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Fat in the Family?

Fat in the Family?

Study could lead to therapeutics that boost metabolism

Thu, 2015-08-20

Image: iStock/Juanmonino

Like many other conditions, obesity is caused by an interplay between genetic and environmental factors. While efforts to combat the obesity epidemic will need to include changes in diet and exercise, insights into the genes involved may also help with prevention and treatment.

Now a research team led by Harvard Medical School investigators at Beth Israel Deaconess Medical Center and MIT reveals the mechanistic explanation behind the strongest genetic association with obesity.

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The findings, published in the New England Journal of Medicine, uncover a genetic circuit that controls whether our bodies burn or store fat. Manipulating that genetic circuit may offer a new approach for obesity treatments.

The strongest genetic association with obesity lies within an unexpressed region of the FTO gene and contains 89 common variants in a region of 47,000 nucelotides.

The obesity-risk version of the region that predisposes individuals to increased body weight is found in 44 percent of individuals in European populations, but its mechanistic basis has until now remained unknown, despite extensive investigation.

Assessing epigenetic modifications

To identify the cell types in which the FTO obesity-risk region might exert its effects, the researchers analyzed information from the Roadmap Epigenomics project, which assesses chemical or “epigenetic” modifications within chromosomes that switch genes on or off.

The project’s data revealed that the strongest epigenetic signal was found in “preadipocyte” cells, the progenitor cells that go on to become fat cells.

Melina Claussnitzer. Image: Lovely Valentine“Previous studies attempted to uncover a link between FTO and the regulation of appetite or propensity to exercise controlled by the brain,” said the study’s lead and corresponding author Melina Claussnitzer, an HMS instructor in medicine and an investigator in the Division of Gerontology at Beth Israel Deaconess and Hebrew SeniorLife, a visiting professor at MIT’s Computer Science and Artificial Intelligence Laboratory (CSAIL), and member of the Broad Institute.

“But an unbiased look across more than one hundred human tissues and cell types indicated that the obesity-associated region acts primarily in adipocyte progenitor cells—not the brain,” she said.

The researchers collected fat, or adipose, tissue samples from individuals who carried the genetic obesity risk variant and compared it with tissue samples of individuals who did not carry the variant; they found increased expression of two distant genes, IRX3 and IRX5, indicating that these genes are under genetic control by the obesity risk variant.

“Despite years of investigating the FTO obesity region, no substantial expression differences were found between obesity-risk and non-risk individuals in brain or other tissue types, making it difficult to trace its mechanism of action,” said Manolis Kellis, professor at MIT’s CSAIL.

“We found a strong difference for both IRX3 and IRX5 in preadipocytes, revealing the target genes, cell type and developmental stage where the genetic variant acts, thus enabling us to begin dissecting its mechanism of action,” said Kellis.

Manipulating the pathways

Elevated expression of these genes resulted in a shift from energy-burning beige fat cells to energy-storing white fat cells. The researchers showed that they could manipulate this new pathway to reverse the signatures of obesity.

“By altering the expression of either gene in human preadipoctyes, we could alter adipocyte metabolism between energy storage and energy dissipation, providing a direct link between IRX3 and IRX5 expression and energy balance,” said Kellis.

To evaluate the effect of IRX3 inhibition on whole-body energy metabolism and body weight, the team inhibited the corresponding gene in the fat cells of mice. The animals’ metabolism increased and they lost weight, even though their physical activity and appetite were unchanged.

“The results at the organism level were dramatic,” said Claussnitzer.

“These mice were 50 percent thinner than the control mice, and they did not gain any weight on a high-fat diet. Instead they dissipated more energy, even in their sleep, suggesting a dramatic shift in their global metabolism. The circuitry underlying the FTO region functions like a master regulatory switch between energy storage and energy dissipation,” she said.

The researchers then sought to connect these differences in metabolism and gene expression to the genetic differences between lean and obese people within the FTO gene.

They predicted that the specific T-to-C single-nucleotide alteration within FTO is responsible for the obesity association by repressing an evolutionarily conserved gene regulator called ARID5B.

Loss of repression turns on IRX3 and IRX5 during early adipocyte differentiation, leading to a shift from beige adipocyte functions and thermogenesis, or energy burning, to white adipocyte lipid accumulation.

“We could narrow down a genetic region spanning 47,000 nucleotides to reveal a single-nucleotide alteration, and explain precisely how it leads to loss of repressor binding, activation of a regulatory region, gain of distal gene expression, a change in adipocyte metabolism, and ultimately, obesity at the organism level,” explained Claussnitzer.

Noncoding variants

“This can serve as a model for understanding the mechanistic basis of other non-coding variants in other diseases and traits. Noncoding variants make up more than 90 percent of top-scoring variants that have emerged from genome-wide association studies, which find association between genetic variants and disease risk, she said.”

Using the genome editing technique known as CRISPR/Cas9, the team found that switching the risk variant to the protective variant in preadipocytes turned off IRX3 and IRX5 and restored thermogenesis, while the reverse change turned on IRX3 and IRX5 and turned off thermogenesis.

“Bidirectional genome editing of the causal nucleotide variant allowed us to demonstrate that a single nucleotide is responsible for flipping this metabolic switch between obese and lean individuals,” said Claussnitzer.

“This is the first time that causality has been demonstrated for a genetic variant in a distal non-coding region, but we hope it will be the first of many such studies to come, now that genome editing is becoming broadly adopted,” she added.

This study was supported, in part, by grants from National Institutes of Health (R01HG004037, R01GM113708 and RC1HG005334).

Adapted from a Beth Israel Deaconess news release. 

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Gene for Mitral Valve Prolapse

Gene for Mitral Valve Prolapse

International network reveals role of gene mutations in families with inherited cardiac disorder

By SUE McGREEVEY
Tue, 2015-08-11

An international research collaboration led by Harvard Medical School investigators at Massachusetts General Hospital has identified the first gene whose mutations cause the common form of mitral valve prolapse, a heart valve disorder that affects almost 2.5 percent of the U. S. population.

In a paper published in Nature, the team reports finding mutations in a gene called DCHS1 in affected members of three families in which mitral valve prolapse is inherited.

“This work provides insights into the pathways regulating valve growth and development and implicates a previously unrecognized basis for the long-term structural integrity of the mitral valve,” said senior author Susan Slaugenhaupt, HMS professor of neurology.

