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Developmental biology - Genetics|
How Junk DNA Switches On Genes
Enhancers switch on the function of other genes. In the mammalian genome [the complete set of genes present in a cell or an entire organism], there are an estimated 200,000 to 1 million enhancers, many located far away on the same DNA strand as the gene they regulate. This raises the question - How do they locate and connect to a target gene?
Many previous studies on enhancers were conducted on non-living cells because of the difficulty in imaging gene activity in living organisms. Such studies give only snapshots in time and can miss important details. In the new study, researchers used imaging techniques developed at Princeton to track the position of an enhancer and its target gene while simultaneously monitoring the gene's activity in living fly (drosophil melanogastor) embryos.
"This study provides the unique opportunity to observe in real time how two regions of DNA interact with each other," explains Michal Levo, postdoctoral research fellow in the Lewis-Sigler Institute. "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."
The videos demonstrate how physical contact between enhancer and gene is needed to activate transcription, which is the first step in reading gene instructions. Enhancers stay connected to their gene the entire time that gene is active. When the enhancer disconnects, gene activity stops.
The team believes a solution for this puzzle may be found in the way DNA is uniquely wrapped within cells. Enhancer and gene may be a half-inch apart if DNA is stretched into a line, but packed tightly into a cell nucleus with other proteins and enhancers, they may be considerably closer.
"Through this study, we can look at the relationship between DNA's structural configurations and gene activation," says Hongtao Chen, postdoctoral research fellow in the Lewis-Sigler Institute, and lead author on the study. The videos provide evidence against a favorite concept - the "hit-and-run model" - where an enhancer doesn't need to be attached to the gene during the transcription process. The team shows that sometimes the enhancer and gene meet and connect, but gene activation doesn't occur, a finding they will continue to explore.
To capture video of an enhancer contacting a gene, Chen attached fluorescent tags to the enhancer and its target gene. The enhancers examined are those of a gene called eve, and gives rise to a pattern of seven stripes on the surface of the developing drosophila embryo after about three hours.
Additionally, Chen 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. Gregor's team at Princeton previously developed a method of adding fluorescent tags to RNA as it is being created to obtain a real-time readout of gene expression in fly embryos.
A long-standing question in gene regulation is how remote enhancers communicate with their target promoters, and specifically how chromatin topology dynamically relates to gene activation. Here, we combine genome editing and multi-color live imaging to simultaneously visualize physical enhancer–promoter interaction and transcription at the single-cell level in Drosophila embryos. 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. We show that sustained proximity of the enhancer to its target is required for activation. Transcription in turn affects the three-dimensional topology as it enhances the temporal stability of the proximal conformation and is associated with further spatial compaction. Furthermore, the facilitated long-range activation results in transcriptional competition at the locus, causing corresponding developmental defects. Our approach offers quantitative insight into the spatial and temporal determinants of long-range gene regulation and their implications for cellular fates.
Hongtao Chen, Michal Levo, Lev Barinov, Miki Fujioka, James B. Jaynes and Thomas Gregor.
We thank K. Bystricky for introducing us to the ParB/parS system, and F. Payre and P. Valenti for sharing a ParB-eGFP plasmid and the parS sequence. We also thank S. Blythe, H. Garcia, H. Grabmayr, T. Fukaya, M. Levine, S. Little, P. Ratchasanmuang, S. Ryabichko, P. Schedl, E.F. Wieschaus, B. Zoller, and the Bloomington Drosophila Stock Center. This study was funded by grants from the National Institutes of Health (U01 EB021239, U01 DA047730, R01 GM097275, R01 GM117458) and from the National Science Foundation (PHY-1734030). H.C. was supported by the Charles H. Revson Biomedical Science Fellowship. M.L. was supported by the Rothschild, EMBO and HFSP fellowships.
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The DNA segment, known as an enhancer (BLUE), as it approaches the gene (GREEN) and activates it (RED). The study reveals that close proximity between enhancer and gene is needed to kickstart gene activity. Image Credit: Hongtao Chen, Princeton University. For 4 videos of these instances scroll to Supplementary Information