Identifying gene-enhancers: New technique

Diane Dickel is the lead author of Nature Methods paper describing a new technique for identifying gene enhancers in the genomes of humans and other mammals. Credit: Roy Kaltschmidt

An international team led by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) has developed a new technique for identifying gene enhancers -- sequences of DNA that act to amplify the expression of a specific gene -- in the genomes of humans and other mammals. Called SIF-seq, for site-specific integration fluorescence-activated cell sorting followed by sequencing, this new technique complements existing genomic tools, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), and offers some additional benefits.

"While ChIP-seq is very powerful in that it can query an entire genome for characteristics associated with enhancer activity in a single experiment, it can fail to identify some enhancers and identify some sites as being enhancers when they really aren't," says Diane Dickel, a geneticist with Berkeley Lab's Genomics Division and member of the SIF-seq development team. "SIF-seq is currently capable of testing only hundreds to a few thousand sites for enhancer activity in a single experiment, but can determine enhancer activity more accurately than ChIP-seq and is therefore a very good validation assay for assessing ChIP-seq results."

Dickel is the lead author of a paper in Nature Methods describing this new technique. The paper is titled "Function-based identification of mammalian enhancers using site-specific integration." The corresponding authors are Axel Visel and Len Pennacchio, also geneticists with Berkeley Lab's Genomics Division.

With the increasing awareness of the important role that gene enhancers play in normal cell development as well as in disease, there is strong scientific interest in identifying and characterizing these enhancers. This is a challenging task because an enhancer does not have to be located directly adjacent to the gene whose expression it regulates, but can instead be located hundreds of thousands of DNA base pairs away. The challenge is made even more difficult because the activity of many enhancers is restricted to specific tissues or cell types.

"For example, brain enhancers will not typically work in heart cells, which means that you must test your enhancer sequence in the correct cell type," Dickel says.

Currently, enhancers can be identified through chroma­tin-based assays, such as ChIP-seq, which predict enhancer elements indirectly based on the enhancer's association with specific epigenomic marks, such as transcription factors or molecular tags on DNA-associated histone proteins. Visel, Pennacchio, Dickel and their colleagues developed SIF-seq in response to the need for a higher-throughput functional enhancer assay that can be used in a wide variety of cell types and devel­opmental contexts.

"We've shown that SIF-seq can be used to identify enhancers active in cardiomyocytes, neural progenitor cells, and embryonic stem cells, and we think that it has the potential to be expanded for use in a much wider variety of cell types," Dickel says. "This means that many more types of enhancers could potentially be tested in vitro in cell culture."

In SIF-seq, hundreds or thousands of DNA fragments to be tested for enhancer activity are coupled to a reporter gene and targeted into a single, reproducible site in embryonic cell genomes. Every embryonic cell will have exactly one potential enhancer-reporter. 

Fluorescence-activated sorting is then used to identify and retrieve from this mix only those cells that display strong reporter gene expression, which represent the cells with the most active enhancers.

"Unlike previous enhancer assays for mammals, SIF-seq includes the integration of putative enhancers into a single genomic locus," says Visel. "Therefore, the activity of enhancers is assessed in a reproducible chromosomal context rather than from a transiently expressed plasmid. Furthermore, by making use of embryonic stem cells and in vitro differentia­tion, SIF-seq can be used to assess enhancer activity in a wide variety of disease-relevant cell types."

Adds Pennacchio, "The range of biologically or disease-relevant enhancers that SIF-seq can be used to identify is limited only by currently available stem cell differentiation methods. Although we did not explicitly test the activity of species-specific enhancers, such as those derived from certain classes of repetitive elements, our results strongly suggest that SIF-seq can be used to identify enhancers from other mammalian genomes where desired cell types are difficult or impossible to obtain."

The ability of SIF-seq to use reporter assays in mouse embryonic stem cells to identify human embryonic stem cell enhancers that are not present in the mouse genome opens the door to intriguing research possibilities as Dickel explains.

"Human and chimpanzee genes differ very little, so one hypothesis in evolutionary genomics holds that humans and chimpanzees are so phenotypically different because of differences in the way they regulate gene expression. It is very difficult to carry out enhancer identification through ChIP-seq that would be useful in studying this hypothesis," she says. 

"However, because SIF-seq only requires DNA sequence from a mammal and can be used in a variety of cell types, it should be possible to compare the neuronal enhancers present in a large genomic region from human to the neuronal enhancers present in the orthologous chimpanzee region. This could potentially tell us interesting things about the genetic differences that differentiate human brain development from that of other primates."

