Master Orchestrator of the Genome Is Discovered

Master Orchestrator of the Genome Is Discovered

Thursday, 07 May 2015

One of developmental biology’s most perplexing questions concerns what signals transform masses of undifferentiated cells into tremendously complex organisms, a process called ontogeny.

UB research suggests a new paradigm, visualized

in this diagram, for developmental global genome

programming by the nuclear FGFR1 protein.

New research by University at Buffalo scientists, published last week in PLOS ONE, provides evidence that it all begins with a single “master” growth factor receptor that regulates the entire genome.

“The finding provides a new level of understanding of the fundamental aspects of how organisms develop,” says senior author Michal K. Stachowiak, PhD, professor in the Department of Pathology and Anatomical Sciences in the UB School of Medicine and Biomedical Sciences and senior author. He also directs the Stem Cell Engraftment and In Vivo Analysis Facility and the Stem Cell Culture and Training Facility at the Western New York Stem Cell Culture and Analysis Center at UB.

“Our research shows how a single growth factor receptor protein moves directly to the nucleus in order to program the entire genome,” he said.

Michal Stachowiak, PhD, Professor, Department

of Pathology and Anatomical Sciences. 

The research challenges a long-held supposition in biology that specific types of growth factors only functioned at a cell’s surface. For two decades, Stachowiak’s team has been intrigued by the possibility that growth factors function from within the nucleus, a point, he says, this current paper finally proves.

A more advanced understanding of how organisms form, based on this work, has the potential to significantly enhance the understanding and treatment of cancers, which result from uncontrolled development as well as congenital diseases, the researchers say. The new research also will contribute to the understanding of how stem cells work.

This work was conducted on mouse embryonic stem cells, not human cells.

Organizing ‘this cacophony of genes’

“We’ve known that the human body has almost 30,000 genes that must be controlled by thousands of transcription factors that bind to those genes,” Stachowiak said, “yet we didn’t understand how the activities of genes were coordinated so that they properly develop into an organism.”

“Now we think we have discovered what may be the most important player, which organizes this cacophony of genes into a symphony of biological development with logical pathways and circuits,” he said.

At the centre of the discovery is a single protein called nuclear Fibroblast Growth Factor Receptor 1 (nFGFR1).

“FGFR1 occupies a position at the top of the gene hierarchy that directs the development of multicellular animals,” said Stachowiak.

The FGFR1 gene is known to govern gastrulation, occurring in early development, where the three-layered embryonic structure forms. It also plays a major role in the development of the central and peripheral nervous systems and the development of the body’s major systems, including muscles and bones.

To study how nuclear FGFR1 worked, the UB team used genome-wide sequencing of mouse embryonic stem cells programmed to develop cells of the nervous system, with additional experiments in which nuclear FGFR1 was either introduced or blocked. The researchers found that the protein was responsible, either alone or with so-called partner nuclear receptors, for ensuring that embryonic stem cells develop into differentiated cells. By targeting thousands of genes, it controls the development of the major points of growth in the body (known as axes) as well as neuronal and muscle development.

The research shows that nuclear FGFR1 binds to promoters of genes that encode transcription factors, the proteins that control which genes are turned on or off in the genome.

“We found that this protein works as a kind of ‘orchestration factor,’ preferably targeting certain gene promoters and enhancers. The idea that a single protein could bind thousands of genes and then organize them into a hierarchy, that was unknown,” Stachowiak said.

“Nobody predicted it.”

Sequencing advances

The discovery that a single protein can exert such a global genomic function stems from recent advances in DNA sequencing technologies, which allow for the sequencing of a complex genome in just hours.

“NextGen DNA sequencing allows us to analyse millions of DNA sequences selected by the interacting protein,” Stachowiak said.

In the UB research, the DNA sequencing data were processed by the supercomputer at the university’s Center for Computational Research (CCR). Stachowiak and his colleagues then spent weeks aligning these data to the genome and conducting further analyses.

“We imposed nuclear FGFR1 on every little corner of genome,” he said.

“The computer spit out which genes are affected by nuclear FGFR1: it was an enormously complex network of genome activity.”

They found that the protein binds to genes that make neurons and muscles as well as to an important oncogene, TP63, which is involved in a number of common cancers.

