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.