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 ......... ZenMaster

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Drosophila Drug Screen for Fragile X Syndrome

Promising compounds and potential drug targets found Monday, 10 March 2008 Scientists using a new drug screening method in Drosophila (fruit flies), have identified several drugs and small molecules that reverse the features of fragile X syndrome – a frequent form of mental retardation and one of the leading known causes of autism. The discovery sets the stage for developing new treatments for fragile X syndrome. The results of the research by lead scientist Stephen Warren, PhD, chair of the Department of Human Genetics at Emory University School of Medicine, are published online in the journal Nature Chemical Biology. Dr. Warren led an international group of scientists that discovered the FMR1 gene responsible for fragile X syndrome in 1991. Fragile X syndrome is caused by the functional loss of the fragile X mental retardation protein (FMRP). Currently there is no effective drug therapy for fragile X syndrome, and previously no assays had been developed to screen drug candidates for the disorder. During the past 17 years, intense efforts from many laboratories have uncovered the fundamental basis for fragile X syndrome. Scientists believe FMRP affects learning and memory through regulation of protein synthesis at synapses in the brain. One leading view, proposed by Dr. Warren and colleagues, suggests that over stimulation of neurons by the neurotransmitter glutamate is partly responsible for the brain dysfunction resulting from the loss of FMRP. In their current experiment, Emory scientists used a Drosophila model lacking the FMR1 gene. These fruit flies have abnormalities in brain architecture and behaviour that parallel abnormalities in the human form of fragile X syndrome. When FMR1-deficient fly embryos were fed food containing increased levels of glutamate, they died during development, which is consistent with the theory that the loss of FMR1 results in excess glutamate signalling. The scientists placed the FMR1-deficient fly embryos in thousands of tiny wells containing food with glutamate. In addition, each well contained one compound from a library of 2,000 drugs and small molecules. The scientists’ uncovered nine molecules that reversed the lethal effects of glutamate, using this screening method. The three top identified compounds were known activators of GABA, a neural pathway already known to inhibit the effects of glutamate. In the study, GABA reversed all the features of fragile X syndrome in the fruit flies, including deficits in the brain's primary learning centre and behavioural deficits. The screening also identified other neural pathways that may have a parallel role in fragile X syndrome and could be targets for drug therapy. "Our discovery of glutamate toxicity in the Drosophila model of fragile X syndrome allowed us to develop this new screen for potential drug targets," notes Dr. Warren. "We believe this is the first chemical genetic screen for fragile X syndrome, and it highlights the general potential of Drosophila screens for drug development. Most importantly, it identifies several small molecules that significantly reverse multiple abnormal characteristics of FMR1 deficiency. It also reveals additional pathways and relevant drug targets. These findings open the door to development of effective new therapies for fragile X syndrome." First author of the article was Shuang Chang, postdoctoral student in Emory's Department of Human Genetics. Other authors included Steven M. Bray and Peng Jin from Emory, Zigang Li from the University of Chicago and Daniela C. Zarnescu from the University of Arizona. The research was supported by the National Institutes of Health, the Fragile X Research Foundation, and the Colonial Oaks Foundation. Dr. Warren is chair of the scientific advisory board for Seaside Therapeutics, which is developing drugs for fragile X syndrome. ......... ZenMaster

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Human ESC's derived from PGD embryos with Fragile X

hESCs derived from PGD embryos with Fragile X Wednesday, 14 November 2007 A human stem cell line derived from embryos that were identified by preimplantation genetic diagnosis (PGD) to carry the mutation for fragile X syndrome has provided an unprecedented view of early events associated with this disease. In addition to giving scientists fresh insight into fragile X, results from this unique model system have emphasized the value of this new source of embryonic stem cells and may have a significant impact on the way that genetic diseases are studied in the future. The research is published in the November issue of the journal Cell Stem Cell, published by Cell Press. Fragile X syndrome, the most common cause of inherited mental impairment and of autism, is caused by the absence of the fragile X mental retardation protein (FMRP). Most individuals with fragile X exhibit a specific mutation in the fragile X mental retardation 1 (FMR1) gene that usually coincides with epigenetic DNA modifications. However, the developmental timing and mechanisms associated with acquisition of these characteristics are not clear due to the absence of appropriate cellular and animal models. To examine developmentally regulated events involved in fragile X pathogenesis, Dr. Nissim Benvenisty and Dr. Rachel Eiges from the Hebrew University Department of Genetics in Jerusalem, Israel, together with Dr. Dalit Ben-Yosef from the IVF unit at the Tel-Aviv Sourasky Medical Center, established a human embryonic stem cell (hESC) line from a preimplantation fragile X-affected embryo identified by PGD. The fragile X cell line, called HEFX, displayed all characteristics typical of an hESC line and possessed the full genetic mutation observed in fragile X patients. The work "highlights the value of [human embryonic stem cells] as a model system for early human embryo development," the study's co-author, Rachel Eiges, told The Scientist. "We show that it can be used as a powerful tool to analyze the effect of a specific mutation on particular developmental events, allowing exploring processes which are otherwise inaccessible for research." The researchers found that undifferentiated HEFX cells transcribed FMR1 and expressed FMRP, suggesting that the fragile X mutation by itself is not sufficient to cause FMR1 inactivation. The research team went on to show that differentiated derivatives of HEFX cells exhibited a decrease in FMRI transcription and FMRP expression along with an increase in epigenetic modifications associated with fragile X syndrome. “The fact that FMR1 inactivation and other modifications take place after differentiation suggests that it might be possible to prevent some of these events as an attempt to rescue the abnormal phenotype in cells with the full fragile X mutation,” suggests Dr. Benvenisty. HEFX cells represent an excellent model for examination of early embryogenesis and will contribute to a clearer understanding of the molecular mechanisms underlying fragile X pathogenesis. This research is also compelling on a more general level in that it validates the usefulness of hESCs derived from embryos that have been screened for specific mutations with PGD. hESC lines derived in this manner represent a potent tool for the study of a variety of human diseases and the development of new therapeutic strategies.

"Certainly, stem cell lines such as this will help science unravel the mechanisms associated with human genetic disorders, and hopefully lead to new therapeutic treatments and interventions in the future," said Robert Lanza of Advanced Cell Technology in Los Angeles, CA, who was not involved in the research. Such an approach has been overshadowed by the focus on developing stem cells as treatments for various disorders, he noted. ......... ZenMaster

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