Other studies in Stachowiak’s laboratory demonstrate that these interactions also take place in the human genome, controlling function and possibly underlying diseases like schizophrenia. Targeting of the nuclear FGFR1 allows for the reactivation of neural development in the adult brain in preclinical studies and thus, Stachowiak says, may offer unprecedented opportunity for regenerative medicine. Nuclear accumulation of nuclear FGFR1 may be altered in some cancer cells, and thus could become a focus in cancer therapy, he added.  

“This seminal discovery lends new perspectives to the origin, nature and treatment of a variety of human disease,” Stachowiak concluded.

Source: University at Buffalo, New York

Contact: Ellen Goldbaum

Reference:

Global Developmental Gene Programing Involves a Nuclear Form of Fibroblast Growth Factor Receptor-1 (FGFR1)

Christopher Terranova,Sridhar T. Narla, Yu-Wei Lee, Jonathan Bard, Abhirath Parikh, Ewa K. Stachowiak, Emmanuel S. Tzanakakis, Michael J. Buck, Barbara Birkaya, Michal K. Stachowiak

PLoS ONE 2015 10(4):e0123380, doi:10.1371/journal.pone.0123380

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Mobile DNA Sequencer Shows Potential for Disease Surveillance

A pocket-sized device that can rapidly determine the sequence of an organism's DNA has shown its potential in disease detection, according to a study published in the open access, open data journal GigaScience

Thursday, 26 March 2015

This is a close up of MinION.
Credit: Andrew Kilianski.

A pocket-sized device that can rapidly determine the sequence of an organism's DNA has shown its potential in disease detection, according to a study published in the open access, open data journal GigaScience.

In the first analysis of its kind, researchers were able to use the device to accurately identify a range of closely-related bacteria and viruses within six hours, demonstrating the potential for this technology to be used as a mobile diagnostic clinic during outbreaks.

The MinION™ 'Nanopore sequencer' is a low-cost palm-sized sequencing device from Oxford Nanopore Technologies that has been made available to some research groups for testing. It is powered and operated via a USB connection plugged into a laptop, which means that it could potentially be used for on-site clinical analyses in remote locations, negating the need for samples to be sent off to laboratories.

Lead author Andrew Kilianski from Edgewood Chemical Biological Center, USA, whose team tested the device in joint collaboration with Signature Science, LLC, said:

"Our findings are important because we have for the first time communicated to the community that this technology can be incredibly useful in its current state.”

"Being able to accurately identify and characterize strains of viruses and bacteria using a mobile platform is attractive to anyone collecting biological samples in the field. And we expect that as the technology improves, the sequencing will generally become cheaper, faster and more accurate, and could have further clinical applications."

This image shows MinIONs and 

laptops. Credit: Scott Edmunds.

The researchers were able to use the MinION™ to accurately identify and differentiate viral and bacterial species from samples. Within six hours, the device generated sufficient data to identify an E. coli sample down to species level, and three poxviruses (cowpox, vaccinia-MVA, and vaccinia-Lister) down to strain level. The device was able to distinguish between the two vaccinia strains despite them being closely related and over 98% similar to each other.

The technology relies on protein 'nanopores' to determine the sequence of a strand of DNA. At the core of the protein is a hollow tube only a few nanometres in diameter, through which a single DNA strands can pass. As the DNA strand passes through the nanopore, it causes characteristic electrical signatures, from which bases can be identified, and the sequence of the strand determined.

Despite MinION™'s observed read error rate of 30%, which is higher than that of other DNA sequencing methods, the team was able to overcome some of the current limitations by utilizing an approach based on amplified DNA (an 'amplicon' approach). This allowed them to confidently differentiate between closely-related strains.

The amplicon approach allows for the analysis of more complex mixed samples containing a range of organisms in a short runtime. For whole genome sequencing approaches in less pure samples, they note that improvements will need to be made as the technology matures.

The authors state it would be difficult to accurately characterize pathogens within a complex sample in six hours without applying the amplicon methodology.

Source: BioMed Central

Contact: Joel Winston

Reference:

Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer

Andy Kilianski, Jamie L Haas, Elizabeth J Corriveau, Alvin T Liem, Kristen L Willis, Dana R Kadavy, C Nicole Rosenzweig and Samuel S Minot

GigaScience 2015, DOI: 10.1186/s13742-015-0051-z

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The ISSCR Issues Statement on Human Germ Line Genome Modification

The International Society for Stem Cell Research has released a statement calling for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice

Sunday, 22 March 2015

In a statement released on Thursday, the International Society for Stem Cell Research called for a moratorium on attempts at clinical application of nuclear genome editing of the human germ line to enable more extensive scientific analysis of the potential risks of genome editing and broader public discussion of the societal and ethical implications.

