ScienceDaily (Feb. 22, 2010) — Scientists from the U.S. Department of Energy’s Lawrence Berkeley National Laboratory have learned how an interval of DNA in an unexplored region of the human genome increases the risk for coronary artery disease, the leading cause of death worldwide.

Their research paints a fuller picture of a genetic risk for the disease that was discovered only three years ago and which lurks in one out of two people.

It also reinforces the tantalizing possibility that many more disease risks — and potential therapies — are hidden in the vast and uncharted part of the genome that doesn’t contain instructions for making proteins.

The research is reported in the February 21 advance online publication of the journal Nature.

The team focused on an interval of DNA in chromosome 9p21. People who carry variations of this interval have an increased chance of developing coronary artery disease, which is an accumulation of plaque in coronary arteries that restricts blood flow to the heart and causes heart attacks.

Determining how this DNA contributes to the disease is difficult because it’s in the poorly understood part of the genome that doesn’t code for proteins, the workhorses of cellular function.

In groundbreaking research, the Berkeley Lab scientists found that the DNA interval regulates a pair of genes that inhibit cell division, and that bad copies of the interval reduce the genes’ expression. Although more work is needed to understand how this mechanism contributes to coronary artery disease, the researchers speculate that the hobbled genes allow vascular cells to proliferate unchecked and narrow coronary arteries.

“We show that this non-coding interval affects the expression of two cell cycle inhibitor genes located almost 100,000 base pairs away. We believe that something goes awry in variants of this interval, causing vascular cells to divide and multiply more quickly than usual,” says Len Pennacchio, a geneticist with Berkeley Lab’s Genomics Division who conducted the research with Axel Visel and several other scientists from Berkeley Lab, as well as Jonathan Cohen of the University of Texas Southwestern Medical Center.

The link between an interval of DNA in chromosome 9p21 and a risk for coronary artery disease was established in several recent studies, one of which was published in the journal Science in 2007. In that study, led by Cohen and co-authored by several scientists including Pennacchio, the researchers scoured the human genome for differences in people who have coronary artery disease versus people who don’t.

This genome-wide association analysis alighted on a stretch of DNA in chromosome 9p21 that spans 58,000 base pairs of DNA. The study found that people with bad copies of this interval have a moderately higher risk of developing coronary artery disease. In addition, 50 percent of people have one bad copy and 25 percent have two bad copies.

“The risk of coronary artery disease isn’t very high in any give person with bad copies. But they are so common that population-wide the effect is significant,” says Pennacchio.

Remarkably, the study also found that the DNA interval isn’t associated with known risks for coronary artery disease such as diabetes, high blood pressure, and high cholesterol level. An unknown mechanism was at work.

“We landed on this risk interval and immediately said ‘wow!’ why doesn’t it link to problems that we know cause coronary artery disease?” says Pennacchio. “So the big question became: what is this DNA doing?”

Adding to the mystery, the DNA interval is among the 98 percent of our genome that doesn’t code for proteins. Most efforts to determine the function of the genome have focused on the two percent of our DNA that overlaps protein-coding genes. Scientists are just now beginning to explore the non-coding region, once referred to as “junk DNA.”

As part of this effort, the Berkeley Lab scientists set out to determine the function of the DNA interval in chromosome 9p21 that’s linked to coronary artery disease. They removed an analogous section of DNA from mice, then tracked what happened.

The expression level of two genes located far away, Cdkn2a and Cdkn2b, plummeted by about 90 percent in the “knock-out” mice compared to normal mice. These genes are important in controlling cell cycles and have been linked to cancer when mutated, but they had never been linked to coronary artery disease.

The scientists also studied heart tissue of the “knock-out” mice and found that the smooth muscle cells from their aortas had increased proliferation, a hallmark of coronary artery disease.

“Our research shows that the DNA interval plays a pivotal role in regulating the expression of two genes that control cell cycles. It also suggests that variants of the interval spur the progression of coronary artery disease by altering the dynamics of vascular cells,” says Pennacchio.

