January 15, 2016 (Vol. 36, No. 2)

Richard A. A. Stein M.D., Ph.D.

Advantageously Epigenetic Analyses Can Capture both Genetic Factors and Environmental Exposures

Over half a century ago, Conrad Hal Waddington introduced his model of the epigenetic landscape. He depicted a differentiating cell as a ball rolling down a landscape of bifurcating valleys and ridges, with each valley representing an alternative developmental path. Just as a ball may roll from valley to valley until it reaches the bottom of the landscape, a cell may progress from one developmental alternative to another until it reaches its fully differentiated state.

The model’s original purpose was to integrate concepts from genetics and developmental biology and to describe mechanisms that connect the genotype to the phenotype. Today, the model remains a compelling metaphor for epigenetics, which has developed into one of the most vibrant biomedical fields. Epigenetics has become indispensable for exploring development, differentiation, homeostasis, and diseases that span virtually every clinical discipline.

Analyzing Methylation Patterns

“Modern efforts toward explaining human disease purely based upon sequencing cannot possibly succeed in isolation,” says Andrew P. Feinberg, M.D., professor of medicine and director of the Center for Epigenetics at Johns Hopkins University School of Medicine. “At least half of human disease is caused by exposure to the environment.”

While the contribution of genetic factors to disease is more predictable and easier to study in the case of highly penetrant Mendelian disorders, most medical conditions involve multiple genes that may interact with one another and with environmental factors. Particularly for these conditions, capturing epigenetic changes becomes a crucial aspect of understanding pathogenesis and designing prophylactic and therapeutic interventions.

“In these cases,” notes Dr. Feinberg, “an approach not including epigenetics will be severely limited in what it can accomplish.”

In a recent study, Dr. Feinberg and colleagues reported that large blocks of the human genome are hypomethylated in the epidermis as a result of sun exposure, which together with aging represents a known risk factor for skin cancer. These hypomethylated regions overlap with regions that have methylation changes in patients with squamous cell carcinoma.

This overlap could explain the causal link between sun exposure and the increased risk of malignancy found in many epidemiological studies. Most of the methylation changes were observed in the epidermis, not in the dermis, pointing toward the combination between the genotype and exposure, acting on specific cell types, as a key factor in shaping disease.

“One of the advantages of epigenetic analyses is that they capture both genetic factors and environmental exposures,” explains Dr. Feinberg. In the study of complex diseases, the existence of many distinct genetic variants identified in different individuals makes it challenging to understand their roles in pathogenesis. “But if genetic variants converge on gene regulatory loci, then measuring methylation can still be informative about these variants,” continues Dr. Feinberg, “even if genetic changes are inconsistent across the patients.”

In combining data from genome-wide association analysis and epigenome-wide analysis, Dr. Feinberg and colleagues revealed that two single-nucleotide polymorphisms on human chromosome 11, located 100 kb apart and involved in different aspects of lipid metabolism, controlled DNA methylation at two CpG sites in a bidirectional promoter situated between two genes encoding the fatty acid desaturases FADS1 and FADS2. Genome-wide association studies alone would not capture the convergence of these two single-nucleotide polymorphisms as they regulate DNA methylation in the shared promoter region.

“Measuring DNA methylation,” concludes Dr. Feinberg, “can pick up the fact that these single nucleotide polymorphisms act through DNA methylation to regulate the genes.”

Identifying Metastable Epialleles

Over the years, genome-wide association studies provided opportunities to establish links between genetic variation and phenotypic changes. For these analyses, genetic material from any of an individual’s cells, such as a peripheral white blood cell, is informative about the individual’s genotype. However, for epigenetic changes, which vary across tissues and within the same tissue among different cells, it is much more challenging to examine associations with disease.

Robert A. Waterland, Ph.D., associate professor of pediatrics and molecular and human genetics at Baylor College of Medicine, thinks that identifying human metastable epialleles will help circumvent some of these challenges. “Getting investigators and the field interested in metastable epialleles is going to be an important first step in helping us understand how epigenetic dysregulation contributes to human disease,” says Dr. Waterland.