Mitral valve prolapse. Image: NIHLBIShe is also scientific director of the Mass General Research Institute, an investigator at the Mass General Center for Human Genetic Research and one of the lead scientists in the collaborative group that conducted the research. 

“This finding can teach us how to prevent this inborn disease from manifesting as an illness in people who inherit mutated forms of this gene,” said Robert Levine, HMS professor of medicine at Mass General and co-senior author of the Nature paper. “Understanding how defects in this gene cause errors in early valve formation can point to ways we can prevent the progression of this condition to keep the valve and the heart healthy and help the patient avoid complications.”

One of four valves controlling the flow of blood through the heart, the mitral valve lies between the left atrium and the left ventricle, which handle oxygenated blood returning from the lungs. The valve consists of two leaflets that open to let blood pass through and close to keep it from moving backwards.

In mitral valve prolapse, the leaflets become thickened, elongated and floppy, preventing the valve from closing completely and allowing blood to leak backward in a process called regurgitation. Patients with serious mitral valve prolapse can develop shortness of breath, cardiac arrhythmia, heart failure or an infection of the heart valves. Mitral valve prolapse is the most common reason for mitral valve surgery.

While mitral valve prolapse can accompany connective tissue disorders such as Marfan syndrome, its most common form runs in families without such syndromes. A specific genetic cause of familial mitral valve prolapse had not previously been identified, and the Mass General research team’s first step was to link the occurrence of mitral valve prolapse in one large family to a genetic risk factor located on chromosome 11.

Their work depended on advanced diagnostics developed by Levine and colleagues in the Mass General Cardiac Ultrasound Laboratory, which in turn relied on their discovery of the three-dimensional shape of the mitral valve.

In the current study, detailed DNA analysis of the affected members of that family identified two rare mutations in DCHS1, a chromosome 11 gene previously studied in fruit flies. Experiments in zebrafish led by David Milan, HMS assistant professor of medicine at Mass General and co-senior author of the paper, revealed that inactivation of an analogous gene led to significant defects in the development of the heart at the site corresponding to the mitral valve. These defects could be prevented by the introduction of the normal copy of the human DCHS1 gene but not the mutated version.

Based on those findings, the Mass General team sought to collaborate with other groups studying mitral valve prolapse in order to determine whether the same mutations had a role in other families with the condition. A major grant from the Leducq Foundation enabled the formation of a network consisting of 11 centers in the U.S. and four European countries. Analysis of DNA from those centers identified two French families in which mitral valve prolapse was caused by another DCHS1 mutation.

Further experiments in cells indicated that mutations associated with mitral valve prolapse significantly reduced the expression of the DCHS1 protein, which helps organize how cells are patterned into tissues.

Co-senior author Russell Norris and his team at the Medical University of South Carolina analyzed the development of mice in which one copy of the DCHS1 gene was mutated, as it is in affected family members. Their findings revealed that the gene plays a critical role in the proper formation of the mitral valve, the first evidence for the gene’s role in cardiac development, and that the mutation led to mitral valve changes resembling the human disease.

“This discovery required the cooperation of multiple disciplines and teams—ranging from clinical cardiology and ultrasound diagnostics to classical genetics, screening of potential mutations in zebrafish and functional studies in our mouse models,” said Levine.

Milan added, “As a follow-up, this same international network has been seeking other genes that cause mitral valve prolapse across the population, which should point us to common pathways that could be targets of therapies designed to prevent progression into symptomatic disease.”

In addition to a grant from the Leducq Foundation, support for the study includes grants from the Doris Duke Medical Foundation and several grants from the National Heart, Lung, and Blood Institute.

Adapted from a Mass General news release.

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Building Muscle

Building Muscle

Lab-grown muscle fibers offer new muscular dystrophy model 

By HALEY BRIDGER
Mon, 2015-08-03

Researchers have been able to grow differentiated muscle fibers (green) in the lab. Nuclei are shown in blue. Image: Olivier Pourquié

Skeletal muscle is one of the most abundant tissue types in the human body, but it has proven difficult to produce in large quantities in the lab.

Unlike other cell types, such as heart cells, neurons and cells found in the gut, previous attempts to efficiently and accurately derive muscle cells from precursor cells or from culture have not been fruitful.

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In a study published Aug. 3 in Nature Biotechnology, a research team led by investigators from Harvard Medical School, Brigham and Women’s Hospital and the Harvard Stem Cell Institute report that by identifying and mimicking important developmental cues, they have been able to drive cells to grow into muscle fibers, producing millimeter-long muscle fibers capable of contracting in a dish and multiplying in large numbers.

This new method of producing muscle cells could offer a better model for studying muscle diseases, such as muscular dystrophy, and for testing potential treatment options.

Previous studies have used genetic modification to create small numbers of muscle cells in the lab, but the research team wanted a technique that would allow them to grow large numbers of muscle cells efficiently for use in clinical applications.

“We took the hard route,” said corresponding author Olivier Pourquié, the Frank Burr Mallory Professor of Pathology at Brigham and Women’s and professor of genetics at HMS. “We wanted to recapitulate all of the early stages of muscle cell development that happen in the body and recreate that in a dish in the lab.”

“We analyzed each stage of early development and generated cell lines that glowed green when they reached each stage,” he said. “Going step by step, we managed to mimic each stage of development and coax cells toward muscle cell fate.”

The team found that a combination of secreted factors that are important at early embryonic stages are also essential for stimulating differentiation—the specialization of stem cells into particular cell types—in the lab. Using just the right recipe for differentiation, the team was able to produce long, mature fibers in a dish, derived from mouse or human pluripotent stem cells.

They also cultured stem cells from a mouse model of Duchenne muscular dystrophy, observing the striking branched pattern that dystrophin-deficient muscle fibers show in the body.

The research team was also able to produce more-immature cells known as satellite cells which, when grafted into the mouse model of Duchenne muscular dystrophy, produced muscle fibers. Further studies will be needed to determine if the new strategy could be optimized to develop cell therapies for treating degenerative diseases in humans.

In addition to developing a better model for Duchenne muscular dystrophy, the new protocol may be useful for studying other muscle diseases such as sarcopenia (degenerative muscle loss), cachexia (wasting away of muscle associated with severe illness) and other muscular dystrophies.

