Monday, 29 September 2014

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.

Contact: Tim Stephens

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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Thursday, 18 September 2014

Stem Cells Use 'First Aid Kits' to Repair Damage

Stem Cells Use 'First Aid Kits' to Repair Damage
Thursday, 18 September 2014

Stem cells hold great promise as a means of repairing cells in conditions such as multiple sclerosis, stroke or injuries of the spinal cord because they have the ability to develop into almost any cell type. Now, new research shows that stem cell therapy can also work through a mechanism other than cell replacement.

Confocal super resolution imaging of the rapid
(2h) up-take of CD63-RFP EVs packed via
target cell fEGFP (green) plasma membrane in
vitro. Credit: Design and artwork by CongJian
In a study published today in Molecular Cell, a team of researchers led by the University of Cambridge has shown that stem cells "communicate" with cells by transferring molecules via fluid filled bags called vesicles, helping other cells to modify the damaging immune response around them.

Although scientists have speculated that stem cells might act rather like drugs – in sensing signals, moving to specific areas of the body and executing complex reactions – this is the first time that a molecular mechanism for this process has been demonstrated. By understanding this process better, researchers can identify ways of maximising the efficiency of stem-cell-based therapies.

"These tiny vesicles in stem cells contain molecules like proteins and nucleic acids that stimulate the target cells and help them to survive – they act like mini "first aid kits".

"Essentially, they mirror how the stem cells respond to an inflammatory environment like that seen during complex neural injuries and diseases, and they pass this ability on to the target cells. We think this helps injured brain cells to repair themselves."

Mice with damage to brain cells – such as the damage seen in multiple sclerosis – show a remarkable level of recovery when neural stem/precursor cells (NPCs) are injected into their circulatory system. It has been suggested that this happens because the NPCs discharge molecules that regulate the immune system and that ultimately reduce tissue damage or enhance tissue repair.

The team of researchers from the UK, Australia, Italy, China and Spain has now shown that NPCs make vesicles when they are in the vicinity of an immune response, and especially in response to a small protein, or cytokine, called Interferon-gamma which is released by immune cells. This protein has the ability to regulate both the immune responses and intrinsic brain repair programmes and can alter the function of cells by regulating the activity of scores of genes.

Their results show that a highly specific pathway of gene activation is triggered in NPCs by IFN-gamma, and that this protein also binds to a receptor on the surface of vesicles. When the vesicles are released by the NPCs, they adhere and are taken up by target cells. Not only does the target cell receive proteins and nucleic acids that can help them self-repair, but it also receives the IFN-gamma on the surface of the vesicles, which activates genes within the target cells.

The researchers, who were funded by the European Research Council and the Italian MS Society, used electron microscopy and super resolution imaging to visualise the vesicles moving between the NPCs and target cells in vitro.

"Our work highlights a surprising novel role for stem-cell-derived vesicles in propagating responses to the environment," added Pluchino.

"It represents a significant advance in understanding the many levels of interaction between stem cells and the immune system, and a new molecular mechanism to explain how stem-cell therapy works."

Contact: Louise Walsh

Extracellular Vesicles from Neural Stem Cells Transfer IFN-γ via Ifngr1 to Activate Stat1 Signaling in Target Cells
Chiara Cossetti, Nunzio Iraci, Tim R. Mercer, Tommaso Leonardi, Emanuele Alpi, Denise Drago, Clara Alfaro-Cervello, Harpreet K. Saini, Matthew P. Davis, Julia Schaeffer, Beatriz Vega, Matilde Stefanini, CongJian Zhao, Werner Muller, Jose Manuel Garcia-Verdugo, Suresh Mathivanan, Angela Bachi, Anton J. Enright, John S. Mattick, Stefano Pluchino
Molecular Cell, September 18, 2014, DOI:

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Tuesday, 9 September 2014

In Directing Stem Cells, Study Shows Context Matters

In Directing Stem Cells, Study Shows Context Matters
Tuesday, 09 September 2014

Figuring out how blank slate stem cells decide which kind of cell they want to be when they grow up — a muscle cell, a bone cell, a neuron — has been no small task for science.