Technologies used to introduce changes into the DNA sequence of cells have advanced rapidly, making genome editing increasingly simple. Genome editing is feasible, not just in the somatic cells of an adult organism, but also in early embryos, as well as the gametes (sperm and egg) that carry the inheritable, germ line DNA. Research involving germ line nuclear genome editing has been performed to date in many organisms, including mice and monkeys, and applications to human embryos are possible.

The ISSCR statement raises significant ethical, societal and safety considerations related to the application of nuclear genome editing to the human germ line in clinical practice. Current genome editing technologies carry risks of unintended genome damage, in addition to unknown consequences. Moreover, consensus is lacking on what, if any, therapeutic applications of germ line genome modification might be permissible.

The statement calls for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice, as scientists currently lack an adequate understanding of the safety and potential long term risks of germ line genome modification. Moreover, the ISSCR asserts that a deeper and more rigorous deliberation on the ethical, legal and societal implications of any attempts at modifying the human germ line is essential if its clinical practice is ever to be sanctioned.

In calling for the above moratorium, the ISSCR is not taking a position on the clinical testing of mitochondrial replacement therapy, a form of germ line modification that entails replacing the mitochondria (found outside the nucleus) in the eggs of women at risk of transmitting certain devastating diseases to their children.

Source: International Society for Stem Cell Research

Contact: Michelle Quivey

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Genome's Tale of 'Conquer and Enslave'

Genome's Tale of 'Conquer and Enslave'

Saturday, 21 February 2015

Toronto scientists uncovered how viral remnants helped shape control of our genes.

If genes were lights on a string of DNA, the genome would appear as an endless flicker, as thousands of genes come on and off at any given time. Tim Hughes, a Professor at the University of Toronto's Donnelly Centre, is set on figuring out the rules behind this tightly orchestrated light-show, because when it fails, disease can occur.

This is Professor Tim Hughes of University of

Toronto's Donnelly Centre for Cellular and

Biomolecular Research. Credit: University of

Toronto.

Genes are switched on or off by proteins called transcription factors. These proteins bind to precise sites on the DNA that serve as guideposts, telling transcription factors that their target genes are nearby.

In their latest paper, published in Nature Biotechnology, Hughes and his team did the first systematic study of the largest group of human transcription factors, called C2H2-ZF.

Despite their important roles in development and disease, these proteins have been largely unexplored because they posed a formidable challenge for researchers.

C2H2-ZF transcription factors count over 700 proteins - around three per cent of all human genes! To make matters more complicated, most human C2H2-ZF proteins are very different from those in other organisms, like those in mice. This means that scientists could not apply insights gained from animal studies to human C2H2-ZFs.

Hughes' team found something remarkable: the reason C2H2-ZFs are so abundant and diverse - which makes them difficult to study - is that many of them evolved to defend our ancestral genome from damage caused by the notorious "selfish DNA."

Selfish DNA are bits of parasitic DNA whose only purpose is to multiply, a kind of virus for our genome. They seize a cell's resources to make copies of themselves, which they insert randomly across the genome - causing harmful mutations along the way.

Almost half the human genome is made of selfish DNA, which probably came from ancient retro-viruses which, similar to modern counterparts, inserted their DNA into the host's genome. When this happens in an egg or sperm, the viral DNA gets passed on to the next generation, and the selfish DNA is then known as endogenous retro-elements (EREs).

Evolutionary biologists believe that selfish DNA was instrumental in making genomes bigger, giving natural selection additional DNA material to tinker with.

But Hughes' data suggest that EREs took centre stage in an evolutionary arms race, and that this change spawned C2H2-ZFs, a new group of proteins.

It is an enthralling tale of "conquer and enslave," one that stretches from before mammals existed to the present day.

Hughes says that C2H2-ZFs initially evolved to switch off EREs. As new EREs invaded the genome of our lizard-like ancestor, new C2H2-ZFs arose to prevent them from disrupting gene function.

This would explain how the C2H2-ZF came to be so abundant but also why they are so diverse among different organisms.

