How to Grow a Spine

Cell model reveals dynamic nature of segmentation clock that drives vertebrae formation

Like a string of pearls, the spine is made of a series of similar vertebrae. A so-called segmentation clock creates this repetitive arrangement in developing embryos: Each time the clock ticks, a vertebra starts to form.

In a paper published Sept. 21 in Cell, Harvard Medical School genetics professor Olivier Pourquié—whose lab discovered the segmentation clock 20 years ago—and colleagues report that they used mouse cells to reconstitute a stable version of this clockwork for the first time in a petri dish, leading to several new discoveries about where the clock is located, what makes it tick and how the vertebral column takes shape.

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The team’s insights not only illuminate normal vertebrate development but also could lead to improved understanding of human spinal defects such as scoliosis, said Pourquié, who is also the HMS Frank Burr Mallory Professor of Pathology at Brigham and Women’s Hospital and a principal faculty member of the Harvard Stem Cell Institute.

Individual cells do not oscillate. Video: Pourquié lab

The researchers found that the segmentation clock lies quiescent in individual embryonic cells that give rise to the vertebrae, then clicks on all at once, collectively, when the cells reach a critical mass.

The researchers further discovered that the clock is controlled by two signals, Notch and Yap, that are sent and received by these cells.

On its own, they found, Notch starts the clock ticking by triggering cellular oscillations that release instructions to build structures that will ultimately become vertebrae. But Notch isn’t the only signal in town.

It turns out that the cells’ Yap chatter determines the amount of Notch required to activate the segmentation clock. If Yap is very low, then the clock runs on its own. If Yap levels are “medium,” said Pourquié, then Notch is needed to start the clock. And if Yap levels are high, even a lot of Notch won’t convince the clock to tick. Scientists call this an excitability threshold.

“If you stimulate the system a little, nothing happens. But if you stimulate it a little more and cross the threshold, then the system has a very strong response,” explained Pourquié.

A critical mass of cells is necessary to activate the segmentation clock and kick off vertebrae formation. Video: Pourquié lab

The researchers theorize that the segmentation clock works like other excitable biological systems that require certain thresholds to be met before sparking an action, such as neurons firing and calcium waves traveling across heart cells.

“There are probably similarities in the underlying circuits,” Pourquié said.

The researchers were surprised to find that they could stop and restart the segmentation clock in several ways—physically, by separating and reaggregating the cells, and chemically, with a Yap-blocking drug.

“For many years, we have been trying to understand the clockwork underlying these oscillations,” said Pourquié. “Now we have a great theoretical framework to understand what generates them and to help us make and test more hypotheses.”

Without enough Notch, the segmentation clock winds down. Video: Pourquié lab

Pourquié shared senior authorship of the paper with Lakshminarayanan Mahadevan, the Lola England de Valpine Professor of Applied Mathematics, of Organismic and Evolutionary Biology, and of Physics at Harvard University and a core member of the Wyss Institute for Biologically Inspired Engineering.

Alexis Hubaud, a former postdoctoral fellow in the Pourquié lab who is now at the Novartis Institutes for Biomedical Research, was first author of the study. Ido Regev, a former postdoctoral fellow in the Mahadevan lab who is currently at Ben-Gurion University of the Negev in Israel, was also an author.

This study was funded by the European Research Council, the National Institutes of Health (grant R01HD085121) and the Human Frontier Science Program (RGP0051/2012). Authors were additionally supported by fellowships from the French Ministry of Higher Education and Research, Fondation pour la Recherche Médicale (FDT20140930947), Schlumberger Foundation and MacArthur Foundation.


Lessons from a Tarantula

Spider muscles reveal details about mutations that disrupt heart relaxation

Tarantulas—those shaggy arachnids the size of your hand, with bristly bodies and eight bushy legs—harbor a secret. The muscles that control each limb bear a remarkable molecular resemblance to the muscle beating in our chests.

For Christine Seidman, professor of genetics at Harvard Medical School and the HMS Thomas W. Smith Professor of Medicine at Brigham and Women’s Hospital, that likeness did not go unnoticed.