With this mechanism identified, scientists can develop therapies that fight coronary artery disease by targeting the two genes and jumpstarting them into action, says Pennacchio. He also believes that the genetic roots of many other diseases will be unearthed as scientists learn how to decipher the function of non-coding DNA.

“Non-coding DNA is a huge area of the genome, waiting to be explored, which could have huge dividends for understanding and treating disease,” says Pennacchio.

The research was funded by the National Institutes of Health.

Other Berkeley Lab scientists involved in the research include Yiwen Zhu, Dalit May, Veena Afzal, Elaine Gong, Cattia Attanasio, Matthew Blow, and Eddy Rubin.

DNAWellnessinfo.com Resource:  http://www.sciencedaily.com/releases/2010/02/100222094801.htm

From Uncharted Region of Human Genome, Clues Emerge About Origins of Coronary Artery Disease is a post from: dnawellnessinfo.com

• By Brandon Keim
• October 8, 2009  |
• 3:16 pm- wired.com

genome folding

By breaking the human genome into millions of pieces and reverse-engineering their arrangement, researchers have produced the highest-resolution picture ever of the genome’s three-dimensional structure.

The picture is one of mind-blowing fractal glory, and the technique could help scientists investigate how the very shape of the genome, and not just its DNA content, affects human development and disease.

“It’s become clear that the spatial organization of chromosomes is critical for regulating the genome,” said study co-author Job Dekker, a molecular biologist at the University of Massachusetts Medical School. “This opens up new aspects of gene regulation that weren’t open to investigation before. It’s going to lead to a lot of new questions.”

As depicted in basic biology textbooks and the public imagination, the human genome is packaged in bundles of DNA and protein on 23 chromosomes, arrayed in a neatly X-shaped form inside each cell nucleus. But that’s only true during the fleeting few moments when cells are poised to divide. The rest of the time, those chromosomes exist in a dense and ever-shifting clump. Of course their constituent DNA strings are clumped, too: If the genome could be laid out end-to-end, it’d be six feet long.

For decades, some cell biologists suspected that the genome’s compression wasn’t just an efficient storage mechanism, but linked to the very function and interaction of its genes. But this wasn’t easy to study: Sequencing the genome destroys its shape, and electron microscopes can barely penetrate its active surface. Though its constituent parts are known, the genome’s true shape has been a mystery.

In April, a paper published in the Proceedings of the National Academy of Sciences linked patterns of gene activation to their physical proximity on chromosomes. It still provided the most persuasive evidence to date that genome shape matters, even though the researchers’ chromosome map was relatively low-resolution. The topography described in the latest research, published Thursday in Science, is far more detailed.

“It’s going to change the way that people study chromosomes. It will open up the black box. We didn’t know the internal organization. Now we can look at it in high resolution, try to link that structure to the activity of genes, and see how that structure changes in cells and over time,” said Dekker.

To determine genome structure without being able to directly see it, the researchers first soaked cell nuclei in formaldehyde, which interacts with DNA like glue. The formaldehyde stuck together genes that are distant from each other in linear genomic sequences, but adjacent to each other in actual three-dimensional genomic space.

The researchers then added a chemical that dissolved the gene-by-gene linear sequence bonds, but left the formaldehyde links intact. The result was a pool of paired genes, something like a frozen ball of noodles that had been sliced into a million fragmentary layers and mixed.

By studying the pairs, the researchers could tell which genes had been near each other in the original genome. With the aid of software that cross-referenced the gene pairs with their known sequences on the genome, they assembled a digital sculpture of the genome. And what a marvelous sculpture it is.

“There’s no knots. It’s totally unentangled. It’s like an incredibly dense noodle ball, but you can pull out some of the noodles and put them back in, without disturbing the structure at all,” said Harvard University computational biologist Erez Lieberman-Aiden, also a study co-author.

In mathematical terms, the pieces of the genome are folded into something similar to a Hilbert curve, one of a family of shapes that can fill a two-dimensional space without ever overlapping — and then do the same trick in three dimensions.

