Posts tagged genes

New technique can rapidly turn genes on and off, helping scientists better understand their function.

Although human cells have an estimated 20,000 genes, only a fraction of those are turned on at any given time, depending on the cell’s needs — which can change by the minute or hour. To find out what those genes are doing, researchers need tools that can manipulate their status on similarly short timescales.

That is now possible, thanks to a new technology developed at MIT and the Broad Institute that can rapidly start or halt the expression of any gene of interest simply by shining light on the cells.

The work is based on a technique known as optogenetics, which uses proteins that change their function in response to light. In this case, the researchers adapted the light-sensitive proteins to either stimulate or suppress the expression of a specific target gene almost immediately after the light comes on.

“Cells have very dynamic gene expression happening on a fairly short timescale, but so far the methods that are used to perturb gene expression don’t even get close to those dynamics. To understand the functional impact of those gene-expression changes better, we have to be able to match the naturally occurring dynamics as closely as possible,” says Silvana Konermann, an MIT graduate student in brain and cognitive sciences.

The ability to precisely control the timing and duration of gene expression should make it much easier to figure out the roles of particular genes, especially those involved in learning and memory. The new system can also be used to study epigenetic modifications — chemical alterations of the proteins that surround DNA — which are also believed to play an important role in learning and memory.

Konermann and Mark Brigham, a graduate student at Harvard University, are the lead authors of a paper describing the technique in the July 22 online edition of Nature. The paper’s senior author is Feng Zhang, the W.M. Keck Assistant Professor in Biomedical Engineering at MIT and a core member of the Broad Institute and MIT’s McGovern Institute for Brain Research.

Shining light on genes

The new system consists of several components that interact with each other to control the copying of DNA into messenger RNA (mRNA), which carries genetic instructions to the rest of the cell. The first is a DNA-binding protein known as a transcription activator-like effector (TALE). TALEs are modular proteins that can be strung together in a customized way to bind any DNA sequence.

Fused to the TALE protein is a light-sensitive protein called CRY2 that is naturally found in Arabidopsis thaliana, a small flowering plant. When light hits CRY2, it changes shape and binds to its natural partner protein, known as CIB1. To take advantage of this, the researchers engineered a form of CIB1 that is fused to another protein that can either activate or suppress gene copying.

After the genes for these components are delivered to a cell, the TALE protein finds its target DNA and wraps around it. When light shines on the cells, the CRY2 protein binds to CIB1, which is floating in the cell. CIB1 brings along a gene activator, which initiates transcription, or the copying of DNA into mRNA. Alternatively, CIB1 could carry a repressor, which shuts off the process.

A single pulse of light is enough to stimulate the protein binding and initiate DNA copying. The researchers found that pulses of light delivered every minute or so are the most effective way to achieve continuous transcription for the desired period of time. Within 30 minutes of light delivery, the researchers detected an uptick in the amount of mRNA being produced from the target gene. Once the pulses stop, the mRNA starts to degrade within about 30 minutes.

In this study, the researchers tried targeting nearly 30 different genes, both in neurons grown in the lab and in living animals. Depending on the gene targeted and how much it is normally expressed, the researchers were able to boost transcription by a factor of two to 200.

Karl Deisseroth, a professor of bioengineering at Stanford University and one of the inventors of optogenetics, says the most important innovation of the technique is that it allows control of genes that naturally occur in the cell, as opposed to engineered genes delivered by scientists.

“You could control, at precise times, a particular genetic locus and see how everything responds to that, with high temporal precision,” says Deisseroth, who was not part of the research team.

Epigenetic modifications

Another important element of gene-expression control is epigenetic modification. One major class of epigenetic effectors is chemical modification of the proteins, known as histones, that anchor chromosomal DNA and control access to the underlying genes. The researchers showed that they can also alter these epigenetic modifications by fusing TALE proteins with histone modifiers.

Epigenetic modifications are thought to play a key role in learning and forming memories, but this has not been very well explored because there are no good ways to disrupt the modifications, short of blocking histone modification of the entire genome. The new technique offers a much more precise way to interfere with modifications of individual genes.