"What I think was not appreciated until this study is that retro-elements are really a driving force in the evolution of transcription factors themselves. All mammals have a whole bunch of custom transcription factors that came about to silence the EREs," says Hughes, who is also a Professor in U of T's Department of Molecular Genetics and a Senior Fellow of the Canadian Institute for Advanced Research.

"But the EREs and these new transcription factors are different even for different vertebrates."

These EREs are now harmless because they are millions of years old. Over time they accumulated mutations, which pepper the genome at a constant rate, and, as a result, lost their ability to multiply and move around.

The C2H2-ZFs, on the other hand, took on new jobs.

C2H2-ZF proteins began using the EREs scattered across the genome as DNA docking sites, from which they could take control of nearby genes. The conquered EREs were finally enslaved.

Hughes describes a neat example of this process. One C2H2-ZF family member, a transcription factor called ZNF189 evolved to silence an ancient retro-element, known as LINE L2, which is a staggering 100 million years old. L2 is now inactive but ZNF189 still binds to it because it uses L2 remnants to reach other genes.

Relics of L2 sequences happen to be near genes that drive brain and heart development. And so ZNF189 could take on a new role in shaping these organs, an arrangement preserved by natural selection because it was beneficial to the embryo.

ZFN189 likely puts "breaks" on the "brain genes", similar to its ancient role with L2. But in heart cells, it may actually turn genes on because it misses the part that makes the "off switch."

This story is a beautiful example of just how malleable genomes are at the hands of evolution.

Source: University of Toronto

Contact: Jovana Drinjakovic

Reference:

C2H2 zinc finger proteins greatly expand the human regulatory lexicon

Najafabadi HS, Mnaimneh S, Schmitges FW, Garton M, Lam KN, Yang A, Albu M, Weirauch MT, Radovani E, Kim PM, Greenblatt J, Frey BJ, Hughes TR

Nat Biotechnol. 2015 Feb 18; doi:10.1038/nbt.3128

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NIH-Supported Researchers Map Epigenome of More Than 100 Tissue and Cell Types

NIH-Supported Researchers Map Epigenome of More Than 100 Tissue and Cell Types

Thursday, 19 February 2015

Much like mapping the human genome laid the foundations for understanding the genetic basis of human health, new maps of the human epigenome may further unravel the complex links between DNA and disease. The epigenome is part of the machinery that helps direct how genes are turned off and on in different types of cells.

Researchers supported by the National Institutes of Health Common Fund's Roadmap Epigenomics Program have mapped the epigenomes of more than 100 types of cells and tissues, providing new insight into which parts of the genome are used to make a particular type of cell. The data, available to the biomedical research community, can be found at the National Center for Biotechnology Information website.

Reference epigenomes are available for more than 100 cell and tissue types.
Credit: Image courtesy of Nature and Roadmap Epigenomics Consortium. 

"This represents a major advance in the ongoing effort to understand how the 3 billion letters of an individual's DNA instruction book are able to instruct vastly different molecular activities, depending on the cellular context," said NIH Director Francis Collins, M.D., Ph.D.

"This outpouring of data-rich publications, produced by a remarkable team of creative scientists, provides powerful momentum for the rapidly growing field of epigenomics."

Researchers from the NIH Common Fund's Roadmap Epigenomics Program published a description of the epigenome maps in the journal Nature. More than 20 additional papers, published in Nature and Nature-associated journals, show how these maps can be used to study human biology.

"What the Roadmap Epigenomics Program has delivered is a way to look at the human genome in its living, breathing nature from cell type to cell type," said Manolis Kellis, Ph.D., professor of computer science at the Massachusetts Institute of Technology, Cambridge, and senior author of the paper.

Understanding epigenomics

Almost all human cells have identical genomes that contain instructions on how to make the many different cells and tissues in the body. During the development of different types of cells, regulatory proteins turn genes on and off and, in doing so, establish a layer of chemical signatures that make up the epigenome of each cell. In the Roadmap Epigenomics Program, researchers compared these epigenomic signatures and established their differences across a variety of cell types. The resulting information can help us understand how changes to the genome and epigenome can lead to conditions such as Alzheimer's disease, cancer, asthma, and foetal growth abnormalities.