How evolution arrived at this solution to the challenge of genome storage is unknown. It might be an intrinsic property of chromatin, the DNA-and-protein mix from which chromosomes are made. But whatever the origin, it’s more than mathematically elegant. The researchers also found that chromosomes have two regions, one for active genes and another for inactive genes, and the unentangled curvatures allow genes to be moved easily between them.

Lieberman-Aiden likened the configuration to the compressed rows of mechanized bookshelves found in large libraries. “They’re like stacks, side-by-side and on top of each other, with no space between them. And when the genome wants to use a bunch of genes, it opens up the stack. But not only does it open the stack, it moves it to a new section of the library,” he said.

The segregation of active and inactive genes adds to evidence that genome structure affects gene function.

“It’s a great description of the structure of the nucleus, and if you put that on top of what we did, it forms the big picture,” said Steven Kosak, a Northwestern University cell biologist and co-author of the April PNAS paper that linked rough outlines of chromosome arrangement to gene activation. Whereas that study only looked at a few chromosomes, the Science paper “looks at fine resolution over the whole genome,” said Kosak.

“Now you can produce these genome maps, and superimpose them with genome-wide analyses of gene expression. You can really start asking how changes in spatial organization relate to changes in genes turning on and off,” said Tom Misteli, a National Cancer Institute cell biologist who studies how glitches in chromosome structure may turn cells cancerous. Neither Misteli nor Kosak were involved in the Science study.

Connecting genome shape to gene function could also help explain the connection between genes and disease, which remain largely unexplained by traditional, sequence-focused genomics.

“It’s perfectly reasonable and almost inevitable that the 3-D structure of DNA is going to influence how it functions,” said Teri Manolio, director of the National Human Genome Research Institute’s Office of Population Genomics.

Researchers also want to study how genome shape is altered. That appears to happen constantly during the transition from stem cell to adult cell, and then during cell function.

“How much variation is there in structure across cell types? What controls it? Exactly how important is it? We don’t know,” said Dekker. “This is a new area of science.”

Image: From Science, a two-dimensional Hilbert curve, and the three-dimensional shape of a genome.

DNAWellnessinfo.com Resource: http://www.wired.com/wiredscience/2009/10/fractal-genome/

The Human Genome in 3 Dimensions is a post from: dnawellnessinfo.com

Posted on Sunday, 06.14.09 on miamiherald.com

By ROBERT S. BOYD

McClatchy Newspapers

New era of gene-based personalized medicine’ dawning is a post from: dnawellnessinfo.com

ScienceDaily (June 11, 2009) — A team of researchers at The Scripps Research Institute and other institutions has discovered a new way by which DNA repairs itself, a process that is critical to the protection of the genome, and integral to prevention of cancer development.

Scientists who study the repair of the DNA bases, which make up the information in the human genome, had known of only one type of method that cells use to fix a specific kind of damage to their DNA, but in the June 11, 2009 issue of Nature, the team found a novel way—one that combines elements from the known mechanisms and an unrelated second method that was previously not known to play a role in this type of DNA repair.

“We found a connection between the known DNA repair processes that people did not know was there,” says Professor John Tainer, a member of the Skaggs Institute for Chemical Biology at Scripps Research, who led the study with Geoffrey P. Margison of the University of Manchester (United Kingdom) and Anthony E. Pegg of the Pennsylvania State University College of Medicine. “This changes the game, and gives us something important to look for in cancers that are resistant to chemotherapy.”

This new mechanism is controlled by alkyltransferase-like proteins (ATLs), whose structure and function had been unknown and which had been identified only in bacteria and yeast. In addition to describing the function of ATLs, in the new study the scientists showed that ATLs exist in a multicellular organism, the sea anemone, which suggests this protein or its cousins in terms of repair activity also exist in other species, including humans.

Known Strategies for DNA Repair

Damage occurs to a cell’s DNA on a continuing basis from outside sources, such as radiation and UV light, and from activities that go on day by day inside the cell. Most of this damage consists of damage to the DNA bases adenine, cytosine, guanine, and thymine. These bases pair up together inside the DNA double helix—adenine and thymine join together, and guanine and cytosine link to each other and their sequence forms the information in the human genome.