Source DOE/Lawrence Berkeley National Laboratory

Birth of four foals from genotyped, cryopreserved embryos: A first in Europe

Genotyping allows scientists to choose the embryos they want to use based on different criteria: sex, like in this experiment, the absence of known genetic disorders, or, perhaps in the future, other traits that are tied to behavior, such as emotivity or sociability. Credit: INRA/C. Maitre

IFCE and INRA announced that, for the first time ever in Europe, four foals were successfully born as the result of the transfer of genotyped and cryopreserved embryos. The goal of this work is to better understand embryonic development, control livestock reproduction, and maintain breed genetic diversity. Furthermore, it is advantageous for the horse industry to be able to determine the traits of a future foal.

The INRA Val de Loire center at Nouzilly is where the technology to maintain embryo viability following genotyping and cryopreservation was honed, and then, last summer, the transfer of several embryos took place at the IFCE Haras du Pin Stud Farm, located in the French department of Orne. The partnership between the two institutions has now been cemented by the birth of the healthy foals.

What were the steps leading up to the birth of these foals?

Seven days after fertilization, embryos were collected from Welsh B ponies that are part of INRA's livestock. The embryos were genotyped: scientists sampled some of the embryos' cells to analyze their genomes. In this experiment, embryos were selected based on sex, the idea being to use sex-based selection to test the technique's feasibility. The embryos were then cryopreserved in liquid nitrogen (at -196°). Last summer, they were transferred into saddlebred mares at the Haras du Pin center. After an 11-month gestation period, the foals were born in May. They were of the expected sex: two females and two males. This is the first time that such an event has taken place in Europe, and it is the product of more than 10 years of various types of embryonic research carried out by INRA and IFCE scientists.

What made it so difficult to apply this technique to horses?

Although embryo preservation techniques are already well developed for bovines, small ruminant species, and even humans, preserving horse embryos is a very complex process. For instance, horse embryos vary greatly in size: 7-day-old embryos range in diameter from 200 to 700 micrometers. It is very difficult to cryopreserve the largest embryos because the liquid inside them forms ice crystals when the embryos are frozen at very cold temperatures. What's more, horse embryos are surrounded by a capsule that interferes with successful cryopreservation.

What is the significance of this event, which is the first of its kind in Europe?

There are several reasons why being able to successfully cryopreserve embryos is important. For example, it allows us to maintain breed genetic diversity, particularly that of breeds with small population sizes, such as the Landais or the Poitevin Mulassier. Furthermore, the factor that currently limits the use of embryo transfer is its cost: the transfer center has to maintain a team of recipient mares that are reproductively synchronized with the donor mares. Cryopreservation means that the transfer doesn't have to take place immediately; it can wait until a recipient mare becomes available to receive the embryo. Finally, it may now be possible to directly repopulate horse herds that have experienced losses as a result of various issues, such as disease-related problems, instead of having to use the indirect technique of crossbreeding.

Why genotype the embryos?

Genotyping allows us to choose the embryos we want to use based on different criteria: sex, like in this experiment, the absence of known genetic disorders, or, perhaps in the future, other traits that are tied to behavior, such as emotivity or sociability. It is advantageous for the horse industry to be able to determine the traits of a future foal. We will next aim to simplify the process -- to make this technology more accessible and user friendly for those in the horse industry.

Source: INRA-France

Genetically identical ants help unlock the secrets of larval fate

Cerapachys biroi ants, native to Asia and introduced globally on tropical and subtropical islands, have no queens and have minimal genetic variation, making them ideal for research on social behavior. Credit: Image courtesy of Rockefeller University

A young animal's genes are not the only genes that determine its fate. The genetic identity of its caretakers matters too. Researchers suspect the interaction between the two can sway the fate of the young animal, but this complex dynamic is difficult to pin down in lab experiments.

However, social insect researchers have found a solution. Rockefeller University's Daniel Kronauer, head of the Laboratory of Insect Social Evolution, and his colleagues are developing a species of small raider ants as a model organism in order to ask questions about the relationships between genes, social behavior and evolution.

In a pair of recent papers, the researchers first explain the unique, and potentially useful, biology of this 2.5-millimeter-long ant. Then, in work with collaborators at the University of Paris 13, they put it to work exploring the interaction between the larvae and their nursemaids, and the influence on the young ants' reproductive success as adults.

Clonal raider ants, the species Cerapachys biroi, reproduce by cloning, and they live in colonies of as many as a few hundred nearly genetically identical workers. This makes these ants ideal for studies testing how a particular genetic makeup responds to different conditions, the researchers write in Current Biology. With the help of collaborators at BGI China, researchers in Kronauer's lab have sequenced the clonal raider ant's genome. This is an important step toward using the ant in the sorts of experiments conducted for years in traditional model organisms, such as mice and fruit flies.

"We have shown that colony mates are extremely closely related to one another, with all of the individuals in a colony being essentially genetically identical. This gives us precise control in experiments because we don't need to account for individual genetic variation," says Peter Oxley, a postdoc in the laboratory who led work establishing the clonal raider ant as a promising new model organism.