An epigenomic signature can be made on the

genome in two ways, both of which play a role in

turning genes off or on. The first occurs when

chemical tags called methyl groups are attached

to a DNA molecule directly (DNA methylation).

The second occurs when a variety of chemical

tags attach to the tails of histone proteins that

package DNA (histone modifications). Credit:

Image courtesy of John Stamatoyannopoulos

and Rae Senarighi. 

The value of epigenomic data

Researchers can now take data from different cell types and directly compare them.

"Today, sequencing the human genome can be done rapidly and cheaply, but interpreting the genome remains a challenge," said Bing Ren, Ph.D., professor of cellular and molecular medicine at the University of California, San Diego, and co-author of the Nature paper and several of the associated papers.

"These 111 reference epigenome maps are essentially a vocabulary book that helps us decipher each DNA segment in distinct cell and tissue types. These maps are like snapshots of the human genome in action."

"This is the most comprehensive catalogue of epigenomic data from primary human cells and tissues to date," said Lisa Helbling Chadwick, Ph.D., project team leader and a program director at the National Institute of Environmental Health Sciences (NIEHS), part of NIH.

"This coordinated effort, along with uniform data processing, makes it much easier for researchers to make direct comparisons across the entire data set."

"Researchers from the 88 projects supported by the program, including those from this recent series of papers, have propelled the development of new epigenomic technologies," said John Satterlee, Ph.D., co-coordinator of the Roadmap Epigenomics Program, and program director at the National Institute on Drug Abuse (NIDA), part of NIH. Satterlee added that the work of this program has served as a foundation for continued exploration of the human epigenome through the International Human Epigenome Consortium.

"With this increased understanding of the full epigenome, and the datasets available to the entire scientific community, the NIH Common Fund is striving to catalyse future research, to aid the understanding of how epigenomics plays a role in human diseases, with the expectation that further studies will identify early indications of disease and targets for therapeutics," said James Anderson, M.D., Ph.D., director of NIH Division of Program Coordination, Planning, and Strategic Initiatives that oversees the NIH Common Fund.

NIDA, NIEHS, and the National Institute on Deafness and Other Communication Disorders are co-administrators of the NIH Common Fund's Epigenomics Program.

Source: National Institute of Environmental Health Sciences

Contact: Robin Mackar

Reference:

Integrative analysis of 111 reference human epigenomes

Roadmap Epigenomics Consortium

Nature, (2015); doi: 10.1038/nature14248

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Why is a Dolphin Not a Cat?

How repurposing non-coding elements in the genome gave rise to the great 'mammalian radiation'

Friday, 30 January 2015

Exploring gene regulation in 20 mammals

provides insights into the mammalian radiation

that occurred over 100 million years ago. Credit:

Spencer Phillips, EMBL-EBI.

New research shows how evolution has given rise to a rich diversity of species by repurposing functional elements shared by all mammals. Published in Cell by scientists at the European Bioinformatics Institute (EMBL-EBI) and the University of Cambridge Cancer Research UK-Cambridge Institute (CRUK CI), the study demonstrates how methods for understanding human biology can be used to understand a broad range of species.

Mammals all share a common ancestor, and they share a lot of the same genes. So what exactly makes a dolphin not a cat, and how did we all start to diverge from one another millions of years ago? Part of the answer lies in how - and when - genes are regulated. This latest research explores the evolution of gene regulation in 20 mammalian species, and provides deep insights into the 'mammalian radiation', a time of rapid morphological evolution that occurred shortly after the asteroid impact that caused the extinction of the dinosaurs.

Leveraging findings from a study comparing the genome sequences of 29 mammals, and with the help of conservation organisations such as the UK Cetacean Strandings Investigation Programme and the Copenhagen Zoo, the team were able to study and compare gene regulation in liver cells from 20 key species including the naked mole rat, human, Tasmanian devil, dolphin and Sei whale.

"What we've shown is that evolution repurposes things that exist in all species, to make each species unique," explains Paul Flicek, head of Vertebrate Genomics at EMBL-EBI.

"By looking at gene promoters and enhancers in many different mammals, we demonstrated that species-specific enhancers come from ancient DNA - that evolution captures DNA that's been around for a long time, and uses it for gene regulation in specific tissues."

