Handle with Care: Telomeres Resemble DNA Fragile Sites

A protein at the ends of chromosomes, helps prevent DNA replication from stalling at telomeres
Friday, 10 July 2009

Telomeres, the repetitive sequences of DNA at the ends of linear chromosomes, have an important function: They protect vulnerable chromosome ends from molecular attack. Researchers at Rockefeller University now show that telomeres have their own weakness. They resemble unstable parts of the genome called fragile sites where DNA replication can stall and go awry. But what keeps our fragile telomeres from falling apart is a protein that ensures the smooth progression of DNA replication to the end of a chromosome.

The research, led by Titia de Lange, head of the Laboratory of Cell Biology and Genetics, and first author Agnel Sfeir, a postdoctoral associate in the lab, suggests a striking similarity between telomeres and common fragile sites, parts of the genome where breaks tend to occur, albeit infrequently. (Humans have 80 common fragile sites, many of which have been linked to cancer.) De Lange and Sfeir found that these newly discovered fragile sites make it difficult for DNA replication to proceed, a discovery that unveils a new replication problem posed by telomeres.

At the centre of the discovery is a protein known as TRF1, which de Lange, in an effort to understand how telomeres protect chromosome ends, discovered in 1995. Using a conditional mouse knockout, de Lange and Sfeir have now revealed that TRF1, which is part of a six-protein complex called shelterin, enables DNA replication to drive smoothly through telomeres with the aid of two other proteins.

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Fragile telomeres. This is a series of images showing chromosomes with fragile telomeres (green). Without the protein TRF1, telomeres resemble common fragile sites, unstable regions on chromosomes that break into segments or stretch due to faulty DNA replication. Credit: Cell.

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“Telomeric DNA has a repetitive sequence that can form unusual DNA structures when the DNA is unwound during DNA replication,” says de Lange.

“Our data suggest that TRF1 brings in two proteins that can take out these structures in the telomeric DNA. In other words, TRF1 and its helpers remove the bumps in the road so that the replication fork can drive through.”

The work, published in the July 10 issue of Cell, began when Sfeir deleted TRF1 and saw that the telomeres resembled common fragile sites, suggesting that TRF1 protects telomeres from becoming fragile. Instead of a continuous string of DNA, the telomeres were broken into fragments of twos and threes. To see if the replication fork stalls at telomeres, de Lange and Sfeir joined forces with

Carl L. Schildkraut, a researcher at Albert Einstein College of Medicine in New York City. Using a technique called SMARD, the researchers observed the dynamics of replication across individual DNA molecules — the first time this technique has been used to study telomeres. In the absence of TRF1, the fork often stalled for a considerable amount of time.

The only other known replication problem posed by telomeres was solved in 1985 when it was shown that the enzyme telomerase elongates telomeres, which shorten during every cell division. The second problem posed by telomeres, the so-called end-protection problem, was solved by de Lange and her colleagues when they found that shelterin protects the ends of linear chromosomes, which look like damaged DNA, from unnecessary repair. Working with TRF1, the very first shelterin protein ever to be identified, de Lange and Sfeir have not only unveiled a completely unanticipated replication problem at telomeres, they have also shown how it is solved.

The research lays new groundwork for the study of common fragile sites throughout the genome, explains de Lange.

“Fragile sites have always been hard to study because no specific DNA sequence precedes or follows them,” she says.

“In contrast, telomeres represent fragile sites with a known sequence, which may help us understand how common fragile sites break throughout the genome — and why.”

Reference:
Mammalian Telomeres Resemble Fragile Sites and Require TRF1 for Efficient Replication
Agnel Sfeir, Settapong T. Kosiyatrakul, Dirk Hockemeyer, Sheila L. MacRae, Jan Karlseder, Carl L. Schildkraut and Titia de Lange
Cell, July 10, 2009, 138(1): 90-103
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Key to Maintaining Embryonic Stem Cells in Lab

Key to Maintaining Embryonic Stem Cells in Lab
Friday, 10 July 2009

In a new study that could transform embryonic stem cell (ES cell) research, scientists at UT Southwestern Medical Center have discovered why mouse ES cells can be easily grown in a laboratory while other mammalian ES cells are difficult, if not impossible, to maintain.

If the findings in mice can be applied to other animals, scientists could have an entirely new palette of research tools to work with, said Dr. Steven McKnight, chairman of biochemistry at UT Southwestern and senior author of the study appearing in the July 9 issue of Science Express.