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Seidman studies a heart disease called hypertrophic cardiomyopathy, or HCM. In HCM, the heart contracts too well and does not relax properly, which increases energy consumption and leads to adverse events such as arrhythmias and heart failure.

Seidman’s past work has identified eight genes encoding muscle proteins that, if mutated, cause HCM. Most of these mutations are in two genes, one of which codes for myosin, a protein crucial to muscle contraction. These myosin mutations simply switch one amino acid for another.

The findings prompted a new question: How could such a subtle change have such profound effects?

In search of an answer, Seidman looked outside of genetics.

During a Howard Hughes Medical Institute science meeting in 2011, Seidman, who is an HHMI Investigator, sought help from Raúl Padrón, a structural biologist at the Venezuelan Institute for Scientific Research, whose journal articles she’d been following. At the time, Padrón was an HHMI international research scholar studying how muscle proteins interact in tarantulas (which he describes as “very friendly”).

“One of the critical proteins that Raúl was studying was—big surprise—myosin,” said Seidman. Armed with the title of Padrón’s meeting poster, she set out to find him.

Padrón recalls their first interaction in detail. “I was in complete shock when Christine came to my poster; I had never met her before, and she was very well-known in my field, as she discovered many of the mutations we were mapping in the myosin model in our poster,” he said. “She walked up to me and said, ‘We need to work together to understand how different mutations affect the myosin motif.’”

And so they did.

With Padrón’s expertise in structural biology and Seidman’s keen knowledge of genetics, the two investigated how HCM-associated mutations change the structural interactions of myosin that occur during cardiac relaxation.

The two scientists and members of their respective laboratories reported their findings June 13 in the online journal eLife.

A baby Venezuelan tarantula. Image courtesy Raúl Padrón

As a nonstructural biologist, Seidman said, it was beautiful to see the actual myosin structure, even at low resolution.

“And there was another piece that was very important to me,” she said. “Raúl could tell me the amino acids that participate in myosin interactions that occur during relaxation.”

It turned out that many of the amino acids involved in the molecular interactions of relaxation are the very ones that are altered by HCM mutations. That, she said, was “an ‘aha!’ moment.”

Now, the two are planning next steps, asking the natural follow-up questions in their respective fields.

They plan to take advantage of the ongoing “cryo-EM resolution revolution” to achieve near-atomic resolution of myosin interactions by using recently improved cryo-electron microscopy.

“We’d very much like to work with Raúl to solve these structures using human specimens, with and without HCM mutations,” Seidman said. “That would be a big step.”

But the clinician side of Seidman hopes the information will help answer another question.

“We also want to know if there is a way to reduce the symptoms and adverse outcomes that occur in HCM by improving relaxation with small molecules in the heart,” she said. “In addition, we know that abnormal relaxation of the heart occurs in a lot of different diseases, not just HCM, so understanding if these structures might contribute to broader cardiovascular disease will also be very, very important.”

This research was supported by the Leducq Foundation (11-CVD-01); Wellcome Trust (107469/Z/15/Z); Medical Research Council; National Heart, Lung, and Blood Institute of the National Institutes of Health (HL084553); and Howard Hughes Medical Institute.

Adapted from an HHMI news story.


Social Networking for the Proteome, Upgraded

New study maps protein interactions for a quarter of the human genome

Harvard Medical School researchers have mapped the interaction partners for proteins encoded by more than 5,800 genes, representing over a quarter of the human genome, according to a new study published online in Nature on May 17.

The network, dubbed BioPlex 2.0, identifies more than 56,000 unique protein-to-protein interactions—87 percent of them previously unknown—the largest such network to date.

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BioPlex reveals protein communities associated with fundamental cellular processes and diseases such as hypertension and cancer, and highlights new opportunities for efforts to understand human biology and disease.

The work was done in collaboration with Biogen, which also provided partial funding for the study.

“A gene isn’t just a sequence of a piece of DNA. A gene is also the protein it encodes, and we will never understand the genome until we understand the proteome,” said co-senior author Wade Harper, the Bert and Natalie Vallee Professor of Molecular Pathology and chair of the Department of Cell Biology at HMS. “BioPlex provides a framework with the depth and breadth of data needed to address this challenge.”