“We want to allow people to prove the causal role of specific epigenetic modifications in the genome,” Zhang says.

So far, the researchers have demonstrated that some of the histone effector domains can be tethered to light-sensitive proteins; they are now trying to expand the types of histone modifiers they can incorporate into the system.

“It would be really useful to expand the number of epigenetic marks that we can control. At the moment we have a successful set of histone modifications, but there are a good deal more of them that we and others are going to want to be able to use this technology for,” Brigham says.

(Source: web.mit.edu)

Scientists at Karolinska Institutet have identified the neuronal circuits in the spinal cord of mice that control the ability to produce the alternating movements of the legs during walking. The study, published in the journal Nature, demonstrates that two genetically-defined groups of nerve cells are in control of limb alternation at different speeds of locomotion, and thus that the animals’ gait is disturbed when these cell populations are missing.

Most land animals can walk or run by alternating their left and right legs in different coordinated patterns. Some animals, such as rabbits, move both leg pairs simultaneously to obtain a hopping motion. In the present study, the researchers Adolfo Talpalar and Julien Bouvier together with professor Ole Kiehn and colleagues, have studied the spinal networks that control these movement patterns in mice. By using advanced genetic methods that allow the elimination of discrete groups of neurons from the spinal cord, they were able to remove a type of neurons characterized by the expression of the gene Dbx1.

"It was classically thought that only one group of nerve cells controls left right alternation", says Ole Kiehn who leads the laboratory behind the study at the Department of Neuroscience. "It was then very interesting to find that there are actually two specific neuronal populations involved, and on top of that that they each control different aspect of the limb coordination."

Indeed, the researchers found that the gene Dbx1 is expressed in two different groups of nerve cells, one of which is inhibitory and one that is excitatory. The new study shows that the two cellular populations control different forms of the behaviour. Just like when we change gear to accelerate in a car, one part of the neuronal circuit controls the mouse’s alternating gait at low speeds, while the other population is engaged when the animal moves faster. Accordingly, the study also show that when the two populations are removed altogether in the same animal, the mice were unable to alternate at all, and hopped like rabbits instead.

There are some animals, such as desert mice and kangaroos, which only hop. The researchers behind the study speculate that the locomotive pattern of these animals could be attributable to the lack of the Dbx1 controlled alternating system.

(Source: ki.se)

A chromosomal deletion is associated with changes in the brain’s white matter and delayed language acquisition in youngsters from Southeast Asia or with ancestral connections to the region, said an international consortium led by researchers at Baylor College of Medicine. However, many such children who can be described as late-talkers may overcome early speech and language difficulties as they grow.

The finding involved both cutting edge technology and two physicians with an eye for unusual clinical findings. Dr. Seema R. Lalani, a physician-scientist at BCM and Dr. Jill V. Hunter, professor of radiology at BCM and Texas Children’s Hospital, worked together to identify this genetic change responsible for expressive language delay and brain changes in children, predominantly from Southeast Asia.

Lalani, assistant professor of molecular and human genetics at BCM, is a clinical geneticist and also signs out diagnostic studies called chromosomal microarray analysis, a gene chip that helps identify abnormalities in specific genes and chromosomes, as part of her work at BCM’s Medical Genetics Laboratory.

"I got intrigued when I kept seeing this small (genomic) change in children from a large sample of more than 15,000 children referred for chromosomal microarray analysis at Baylor College of Medicine. These children were predominantly Burmese refugees or of Vietnamese ancestry living in the United States. It started with two children whom I evaluated at Texas Children’s Hospital and soon realized that there was a pattern of early language delay and brain imaging abnormalities in these individuals carrying this deletion from this part of the world. Within a period of two to three years, we found 13 more families with similar problems, having the same genetic change. There were some children who obviously were more affected than the others and had cognitive and neurological problems, but many of them were identified as late-talkers who had better non-verbal skills compared to verbal performance," said Lalani. Hunter, helped in determining the specific pattern of white matter abnormalities in the MRI (magnetic resonance imaging) scans in children and their parents carrying this deletion. Most of the children either came from Southeast Asia or were the offspring of people from that area. (White matter is the paler material in the brain that consists of nerve fibers covered with myelin sheaths.)