"This might change the way medical research is done. But it's still a big 'if,'" he said.

According to the research, the activation of a gene called TDH in mouse ES cells results in the cells entering a unique metabolic state that is similar to that of rapidly growing bacterial cells. The gene controls the production of the threonine dehydrogenase (TDH) enzyme in mouse ES cells. This enzyme breaks down an amino acid called threonine into two products. One of the two products goes on to control a cellular process called one carbon metabolism; the other provides ES cells with an essential metabolic fuel.

Both of the threonine breakdown products are necessary to keep the ES cells growing and dividing rapidly in a Petri dish without differentiating into specific tissues.

The various substances currently used by scientists to keep mouse ES cells alive in the laboratory were found by trying many different combinations until something worked, Dr. McKnight said. But until now, it wasn't known that these culture conditions keyed into keeping the TDH gene actively expressed.

"Scientists added this and that until they got the right 'soup,' one that works in the mouse ES cells to somehow activate the TDH gene," he said, adding that exactly how that gene is regulated is still unknown.

Other mammalian species have a functional version of the TDH gene, suggesting the possibility that the process could also be activated in them.

"You would think that the 'mouse soup' would then work for all species, but it doesn't. Researchers have been trying for 20 years to get the right formula for maintaining ES cells from other species. With few exceptions, however, they still haven't gotten it right," Dr. McKnight said.

The research was funded by a National Institutes of Health Director's Pioneer Award, which Dr. McKnight received in 2004. The program encourages investigators to take on creative, unexplored avenues of research that carry a relatively high potential for failure but that also possess a greater chance for truly groundbreaking discoveries.

"By applying a highly innovative technique to manipulate the TDH gene, McKnight's work could be an important breakthrough with a profound impact on future research," said Dr. Raynard S. Kington, acting director of the NIH.

"This research, which was partially funded by our Pioneer Award program, shows the value of supporting exceptionally creative approaches to major challenges in biomedical and behavioral research."

Embryonic stem cells are "blank slate" cells – derived from embryos – that go on to develop into any of the more than 200 types of cells in the adult body.

Because mouse ES cells are easily maintained in the lab, they can be manipulated genetically to produce adult mice in which various genes are either modified or eliminated. So-called "knockout mice" allow scientists to study the genetic aspects of many diseases and conditions, including cancer, Alzheimer's, Parkinson's and paralysis.

In the living mouse, and in other species, ES cells exist for only a short time. In that time, they need to grow rapidly in order to accumulate enough cells to begin the process of differentiating into all the body's cell types. Dr. McKnight hypothesizes that the TDH gene tightly controls this process in the animal, allowing the ES cells to grow, but then it shuts off when it's time to differentiate.

"If we can tweak conditions and determine how to keep the gene turned on in other animals, we might be able to grow and maintain ES cells for study in many species. It's still speculative at this point whether it will work, but if it does, then this may prove to represent a transformational discovery," Dr. McKnight said.

Interestingly, although humans carry a form of the TDH gene, it contains three inactivating mutations. As such, human ES cells do not produce the TDH enzyme.

"In the human embryo, something else is taking the place of this TDH-mediated form of rapid cell growth," Dr. McKnight said.

"Human ES cells may exist in a unique metabolic state, but it would not appear to involve threonine breakdown."

Human ES cells grow slowly and are difficult to maintain in the laboratory, which is a huge impediment to this field of study, Dr. McKnight said.

"If scientists could repair the mutated human TDH gene and replace it into human ES cells, could they make those cells grow faster in culture? I don't know whether this will work or not – it's highly speculative. But if so, it would be profound," he said.

Reference:
Dependence of Mouse Embryonic Stem Cells on Threonine Catabolism
Jian Wang, Peter Alexander, Leeju Wu, Robert Hammer, Ondine Cleaver, and Steven L. McKnight
Science Published online July 9 2009; DOI: 10.1126/science.1173288
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Newborn Brain Cells Show the Way

Newborn Brain Cells Show the Way
Friday, 10 July 2009

Although the fact that we generate new brain cells throughout life is no longer disputed, their purpose has been the topic of much debate. Now, an international collaboration of researchers made a big leap forward in understanding what all these newborn neurons might actually do. Their study, published in the July 10, 2009, issue of the journal Science, illustrates how these young cells improve our ability to navigate our environment.