“This has been the missing piece. The ability to produce muscle cells in the lab could give us the ability to test out new treatments and tackle a spectrum of muscle diseases,” said Pourquié.

This work was supported by the European Research Council, the Stowers Institute for Medical Research, the Howard Hughes Medical Institute, the FP7 EU grant Plurimes (agreement no. 602423) and a strategic grant from the French Muscular Dystrophy Association (AFM).

Adapted from a Brigham and Women’s news release.

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Delivering Hope in Ovarian Cancer

Delivering Hope in Ovarian Cancer

Gene therapy blocked chemoresistant tumor growth in mice

By SUE McGREEVEY
Wed, 2015-08-05

HMS researchers at Massachusetts General Hospital used gene therapy to improve survival of subjects with recurrent ovarian cancer. Image: Wikimedia Commons

Using gene therapy to deliver a protein that suppresses the development of female reproductive organs may improve the survival of patients with ovarian cancer that has recurred after chemotherapy. Recurrence happens 70 percent of the time and is invariably fatal. 

In their report published in PNAS Early Edition, Harvard Medical School researchers at Massachusetts General Hospital describe how a single injection of a modified version of Mullerian Inhibiting Substance, a protein critical to sexual development, suppressed the growth of chemotherapy-resistant ovarian tumors in a mouse model. 

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While not all the tested tumors, which were grown from cells grafted from patient tumors, were sensitive to this treatment, the investigators also outlined a noninvasive way to screen cancer cells in vitro for treatment responsiveness.

“Our findings are important because there are currently no therapeutic options for recurrent, chemoresistant ovarian cancer,” said David Pépin, HMS instructor in surgery at Mass General and lead author of the report. “This is also a proof of concept that gene therapies with the AAV9 vector can be used to deliver biologics for the treatment of ovarian cancer and represents the first time this approach has been tested in this type of ovarian cancer model.” 

Potential to treat cancer

During embryonic development, Mullerian Inhibiting Substance is secreted by tissues in male embryos to prevent maturation of the Mullerian duct, which otherwise would give rise to female reproductive organs. The potential of MIS to treat ovarian cancer and other reproductive tumors has been studied for many years by Patricia Donahoe,  Marshall K. Bartlett Distinguished Professor of Surgery at HMS, director of Mass General’s Pediatric Surgical Research Laboratories and senior author of the current study. 

Previous investigations by Donahoe’s team showed that MIS suppresses ovarian cancer growth, both in animals and in human cell lines, by targeting the cancer stem cells that survive chemotherapy. But previous methods of producing MIS were unable to generate sufficient quantities of high-quality protein for pre-clinical testing.

The current study employed a modified form of the MIS gene, developed by Pépin to generate proteins of greater purity and effectiveness, combined with the accepted viral vector AAV9 for delivery into the peritoneal cavity, a common site for the recurrence of ovarian cancer. The modified MIS/AAV9 construct was tested against tumor cells taken from ascites fluid that had accumulated within the abdomens of several patients with recurrent ovarian cancer. 

Testing effectiveness

Initial experiments confirmed that these cells expressed the MIS receptor protein, that they carried markers indicating their identity as cancer stem cells, and that their growth was inhibited in vitro by MIS. A single injection of the MIS/AAV9 construct into the peritoneal cavity of mice resulted in elevated expression of MIS by multiple tissues throughout the abdominal cavity and in adjacent muscles.

The effectiveness of the MIS/AAV9 construct was tested in mice into which ovarian cancer cells were implanted. Those efforts revealed that treatment with MIS/AAV9 three weeks before tumor implantation significantly inhibited tumor growth. 

In a more clinically relevant experiment, the therapy was applied to mice in which tumors already had been induced by implantation of cancer cells from five different patients. The therapy resulted in significant inhibition of further growth of tumors generated from the cells of three of the five patients. 

Analysis of tumor samples from more than 200 patients revealed that 88 percent expressed some level of the MIS receptor, with 65 percent expressing moderate or high protein expression.

Need for biomarkers

“Since the response to MIS gene therapy is not the same for all patients, it will be important to first screen each patient’s tumors to ensure they will respond” said Pépin. “While we have not yet identified biomarkers of treatment response—something we are currently searching for—we have described a way to rapidly grow tumor cells from ascites to be evaluated for drug sensitivity. If further study confirms the susceptibility of chemoresistant tumors to this MIS gene therapy, the ability to inhibit tumor recurrence could significantly extend patient survival.”

The MIS/AAV9 construct was prepared by Guangping Gao, director of the Gene Therapy Center at the University of Massachusetts Medical School.  The single, long-acting injection makes the use of this effective but complex protein both clinically feasible and patient friendly, Donahoe said.

“All of the implanted tumor cells were from patients who failed all previous therapies, so a 60 percent response rate is quite significant for a single agent,” said Donahoe. “Our results provide proof of concept and predict a translation into patient care that was not previously possible.”

Support for the study includes grants from the Ovarian Cancer Research Fund, the Sudna Gar Foundation, Department of Defense Award OC110078 and National Institutes of Health grant R01-CA17393.

Adapted from a Mass General news release.

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American History 201

American History 201

Genetic studies link indigenous peoples in the Amazon and Australasia

By STEPHANIE DUTCHEN
Tue, 2015-07-21

First author Pontus Skoglund explains his team’s new hypothesis about the ancient migrations that gave rise to today’s Native Americans. Video: Stephanie Dutchen

Native Americans living in the Amazon bear an unexpected genetic connection to indigenous people in Australasia, suggesting a previously unknown wave of migration to the Americas thousands of years ago, a new study has found.

“It’s incredibly surprising,” said David Reich, Harvard Medical School professor of genetics and senior author of the study. “There’s a strong working model in archaeology and genetics, of which I have been a proponent, that most Native Americans today extend from a single pulse of expansion south of the ice sheets—and that’s wrong. We missed something very important in the original data.”

Previous research had shown that Native Americans from the Arctic to the southern tip of South America can trace their ancestry to a single “founding population” called the First Americans, who came across the Bering land bridge about 15,000 years ago. In 2012, Reich and colleagues enriched this history by showing that certain indigenous groups in northern Canada inherited DNA from at least two subsequent waves of migration.

The new study, published July 21 in Nature, indicates that there’s more to the story.