“This project is an atlas of human protein interactions, spanning almost every aspect of biology,” said co-senior author Steven Gygi, professor of cell biology and director of the Thermo Fisher Center for Multiplexed Proteomics at HMS. “It creates a social network for each protein and allows us to see not only how proteins interact, but also possible functional roles for previously unknown proteins.”

HMS scientists are mapping interaction networks throughout the human proteome. Edward Huttlin, instructor in cell biology, explains. Video: Elizabeth Cooney

Bait and prey

Of the roughly 20,000 protein-coding genes in the human genome, scientists have studied only a fraction in detail. To work toward a description of the entire cast of proteins in a cell and the interactions between them—known as the proteome and interactome, respectively—a team led by Harper and Gygi developed BioPlex, a high-throughput approach for the identification of protein interplay.

BioPlex uses so-called affinity purification, in which a single tagged “bait” protein is expressed in human cells in the laboratory. The bait protein binds with its interaction partners, or “prey” proteins, which are then fished out from the cell and analyzed using mass spectrometry, a technique that identifies and quantifies proteins based on their unique molecular signatures. In 2015, an initial effort (BioPlex 1.0) used approximately 2,600 different bait proteins, drawn from the Human ORFeome database, to identify nearly 24,000 protein interactions.

In the current study, the team expanded the network to include a total of 5,891 bait proteins, which revealed 56,553 interactions involving 10,961 different proteins. An estimated 87 percent of these interactions have not been previously reported.

Guilt by association

By mapping these interactions, BioPlex 2.0 identifies groups of functionally related proteins, which tend to cluster into tightly interconnected communities. Such “guilt-by-association” analyses suggested possible roles for previously unknown proteins, as these communities often commingle proteins with both known and unknown functions.

The team mapped numerous protein clusters associated with basic cellular processes, such as DNA transcription and energy production, and a variety of human diseases. Colorectal cancer, for example, appears to be linked to protein networks that play a role in abnormal cell growth, while hypertension is linked to protein networks for ion channels, transcription factors and metabolic enzymes.

“With the upgraded network, we can make stronger predictions because we have a more complete picture of the interactions within a cell,” said first author Edward Huttlin, instructor of cell biology at HMS. “We can pick out statistical patterns in the data that might suggest disease susceptibility for certain proteins, or others that might suggest function or localization properties. It makes a significant portion of the human proteome accessible for study.”

The interaction network for CCDC151, a protein that is involved in cilia function in cells. Dysfunction in cilia function can be marked by chronic respiratory tract infections, abnormally positioned internal organs, and infertility. Image: HMS

Launching point

The entire BioPlex network and accompanying data are publicly available, supporting both large-scale studies of protein interaction and targeted studies of the function of specific proteins.

Although the network serves as the largest collection of such data gathered to date, the authors caution it remains an incomplete model. The current pipeline expresses bait proteins in only one cell type (human embryonic kidney cells) grown under one set of conditions, for example, and distinct interactions may occur in different cell types or microenvironments.

As the network increases in size and more human proteins are used as baits, scientists can better judge the accuracy of each individual protein interaction by considering its context in the larger network. Isolating the same protein complex several times, each time using a different member as a bait, can provide multiple independent experimental observations to confirm each protein’s membership.

Moreover, by using prey proteins as bait, many protein interactions can be observed in the opposite direction as well. Both of these scenarios greatly reduce the likelihood that particular interactions were identified due to chance. The team continues to add to BioPlex, with a target goal of around 10,000 bait proteins, which would cover half of the human genome and would further increase the predictive power of the network.

“We certainly aren’t seeing all the interactions, but it’s a launching point. We think it’s important to continue to build this map, to see how much of it is reproduced in other cell types under different conditions, to see whether the interactions are similar or dynamic,” Gygi said. “Because whether you’re interested in cancer or neurodegenerative disease, basic development or evolutionary fitness—you can make new hypotheses and learn something from this network.”

