The fascination with epigenetics stems not only from the profound impact that it has exerted on the biomedical, medical, and social sciences, but also from the somewhat debated and elusive definition of the term itself. It’s shifted multiple times over the years.
A key feature of epigenetic changes— their potential heritability—brings new dimensions to an already vibrant and thought-provoking field, but this aspect has received relatively little attention until recently. One of the prerequisites for epigenetic inheritance is that a specific gene expression pattern be re-established after DNA replication, in daughter cells, to ensure the faithful inheritance of the chromatin architecture.
“Until now, all our knowledge about the inheritance of epigenetic markings has been largely hypothetical, because no methods were available to look at what is happening with proteins just after DNA replication,” says Alexander M. Mazo, Ph.D., professor of biochemistry and molecular biology at the Jefferson Medical College, Thomas Jefferson University.
Previously, it was proposed that histone post-translational modifications were the ones responsible for epigenetic inheritance, but to a large extent these models were based on theoretical assumptions and were not fully supported experimentally. As an additional shortcoming, they did not clearly explain the ability of histones, which cover huge regions of DNA, to recruit their binding partners in a sequence-specific way.
By surveying in vivo protein-protein interactions on nascent DNA sequences at replication forks and examining their posttranslational modifications, Dr. Mazo and colleagues showed that trimethylated H3K4 and H3K27 are replaced by unmethylated histones after DNA replication and are not transferred from parental to daughter nucleosomes.
“It seems that the current model, that methylated histones are stably associated with DNA during replication and are transferred from parental to daughter strands, may not be true, but certain histone-modifying proteins seem to be working as epigenetic marks,” continues Dr. Mazo.
Enhancer-of-Zeste, an H3K27 methylase, Trithorax, an H3H4 methylase, and Polycomb were stably bound to nascent DNA, and their association with the nascent DNA was constitutive and continuous through the S phase, in the absence of trimethylated histones. “Our findings also revealed that epigenetic markings are present at very specific regions, which is one of the key conditions for epigenetic inheritance,” explains Dr. Mazo.
Epigenome-Wide Association Studies
Since the first genome-wide association study in 2005, over 1,400 articles were published, reporting almost 8,000 SNPs.
Although genome-wide association studies revolutionized our understanding of the genetic basis of complex diseases and traits, the relatively small effect exerted by each allele, and the small proportion of the heritability that they explain collectively, caused some disappointment.
“That has created discussions on missing inheritance and possible nongenetic contributions, where 80–90% of the disease phenotype cannot be explained by all the genetic variants that have been identified so far,” says Stephan Beck, Ph.D., professor of medical genomics at the University College London Cancer Institute.
Complementing these observations, studies on monozygotic but disease-discordant twins also revealed that on average only about 50% of the phenotype can be explained by genetic changes.
“These are essentially the reasons why we have set out the concept of epigenome-wide association studies, to track down the missing factors that must contribute to these diseases but cannot be explained by genetic variations,” explains Dr. Beck.
The principle of epigenome-wide association studies involves scanning cases and controls to identify epigenetic variations associated with a specific trait or disease. Although similar to genome-wide association studies, two major differences need to be considered.
One is that unlike in genetic studies, where peripheral blood DNA can serve as a surrogate for all tissues, the highly tissue-specific nature of epigenetic modifications cannot be captured by using a single source of DNA.
“One has to chose the source of the study material very carefully, to make it informative for the phenotype to be studied,” explains Dr. Beck.
The second aspect is that, unlike in genetic studies, where interpreting a mutation is based on the knowledge that common diseases are not genotoxic and do not change the gene sequence, an association found in epigenetic studies is not necessarily the cause of the phenotype, but may be the consequence of it, a phenomenon known as reverse causation.
Therefore, it is not suitable to simply analyze a single case/control cohort, as in genome-wide association studies.
“We suggested a powerful two-tiered study design, in which one should first look at disease-discordant monozygotic twins to exclude genetic changes, and after identifying epigenetic differences, verify and replicate them in unrelated individuals that have been prospectively followed,” explains Dr. Beck.
Integrate Studies with Approaches
The prospective study design ensures that samples are collected and examined before and after a phenotype develops, allowing reverse causation to be excluded.
“An important point, now that we have an additional way to analyze common disease variation, is that we should not replace, but integrate epigenetic studies with genetic approaches, and together they should provide more explanations of what could cause the disease,” emphasizes Dr. Beck.
“Over the last couple of years, a revolution in our ability to not only sequence, but also synthesize vast amounts of DNA, has enabled us to study the relationship between DNA sequences, epigenetic marks, and gene regulatory activities in a directed and hypothesis-driven manner,” notes Tarjei S. Mikkelsen, Ph.D., principal investigator at the Broad Institute and Harvard Stem Cell Institute.
Dr. Mikkelsen and colleagues used a strategy that combines bioinformatics, synthetic biology, and experimental approaches to examine histone methylation changes that occur over time during the differentiation of human mesenchymal stem cells into adipocytes.
The genome-wide chromatin state maps that were created allowed the dynamic chromatin signatures characteristic for specific stages during differentiation to be visualized and facilitated the identification of key regulatory elements.
“This strategy is very informative for identifying active gene promoters and other functional elements in the genome in a context-dependent way,” explains Dr. Mikkelsen.
In a subsequent study, Dr. Mikkelsen and colleagues designed a massively parallel reporter assay to facilitate the functional analysis of individual regulatory sequences from the human genome at a higher resolution than currently existing approaches. This strategy, which can be adapted to other experimental settings, involves the synthesis of tens of thousands of tagged oligonucleotides that contain a library of regulatory elements.
Each oligonucleotide is cloned on a plasmid containing an optional promoter, a regulatory element, and an open reading frame. After transfecting the plasmid pool into cells, the tags on the reporter mRNAs are sequenced and counted to determine their relative activities.
“We can generate many carefully defined mutations of a natural enhancer and determine in parallel, using the sequencing readout, how each mutation changes its activity,” continues Dr. Mikkelsen.
Investigators in Dr. Mikkelsen’s lab illustrated the strength of this strategy with two inducible enhancers, a synthetic cAMP-regulated enhancer, and a virus-inducible enhancer of the human interferon beta gene. After mapping the transcription factor binding sites at single-nucleotide resolution, quantitative models helped identify mutations that increase enhancer inducibility.
Going forward, they plan to insert these synthetic sequences into the genome to examine how they interact with nearby epigenetic marks.
“We still do not understand whether the genetic information always determines the epigenetic landscape, or whether there is inheritance at the epigenetic level that has no basis in the genetic information. The synthetic biology approach is emerging as a very powerful tool for probing these questions,” says Dr. Mikkelsen.