Now, in a report that appears online in the American Journal of Human Genetics, Lalani, Hunter and an international group of collaborators identify a genomic deletion on chromosome 2 that is associated with bright white spots that show up in an MRI in the white matter of the brain . The chromosomal deletion removes a portion of a gene known as TM4SF20 that encodes a protein that spans the cellular membrane. They do not know yet what the function of the protein is. They found this genetic change in children from 15 unrelated families mainly from Southeast Asia.

"This deletion could be responsible for early childhood language delay in a large number of children from this part of the world," says Lalani.

She credits Dr. Wojciech Wiszniewski, an assistant professor of molecular and human genetics at BCM with doing much of the work. Wiszniewski has an interest in genomic disorders and is working under the mentorship of Dr. James R. Lupski, vice chair of the department of molecular and human genetics.

Lupski said, “Professor Lalani has made a stunning discovery in that she provides evidence that population-specific intragenic CNV (copy number variation – a deletion or duplication of the chromosome) can contribute to genetic susceptibility of even common complex disease such as speech delay in children.”

"In a way, this is a good news story," said Hunter. There is evidence from family studies that some of these children may do quite well in the future, said Lalani.

Lalani elaborates. “This is a genetic change that is present in 2 percent of Vietnamese Kinh population (an ethnic group that makes up 90 percent of the population in that country),” she said. “In the 15 families we have identified, all children have early language delay. Some are diagnosed with autism spectrum disorder, and if you do a brain MRI study, you find white matter changes in about 70 percent of them. We have found this change in children who are Vietnamese, Burmese, Thai, Indonesian, Filipino and and Micronesian. It is very likely that children from other Southeast Asian countries within this geographical distribution also carry this genetic change.”

Because these are all within a geographic location, she suspects that there is an ancient founder effect, meaning that at some point in the distant past, the gene deletion occurred spontaneously in an individual, who then passed it on to his or her children and to succeeding generations.

"It is important to follow these children longitudinally to see how these late-talkers develop as they grow," said Lalani. "We have also seen this deletion in children whose parents clearly were late-talkers themselves, but overcame the earlier problems to become doctors and professionals. The variability within the deletion carriers is fascinating and brings into question genetic and environmental modifiers that contribute to the extent of disease in these children.

Language delays mean that they may speak only two or three words at age 2, in comparison to other children who would generally have between 75-100 word vocabulary by this age. While there is evidence that children with this deletion may catch up, it is unclear if they continue to have better non-verbal skills than verbal skills. It is also unclear how the specific brain changes correlate with communication disorders in these children.

In fact, when doctors check the parents of these children, they often find similar white matter changes in the parent carrying the deletion. “Young parents in their 30s should not have age-related white matter changes in the brain and these changes should definitely not be present in healthy children,” said Lalani. Hunter said they are not sure how the gene variation relates to the changes in brain white matter and how all of these result in delay in language.

(Source: eurekalert.org)

Finding of disrupted brain gene orchestration gives first direct evidence of circadian rhythm changes in depressed brains, opens door to better treatment

Every cell in our bodies runs on a 24-hour clock, tuned to the night-day, light-dark cycles that have ruled us since the dawn of humanity. The brain acts as timekeeper, keeping the cellular clock in sync with the outside world so that it can govern our appetites, sleep, moods and much more.

But new research shows that the clock may be broken in the brains of people with depression — even at the level of the gene activity inside their brain cells.

It’s the first direct evidence of altered circadian rhythms in the brain of people with depression, and shows that they operate out of sync with the usual ingrained daily cycle. The findings, in the Proceedings of the National Academy of Sciences, come from scientists from the University of Michigan Medical School and other institutions.

The discovery was made by sifting through massive amounts of data gleaned from donated brains of depressed and non-depressed people. With further research, the findings could lead to more precise diagnosis and treatment for a condition that affects more than 350 million people worldwide.