The term metastable epialleles refers to genomic loci with differential epigenetic regulation that are variably expressed in genetically identical individuals, and where the epigenetic state is established stochastically in the very early embryo, before gastrulation, and subsequently maintained. This leads to systemic (non-tissue-specific) interindividual epigenetic differences that are not genetically mediated.

The fact that DNA methylation at metastable epialleles is particularly sensitive to environmental influences makes these loci valuable in mechanistically exploring the developmental origins hypothesis, the concept that environmental exposures during critical periods of prenatal and early postnatal development can have long-term implications in the risk of disease. Previous studies have implicated epigenetic modifications as a mechanism by which environmental changes during pregnancy may lead to epigenetic changes that influence health later in life.

In the most recent genome-wide screen meant to identify metastable epialleles in humans, Dr. Waterland teamed up with Dr. Andrew Prentice and colleagues at the London School of Hygiene and Tropical Medicine and used two independent and complementary experimental approaches to identify DNA methylation changes that occur in the cleavage-stage embryo (shortly after the time of conception). The first approach involved a genome-wide screen for DNA methylation in multiple tissues from two healthy Caucasian adults. In parallel, genome-wide DNA methylation profiling was performed in a rural population from The Gambia to examine the link between the season of conception (a proxy for maternal nutritional status) and DNA methylation in the offspring and sought to capture the effect of maternal nutritional status on the epigenetic profile of the offspring.

“We identified the same genomic locus as the top hit in both screens, suggesting that this is likely to be a key indicator of early environmental influences on the epigenome,” explains Dr. Waterland. Both approaches identified VTRNA2-1 as the lead candidate for an environmentally-responsive epiallele.

VTRNA2-1, a genomically imprinted small noncoding RNA and a putative tumor suppressor gene, is preferentially methylated on the maternally inherited allele, and loss of imprinting at this locus promises to link the early embryonic environment to epigenetic changes that shape disease risk later in life. Besides VTRNA2-1, over 100 metastable epialleles were identified in the study.

“At metastable epialleles such as VTRNA2-1, DNA methylation in peripheral blood or in any easily accessible tissue can give an indication about the epigenetic regulation throughout the body,” concludes Dr. Waterland. “That is what is really different.”

The image shows a cleavage-stage human embryo. This is around the same stage that DNA methylation is ‘set’ at metastable epialleles. [Instituto Bernabeu]

Mapping Heterochromatin Domains

“For the first time, we found that a histone chaperone is implicated in organizing chromatin at a large scale,” says David Bazett-Jones, Ph.D., professor of biochemistry at the University of Toronto and senior scientist at the Hospital for Sick Children. The discovery and characterization of histone variants has been a vital facet of understanding chromatin organization and dynamics.

One of the most extensively studied histone variants is H3.3. Although H3.3 is 96% identical at the amino acid level to histone H3.1, histones H3.3 and H3.1 are functionally distinct. Histone H3.3 is expressed throughout the cell cycle, and it is enriched in transcriptionally active chromatin and in certain types of post-translational modifications. The death domain-associated protein DAXX, one of the proteins associated with histone H3.3 deposition, was recently identified as its chaperone.

Dr. Bazett-Jones and colleagues, including his graduate student Lindsy Rapkin, revealed that the loss of DAXX led to a global structural change in the chromatin landscape, characterized by genomic regions enriched in the trimethylated H3K9 epigenetic mark that were juxtaposed to large chromatin domains devoid of this modification.

“These major changes probably occur because the boundaries between heterochromatin domains and other regions were not being respected, leading to the inappropriate insertions of histone H3.3, and this exerted quite profound effects,” explains Dr. Bazett-Jones. The loss of DAXX led to the uncoupling of the epigenetic marks from the global chromatin architecture. “This shows that a major global reorganization of the chromatin was taking place,” Dr. Bazett-Jones continues.

To visualize chromatin changes that result from the loss of DAXX, Dr. Bazett-Jones and colleagues used electron spectroscopic imaging, an experimental approach that is based on the principle of electron energy loss spectroscopy. When a biological specimen is targeted with electrons and its atoms become ionized, the ionization energy is equal to the energy that is lost by the incident electrons that generated the event.

The electron microscope technique generates nitrogen and phosphorus maps, which are used to discriminate between nucleic-acid-rich and protein-rich cellular structures. These maps offer high-contrast images of chromatin and its three-dimensional organization in intact cells.