In the second study, one of the first to make use of the clonal raider ant, a team led by Serafino Teseo of the University of Paris 13 used the unique aspects of the ants' biology to test the indirect role genes play in shaping the future identity of larvae and whole colonies by looking at the interaction between larvae and adults. They did so by observing the success of two ant clones, A and B, in pure colonies or mixed together into chimeric colonies. They also swapped broods, so A adults raised B larvae and vice versa.

It turned out that A and B larvae developed differently depending on whether A or B nurses raised them. Left alone, pure A colonies produced the most young after six generations, making them more successful than B. However, in mixed colonies, B did better because its larvae more frequently turned into large adults that specialize in egg-laying rather than smaller, foraging-focused individuals.

The researchers suspect an indirect genetic effect -- specifically, a social influence. To begin to tease apart the dynamic, they had adults from one clone raise larvae from the other. Again, B did better when raised by A nurses than any of the other combinations. The results were published in Nature Communications.

The B colony's strategy of favoring reproduction over foraging when raised by A colony nurses smacks of social parasitism, in which one organism exploits another's social behavior for its own benefit. "This doesn't mean B is a parasite in the making, just that uncoupling the normal interaction between larvae and their nearly identical adult nursemaids reveals the presence of this mechanism," Kronauer says.

The study shows that, in social species, genetic makeup alone does not provide enough information to predict social behavior. Instead, interactions between social partners, such as larvae and their caregivers, are crucial determinants and can lead to surprising outcomes.

Source: Rockefeller University

PHS gene prevents wheat from sprouting: Fewer crop losses anticipated

Preharvest sprouting can cause significant losses in wheat crops, particularly in white wheat crops. Credit: Kansas State University Photo Services

A new study about the common problem of preharvest sprouting, or PHS, in wheat is nipping the crop-killing issue in the bud.

Researchers at Kansas State University and the U.S. Department of Agriculture-Agricultural Research Service, or USDA-ARS, found and cloned a gene in wheat named PHS that prevents the plant from preharvest sprouting. Preharvest sprouting happens when significant rain causes the wheat grain to germinate before harvest and results in significant crop losses.

"This is great news because preharvest sprouting is a very difficult trait for wheat breeders to handle through breeding alone," said Bikram Gill, university distinguished professor of plant pathology and director of the Wheat Genetics Resource Center. "With this study, they will have a gene marker to expedite the breeding of wheat that will not have this problem."

Gill conducted the study with Guihau Bai, a researcher with the Hard Winter Wheat Genetics Research Unit of the USDA-ARS, adjunct professor of agronomy at Kansas State University and the study's lead author. Also involved were Harold Trick, professor of plant pathology; Shubing Liu, research associate in agronomy; Sunish Sehgal, senior scientist in plant pathology; Jiarui Li, research assistant professor; and Meng Lin, doctoral student in agronomy, all from Kansas State University; and Jianming Yu, Iowa State University.

Their study, "Cloning and Characterization of a Critical Regulator for Pre-Harvest Sprouting in Wheat," appears in a recent issue of the scientific journal Genetics.

The finding will to be most beneficial to white wheat production, which loses $1 billion annually to preharvest sprouting, according to Gill.

He said consumers prefer white wheat to the predominant red wheat because white wheat lacks the more bitter flavor associated with red wheat. Millers also prefer white wheat to red because it produces more flour when ground. The problem is that white wheat is very susceptible to preharvest sprouting.

"There has been demand for white wheat in Kansas for more than 30 years," Gill said. "The very first year white wheat was grown in the state, though, there was rain in June and then there was preharvest sprouting and a significant loss. The white wheat industry has not recovered since and has been hesitant to try again. I think that this gene is a big step toward establishing a white wheat industry in Kansas."

Gill said identifying the PHS gene creates a greater assurance before planting a crop that it will be resistant to preharvest sprouting once it grows a year later. Wheat breeders can now bring a small tissue sample of a wheat plant into a lab and test whether it has the preharvest sprouting resistance gene rather than finding out once the crop grows.

Much of the work to isolate the PHS gene came from Gill and his colleagues' efforts to fully sequence the genome -- think genetic blueprint -- of common wheat. Wheat is the only major food plant not to have its genome sequenced. The genome of wheat is nearly three times the size of the human genome.

Researchers were able to study sequenced segments of the common wheat genome and look for a naturally occurring resistance gene. Gill said without the sequenced segments, finding the PHS gene would have been impossible.