Pontus Skoglund, first author of the paper and a postdoctoral researcher in the Reich lab, was studying genetic data gathered as part of the 2012 study when he noticed a strange similarity between one or two Native American groups in Brazil and indigenous groups in Australia, New Guinea and the Andaman Islands.

“That was an unexpected and somewhat confusing result,” said Reich, who is also an associate member of the Broad Institute of Harvard and MIT and a Howard Hughes Medical Investigator. “We spent a really long time trying to make this result go away and it just got stronger.”

Skoglund and colleagues from HMS, the Broad and several universities in Brazil analyzed publicly available genetic information from 21 Native American populations from Central and South America. They also collected and analyzed DNA from nine additional populations in Brazil to make sure the link they saw hadn’t been an artifact of how the first set of genomes had been collected. The team then compared those genomes to the genomes of people from about 200 non-American populations.

The link persisted. The Tupí-speaking Suruí and Karitiana and the Ge-speaking Xavante of the Amazon had a genetic ancestor more closely related to indigenous Australasians than to any other present-day population. This ancestor doesn’t appear to have left measurable traces in other Native American groups in South, Central or North America.

An indigenous Xavante midwife. Image: Agência Brasil

The genetic markers from this ancestor don’t match any population known to have contributed ancestry to Native Americans, and the geographic pattern can’t be explained by post-Columbian European, African or Polynesian mixture with Native Americans, the authors said. They believe the ancestry is much older—perhaps as old as the First Americans.

In the ensuing millennia, the ancestral group has disappeared.

“We’ve done a lot of sampling in East Asia and nobody looks like this,” said Skoglund. “It’s an unknown group that doesn’t exist anymore.”

The team named the mysterious ancestor Population Y, after the Tupí word for ancestor, “Ypykuéra.”

Reich, Skoglund and colleagues propose that Population Y and First Americans came down from the ice sheets to become the two founding populations of the Americas.

“We don’t know the order, the time separation or the geographical patterns,” said Skoglund.

Researchers do know that the DNA of First Americans looked similar to that of Native Americans today. Population Y is more of a mystery.

“About 2 percent of the ancestry of Amazonians today comes from this Australasian lineage that’s not present in the same way elsewhere in the Americas,” said Reich.

However, that doesn’t establish how much of their ancestry comes from Population Y. If Population Y were 100 percent Australasian, that would indeed mean they contributed 2 percent of the DNA of today’s Amazonians. But if Population Y mixed with other groups such as the First Americans before they reached the Americas, the amount of DNA they contributed to today’s Amazonians could be much higher—up to 85 percent.

To answer that question, researchers would need to sample DNA from the remains of a person who belonged to Population Y. Such DNA hasn’t been obtained yet. One place to look might be in the skeletons of early Native Americans whose skulls some researchers say have Australasian features. The majority of these skeletons were found in Brazil.

Reich and Skoglund think that some of the most interesting open questions about Native American population history are about the relationships among groups after the initial migrations.

“We have a broad view of the deep origins of Native American ancestry, but within that diversity we know very little about the history of how those populations relate to each other,” said Reich.

This work was supported by the National Science Foundation (HOMINID grant BCS-1032255), the National Institutes of Health (GM100233), the Simons Foundation (grant 280376), the Howard Hughes Medical Institute, the Conselho Nacional de Desenvolvimento Científico e Tecnológico and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil), the Wenner-Gren Foundation and the Swedish Research Council (VR grant 2014-453). 

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Evolving CRISPR

Evolving CRISPR

Engineered variants double the target range and improve the precision of gene-cutting enzyme

By SUE McGREEVEY
Mon, 2015-06-22

CRISPR-Cas9 gene-editing complex from Streptococcus pyogenes. Image: iStock

A team of Harvard Medical School researchers at Massachusetts General Hospital has found a way to expand the use and precision of powerful gene-editing tools called CRISPR-Cas9 RNA-guided nucleases.

In their report in Nature, the investigators describe evolved versions of the DNA-cutting Cas9 enzyme that can recognize a different range of nucleic acid sequences than is now possible with the naturally occurring form of Cas9 scientists have been using.

“In our paper we show that sites in human and zebrafish genes that could not previously be modified by wild-type Cas9 can now be targeted with the new variants we have engineered,” said Benjamin Kleinstiver, HMS research fellow in pathology at Mass General and lead author of the Nature paper. “This will allow researchers to target an expanded range of sites in a variety of genomes, which will be useful for applications requiring highly precise targeting of DNA sequences.”

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CRISPR-Cas9 nucleases consist of the Cas9 bacterial enzyme and a short, 20-nucleotide RNA molecule that matches the target DNA sequence. In addition to the RNA/DNA match, the Cas9 enzyme needs to recognize a specific nucleotide sequence called a protospacer adjacent motif (PAM) adjacent to the target DNA.

The most commonly used form of Cas9, derived from the bacteria Streptococcus pyogenes and known as SpCas9, recognizes PAM sequences in which any nucleotide is followed by two guanine DNA bases. This limits the DNA sequences that can be targeted using SpCas9 to only those that include two sequential guanines.

To get around this limitation, the team set up an engineering system that allowed them to rapidly evolve the ability of SpCas9 to recognize different PAM sequences. From a collection of randomly mutated SpCas9 variants, they identified combinations of mutations that enabled SpCas9 to recognize new PAM sequences.

These evolved variants essentially double the range of sites that can now be targeted for gene editing using SpCas9. They also identified an SpCas9 variant that was less likely to induce the off-target gene mutations sometimes produced by CRISPR-Cas9 nucleases, a problem originally described in a 2013 study led by J. Keith Joung, HMS professor of pathology at Mass General and senior author of the current study.

“This additional evolved variant with increased specificity should be immediately useful to all researchers who currently use wild-type SpCas9 and should reduce the frequencies of unwanted off-target mutations,” Joung said. “Perhaps more important, our findings provide the first demonstration that the activities of SpCas9 can be altered by directed protein evolution.”

The scientists showed in their paper that the forms of Cas9 found in two other bacteria—Staphylococcus aureus and Streptococcus thermophiles—can also function in their bacterial evolution system, suggesting that their functions can be modified as well, Joung said.

“This work just scratches the surface of the range of PAMs that can be targeted by Cas9,” Joung said. “We believe that other useful properties of the enzyme may be modified by a similar approach, allowing potential customization of many important features.”