Another component of the DAXX deletion phenotype included the loss of nucleolar structural integrity, resulting in an increased number of cells containing mini-nucleoli, and the dispersal of ribosomal DNA genes outside the nucleolus. Collectively, these findings pointed toward a novel role that DAXX plays in the subnuclear organization of chromatin and in maintaining nucleolar structural integrity.

“Historically, we thought that the well-known epigenetic modifications dictate the compact character of heterochromatin,” notes Dr. Bazett-Jones. “But our findings, and those from other groups, reveal that a heterochromatin domain epigenetically marked with H3K9 trimethylation, for example, can be found in a structurally ‘open’ state, similar to euchromatin.”

This indicates that the boundaries between heterochromatin and euchromatin are much more fluid than previously envisioned, a concept that is crucial for understanding factors that dynamically shape the three-dimensional interaction between epigenetic changes. A key implication of these findings is that the epigenetic marks at a specific genomic locus depend on both the local environment and the three-dimensional context.

“We need to look at what loci come together in specific regions of the nucleus in three dimensions and how they affect each other,” concludes Dr. Bazett-Jones. “This is on top of capturing epigenetic marks, which are on top of the genomic sequences that we need to explore.”

Electron spectroscopic image of a region of the nucleus of a mouse embryonic fibroblast. Phosphorus and nitrogen maps allow chromatin (yellow) to be distinguished from protein-based structures (cyan). The arrow indicates the nuclear envelope. The large structure in the middle of the field, a chromocentre, is an accumulation of pericentric heterochromatin. It is surrounded by dispersed chromatin fibers. The heterochromatin mark, trimethlated H3K9, is immunolabelled and visualized with gold tags (white foci). [David Bazett-Jones]

Identifying Druggable Epigenetic Processes

“There is a big gap in understanding the biology of epigenetics,” says Chris J. Burns, Ph.D., laboratory head, Division of Chemical Biology, Walter and Eliza Hall Institute of Medical Research, Melbourne. “And this goes hand in hand with the need to learn how to generate small molecule probes or drugs.”

When interrogating epigenetic processes, researchers find it useful to integrate biological and chemical perspectives. For example, researchers have generated a large body of literature demonstrating that many epigenetic processes involve highly complicated protein complexes.

Historically, genetics studies have typically relied on knocking down or knocking out a gene and its protein product to examine the resulting phenotype. “In contrast, knocking down a protein that is part of a protein complex fundamentally alters that complex, and the phenotype could be quite different from the one that can be seen with a small molecule inhibition of a catalytic component of the protein complex,” notes Dr. Burns. This opens an acute need to identify small molecules that can selectively impact just one particular aspect of these protein complexes.

A major effort in Dr. Burns’ lab is focusing on identifying therapeutic agents that could target epigenetic processes. “Epigenetics in terms of drug discovery and development is still in an early stage,” explains Dr. Burns. While several drugs that target epigenetic processes have become available in recent years—drugs such as HDAC inhibitors and DNA methyl transferase inhibitors—many other drugs are still at early stages of development.

“Some epigenetic processes have not yet been drugged,” Dr. Burns points out. “For some of them, there may not be any therapeutic agents that are particularly good.”

Dr. Burns’ lab has collaborated with investigators led by Carl Walkley, Ph.D., joint head, Stem Cell Regulation Unit, St. Vincent’s Institute of Medical Research, Melbourne. Together, the research teams revealed that several bromodomain inhibitors exert powerful antitumor activity in human osteosarcoma cell lines and in osteosarcoma primary cells from mouse models of the disease.

The researchers’ findings were surprising. JQ1, one the bromodomain inhibitors tested, exerted its antiproliferative activity by inducing apoptosis, and not by mediating cell cycle arrest, as expected. Moreover, even though previous studies identified MYC as an oncogenic driver in osteosarcoma, the activity of JQ1 was exerted independently of MYC downregulation.

At the same time, this work revealed that downregulation of FOSL1, a gene previously implicated in osteoblast differentiation, is an important contributor to the effects of JQ1, marking the first time when this gene was implicated in osteosarcoma.