Source: Kansas State University

Advanced biological computer developed

Microprocessor with DNA (illustration). Scientists have developed and constructed an advanced biological transducer, a computing machine capable of manipulating genetic codes, and using the output as new input for subsequent computations Credit: © Giovanni Cancemi / Fotolia

Using only biomolecules (such as DNA and enzymes), scientists at the Technion-Israel Institute of Technology have developed and constructed an advanced biological transducer, a computing machine capable of manipulating genetic codes, and using the output as new input for subsequent computations. The breakthrough might someday create new possibilities in biotechnology, including individual gene therapy and cloning.

The findings appear today (May 23, 2013) in Chemistry & Biology (Cell Press).
Interest in such biomolecular computing devices is strong, mainly because of their ability (unlike electronic computers) to interact directly with biological systems and even living organisms. No interface is required since all components of molecular computers, including hardware, software, input and output, are molecules that interact in solution along a cascade of programmable chemical events.

"Our results show a novel, synthetic designed computing machine that computes iteratively and produces biologically relevant results," says lead researcher Prof. Ehud Keinan of the Technion Schulich Faculty of Chemistry. "In addition to enhanced computation power, this DNA-based transducer offers multiple benefits, including the ability to read and transform genetic information, miniaturization to the molecular scale, and the aptitude to produce computational results that interact directly with living organisms."

The transducer could be used on genetic material to evaluate and detect specific sequences, and to alter and algorithmically process genetic code. Similar devices, says Prof. Keinan, could be applied for other computational problems.

"All biological systems, and even entire living organisms, are natural molecular computers. Every one of us is a biomolecular computer, that is, a machine in which all components are molecules "talking" to one another in a logical manner. The hardware and software are complex biological molecules that activate one another to carry out some predetermined chemical tasks. The input is a molecule that undergoes specific, programmed changes, following a specific set of rules (software) and the output of this chemical computation process is another well defined molecule."

Also contributing to the research were postdoctoral fellows Dr. Tamar Ratner and Dr. Ron Piran of the Technion's Schulich Faculty of Chemistry, and Dr. Natasha Jonoska of the Department of Mathematics at the University of South Florida.

Source:  American Technion Society

Scientists produce cloned embryos of extinct frog

This is a gastric-brooding frog, Rheobatrachus silus, giving oral birth in the lab of Mike Tyler of the University of Adelaide. Credit: Mike Tyler, University of Adelaide

The genome of an extinct Australian frog has been revived and reactivated by a team of scientists using sophisticated cloning technology to implant a "dead" cell nucleus into a fresh egg from another frog species.

The bizarre gastric-brooding frog, Rheobatrachus silus -- which uniquely swallowed its eggs, brooded its young in its stomach and gave birth through its mouth -- became extinct in 1983.

But the Lazarus Project team has been able to recover cell nuclei from tissues collected in the 1970s and kept for 40 years in a conventional deep freezer. The "de-extinction" project aims to bring the frog back to life.

In repeated experiments over five years, the researchers used a laboratory technique known as somatic cell nuclear transfer. They took fresh donor eggs from the distantly related Great Barred Frog, Mixophyes fasciolatus, inactivated the egg nuclei and replaced them with dead nuclei from the extinct frog. Some of the eggs spontaneously began to divide and grow to early embryo stage -- a tiny ball of many living cells.

Although none of the embryos survived beyond a few days, genetic tests confirmed that the dividing cells contain the genetic material from the extinct frog.
The results are yet to be published.

"We are watching Lazarus arise from the dead, step by exciting step," says the leader of the Lazarus Project team, Professor Mike Archer, of the University of New South Wales, in Sydney. "We've reactivated dead cells into living ones and revived the extinct frog's genome in the process. Now we have fresh cryo-preserved cells of the extinct frog to use in future cloning experiments.

"We're increasingly confident that the hurdles ahead are technological and not biological and that we will succeed. Importantly, we've demonstrated already the great promise this technology has as a conservation tool when hundreds of the world's amphibian species are in catastrophic decline."

The technical work was led by Dr Andrew French and Dr Jitong Guo, formerly of Monash University, in a University of Newcastle laboratory led by frog expert, Professor Michael Mahony, along with Mr Simon Clulow and Dr John Clulow. The frozen specimens were preserved and provided by Professor Mike Tyler, of the University of Adelaide, who extensively studied both species of gastric-brooding frog -- R. silus and R. vitellinus -- before they vanished in the wild in 1979 and 1985 respectively.

UNSW's Professor Archer spoke publicly for the first time today about the Lazarus Project and also about his ongoing interest in cloning the extinct Australian thylacine, or Tasmanian tiger, at the TEDx DeExtinction event in Washington DC, hosted by Revive and Restore and the National Geographic Society.

Researchers from around the world are gathered there to discuss progress and plans to 'de-extinct' other extinct animals and plants. Possible candidate species include the woolly mammoth, dodo, Cuban red macaw and New Zealand's giant moa.

Source: University of New South Wales