This work was supported by the National Institutes of Health (HG006673, DK098285), Biogen and the Canadian Institutes of Health Research.

Co-authors on the study included Raphael J. Bruckner, Joao A. Paulo, Joe R. Cannon, Lily Ting, Kurt Baltier, Greg Colby, Fana Gebreab, Melanie P. Gygi, Hannah Parzen, John Szpyt, Stanley Tam, Gabriela Zarraga, Laura Pontano-Vaites, Sharan Swarup, Anne E. White, Devin K. Schweppe, Ramin Rad, Brian K. Erickson, Robert A. Obar, K.G. Guruharsha, Kejie Li and Spyros Artavanis-Tsakonas.


Unraveling the Mysteries of Aging

Experiments suggest way to thwart DNA damage from aging, radiation

Video: Rick Groleau

DNA repair is essential for cell vitality, cell survival and cancer prevention, yet cells’ ability to patch up damaged DNA declines with age for reasons not fully understood.

Now, research led by scientists at Harvard Medical School reveals a critical step in a molecular chain of events that allows cells to mend their broken DNA.

The findings, published March 24 in Science, offer a critical insight into how and why the body’s ability to fix DNA dwindles over time and point to a previously unknown role for the signaling molecule NAD as a key regulator of protein-to-protein interactions in DNA repair. NAD, identified a century ago, is already known for its role as a controller of cell-damaging oxidation.

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Additionally, experiments conducted in mice show that treatment with the NAD precursor NMN mitigates age-related DNA damage and wards off DNA damage from radiation exposure. 

The scientists caution that the effects of many therapeutic substances are often profoundly different in mice and humans owing to critical differences in biology. However, if affirmed in further animal studies and in humans, the findings can help pave the way to therapies that prevent DNA damage associated with aging and with cancer treatments that involve radiation exposure and some types of chemotherapy, which along with killing tumors can cause considerable DNA damage in healthy cells. Human trials with NMN are expected to begin within six months, the researchers said.

Disarming a rogue agent: When the NAD molecule (red), binds to the DBC1 protein (beige), it prevents DBC1 from attaching to and incapacitating a protein critical for DNA repair. Image: David Sinclair“Our results unveil a key mechanism in cellular degeneration and aging but beyond that they point to a therapeutic avenue to halt and reverse age-related and radiation-induced DNA damage,” said senior author David Sinclair, professor in the Department of Genetics at HMS, co-director of the Paul F. Glenn Center for the Biology of Aging and professor at the University of New South Wales School of Medicine in Sydney, Australia.

previous study led by Sinclair showed that NMN reversed muscle aging in mice.

A plot with many characters

The investigators started by looking at a cast of proteins and molecules suspected to play a part in the cellular aging process. Some of them were well-known characters, others more enigmatic figures.

The researchers already knew that NAD, which declines steadily with age, boosts the activity of the SIRT1 protein, which delays aging and extends life in yeast, flies and mice. Both SIRT1 and PARP1, a protein known to control DNA repair, consume NAD in their work.

Another protein DBC1, one of the most abundant proteins in humans and found across life forms from bacteria to plants and animals, was a far murkier presence. Because DBC1 was previously shown to inhibit vitality-boosting SIRT1, the researchers suspected DBC1 may also somehow interact with PARP1, given the similar roles PARP1 and SIRT1 play.

“We thought if there is a connection between SIRT1 and DBC1, on one hand, and between SIRT1 and PARP1 on the other, then maybe PARP1 and DBC1 were also engaged in some sort of intracellular game,” said Jun Li, first author on the study and a research fellow in the Department of Genetics at HMS.

They were.

To get a better sense of the chemical relationship among the three proteins, the scientists measured the molecular markers of protein–to-protein interaction inside human kidney cells. DBC1 and PARP1 bound powerfully to each other. However, when NAD levels increased, that bond was disrupted. The more NAD present inside cells, the fewer molecular bonds PARP1 and DBC1 could form. When researchers inhibited NAD, the number of PARP1-DBC1 bonds went up. In other words, when NAD is plentiful, it prevents DBC1 from binding to PARP1 and meddling with its ability to mend damaged DNA. 