When blank slate stem cells are exposed to a soft
as opposed to a hard surface on which to grow,
they begin to transform themselves into neurons,
the large, complex cells of the central nervous
system. Absent any soluble factors to direct cell
differentiation, surface matters, according to
new research from the lab of University of
Wisconsin-Madison chemist and biochemist
Laura Kiessling. Credit: Kiessling Lab/UW-
Human pluripotent stem cells, the undifferentiated cells that have the potential to become any of the 220 types of cells in the body, are influenced in the lab dish by the cocktail of chemical factors and proteins upon which they are grown and nurtured. Depending on the combination of factors used in a culture, the cells can be coaxed to become specific types of cells.

Now, in a new study published today, Sept. 8, in the Proceedings of the National Academy of Sciences, a team of researchers from the University of Wisconsin-Madison has added a new wrinkle to the cell differentiation equation, showing that the stiffness of the surfaces on which stem cells are grown can exert a profound influence on cell fate.

"To derive lineages, people use soluble growth factors to get the cells to differentiate," explains Laura Kiessling, a UW-Madison professor of chemistry and biochemistry and stem cell expert.

Past work, she notes, hinted that the qualities of the surface on which a cell lands could exert an influence on cell fate, but the idea was never fully explored in the context of human pluripotent stem cell differentiation.

In the lab, stem cells are grown in plastic dishes coated with a gel that contains as many as 1,800 different proteins. Different factors can be introduced to obtain certain types of cells. But even in the absence of introduced chemical or protein cues, the cells are always working to differentiate — but in seemingly random, undirected ways.

The Wisconsin group, directed by Kiessling and led by chemistry graduate student Samira Musah, decided to test the idea that the hardness of a surface can make a difference. After all, in a living body, cells seek different niches with different qualities and transform themselves accordingly.

"Many cell types grow on a surface. If a cell is near bone, the environment has certain features," says Kiessling, whose group — collaborating with UW-Madison colleagues Sean Palecek, Qiang Chang and William Murphy — has been working to produce precise, chemically defined surfaces on which to grow stem cells.

"A cell will react differently if it lands near soft tissue like the brain."

To fully explore the idea that surface matters to a stem cell, Kiessling's group created gels of different hardness to mimic muscle, liver and brain tissues. The study sought to test whether the surface alone, absent any added soluble factors to influence cell fate decisions, can have an effect on differentiation.

Results, according to Kiessling, showed that a soft, brain tissue-like surface, independent of any soluble factors, was catalyst enough to direct cells to become neurons, the large elaborate cells that make up the central nervous system. Stiffer surfaces favoured the stem cell state.

"We didn't change anything but switch from a hard surface to a soft surface," Kiessling says.

"They all started looking like neurons. It was stunning to me that the surface had such a profound effect."

In the case of the soft, brain-like surface, the Wisconsin researchers believe that the mechanical properties of a surface are influencing a protein called YAP. YAP can be found in the cytoplasm but also the nucleus of a cell, and when it is in the nucleus, YAP regulates gene expression. According to the study results, YAP is excluded from the nucleus on the soft gels, and its depletion there helps drive the stem cells onto a brain cell developmental pathway.

The finding, that the simple mechanical properties of a surface can play a big role in helping stem cells decide what to be, promises to help scientists better define the experimental conditions to direct stem cell fate. It may also ultimately inform the methods that will be used for producing large quantities of cells for therapeutic use and other applications such as the high-throughput screening of chemicals for brain toxicity or therapeutics.

Contact: Laura Kiessling

Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification
Samira Musah, Paul J. Wrighton, Yefim Zaltsman, Xiaofen Zhong, Stefan Zorn, Matthew B. Parlato, Cheston Hsiao, Sean P. Palecek, Qiang Chang, William L. Murphy and Laura L. Kiessling
PNAS 2014, September 8, doi: 10.1073/pnas.1415330111

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