These bases can be chemically modified in a number of ways, including by alkylation, in which an alkyl group (or “adduct”) is transferred onto a guanine base. When this happens, one of the hydrogen bonds holding guanine and cytosine together is removed, increasing the chances that thymine will be inserted across from guanine during DNA replication. If DNA is replicated with this “transition” error, a mutated gene results, so the information is changed. This can lead to harmful results, like cell death or cancer.

As shown in the reported work, this kind of damage occurs, for example, when chemicals derived from cigarette smoke stick to guanine, or when chemotherapy agents put an alkyl adduct onto guanine.

But that is where DNA repair mechanisms come in, which is good in the case of chemicals from cigarettes, but not so desirable when they repair genetic damage purposely induced by chemotherapy drugs intended to kill cancer cells.

The DNA repair process that removes such toxic “lesions” is known as base repair, and uses a protein called AGT (O6-alkylguanine DNA-alkytransferase) to remove the alkyl group before DNA replicates. The protein essentially sticks a chemical finger inside the DNA to flip the damaged guanine out from the DNA helix structure so that its adduct is exposed and can be transferred from the guanine to a part of its protein structure. The guanine is now repaired and can rejoin cytosine with three hydrogen bonds linking them.

AGT is believed to act alone, but there is another, unrelated repair process—nucleotide excision repair (NER)—that uses lots of proteins in its pathway. This repair occurs when bulky adducts stuck to bases distort the sleek shape of the DNA helix. Then a whole group of proteins come in and remove a patch of bases that includes the adduct, and DNA polymerase follows and fills in the patch while adding the correct base back.

A New Way

Before the new study, ATLs were believed to be involved in DNA damage responses, because they protected cells from DNA alkylation damage in lab experiments, but no one understood how they worked or what they did. In the new study, the team describes ATLs’ role.

The scientists undertook a series of structural, genetic, and biochemistry experiments on the protein and determined its structure, both alone and with a guanine that had a methyl adduct and another with a smoking-derived adduct stuck on it. They found that the ATL structure looks like AGT. It, too, had a chemical finger that can rotate a damaged guanine base out from the DNA helix, but it doesn’t remove the adduct like AGT does. Instead, ATL binds tightly to the damaged guanine and bends the DNA in a way that is more pronounced than what AGT does for repair.

“Base flipping by ATL is like a switch that activates the NER pathway, which then removes the alkyl adduct from the guanine,” says first author Julie Tubbs, a research associate at Scripps Research. “So we believe that ATL is conceptually acting like a bridge, connecting the two DNA repair pathways—base and NER—together. This is a surprisingly general mechanism to channel specific base damage into the general NER pathway.”

Before the new study, scientists also didn’t know if ATLs functioned outside of single celled organisms. In the new study, however, the scientists discovered ATLs in two types of ancient organisms, archaeal bacteria and in sea anemone, suggesting this new bridging pathway may be general to most cells and organisms.

“What’s especially important about these newly discovered ATLs is that we now know that ATLs exist in all domains of life, so it is very likely that ATL was common to the evolutionary branches before complex eukaryotes [single-celled or multicellular organisms whose cells contain a distinct membrane-bound nucleus],” Tainer says. “This suggests higher eukaryotes, including mammals and humans, will either have an ATL or have lost or replaced it with a protein of analogous function.”

If ATLs are found in humans, Tainer sees that either inhibiting or bolstering their function could aid cancer therapy. Inhibiting DNA repair would help chemotherapy effectively destroy cancer cells. Augmenting ATL function could help protect sensitive tissue, such as bone marrow, that is easily destroyed during cancer treatment.

“There are all kinds of exciting ideas to emerge from this research,” says Tainer. “For one thing, we now know what to look for when we see resistance to some chemotherapies.”