Evolution has two ways to turn changes in the genome into differences between species: it can change a protein sequence, or it can change the way promoters or enhancers control that protein's expression. Today's study also shows that in some cases evolution uses both strategies at once. When amino acid sequences evolve very quickly, important regulation changes occur at the same time: the protein-coding sequence and the corresponding regulatory sequence change synergistically.

Gathering the samples - the experimental efforts were led by Diego Villar of CRUK CI - took well over two years, and the experiments themselves produced a staggering volume of data. Analysing the results brought the team to a new frontier in bioinformatics.

"People spend a lot of time and money trying to understand human biology, so most of the tools we have are designed to study human genomes," explains Camille Berthelot of EMBL-EBI, who led the computational work.

"The reference data we have for the less studied species, like the Sei whale or Tasmanian devil, are nothing like the pored-over datasets we have for the human genome. A lot of what we did involved benchmarking, and making sure the methods and algorithms were fit for this kind of comparison."

"What inspired this work was a desire to get on top of the mountain, look out and see what is going on in the landscape of molecular evolution across the breadth of mammalian space," says Duncan Odom of CRUK CI and Wellcome Trust Sanger Institute.

"What's exciting about this study is that we now know we can start to answer questions about the functional genetics of many under-explored species - questions we usually can ask only of humans and mice. We can use tools developed to study humans to understand the biology of all kinds of animals, whether they're blackbirds or elephants, and explore their relationship with one another. This research has given us new insights into mammalian evolution, and proven how powerful these methods can be."

Source: EMBL

Contact: Mary Todd Bergman

Reference:

Enhancer Evolution across 20 Mammalian Species

Diego Villar, Camille Berthelot, Sarah Aldridge, Tim F. Rayner, Margus Lukk, Miguel Pignatelli, Thomas J. Park, Robert Deaville, Jonathan T. Erichsen, Anna J. Jasinska, James M.A. Turner, Mads F. Bertelsen, Elizabeth P. Murchison, Paul Flicek, Duncan T. Odom

Cell, Volume 160, Issue 3, p554–566, 29 January 2015, DOI: http://dx.doi.org/10.1016/j.cell.2015.01.006

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Human Genome Was Shaped by an Evolutionary Arms Race with Itself

New study of primate genomes reveals an ongoing battle to control 'jumping genes,' driving the evolution of greater genomic complexity

Sunday, 28 September 2014

New findings by scientists at the University of California, Santa Cruz, suggest that an evolutionary arms race between rival elements within the genomes of primates drove the evolution of complex regulatory networks that orchestrate the activity of genes in every cell of our bodies.

The arms race is between mobile DNA sequences known as "retrotransposons" (a.k.a. "jumping genes") and the genes that have evolved to control them. The UC Santa Cruz researchers have, for the first time, identified genes in humans that make repressor proteins to shut down specific jumping genes. The researchers also traced the rapid evolution of the repressor genes in the primate lineage.

Their findings, published September 28 in Nature, show that over evolutionary time, primate genomes have undergone repeated episodes in which mutations in jumping genes allowed them to escape repression, which drove the evolution of new repressor genes, and so on. Furthermore, their findings suggest that repressor genes that originally evolved to shut down jumping genes have since come to play other regulatory roles in the genome.

"We have basically the same 20,000 protein-coding genes as a frog, yet our genome is much more complicated, with more layers of gene regulation. This study helps explain how that came about," said Sofie Salama, a research associate at the UC Santa Cruz Genomics Institute who led the study.

Retrotransposons are thought to be remnants of ancient viruses that infected early animals and inserted their genes into the genome long before humans evolved. Now they can only replicate themselves within the genome. Depending on where a new copy gets inserted into the genome, a jumping event can disrupt normal genes and cause disease. Often the effect is neutral, simply adding to the overall size of the genome. Very rarely the effect might be advantageous, because the added DNA can itself be a source of new regulatory elements that enhance gene expression. But the high probability of deleterious effects means natural selection favours the evolution of mechanisms to prevent jumping events.

Scientists estimate that jumping genes or "transposable elements" account for at least 50 percent of the human genome, and retrotransposons are by far the most common type.

"There have been successive waves of retrotransposon activity in primate evolution, when a transposable element changed to become expressed and replicated itself throughout the genome until something turned it off," Salama said.

"We've discovered a major mechanism by which the genome is able to shut down these mobile DNA elements."

