After a segment of non-protein–coding DNA called an enhancer gets close to its target gene, it tends to stick around, staying attached to the gene’s promoter while the gene’s transcription proceeds. This finding, which comes from an analysis of real-time imaging of an enhancer–promoter interaction, provides evidence against a favored model of gene activation known as the hit-and-run model, which holds that an enhancer need not stay attached to its target gene’s promoter while the gene is transcribed.
Many studies on enhancers have been conducted on non-living cells because of the difficulty in imaging genetic activity in living organisms. Such studies give only snapshots in time and can miss important details. To acquire moving images, not just snapshots, researchers based at Princeton University combined genome editing and multicolor live imaging to simultaneously visualize physical enhancer–promoter interaction and transcription at the single-cell level in Drosophila embryos.
This work allowed the Princeton researchers to visualize how enhancers find and activate a target gene in the crowded environment of a cell's nucleus. Detailed results from this work appeared July 23 in the journal Nature Genetics, in an article titled, “Dynamic interplay between enhancer–promoter topology and gene activity.”
“By examining transcriptional activation of a reporter by the endogenous even-skipped enhancers, which are located 150 kb away, we identify three distinct topological conformation states and measure their transition kinetics,” wrote the article’s authors. “We show that sustained proximity of the enhancer to its target is required for activation.”
Essentially, the researchers obtained video showing that physical contact between the enhancer and the gene is necessary to activate transcription, the first step in reading genetic instructions. The enhancers stay connected to the gene the entire time it is active. When the enhancer disconnects, gene activity stops.
The researchers also found that during transcription, the structure formed by the enhancer and gene becomes more compact, suggesting a change in the DNA in that region. “Transcription,” the authors of the Nature Genetics article noted, “affects the three-dimensional topology as it enhances the temporal stability of the proximal conformation and is associated with further spatial compaction.”
Enhancers, as their name suggests, switch on the expression of other genes. In the mammalian genome, there are an estimated 200,000 to 1 million enhancers, and many are located far away on the DNA strand from the gene they regulate, raising the question of how the regulatory segments can locate and connect with their target genes.
Given that there can be numerous genes between the enhancer and its target, it is remarkable that enhancers can reach the exact target at the right time for that gene to become active, the researchers said.
The team believes that the solution may be found in the DNA's unique wrapping within our cells. The enhancer and gene may be a half-inch apart when DNA is stretched out in a line, but when packed into the cell, with specific proteins facilitating physical interactions, they could be considerably closer.
“This study provides the unique opportunity to observe in real time how two regions of DNA interact with each other,” said Michal Levo, a coauthor of the current study and a postdoctoral research fellow at the Lewis-Sigler Institute for Integrative Genomics. “We can monitor in time where the enhancer and the gene are physically located and simultaneously measure the gene's activity in an attempt to relate these processes.”
To capture video of an enhancer contacting a gene, the researchers attached fluorescent tags to the enhancer and its target gene. The enhancers examined are those of a gene called eve, and they give rise to a pattern of seven stripes that forms on the surface of the developing embryo after about three hours.
Additionally, the researchers attached a separate fluorescent tagging system to the target gene that lights up when the gene is activated and undergoes transcription to produce an intermediary readout of the genetic code, a molecule called RNA.
Analyses of how enhancers activate genes can aid in the understanding of normal development, when even small genetic missteps can result in birth defects. The timing of gene activation also is important in the development of many diseases including cancer.
“The key to curing such conditions is our ability to elucidate underlying mechanisms,” said Thomas Gregor, the current study’s senior author and an associate professor of physics at the Lewis-Sigler Institute for Integrative Genomics. “The goal is to use these rules to regulate and re-engineer the programs underlying development and disease processes.”
“Facilitated long-range activation results in transcriptional competition at the locus, causing corresponding developmental defects,” the authors of the current study concluded. “Our approach offers quantitative insight into the spatial and temporal determinants of long-range gene regulation and their implications for cellular fates.”