In addition to Tainer, Margison, Pegg, and Tubbs, authors of the new study are Vitaly Latypov, Amna Butt, Andrew Marriott, Amanda J. Watson, Barbara Verbeek, Gail McGown, and Mary Thorncroft of the University of Manchester; Sreenivas Kanugula of the Pennsylvania State University College of Medicine; Manana Melikishvili and Michael G. Fried of the University of Kentucky; Rolf Kraehenbuehl and Oliver Fleck of Bangor University; Mauro F. Santibanez-Koref of the University of Newcastle-upon-Tyne; Christopher Millington and David M. Williams of the University of Sheffield; Lisa A. Peterson of the University of Minnesota; and Andrew S. Arvai and Matthew D. Kroeger of Scripps Research. Tainer also holds a position at Lawrence Berkeley National Laboratory.

The study was supported by the National Institutes of Health, The Skaggs Institute for Chemical Biology, U.S. Department of Energy, the North West Cancer Research Fund, Cancer Research-UK, and CHEMORES.

Journal reference:

1. Julie L. Tubbs, Vitaly Latypov, Sreenivas Kanugula, Amna Butt, Manana Melikishvili, Rolf Kraehenbuehl, Oliver Fleck, Andrew Marriott, Amanda J. Watson, Barbara Verbeek, et al. Flipping of alkylated DNA damage bridges base and nucleotide excision repair. Nature, 459, 808-813; June 11, 2009 DOI: 10.1038/nature08076

New Way That Cells Fix Damage To DNA Discovered is a post from: dnawellnessinfo.com

ScienceDaily (May 30, 2009) — Researchers at K.U. Leuven and Harvard University show that stretches of DNA previously believed to be useless ‘junk’ DNA play a vital role in the evolution of our genome. They found that unstable pieces of junk DNA help tuning gene activity and enable organisms to quickly adapt to changes in their environments. The results will be published in the journal Science.

Junk DNA

“Most people do not realize that all our genes only comprise about 3% of the total human genome. The rest is basically one large black box,” says Kevin Verstrepen, heading the research team. “Why do we have this DNA, what is it doing?”

Scientists used to believe that most of the DNA outside of genes, the so-called non-coding DNA, is useless trash that has sneaked into our genome and refuses to leave. One commonly known example of such ‘junk DNA’ are the so-called tandem repeats, short stretches of DNA that are repeated head-to-tail. “At first sight, it may seem unlikely that this stutter-DNA has any biological function,” says Marcelo Vinces, one of the lead authors on the paper. “On the other hand, it seems hard to believe that nature would foster such a wasteful system.”

Unstable repeats

The international team of scientists found that stretches of tandem repeats influence the activity of neighboring genes. The repeats determine how tightly the local DNA is wrapped around specific proteins called ‘nucleosomes’, and this packaging structure dictates to what extent genes can be activated. Interestingly, tandem repeats are very unstable — the number of repeats changes frequently when the DNA is copied. These changes affect the local DNA packaging, which in turn alters gene activity. In this way, unstable junk DNA allows fast shifts in gene activity, which may allow organisms to tune the activity of genes to match changing environments — a vital principle for survival in the endless evolutionary race.

Evolution in test tubes

To further test their theory, the researchers conducted a complex experiment aimed at mimicking biological evolution, using yeast cells as Darwinian guinea pigs. Their results show that when a repeat is present near a gene, it is possible to select yeast mutants that show vastly increased activity of this gene. However, when the repeat region was removed, this fast evolution was impossible. “If this was the real world,” the researchers say, “only cells with the repeats would be able to swiftly adapt to changes, thereby beating their repeat-less counterparts in the game of evolution. Their junk DNA saved their lives.”

The research has been funded by Human Frontier Science Program, Fund for Scientific Research Flanders, NIH, K.U. Leuven and VIB (the Flanders Institute for Biotechnology).

1. Marcelo D. Vinces, Matthieu Legendre, Marina Caldara, Masaki Hagihara, and Kevin J. Verstrepen. Unstable Tandem Repeats in Promoters Confer Transcriptional Evolvability. Science, 2009; 324 (5931): 1213 DOI: 10.1126/science.1170097

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Saved By Junk DNA: Vital Role In The Evolution Of Human Genome is a post from: dnawellnessinfo.com