What’s more, the research also reveals a previously unknown daily rhythm to the activity of many genes across many areas of the brain – expanding the sense of how crucial our master clock is.

In a normal brain, the pattern of gene activity at a given time of the day is so distinctive that the authors could use it to accurately estimate the hour of death of the brain donor, suggesting that studying this “stopped clock” could conceivably be useful in forensics. By contrast, in severely depressed patients, the circadian clock was so disrupted that a patient’s “day” pattern of gene activity could look like a “night” pattern — and vice versa.

The work was funded in large part by the Pritzker Neuropsychiatric Disorders Research Fund, and involved researchers from the University of Michigan, University of California’s Irvine and Davis campuses, Weill Cornell Medical College, the Hudson Alpha Institute for Biotechnology, and Stanford University.

The team uses material from donated brains obtained shortly after death, along with extensive clinical information about the individual. Numerous regions of each brain are dissected by hand or even with lasers that can capture more specialized cell types, then analyzed to measure gene activity. The resulting flood of information is picked apart with advanced data-mining tools.

Lead author Jun Li, Ph.D., an assistant professor in the U-M Department of Human Genetics, describes how this approach allowed the team to accurately back-predict the hour of the day when each non-depressed individual died – literally plotting them out on a 24-hour clock by noting which genes were active at the time they died. They looked at 12,000 gene transcripts isolated from six regions of 55 brains from people who did not have depression.

This provided a detailed understanding of how gene activity varied throughout the day in the brain regions studied. But when the team tried to do the same in the brains of 34 depressed individuals, the gene activity was off by hours. The cells looked as if it were an entirely different time of day.

“There really was a moment of discovery,” says Li, who led the analysis of the massive amount of data generated by the rest of the team and is a research assistant professor in U-M’s Department of Computational Medicine at Bioinformatics. “It was when we realized that many of the genes that show 24-hour cycles  in the normal individuals were well-known circadian rhythm genes – and when we saw that the people with depression were not synchronized to the usual solar day in terms of this gene activity. It’s as if they were living in a different time zone than the one they died in.”

Huda Akil, Ph.D., the co-director of the U-M Molecular & Behavioral Neuroscience Institute and co-director of the U-M site of the Pritzker Neuropsychiatric Disorders Research Consortium, notes that the findings go beyond previous research on circadian rhythms, using animals or human skin cells, which were more easily accessible than human brain tissues.

“Hundreds of new genes that are very sensitive to circadian rhythms emerged from this research — not just the primary clock genes that have been studied in animals or cell cultures, but other genes whose activity rises and falls throughout the day,” she says. “We were truly able to watch the daily rhythm play out in a symphony of biological activity, by studying where the clock had stopped at the time of death. And then, in depressed people, we could see how this was disrupted.”

Now, she adds, scientists must use this information to help find new ways to predict depression, fine-tune treatment for each depressed patient, and even find new medications or other types of treatment to develop and test. One possibility, she notes, could be to identify biomarkers for depression – telltale molecules that can be detected in blood, skin or hair.

And, the challenge of determining why the circadian clock is altered in depression still remains. “We can only glimpse the possibility that the disruption seen in depression may have more than one cause. We need to learn more about whether something in the nature of the clock itself is affected, because if you could fix the clock you might be able to help people get better,” Akil notes.

The team continues to mine their data for new findings, and to probe additional brains as they are donated and dissected. The high quality of the brains, and the data gathered about how their donors lived and died, is essential to the project, Akil says. Even the pH level of the tissue, which can be affected by the dying process and the time between death and freezing tissue for research, can affect the results. The team also will have access to blood and hair samples from new donors.

(Source: uofmhealth.org)

Many medical issues affect nerves, from injuries in car accidents and side effects of chemotherapy to glaucoma and multiple sclerosis. The common theme in these scenarios is destruction of nerve axons, the long wires that transmit signals to other parts of the body, allowing movement, sight and sense of touch, among other vital functions.