“Because we used primary cell from animals, these findings reflect the disease process better than cell lines, which may take on a number of other mutations,” concludes Dr. Burns. “This explains why our findings are contrary to previous reports in the literature.”

“We have shown that epigenetic drugs may work not only on protein-coding genes but also on the noncoding part of the genome,” says Claes Wahlestedt, M.D., Ph.D., professor and associate dean for therapeutic innovation at the University of Miami Miller School of Medicine.

A therapeutically promising class of epigenetic compounds consists of bromodomain inhibitors. These compounds have received increasing attention in recent years, and several leads have entered clinical trials for malignancies, atherosclerosis, and type 2 diabetes.

”One of our interests is to see if bromodomain inhibitors could be used for diseases of the nervous system,” notes Dr. Wahlestedt.

Using in vitro and in vivo approaches, investigators in Dr. Wahlestedt’s group, in collaboration with investigators led by Nagi Ayad, Ph.D., found that BET bromodomain inhibitors can inhibit glioblastoma cell proliferation by inducing a cyclin-dependent kinase inhibitor. These findings set the stage for subsequent experiments that used single molecule sequencing to profile long noncoding RNAs (lncRNAs) differentially expressed in glioblastoma multiforme. This helped identify a set of transcripts that are specific for this malignancy and could be regulated by bromodomain inhibitors.

In glioblastoma multiforme cells, the I-BET151 bromodomain inhibitor localized to the promoter of HOTAIR, a tumor-promoting lncRNA that acts as an epigenetic silencer and has been implicated in several cancers, decreased its expression, and restored the expression of several lncRNA species that are downregulated in this malignancy.

In another collaborative endeavor, Dr. Wahlestedt and colleagues conducted a semi-high-throughput gene-expression-based screen to identify small molecules that could increase the expression of C9ORF72. A GGGGCC hexanucleotide repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic cause for amyotrophic lateral sclerosis. Individuals without this condition harbor 2 to 25 of these repeats, but their number can reach up to several hundreds in ALS patients, reducing C9ORF72 expression, which has been implicated in the pathogenesis of this condition.

The gene-expression-based screen identified, in fibroblasts from affected and unaffected individuals, small interfering RNAs against the BRD3 bromodomain protein and several small molecule bromodomain inhibitors that were able to increase C9ORF72 expression. This effect occurred without changes in promoter CpG hypermethylation and trimethylated H3K9 marks, which are heterochromatin markers of the expanded C9ORF72 alleles.

“The mechanism of action of these compounds is probably broader than we thought before,” concludes Dr. Wahlestedt.

RNA-Based Therapeutics Can Up- or Downregulate Genetic Activity

“We are pursuing RNA-based therapeutics for commercially attractive diseases with unmet medical need,” says Joseph E. Payne, president and CEO of Arcturus Therapeutics. “As the future unfolds, we anticipate many more opportunities in this space.”

The possibility of manipulating gene expression in both directions, with technologies that allow medically relevant genes to be knocked down with small interfering RNA or upregulated with messenger RNA, sets Arcturus Therapeutics apart in the RNA therapeutics arena. ”We are the only company in the world that has executed license and collaborations for both siRNA and mRNA therapeutics,” notes Payne. “Arcturus’ proprietary technologies can upregulate or downregulate genetic activity, and this has led to partnerships with top-tier companies in both areas.”

Arcturus collaborations with Johnson & Johnson and Ultragenyx have enabled investigators at Arcturus Therapeutics to collect preclinical data for RNA-based therapeutic compounds against large patient population diseases and rare disorders. Two factors imperative to succeed in the RNA therapeutics arena are the development of reliable chemistry, combined with safe and efficient delivery technologies.

“Our patented UNA Oligomer™ chemistry can improve the pharmaceutical properties of all types of RNA medicines, including small, medium, and large RNA molecules, whether single- or double-stranded,” asserts Payne. The company’s LUNAR™ delivery technology, developed in-house by Arcturus Therapeutics’ investigators, is efficient for several types of RNA species, including siRNA, messenger RNA, and gene-editing RNA. “By combining UNA chemistry with LUNAR delivery,” concludes Payne, “we are able to design effective drugs toward a suite of pharmaceutical therapeutic areas, including mainstream and rare diseases.”

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