclear blood cells in nonagenerians and young adults, which adjusted for leukocyte subtypes, age-related hypo- and hypermethylation were observed but only hypermethylation was associated with specific functionally related groups of genes (especially development-related DNA-binding proteins) [4] . Our understanding of the contribution of changes in genomic 5hmC to disease and physiology is dramatically expanding. This is especially for true for brain, which has very much higher levels of genomic 5hmC than any other organ. For example, a recent study of mice implicates cocaine exposure with enrichment of 5hmC at enhancers, promoter-adjacent regions and gene bodies in the basal forebrain, including at genes that are involved in addiction [5] .
QQ Are there any exciting research projects you are working on within the field of epigenomics that would be of note to our readers? We are investigating the biological significance of tissue-specific DNA hypermethylation at the borders of active promoter regions for developmentally important genes. We are testing the hypothesis that one reason that such hypermethylated DMRs have a positive relationship with gene expression is that they may limit the size and strength of promoters to downmodulate, but not turn off, gene expression or to influence the choice of transcription start sites. This study involves transfection experiments with DNA inserts in CpG-free reporter vectors and in vivo-like in vitro-introduced DNA methylation of the inserts. Among the other types of intriguing tissue-specific DMRs that we are studying are fascinating examples of enhancer chromatin regions inside one gene that appear to be regulating a neighboring gene. In the exciting research area of tissue-differential hydroxymethylation of DNA, we are collaborating with Sriharsa Pradhan to identify and study the functionality of DNA sequences that display exceptionally high levels of 5hmC in skeletal muscle tissue relative to various tissues, including brain.
QQ In what ways has research into
development-linked changes in DNA methylation & hydroxymethylation in humans impacted our understanding & management of human health & disease thus far?
Recent findings from our laboratory and others indicate that the physiological consequences of tissue-specific differential DNA modification in humans are more heterogenous and nuanced than was previously appreciated. For example, evidence indicates that development-linked changes in DNA methylation often finetune gene expression from many different intragenic
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Interview
or intergenic locations far from the transcription start site. In comparison, tissue-specific DMRs much less frequently serve to repress or maintain repression at canonical gene promoters. This is a very important issue in studying the dysregulation of gene expression linked to changes in DNA methylation in disease, especially, in cancer. Most studies of cancer-associated abnormalities of DNA methylation in gene regions have focused exclusively on promoter regions. The contribution of nonpromoter epigenetic changes to cancer is illustrated in a report this year by Rasmussen et al. [6] . Their study implicates the loss of activity of the 5hmC-forming TET2 methylcytosine dioxygenase during the formation of certain leukemias by its decreasing genomic 5hmC, increasing 5mC and altering chromatin structure, especially at many enhancer regions. Results from another study of cancer-associated DNA hypomethylation indicate that the loss of DNA methylation over large regions may trigger chromatin-mediated epigenetic silencing [7] . This finding is at first glance paradoxical because of the abundant association between aberrant promoter hypermethylation and gene repression in cancer. We and others found an association between DNA hypomethylation and chromosomal rearrangements. However, this probably makes a much less frequent contribution to cancer than does dysregulation of gene expression by DNA hypomethylation at or near cis-acting transcription control elements or DNA-binding sites for chromatin-looping proteins or nuclear lamina proteins. Cancer-linked DNA hypo- or hypermethylation may affect transcription initiation, elongation, termination or post-transcriptional processing. The growing understanding of the roles of DNA hypomethylation and hypermethylation in cancer explain why DNA epigenetic markers are among the best diagnostic and prognostic markers of malignancy.