Now, researchers at Washington University School of Medicine in St. Louis have found a way the body can remove injured axons, identifying a potential target for new drugs that could prevent the inappropriate loss of axons and maintain nerve function.

“Treating axonal degeneration could potentially help a lot of patients because there are so many diseases and conditions where axons are inappropriately lost,” says Aaron DiAntonio, MD, PhD, professor of developmental biology. “While this would not be a cure for any of them, the hope is that we could slow the progression of a whole range of diseases by keeping axons healthy.”

DiAntonio is senior author of the study that appears online May 9 in the journal Cell Reports.

While axonal degeneration appears to be a major culprit in diseases like multiple sclerosis, it also paradoxically plays an important role in properly wiring the nervous systems of developing embryos.

“When an embryo is building its nervous system, there can be inappropriate or excessive axonal sprouts, or axons that are only needed at one time in development and not later,” DiAntonio says. “These axons degenerate, and that’s very important for wiring the nervous system. And in adult organisms, it might be useful to have a clean and quick way to remove a damaged axon from a healthy nerve, instead of letting it decay and potentially damage its neighboring axons.”

DiAntonio compares the process to programmed cell death, or apoptosis, which is also important in embryonic development. Apoptosis culls unnecessary or damaged cells from the body. If cell death programs become overactive, they can kill healthy cells that should remain. And if apoptosis fails to destroy damaged cells in adults, it can lead to cancer.

The new discovery also underscores the relatively recent understanding that loss of axons is not a passive decay process resulting from injury. Just as apoptosis actively destroys cells, axonal degeneration results from a cellular program that actively removes the damaged axon. In certain diseases, the program may be inappropriately triggered.

“We want to understand axonal degeneration at the same level that we understand programmed cell death, in the hopes of developing drugs to block the process when it becomes overactive,” DiAntonio says.

DiAntonio’s major collaborators in this project include Jeffrey D. Milbrandt, MD, PhD, the James S. McDonnell Professor and head of the Department of Genetics, and first author Elisabetta Babetto, PhD, postdoctoral research scholar.

Studying mice, the researchers found that a gene called Phr1 plays a major role in governing the self-destruction of injured axons. When they removed Phr1 from adult mice, the severed portion of the axons remained intact for much longer than in genetically normal mice.

In the normal mice, a severed axon degenerated entirely after two days. In mice without Phr1, they found that about 75 percent of the severed axons remained at five days, with a quarter persisting at least 10 days after being cut. The mice showed no side effects and suffered no obvious problems due to the missing Phr1.

The findings raise the possibility that blocking the Phr1 protein with a drug could keep damaged axons alive and functional when the body would normally cause the axons to self-destruct.

DiAntonio emphasizes that he is not trying to save axons that have no connection to the rest of the nerve. The paradigm is simply a good way to model nerve injury. In many instances, such as a crush injury or disease processes in which the axon is not severed, blocking the Phr1 protein could potentially preserve an attached axon that would otherwise self-destruct.

Importantly, the research team also looked at optic nerves of the central nervous system, which are damaged in glaucoma, and found similar protective effects from the loss of Phr1.

“This is not the first gene identified whose loss protects mammalian axons from degeneration,” DiAntonio says. “But it is the first one that shows evidence of working in the central nervous system. So it could be important in conditions like glaucoma, multiple sclerosis and other neurodegenerative diseases where the central nervous system is the primary problem.”

DiAntonio also points out possible ways to help cancer patients. Many chemotherapy drugs cause damage to peripheral axons, which may limit the doses a patient can tolerate.

As part of the new study, the researchers showed that intact axons without Phr1 were protected from the damage caused by vincristine, a chemotherapy drug used to treat leukemia, neuroblastoma, Hodgkin’s disease and non-Hodgkin’s lymphoma, among other cancers.

“In this case, the loss of axons is not caused by disease,” DiAntonio says. “It’s caused by the drug doctors are giving. You know the date it will start. You know the date it will stop. This is probably where I am most optimistic that we could make an impact.”

(Source: news.wustl.edu)