The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM107427 and R01 GM088040, a Jim and Ann Orr Research Scholar Award, and a National Sciences and Engineering Research Council of Canada Postdoctoral Fellowship. The MGH has filed a patent application on the use of the SpCas9 variants described in this paper.

Adapted from a Mass General news release.

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FISHing for Insight

FISHing for Insight

Improved imaging illuminates chromosomes in detail

By STEPHANIE DUTCHEN
Thu, 2015-05-21

HMS professor of genetics Ting Wu explains why she’s excited about new chromosomal imaging techniques. Video: Stephanie Dutchen

Geneticists just got a new pair of glasses.

By improving an imaging technology called FISH, they’ve made it possible to view genetic material in more detail than ever before.

One modification achieves “super resolution” imaging. The other can distinguish maternal from paternal chromosomes.

What the researchers are starting to see promises to help them better understand how DNA gets packaged into chromosomes, how that structure relates to health and disease, what the biological significance may be when genetic material is inherited from one parent versus the other—and more.

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“Geneticists have looked at chromosomes for over a century, but they’ve longed for higher resolution and an easier and more affordable strategy for looking at any part of any genome, not just the most accessible regions,” said Ting Wu, professor of genetics at Harvard Medical School and senior author of the study, published in Nature Communications. “With this new technology, we’ve enabled significant steps forward in all those regards.”

“Scientists have a lot of models where we draw in cartoon form what we think is happening,” said the study’s first author, Brian Beliveau, who conducted the work as a graduate student in the Wu lab. “When a region of the genome is silenced, we draw it as compacted. When a gene is expressed, we draw it as more open or active. But we don’t actually know yet what any of these look like.”

“Now we have a tool to start looking at this systematically, which is very exciting,” said Beliveau, currently a research fellow in the lab of HMS associate professor of systems biology Peng Yin at the Wyss Institute for Biologically Inspired Engineering.

Brian Beliveau describes the advances Oligopaints have made possible. Video: Stephanie Dutchen

Teach a person to FISH…

FISH, short for fluorescence in situ hybridization, is a decades-old visualization technique that locates and illuminates specific genes in the nuclei of cells.

Scientists use FISH to find out things like how many chromosomes a person has or where a particular gene is located on a chromosome.

But conventional FISH has its limits, and scientists want to look closer.

For instance, it hasn’t been able to show the details of how chromosomes are folded, whether there are different ways of folding or how two genome segments interact—all of which would help elucidate how our bodies work.

Researchers have had some success getting high-resolution FISH images of small segments of the genome. But the cost to look at such questions genome-wide has been “beyond anybody’s purse,” said Wu. “The flexibility to target a specific region on the genome has been very difficult also.”

In 2012, Beliveau devised an enhancement to FISH called Oligopaint that made this possible at low cost.

Traditional FISH works by attaching a fluorescent tag to a short, single strand of DNA called a probe, which has a sequence complementary to the segment of DNA a researcher wants to study. When released into a cell, the probe binds to the desired sequence in the nucleus and lights it up so it can be seen under a microscope.

A limiting factor to scaling up FISH has been that “it’s a ton of work to try to find those sequences in nature to make the necessary DNA probes and isolate them in a way that would be compatible with the tool,” said Beliveau.

He solved the problem by developing computer software that lets scientists design the probes they need and then build them with synthetic components—hundreds or thousands at a time.

Super resolution

In the new paper, Beliveau taught Oligopaint two new tricks.

First, he paired it up with two other technologies—STORM from the lab of Xiaowei Zhuang at Harvard University, and DNA-PAINT from Peng Yin’s lab—to zoom in on chromosomes at “super resolution.”

Conventional (left) and STORM (right) images depict the same field of view at the same magnification within a cell. STORM reveals two loop-like protrusions in the cell’s genetic material. Image: Alistair Boettiger, Zhuang lab

The DNA double helix is about 2 nanometers wide. When it gets wound around a bundle called a nucleosome, one of the simplest building blocks of chromosomes, it grows to about 10 nanometers. Since conventional microscopy can resolve images to only about 200 to 300 nanometers, it’s been impossible to see such infinitesimal structures.

The new Oligopaint combinations can bring chromosomes into focus down to about 20 nanometers. That’s possible because each DNA probe is able to bind to a second probe; the resulting fluorescent output blinks, enabling researchers to distinguish the fluorescent tags one at a time and achieve a finer resolution.

“This is an unprecedented level of detail,” said Wu.

“Right now when you do FISH on structures below a certain size, you get a spot,” said Beliveau. “With these technologies, we’re seeing interesting shapes and structures, loops and protrusions, start to drop out of these things.”

Wu, Beliveau and their colleagues have already begun to find “intriguing organizational themes” in the chromatin of mammalian and fruit fly cells. But they consider the current paper mostly a proof of concept.

“The goal is to communicate these techniques to the research community as quickly as possible so everyone can start making discoveries,” said Beliveau.

They hope researchers generate “a huge bolus of images” that will reveal more about what our genetic structure looks like, Wu added.

“Of course, naturally, if we get down to 20 nanometers then people will want 10 nanometers, and then 5,” she said wryly.

Distinguishing mom from dad

We normally inherit our chromosomes pairs: one from our mother and one from our father. The second trick Beliveau taught Oligopaint was to tell them apart.

“This was not thought possible but, to our surprise, our technology managed to break this barrier too,” said Wu.

Beliveau modified his Oligopaint probes so they could detect the presence or absence of the many single nucleotide variants, or SNPs, that distinguish maternal from paternal chromosomes.

Advances in Oligopaint imaging can distinguish maternal (green) from paternal (magenta) chromosomes in a fruit fly. By contrast, conventional Oligopaint probes (blue) bind to both chromosomes. Image: Eric Joyce, Wu lab

As long as researchers know which SNPs to look for and have hundreds or thousands of them to target—just a few isn’t enough—Oligopaint can now light up a chromosome to identify “mom” or “dad.”

Wu, for one, is excited to use this new ability to study things like how one of two X chromosomes gets inactivated in female mammals. It’s also become clear to scientists that maternal and paternal genes don’t behave the same way, and that this might have effects on human health and disease. Wu is particularly interested in Down syndrome, in which children inherit three instead of two copies of chromosome 21.