The repressors identified in the new study belong to a large family of proteins known as "KRAB zinc finger proteins." These are DNA-binding proteins that repress gene activity, and they constitute the largest family of gene-regulating proteins in mammals. The human genome has over 400 genes for KRAB zinc finger proteins, and about 170 of them have emerged since primates diverged from other mammals.

According to Salama, her team's findings support the idea that expansion of this family of repressor genes occurred in response to waves of retrotransposon activity. Because repression of a jumping gene also affects genes located near it on the chromosome, the researchers suspect that these repressors have been co-opted for other gene-regulatory functions, and that those other functions have persisted and evolved long after the jumping genes the repressors originally turned off have degraded due to the accumulation of random mutations.

"The way this type of repressor works, part of it binds to a specific DNA sequence and part of it binds other proteins to recruit a whole complex of proteins that creates a repressive landscape in the genome. This affects other nearby genes, so now you have a potential new layer of regulation available for further evolution," Salama said.

KRAB zinc finger proteins are the subject of intensive research as scientists try to sort out their many regulatory roles within the genome. The idea that they are involved in repression of jumping genes is not new – previous studies by other researchers have shown that these proteins silence jumping genes in mouse embryonic stem cells. But until now, no one had been able to demonstrate that the same thing occurs in human cells.

The UC Santa Cruz team developed a novel assay to test whether a particular KRAB zinc finger protein could shut down certain jumping genes. The first authors of the paper, postdoctoral researcher Frank Jacobs and graduate student David Greenberg, came up with the strategy of testing primate retrotransposons in non-primate cells by using mouse embryonic stem cells that contain a single human chromosome. In the environment of a mouse cell, jumping genes that were repressed in primate cells became active. Greenberg then developed an assay for testing individual zinc finger proteins for their ability to turn off a primate jumping gene in the mouse cell environment.

"We did all our tests in mouse cells because they lack all of the primate zinc finger proteins, so when you put primate retrotransposons into a mouse cell they're all active," Salama explained.

The results demonstrated that two human proteins called ZNF91 and ZNF93 bind and repress two major classes of retrotransposons (known as SVA and L1PA) that are currently or recently active in primates. Assistant research scientist Benedict Paten directed graduate student Ngan Nguyen in a painstaking analysis of primate genomes, including the reconstruction of ancestral genomes, which showed that ZNF91 underwent structural changes 8 to 12 million years ago that enabled it to repress SVA elements.

Experiments with ZNF 93, which shuts down L1PA retrotransposons, provided a striking illustration of the arms race between jumping genes and repressors. The researchers found that, while it is good at shutting down many L1PA elements, there is one subset of a recently evolved lineage of L1PA that has lost a short section of DNA that includes the ZNF93 binding site. Without the binding site, these jumping genes evade repression by ZNF93. Interestingly, when the researchers put the missing sequence back into one of these genes and put it in a mouse cell without ZNF93, they found that it was better at jumping. So even though the sequence helps with jumping activity, losing it gives the jumping gene an advantage in primates by allowing it to escape repression by ZNF93.

"That's kind of the icing on the cake for aficionados of molecular evolution, because it demonstrates that this is a never-ending race," Salama said.

"KRAB zinc finger proteins are a rare class of proteins that is rapidly expanding and evolving in mammalian genomes, which makes sense because the transposable elements are themselves continually evolving to escape repression."

Corresponding author David Haussler, professor of biomolecular engineering and director of the UC Santa Cruz Genomics Institute, said the study involved close collaboration between his group's "wet lab," directed by Salama, and the "dry lab" where researchers under Paten's direction used the computational tools of genome bioinformatics to reconstruct the evolutionary history of primate genomes. Haussler, a Howard Hughes Medical Institute investigator who has used his background in computer science to do pioneering work in genomics, said he established the wet lab to enable just this kind of collaboration.

"Both parts were integral to this study, and there was a lot of back and forth between them. This paper shows how important it is to integrate computational and experimental approaches to fundamental scientific problems, such as how and why we continuously evolve to be more complex," Haussler said.

