The strategy promises to clarify the longstanding mystery of the role played by vast stretches of DNA sequence that do not code for the functional units—genes—that nevertheless may have a powerful regulatory influence. The research is described in the 12 February edition of the journal Nature.
"Our approach employs next generation sequencing technology to find regulatory regions, the 'switches' on a genome-wide scale and much more cost effectively," said Pennacchio. "It's the next layer of knowledge that's been missing."
The DOE JGI was founded in 1997 to accelerate the completion of the HGP and completed the DOE's commitment to sequence three (5, 16, 19) of the 23 chromosomes, totaling 11 percent of the human genome, and published the analysis in Nature back in 2004.
In this newly published study, Pennacchio, lead authors DOE scientist Axel Visel and postdoctoral fellow Matthew Blow, and their colleagues, describe a shortcut for identifying gene regulatory regions or the molecular switches that turn on or off gene expression.
Using what's called ChIP-Sequencing or ChIP-Seq, chromatin immunoprecipitation (ChIP) is combined with massively parallel DNA sequencing to identify binding sites of DNA-associated proteins.
Traditionally researchers have relied on evolution to guide them to non-coding sequences that are likely to have a function—such as enhancing the expression of genes. Via the public genome databases, they would align the entire human genome code with that of other vertebrate species (e.g. other mammals, birds, frogs, fish) and then look for sequences that are conserved in evolution.
"Most protein-coding sequences show signs of conservation between species, but there is also a large number of non-coding sequences that have been surprisingly well conserved for tens or even hundreds of millions of years," said Visel. "This suggests that these regions, formerly thought to be "junk" DNA, actually have some functional relevance and are under selection because sequence changes reduce fitness of affected individuals. Using such sequence conservation, we have in previous studies identified enhancer candidate regions and shown in transgenic mouse experiments that these conserved non-coding regions are in fact often enhancers that are active during embryonic development. Conservation-based methods are relatively good at finding enhancers in the genome, but an important limitation is that they don't tell us where and when that particular enhancer would be active and thereby drive the expression of its neighboring target gene(s).
The older methods lacked specificity, Blow said. "For example, if we have a gene that is important both for brain and for limb development, we would not have been able to specifically identify the enhancer sequences near that gene that would drive the expression in the brain or limb, the only way to find out was to test these activities in experiments one-by-one, which is slow and can't be done on a genomic scale.
"Using this new method, we can directly identify a genome-wide set of enhancers that are active in a particular anatomical region or tissue at a particular time-point, which is an important advantage over conservation-based methods because in addition to telling us where an enhancer is located in the genome, it also provides an initial experimental characterization where we should expect this enhancer to be active."
The team used ChIP linked with a particular enhancer-associated protein, p300, then directed DOE JGI's massively parallel next generation sequencing capacity to map several thousand sites in mouse embryonic forebrain, midbrain and limb tissue. Over 80 of these fragments were tested in transgenic mouse experiments indicating an almost perfect success rate of p300-ChIP-Seq for identifying enhancers active in vivo.
"Enhancers are especially important for regulating genes during embryonic development," said Pennacchio. "They can regulate genes over long distances and switch on their target genes during very specific time-points and in very specific anatomical structures during development. There are several examples of mutations in such enhancers that cause disease in humans because genes are not expressed at the right time or in the right place anymore. A fundamental problem in studying such enhancers is that until recently we did not have effective tools to even find them in the genome on a large scale.
Pennacchio said that this new method will prove useful to the greater genomics and biomedical community for characterizing the role of the vast non-coding regions—dubbed genome "dark matter"—about which little is known.
"These datasets will also help to identify mutations in enhancers that play a role in human disease," Pennacchio said. "Human genetic studies indicate that in many cases disease is caused by mutations in non-coding sequences, but it has been difficult to study this in detail because the function of most non-coding sequences is poorly understood. Eventually, this will be useful for purposes including disease detection and personalized medicine."
With the rapidly increasing efficiency and cost-savings of the next generation sequencing technologies, a deluge of data from individual human genomes are being to come to light, to the point where whole-genome sequencing of patients may soon become a standard diagnostic tool.
"While progress is being made towards this goal, it is important to keep in mind that our current understanding of the genome has focused on protein-coding sequences," said Pennacchio. "Datasets like the one provided through this study will be important to understand the remaining 98 percent of the genome and what its role in health and disease is."
The published study provides an important proof of principle to establish and validate a new method in three different mouse tissues at a single embryonic time-point, Pennacchio said. "We can now generate genome-wide enhancer datasets directly from human tissues and compare genome-wide sets of enhancer activities between healthy people and people suffering from disease, which may reveal how enhancer activities change on a global scale in these disease states."
David Gilbert | EurekAlert!
Further reports about: > ChIP-Seq > ChIP-Sequencing > DNA > DNA sequence > DNA-associated proteins > Genom > Nature Immunology > biofuels production > chromatin immunoprecipitation > embryonic development > human genome > molecular tools > multicellular organisms > non-coding sequence > on-off switches in genomes > protein-coding genes
Make way for the mini flying machines
21.03.2018 | American Chemical Society
New 4-D printer could reshape the world we live in
21.03.2018 | American Chemical Society
In just a few weeks from now, the Chinese space station Tiangong-1 will re-enter the Earth's atmosphere where it will to a large extent burn up. It is possible that some debris will reach the Earth's surface. Tiangong-1 is orbiting the Earth uncontrolled at a speed of approx. 29,000 km/h.Currently the prognosis relating to the time of impact currently lies within a window of several days. The scientists at Fraunhofer FHR have already been monitoring Tiangong-1 for a number of weeks with their TIRA system, one of the most powerful space observation radars in the world, with a view to supporting the German Space Situational Awareness Center and the ESA with their re-entry forecasts.
Following the loss of radio contact with Tiangong-1 in 2016 and due to the low orbital height, it is now inevitable that the Chinese space station will...
Fraunhofer Institute for Organic Electronics, Electron Beam and Plasma Technology FEP, provider of research and development services for OLED lighting solutions, announces the founding of the “OLED Licht Forum” and presents latest OLED design and lighting solutions during light+building, from March 18th – 23rd, 2018 in Frankfurt a.M./Germany, at booth no. F91 in Hall 4.0.
They are united in their passion for OLED (organic light emitting diodes) lighting with all of its unique facets and application possibilities. Thus experts in...
A new scenario seeking to explain how Mars' putative oceans came and went over the last 4 billion years implies that the oceans formed several hundred million...
For the first time, an interdisciplinary team from the University of Basel has succeeded in integrating artificial organelles into the cells of live zebrafish embryos. This innovative approach using artificial organelles as cellular implants offers new potential in treating a range of diseases, as the authors report in an article published in Nature Communications.
In the cells of higher organisms, organelles such as the nucleus or mitochondria perform a range of complex functions necessary for life. In the networks of...
Animal photoreceptors capture light with photopigments. Researchers from the University of Göttingen have now discovered that these photopigments fulfill an...
19.03.2018 | Event News
16.03.2018 | Event News
13.03.2018 | Event News
21.03.2018 | Physics and Astronomy
21.03.2018 | Materials Sciences
21.03.2018 | Life Sciences