QQ How have technological advancements aided research in this field? Are there any new tools in development that will further the capacity for research in this field? Improved methods for whole-genome profiling of DNA methylation, DNA hydroxymethylation, histone modification, transcription factor binding, chromatin-interactions and transcription have revolutionized our understanding of the highly varied interrelationships within the epigenome and between the epigenome and the transcriptome. For instance, they have provided evidence for DNA methylation and hydroxymethylation at certain DNA regions being involved in guiding alternative splicing and the expression of transcription-regulatory noncoding RNAs (ncRNAs). In addition, the ongoing development of tools for DNA modification targeted to specific
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Interview Ehrlich sequences is critical for epigenetics research and its clinical applications. The targeting methods include the use of bioengineered transcription-activator-like effectors (TALEs) and customized zinc finger proteins for recognizing 5mC or 5hmC in specific sequences in the genome (e.g., [8]) and precisely delivering DNA methyltransferase or hydroxymethyltransferase catalytic domains to these sequences. Similarly, the new biotechnology of clustered regularly interspersed short palindromic repeats (CRISPRs) is another promising sequence-specific delivery system for targeted DNA modifications.
QQ What direction is this line of research taking at present & where do you see the field of epigenomics in general heading in the next 5–10 years? The ways in which the context of the DNA sequence, local chromatin structure, regional chromatin structure, intranuclear localization, cell type, physiology, aging and disease influence DNA epigenetics and how DNA epigenetics influences chromatin epigenetics will be better understood and harnessed for biotechnology, clinical applications and advancing basic science.
QQ What are the main obstacles that the field of epigenomics must overcome in its endeavor to improve effective diagnosis & management of diseases such as cancer? Blunt-force pharmacological approaches for reversing neoplastic abnormalities in DNA methylation or hydroxymethylation used alone or in combination therapy need to be replaced by DNA sequence-targeted methods with greater specificity, efficacy and durability. Improvements in cost-effective targeting of epigenetic changes to the desired parts of the genome and minimizing off-target effects are needed. In addition, the delivery of the agents effecting these changes to specific tissues, organs or cell types is usually a major References 1
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Ehrlich M. The controversial denouement of vertebrate DNA methylation research. Biochemistry (Mosc.) 70, 568–575 (2005).
2
Epigenetics Society. http://epigeneticssocietyint.com/
3
Somers EC, Richardson BC. Environmental exposures, epigenetic changes and the risk of lupus. Lupus 23, 568–576 (2014).
4
Marttila S, Kananen L, Hayrynen S et al. Ageing-associated changes in the human DNA methylome: genomic locations and effects on gene expression. BMC Genomics 16, 179 (2015).
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obstacle. Also important is a much more complete understanding of the nature of tissue-specific, development-associated, physiology-related and diseaseassociated epigenetics, so that the epigenetic parameters to be targeted for clinical purposes are chosen for maximum efficacy and minimal side effects. Diagnostic and prognostic epigenetics is at hand and much less difficult than epigenetic interventions. However, with the extreme sensitivity of PCR-based methods to detect epigenetic changes, even in solid tumors from shed cells or DNA, comes the problem of false positives. Such misleading results can be derived from chemical contamination or from a background of rare changes in normal cells that by themselves will not lead to carcinogenesis. A PCRbased study of the follicular lymphoma-associated BCL2/JH translocation that we published in 1995 about detection of this translocation in normal blood samples with an age-dependent increase in its occurrence exemplifies the caution needed in interpreting PCR-based findings. Nonetheless, with sufficient technical care and related comprehensive basic scientific studies, the future for epigenetics in medicine is bright and exciting. Disclaimer The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of Future Medicine Ltd.
Financial & competing interests disclosure M Ehrlich has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. 5
Feng J, Shao N, Szulwach KE et al. Role of TET1 and 5-hydroxymethylcytosine in cocaine action. Nat. Neurosci. 18, 536–544 (2015).
6
Rasmussen KD, Jia G, Johansen JV et al. Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis. Genes Dev. 29(9), 910–922 (2015).
7
Hon GC, Hawkins RD, Caballero OL et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res. 22, 246–258 (2012).
8
Kubik G, Batke S, Summerer D. Programmable sensors of 5-hydroxymethylcytosine. J. Am. Chem. Soc. 137, 2–5 (2015).
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