“I’d love to look at a cell and ask, are two copies from the mother? From the father? How do they behave relative to each other?” she said.

This study was supported by the National Institutes of Health (R01GM61936, R01GM090278, 1R01EB018659, 5DP1GM106412, F32CA157188, 1DP2OD007292, 5R21HD072481, 1DP2OD004641), National Science Foundation (CCF1054898, CCF1162459), Office of Naval Research (N000141110914, N000141010827, N000141310593), Harvard Medical School, Wyss Institute for Biologically Inspired Engineering, Centre National de la Recherche Scientifique, Howard Hughes Medical Institute, Damon Runyon Cancer Research Foundation, Fulbright Visiting Scholar Program and Alexander von Humboldt-Foundation.

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Beyond Average

Beyond Average

New platforms genetically barcode tens of thousands of cells at a time

By STEPHANIE DUTCHEN
Thu, 2015-05-21

“We’re really excited by the questions that this technology is now opening up for us.” Allon Klein, Marc Kirschner and David Weitz describe inDrop sequencing. Video: Rick Groleau

Imagine someone hands you a smoothie and asks you to identify everything that went into it.

You might be able to discern a hint of strawberry or the tang of yogurt. But overall it tastes like a blend of indiscernible ingredients.

Now imagine that the smoothie is made of 20,000 ground-up cells from, say, the brain.

You could run tests to determine what molecules are in the sample, which is what scientists do now. That would certainly give you useful information, but it wouldn’t tell you which cells those molecules originally came from. It would provide only an average cell profile for the whole smoothie.

And when it comes to the tissues in our bodies, averages are almost always misleading. Just as you know there isn’t an “average” food called strawbanaspinach-orangegurt, scientists know there isn’t just one cell type in the brain.

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“If you take a hunk of tissue and grind it up and analyze the RNA, you have no idea if it represents what every cell in that population is doing or what no cell in the population is doing,” said Marc Kirschner, the John Franklin Enders University Professor of Systems Biology and chair of the Department of Systems Biology at Harvard Medical School. “Imagine if you had a population of men and women. If you assume everyone is an average of men and women, you [probably] wouldn’t represent a single person in that population.”

The trouble is, it’s expensive, time-consuming and tricky to characterize tissues one cell, or cell type, at a time.

Kirschner and Steven McCarroll, assistant professor of genetics at HMS, reported this week in separate papers that their labs have developed high-throughput techniques to quickly, easily and inexpensively give every cell in a sample a unique genetic barcode before it goes into the blender.

As a result, scientists can analyze complex tissues by profiling each individual cell—no averaging required.

“Different cells in a tissue use the same genome in amazingly diverse ways: to engineer specialized cell shapes, accomplish diverse feats of physiology, and mount distinct functional responses to the same stimulus. These techniques will finally let science understand how biological systems operate at that single-cell level,” said McCarroll, who is also director of genetics for the Stanley Center for Psychiatric Research at the Broad Institute of Harvard and MIT. “We are so excited about the work ahead.”

To make their tools, both teams collaborated with David Weitz, the Mallinckrodt Professor of Physics and Applied Physics at Harvard’s School of Engineering and Applied Sciences and a pioneer in the field of microfluidics.

The teams expect that their techniques, published concurrently in the journal Cell, will equip biologists to discover and classify cell types in the body in much greater depth, map cell diversity in complex tissues such as the brain, better understand stem cell differentiation and gain more insights into the genetics of disease.

“We can really look at complex systems now, like the brain or the immune system.” Evan Macosko, Steven McCarroll and Anindita Basu describe Drop-seq. Video: Boston Science Communications

Harvard’s Office of Technology Development has been working closely with the researchers to develop patent applications for various aspects of the technology, all with an eye toward commercialization.

‘Two roads diverged in a yellow wood’

Evan Macosko and Allon Klein met in a microfluidics class a few years ago. Then they went their separate ways.

Unbeknownst to each other, they decided to develop methods to answer the same question: How could they obtain gene expression profiles for thousands of individual cells to better understand the complexity of gene expression within a tissue?

Gene expression—the pattern of gene activity in a particular cell—underlies every process in biology, from cognition in the brain to development in the egg. Scientists have known for 50 years that gene expression varies from cell to cell like a fingerprint, making skin cells different from liver cells and making some liver cells different from others. But they haven’t been able to measure it efficiently at the single-cell level in samples with many cell types.

Macosko, HMS instructor in psychiatry at Massachusetts General Hospital and a Stanley Neuroscience Fellow in the McCarroll lab, came up with a technique he called Drop-seq. Klein, assistant professor of systems biology at HMS, devised a method he called indexing droplets for sequencing, or inDrops.

Last fall, they learned about each other’s work through the scientific conference circuit.

“It was kind of like meeting your doppelgänger,” said Macosko. “He had been thinking about the same things I had for two years. Human beings have different ways of solving problems, and it was really cool to see how he did it.”

How they work

The teams each developed ways of using tiny beads to deliver vast numbers of different DNA barcodes into hundreds of thousands of nanometer-sized water droplets simultaneously.

Thanks to Weitz’s expertise, both methods were able to use microfluidic devices to co-encapsulate cells in these droplets along with the beads. The droplets get created in a tiny assembly line, streaming along a channel the width of a human hair.

The bead barcodes get attached to the genes in each cell, so that scientists can sequence the genes all in one batch and still trace each gene back to the cell it came from.

Macosko and Klein make their beads in different ways. The droplets get broken up at different steps in the process. Other aspects of the chemistry diverge. But the result is the same.

After running a single batch of cells through Drop-seq or inDrops, scientists “can see which genes are expressed in the entire sample—and can sort by each individual cell,” said Klein.

They can then use computer software to uncover patterns in the mix, including which cells have similar gene expression profiles. That provides a way to classify what cell types were in the original tissue—and to possibly discover new ones.

Current methods allow researchers to generate 96 single-cell expression profiles in a day for several thousand dollars. Drop-seq, by comparison, enables 10,000 profiles a day for 6.5 cents each.

“If you’re a biologist with an interesting question in mind, this approach could shine a light on the problem without bankrupting you,” said Macosko. “It finally makes gene expression profiling on a cell-by-cell level tractable and accessible. I think it’s something biologists in a lot of fields will want to use.”

Rather than competing with each other, the teams believe that having two options available in Drop-seq and inDrops will benefit the scientific community.