"We believe that new brain cells help us to distinguish between memories that are closely related in space," says senior author Fred H. Gage, Ph.D., a professor in the Laboratory for Genetics at the Salk Institute. He is also the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Diseases, who co-directed the study with Timothy J. Bussey, Ph.D., a senior lecturer in the Department of Experimental Psychology at the University of Cambridge, UK, and Roger A. Barker, PhD., honorary consultant in Neurology at Addenbrookes Hospital and Lecturer at the University of Cambridge.

When the first clues emerged that adult human brains continually sprout new neurons, one of the central tenets of neuroscience — we are born with all the brain cells we'll ever have — was about to be overturned. Although it is never easy to shift a paradigm, a decade later the question is no longer whether neurogenesis exists but rather what all these new cells are actually good for.

"Adding new neurons could be a very problematic process if they don't integrate properly into the existing neural circuitry," says Gage.

"There must be a clear benefit to outweigh the potential risk."

The most active area of neurogenesis lies within the hippocampus, a small seahorse-shaped area located deep within the brain. It processes and distributes memory to appropriate storage sections in the brain after readying the information for efficient recall.

"Every day, we have countless experiences that involve time, emotion, intent, olfaction and many other dimensions," says Gage.

"All the information comes from the cortex and is channelled through the hippocampus. There, they are packaged together before they are passed back out to the cortex where they are stored."

Previous studies by a number of laboratories including Gage's had shown that new neurons somehow contribute to hippocampus-dependent learning and memory but the exact function remained unclear.

The dentate gyrus is the first relay station in the hippocampus for information coming from the cortex. While passing through, incoming signals are split up and distributed among 10 times as many cells. This process, called pattern separation, is thought to help the brain separate individual events that are part of incoming memories.

"Since the dentate gyrus also happens to be the place where neurogenesis is occurring, we originally thought that adding new neurons could help with the pattern separation," says Gage.

This hypothesis allowed graduate student Claire Clelland, who divided her time between the La Jolla and the Cambridge labs, to design experiments that would specifically challenge this function of the dentate gyrus using different behavioural tasks and two distinct strategies to selectively shut down neurogenesis in the dentate gyrus.

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This image depicts a Paired Associates Learning (PAL) task, in which mice have to choose a specific object in its correct location on a touch screen to obtain a reward. Adult mice deficient in adult neurogenesis showed a specific impairment in pattern separation, identifying a dentate gyrus-specific function for newborn neurons in the adult brain. Adult born neurons are shown in red the background. Credit: Courtesy of Jamie Simon, Salk Institute for Biological Studies.

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In the first set of experiments, mice had to learn the location of a food reward that was presented relative to the location of an earlier reward within an eight-armed radial maze.

"Mice without neurogenesis had no trouble finding the new location as long as it was far enough from the original location," says Clelland, "but couldn't differentiate between the two when they were close to each other."

A touch screen experiment confirmed the inability of neurogenesis-deficient mice to discriminate between locations in close proximity to each other but also revealed that these mice had no problem recalling spatial information in general.

"Neurogenesis helps us to make finer distinctions and appears to play a very specific role in forming spatial memories," says Clelland.

Adds Gage, "There is value in knowing something about the relationship between separate events and the closer they get the more important this information becomes."

But pattern separation might not be the only role that new neurons have in adult brain function: a computer model simulating the neuronal circuits in the dentate gyrus based on all available biological information suggested an additional function.

"To our surprise, it turned out that newborn neurons actually form a link between individual elements of episodes occurring closely in time," says Gage.

Given this, he and his team are now planning experiments to see whether new neurons are also critical for coding temporal or contextual relationships.

About the Salk Institute for Biological Studies:
The Salk Institute for Biological Studies is one of the world's preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused on both discovery and mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer's, diabetes, and cardiovascular disorders by studying neuroscience, genetics, cell and plant biology, and related disciplines.

Faculty achievements have been recognized with numerous honours, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent non-profit organization and architectural landmark.

Reference:
A Functional Role for Adult Hippocampal Neurogenesis in Spatial Pattern Separation
C. D. Clelland, M. Choi, C. Romberg, G. D. Clemenson, Jr., A. Fragniere, P. Tyers, S. Jessberger, L. M. Saksida, R. A. Barker, F. H. Gage, T. J. Bussey
Science 10 July 2009: Vol. 325. no. 5937, pp. 210 – 213, DOI: 10.1126/science.1173215
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Stem Cells' 'Suspended' State Preserved by Key Step in Chromatin Remodelling

Stem Cells' 'Suspended' State Preserved by Key Step in Chromatin Remodelling
Thursday, 09 July 2009

Scientists have identified a gene that is essential for embryonic stem cells to maintain their all-purpose, pluripotent state. Exploiting the finding may lead to a greater understanding of how cells acquire their specialized states and provide a strategy to efficiently reprogram mature cells back into the pluripotent state, an elusive step in stem cell research but one crucial to a range of potential clinical treatments.