Source: University of California - Santa Cruz

Contact: Tim Stephens

Reference:

An evolutionary arms race between KRAB zinc-finger genes ZNF91/93 and SVA/L1 retrotransposons

Frank M. J. Jacobs, David Greenberg, Ngan Nguyen, Maximilian Haeussler, Adam D. Ewing, Sol Katzman, Benedict Paten, Sofie R. Salama & David Haussler

Nature, 28 September  2014, doi:10.1038/nature13760

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New Material Could Enhance Fast and Accurate DNA Sequencing

New Material Could Enhance Fast and Accurate DNA Sequencing

Wednesday, 13 August 2014

Gene-based personalized medicine has many possibilities for diagnosis and targeted therapy, but one big bottleneck: the expensive and time-consuming DNA-sequencing process.

A DNA molecule passes through a nanopore in a

sheet of molybdenum disulphide, a material that

researchers have found to be better than

graphene at reading the DNA sequence. 

Credit: Narayana Aluru, University of Illinois.

Now, researchers at the University of Illinois at Urbana-Champaign have found that nanopores in the material molybdenum disulphide (MoS2) could sequence DNA more accurately, quickly and inexpensively than anything yet available.

"One of the big areas in science is to sequence the human genome for under $1,000, the 'genome-at-home,'" said Narayana Aluru, a professor of mechanical science and engineering at the U. of I. who led the study.

"There is now a hunt to find the right material. We've used MoS2 for other problems, and we thought, why don't we try it and see how it does for DNA sequencing?"

As it turns out, MoS2 outperforms all other materials used for nanopore DNA sequencing – even graphene.

A nanopore is a very tiny hole drilled through a thin sheet of material. The pore is just big enough for a DNA molecule to thread through. An electric current drives the DNA through the nanopore, and the fluctuations in the current as the DNA passes through the pore tell the sequence of the DNA, since each of the four letters of the DNA alphabet – A, C, G and T – are slightly different in shape and size.

Illinois researchers found that the material

molybdenum disulphide could be the most

efficient yet found for DNA sequencing, making

personalized medicine more accessible. From

left: Amir Barati Farimani, Kyongmin Min and

Narayana Aluru. Credit: L. Brian Stauffer.

Most materials used for nanopore DNA sequencing have a sizable flaw: They are too thick. Even a thin sheet of most materials spans multiple links of the DNA chain, making it impossible to accurately determine the exact DNA sequence.

Graphene has become a popular alternative, since it is a sheet made of a single layer of carbon atoms – meaning only one base at a time goes through the nanopore. Unfortunately, graphene has its own set of problems, the biggest being that the DNA sticks to it. The DNA interacting with the graphene introduces a lot of noise that makes it hard to read the current, like a radio station marred by loud static.

MoS2 is also a single-layer sheet, thin enough that only one DNA letter at a time goes through the nanopore. In the study, the Illinois researchers found that DNA does not stick to MoS2, but threads through the pore cleanly and quickly. See an animation online.

"MoS2 is a competitor of graphene in terms of transistors, but we showed here a new functionality of this material by showing that it is capable of biosensing," said graduate student Amir Barati Farimani, the first author of the paper.

Most exciting for the researchers, the simulations yielded four distinct signals corresponding to the bases in a double-stranded DNA molecule. Other systems have yielded two at best – A/T and C/G – which then require extensive computational analysis to attempt to distinguish A from T and C from G.

The key to the success of the complex MoS2 simulation and analysis was the Blue Waters supercomputer, located at the National Center for Supercomputing Applications at the U. of Illinois.

"These are very detailed calculations," said Aluru, who is also a part of the Beckman Institute for Advanced Science and Technology at the U. of I.

"They really tell us the physics of the actual mechanisms, and why MoS2 is performing better than other materials. We have those insights now because of this work, which used Blue Waters extensively."

Now, the researchers are exploring whether they can achieve even greater performance by coupling MoS2 with another material to form a low-cost, fast and accurate DNA sequencing device.

"The ultimate goal of this research is to make some kind of home-based or personal DNA sequencing device," Barati Farimani said.

"We are on the path to get there, by finding the technologies that can quickly, cheaply and accurately identify the human genome. Having a map of your DNA can help to prevent or detect diseases in the earliest stages of development. If everybody can cheaply sequence so they can know the map of their genetics, they can be much more alert to what goes on in their bodies."

Source: University of Illinois at Urbana-Champaign 

Contact: Liz Ahlberg

Reference:

DNA Base Detection Using a Single-Layer MoS2

Amir Barati Farimani, Kyoungmin Min, and Narayana R. Aluru 

ACS Nano, July 9, 2014, DOI: 10.1021/nn5029295

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