“Each method has unique elements that makes it better for different applications. Biologists will be able to choose which one is most appropriate for them,” said Macosko.

Different goals

McCarroll, Macosko and their colleagues are excited to explore the brain with Drop-seq.

With luck, that will include discovering new cell types, constructing a global architecture of those cell types in the brain and understanding brain development and function as they relate to disease.

Among the questions they want to pursue are: What are all the cell types that make the brain work? How do these cell types vary in their functions and responses to stimuli? What cell populations are missing or malfunctioning in schizophrenia, autism and other disorders of the brain?

Classifying cell types may not sound exciting, said Joshua Sanes, the Jeff C. Tarr Professor of Molecular and Cellular Biology and the Paul J. Finnegan Family Director of the Center for Brain Science at Harvard University and a co-author of the Drop-seq paper, but it lays the foundation for mapping neuronal circuits and one day being able to probe the mystery of how the “wetware” of the brain gives rise to thoughts, emotions and behaviors.

In the shorter term, Sanes looks forward to completing a catalog of cell types in the mouse retina. Drop-seq has already revealed several new ones.

Kirschner, Klein and their colleagues, meanwhile, are keenly interested in other areas, including stem cell development.

“Does a population of cells that we initially think is uniform actually have some substructure?” Klein wants to know; he’s trying to find out by studying immune cells and different kinds of adult stem cells. “What is the nature of an early developing stem cell? What endows those cells with a pluripotent state? Is gene expression more plastic or does it have a well-defined state that’s different from a more mature cell? How is its fate determined?”

Using inDrops, Klein and team have confirmed prior findings that suggest even embryonic stem cells are not uniform. They found previously undiscovered cell types in the population they studied, as well as cells in intermediate stages that they suspect are converting from one type to another.

Although both teams are excited by the massive amounts of data they and other researchers will obtain from Drop-seq and inDrops, they realize the sheer volume of information poses a problem as well.

“We have thousands of cells expressing tens of thousands of genes. We can’t look in 20,000 directions to pick out interesting features,” said Klein.

Machine learning is able to do some of that, and the teams have already employed new statistical techniques. Still, Kirschner has called on mathematicians and computer scientists to develop new ideas about how to analyze and extract useful information about our biology from the mountains of data that are on the horizon.

Financial disclosures and funding information

Allon Klein, Linas Mazutis, Ilke Akartuna, David Weitz and Mark Kirschner have submitted patent applications (US62/065,348, US62/066,188, US62/072,944) for the work described.

A patent application has also been filed for the work described by Macosko et al.

The Kirschner lab’s study was supported by the National Institutes of Health (SCAP Grant R21DK098818), a Career Award at the Scientific Interface from the Burroughs-Wellcome Fund, and a Marie Curie International Outgoing Fellowship (300121).

The McCarroll lab’s work was supported by the Stanley Center for Psychiatric Research, the Simons Foundation, the National Institutes of Health (P50HG006193, U01MH105960, R25MH094612, F32HD075541), the Klarman Cell Observatory, a Stewart Trust Fellows Award and the Howard Hughes Medical Institute.

Microfluidic device fabrication was performed at the Harvard Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network, with support from the National Science Foundation and the Harvard Materials Research Science and Engineering Center. 

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Splitting Hair Cells

Splitting Hair Cells

‘Parts list’ for inner-ear hair cells advances understanding of deafness and hearing loss

By ELIZABETH COONEY
Thu, 2015-05-07

Hair cells (green) inside the cochlea in the inner ear vibrate with sound waves. Image: Courtesy of Nelson Kiang, MEE

Tiny hair cells in the inner ear play an outsized role.

For balance, five separate patches of hair cells sense movement and tell the brain where the head is in space while translating the pull of gravity.

For hearing, a five cell-wide ribbon of 16,000 hair cells spirals inside the cochlea, the snail-shaped structure where hair cells vibrate in response to sound waves. Every cycle of sound waves sends microscopic cilia on the tips of these cells back and forth, riding a trampoline of cells suspended between two fluid-filled spaces.

The movement opens pores in the cells, allowing electrical current to flow inside.  This conversion of mechanical to electrical signals sends nerve impulses to the brain, which then “hears” the sound.

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David Corey, the Bertarelli Professor of Translational Medical Science at Harvard Medical School and a Howard Hughes Medical Institute investigator, has spent his scientific life studying this mechanosensory apparatus, asking which proteins are involved in converting sounds into nerve impulses. So far, only about one-third of those proteins are known. Déborah Scheffer, HMS research associate in the Corey lab, is interested in what makes hair cells different from the cells that surround them in the inner ear.

Their most recent work, published in The Journal of Neuroscience, has revealed that many genes implicated in hereditary deafness are much more active in hair cells than in surrounding cells. This suggests that other genes that produce proteins only in hair cells might also cause inherited deafness. About 1 in 1,000 children are born deaf, and mutations in as many as 300 different genes might cause deafness.

Their findings may also have implications for age-related hearing loss, which affects about half of adults aged 75 or older. Sometimes impairment occurs much earlier, after exposure to harmful amounts of noise.

“This work gives us a parts list that the hair cell uses to assemble different components, helping us figure out the molecular mechanism of sensing sound,” Corey said. “It also tells us which genes drive the unique development of a hair cell, raising the hope that this information can be used to create new hair cells to restore hearing in cases of age- or noise-related hearing loss.”

To understand the hair cells of the inner ear, Corey and Scheffer collaborated with colleagues Jun Shen, HMS instructor in pathology at Brigham and Women’s Hospital, and Zheng-Yi Chen, associate professor of otolaryngology at Massachusetts Eye and Ear, to find out which genes hair cells use that neighboring cells do not. What makes one cell different from another is the choice and the timing of the genes expressed by a cell. 

Working in mice engineered to make green fluorescent hair cells, Scheffer devised a way to separately purify hair cells and surrounding cells at different points over about two weeks of mouse inner-ear development. At each point and for each sample, she sequenced the RNA used to make proteins for all 20,000 genes in the mouse genome. 

“Now we have a panel of all the genes that are involved in hair cell development,” Scheffer said.

Scheffer and Corey deposited their data in a publicly available database that Shen established three years ago. The Shared Harvard Inner Ear Laboratory Database, or SHIELD, holds gene expression data integrated with comprehensive annotation, including potential locations for deafness genes. Scientists from around the world access the data more than 400 times a day.