The research was led by University of California, San Francisco scientists. It is being reported Wednesday, July 8, 2009, in the advanced online edition of the journal Nature, and will be published in the journal's print edition at the end of July.

Embryonic stem cells are suspended in an "open" state, uniquely poised to become any one of many types of specialized cells, as genetic instructions dictate. Directing the specialization of embryonic stem cells to cells needed by patients is an area of enormous promise in stem cell research. Reversing the natural process – converting specialized cells back into the all-purpose stem cell stage – is another great promise of stem cell research.

Reprogramming specialized cells from Parkinson's patients, for example, would allow scientists to study the mechanisms that cause neurons in the brain to develop the disease. It also could lead to treatments by directing the restored stem cells to produce healthy neurons to introduce into patients.

The new research, conducted on mouse embryo cells, revealed that a gene known as Chd1 loosens the packaging that normally protects DNA in the cell nucleus. This step, known as chromatin remodelling, allows the cell's protein-making machinery to gain access to the DNA and transform progenitor cells into specialized cells and tissue, such as neurons, muscle and bone.

A number of genes are known to trigger chromatin remodelling, allowing small sections of DNA to become accessible in order to make specific proteins. Chd1 is the first gene found to regulate a "global" loosening of the DNA in embryonic stem cells, the scientists report. The global condition sets the stage for turning on many different genes to make a broad range of specialized cells.

"Embryonic stem cells are characterized by this open state, but, up to now, we didn't know the mechanisms that maintain this state, or even if it is necessary for the full stem cell potential," said Alexandre Gaspar-Maia, lead author of the paper.

"We found that Chd1 is critical for both, and for allowing an efficient reprogramming. Chd1 is important for allowing the normal differentiation process, and it is essential for playing the 'differentiation tape' backwards – bringing differentiated cells back to pluripotency."

Gaspar-Maia is a graduate student (from the PhD Program in Experimental Biology and Biomedicine, at the University of Coimbra, Portugal) in the lab of senior author Miguel Ramalho-Santos, PhD, of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

The scientists discovered the pivotal role of Chd1 by using the powerful technique of RNA interference, or RNAi, to screen this gene and 40 other candidate genes. (RNAi is a naturally occurring process in which small RNAs bind to other RNAs to increase or decrease their activity.) In this case, the scientists used the technique to silence Chd1. When they did so, embryonic stem cells could not make the full range of specialized cells.

In a laboratory test used to simulate normal cell specialization, the scientists detected no differentiation of cardiac muscle, and also no formation of a tissue known as primitive endoderm, which is essential for the embryo to survive and develop.

Chd1 also was shown by the research team to be necessary for the reprogramming of specialized cells back to the pluripotent stem cell state. The team plans to study chromatin remodelling in still more detail to clarify what other molecules work in concert with the Chd1 gene to direct the process. This would aid efforts to increase the efficiency and safety of reprogramming cells. This research may also shed light on how cells transition from one type to another, a process that happens normally during embryonic development and goes astray in cancer.

"We now know that Chd1 is essential, and, so far, appears unique in its global effect, but we expect that there are major players yet to be discovered," said senior author Ramalho-Santos, UCSF assistant professor of obstetrics, gynaecology and reproductive sciences, and pathology.

"If we can understand how Chd1 works, that will also tell us more about how the cells regulate their precise specialization during development, and turn on their pluripotency program during reprogramming."

The scientists conclude that embryonic stem cells exist in a dynamic state, poised between the open condition that may assure the cell's full potential, and the more constrained state that allows only certain kinds of cells to progress. Chd1, they say, is central to maintaining the open, pluripotent stem cell state.

Reference:
Chd1 regulates open chromatin and pluripotency of embryonic stem cells
Alexandre Gaspar-Maia, Adi Alajem, Fanny Polesso, Rupa Sridharan, Mike J. Mason, Amy Heidersbach, João Ramalho-Santos, Michael T. McManus, Kathrin Plath, Eran Meshorer & Miguel Ramalho-Santos
Nature advance online publication 8 July 2009 doi:10.1038/nature08212
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