Some scientists interested in the molecular biology of hearing and deafness can use SHIELD to identify new deafness genes, which may lead to specific gene therapies. Others want to know what makes a hair cell a hair cell, so they can find a way to make surrounding cells in the inner ear turn into hair cells. These cells do not normally divide, so once they are lost, the only hope is to somehow induce them to divide or to turn neighboring cells into hair cells.

Next, Scheffer will explore microRNA expression in hair cells to see which genes they regulate. That will yield a genetic network of gene expression, messenger RNA and protein production.

“I want to know all the genes that interact with each other and what transcription factors are involved at each step,” she said.

Corey said their work is only the foundation.

“Someday this work will go to the clinic, but first you have to know the parts list.”

This research was supported by National Institutes of Health grants R01-DC000304, R01-DC002281, R03-DC013866 and R01-DC006908; the Frederick and Ines Yeatts Hair Cell Regeneration Grant; P30 DC05209 to the Eaton-Peabody Laboratory of Massachusetts Eye and Ear and a Hearing Health Foundation Emerging Research Grant.

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Evolutionary Relic

Evolutionary Relic

Pseudogenes in the human genome may lead to cancer development

By BONNIE PRESCOTT
Thu, 2015-04-02

Pseudogenes, a subclass of long noncoding RNA (lncRNA) that developed from the human genome’s 20,000 protein-coding genes but has lost the ability to produce proteins, have long been considered nothing more than genomic “junk.”

Yet the retention of these 20,000 mysterious remnants during evolution suggests that they may in fact possess biological functions and contribute to the development of disease. 

Pier Paolo Pandolfi. Image: BIDMC Media ServicesNow, a team led by investigators at Harvard Medical School and the Cancer Center at Beth Israel Deaconess Medical Center has provided some of the first evidence that one of these noncoding “evolutionary relics” actually has a role in causing cancer.

In a new study published in the journal Cell on April 2, the scientists report that, independent of any other mutations, abnormal amounts of the BRAF pseudogene led to the development of an aggressive lymphoma-like disease in a mouse model, a discovery suggesting that pseudogenes may play a primary role in a variety of diseases.

The new discovery also suggests that with the addition of this vast “dark matter” the functional genome could be tremendously larger than previously thought—three or four times its current known size.

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“Our mouse model of the BRAF pseudogene developed cancer as rapidly and aggressively as it would if you were to express the protein-coding BRAF oncogene,” explained senior author Pier Paolo Pandolfi, the HMS George C. Reisman Professor of Medicine and co-founder of the Institute for RNA Medicine in the Cancer Center at Beth Israel Deaconess.

“It’s remarkable that this very aggressive phenotype, resembling human diffuse large B-cell lymphoma, was driven by a piece of so-called ‘junk RNA,'" he said.

"In the past, we have found noncoding RNA to be overexpressed, or misexpressed, but because no one knew what to do with this information, it was swept under the carpet. Now we can see that it plays a vital role. We have to study this material, we have to sequence it and we have to take advantage of the tremendous opportunity that it offers for cancer therapy,” Pandolfi said.

Competing endogenous RNAs

The new discovery hinges on the concept of competing endogenous RNAs (ceRNA), a functional capability for pseudogenes first described by Pandolfi almost five years ago when his laboratory discovered that pseudogenes and other noncoding RNAs could act as decoys to divert and sequester tiny pieces of RNA, known as microRNAs, away from their protein-coding counterparts to regulate gene expression.

In this new paper, the authors wanted to determine whether this same ceRNA “cross talk” took place in a living organism and whether it would result in similar consequences.

“We conducted a proof-of-principle experiment using the BRAF pseudogene,” explained first author Florian Karreth, who conducted this work as a postdoctoral fellow in the Pandolfi laboratory.

“We investigated whether this pseudogene exerts critical functions in the context of a whole organism and whether its disruption contributes to the development of disease,” Karreth said.

The investigators focused on the BRAF pseudogene because of its potential ability to regulate the levels of the BRAF protein, a well-known proto-oncogene linked to numerous types of cancer.

In addition, said Karreth, the BRAF pseudogene is known to exist in both humans and mice.

The investigators began by testing the BRAF pseudogene in tissue culture. Their findings demonstrated that when overexpressed, the pseudogene did indeed operate as a microRNA decoy that increased the amounts of the BRAF protein.

This, in turn, stimulated the MAP-kinase signaling cascade, a pathway through which the BRAF protein controls cell proliferation, differentiation and survival and which is commonly found to be hyperactive in cancer.

Aggressive lymphoma-like cancer

When the team went on to create a mouse model in which the BRAF pseudogene was overexpressed, they found that the mice developed an aggressive lymphoma-like cancer.

Similar to their findings in their cell culture experiments, the investigators found that the mice overexpressing the BRAF pseudogene displayed higher levels of the BRAF protein and hyperactivation of the MAP kinase pathway, which suggests that this axis is indeed critical to cancer development.

They confirmed this by inhibiting the MAP kinase pathway with a drug that dramatically reduced the ability of cancer cells to infiltrate the liver in transplantation experiments.

The Pandolfi team further validated the microRNA decoy function of the BRAF pseudogene by creating two additional transgenic mice, one overexpressing the front half of the BRAF pseudogene and the other overexpressing the back half.

Both of these mouse models developed the same lymphoma phenotype as the mice overexpressing the full-length pseudogene, a result that the authors described as “absolutely astonishing.”

 The investigators also found that the BRAF pseudogene is overexpressed in human B-cell lymphomas and that the genomic region containing the BRAF pseudogene is commonly amplified in a variety of human cancers. Moreover, the authors said, silencing the BRAF pseudogene in human cancer cell lines that expressed higher levels led to reduced cell proliferation, a finding that highlights the importance of the pseudogene in these cancers and suggests that a therapy that reduces BRAF pseudogene levels may be beneficial in cancer patients.

This work was supported, in part, by the National Institutes of Health (CA170158-01), the Department of Defense Prostate Cancer Research Program, the American Cancer Society, the German National Academy of Sciences Leopoldina, the Italian Association for Cancer Research, the International Association for Cancer Research, Cancer Research UK and the Wellcome Trust.

Adapted from a Beth Israel Deaconess news release.

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