What this suggests, the researchers said, is that as NAD declines with age, fewer and fewer NAD molecules are around to stop the harmful interaction between DBC1 and PARP1. The result: DNA breaks go unrepaired and, as these breaks accumulate over time, precipitate cell damage, cell mutations, cell death and loss of organ function.

Averting mischief

Next, to understand how exactly NAD prevents DBC1 from binding to PARP1, the team homed in on a region of DBC1 known as NHD, a pocket-like structure found in some 80,000 proteins across life forms and species whose function has eluded scientists. The team’s experiments showed that NHD is an NAD binding site and that in DBC1, NAD blocks this specific region to prevent DBC1 from locking in with PARP1 and interfering with DNA repair. 

And, Sinclair added, since NHD is so common across species, the finding suggests that by binding to it, NAD may play a similar role averting harmful protein interactions across many species to control DNA repair and other cell survival processes.

To determine how the proteins interacted beyond the lab dish and in living organisms, the researchers treated young and old mice with the NAD precursor NMN, which makes up half of an NAD molecule. NAD is too large to cross the cell membrane, but NMN can easily slip across it. Once inside the cell, NMN binds to another NMN molecule to form NAD.

As expected, old mice had lower levels of NAD in their livers, lower levels of PARP1 and a greater number of PARP1 with DBC1 stuck to their backs.

However, after receiving NMN with their drinking water for a week, old mice showed marked differences both in NAD levels and PARP1 activity. NAD levels in the livers of old mice shot up to levels similar to those seen in younger mice. The cells of mice treated with NMN also showed increased PARP1 activity and fewer PARP1 and DBC1 molecules binding together. The animals also showed a decline in molecular markers that signal DNA damage. 

In a final step, scientists exposed mice to DNA-damaging radiation. Cells of animals pre-treated with NMN showed lower levels of DNA damage. Such mice also didn’t exhibit the typical radiation-induced aberrations in blood counts, such as altered white cell counts and changes in lymphocyte and hemoglobin levels. The protective effect was seen even in mice treated with NMN after radiation exposure.

Taken together, the results shed light on the mechanism behind cellular demise induced by DNA damage. They also suggest that restoring NAD levels by NMN treatment should be explored further as a possible therapy to avert the unwanted side effects of environmental radiation, as well as radiation exposure from cancer treatments. 

In December 2016, a collaborative project between the Sinclair Lab and Liberty Biosecurity became a national winner in NASA’s iTech competition for their concept of using NAD-boosting molecules as a potential treatment in cosmic radiation exposure during space missions.

Co-authors on the research included Michael Bonkowski, Basil Hubbard, Alvin Ling, Luis Rajman, Sebastian Moniot, Clemens Steegborn, Dapeng Zhang, L. Aravind, Bo Qin, Zhenkun Lou, and Vera Gorbunova.

The work was funded by the Glenn Foundation for Medical Research, the American Federation for Aging Research, Edward Schulak, grants from the National Institute on Aging and the National Institutes of Health, by the National Library of Medicine/NIH intramural program, the National Cancer Institute, and by Deutsche Forschungsgemeinschaft.   

This research project was dedicated to David Sinclair’s mother, Diana Sinclair, who bravely survived cancer for two decades.

Relevant disclosures:

Sinclair is an inventor on patent applications held by The University of New South Wales that cover the use of NAD precursors to modulate DNA repair, fertility, and blood flow, and by Harvard University for the treatment of diseases of aging and mitochondrial disorders. Li and Sinclair are inventors on a patent application held by Harvard University that covers the modulation of Nudix hydrolase domain proteins by small molecules. Co-author Michael Bonkowski, of Harvard Medical School, is a paid consultant to OvaScience. Sinclair is an unpaid consultant, board member and holds equity in companies developing NAD precursor-based medicines (EdenRoc, Liberty Biosecurity, Metrobiotech, and Jumpstart Fertility) and is a paid consultant and inventor on a patent application licensed to OvaScience for improving in vitro fertilization. Patents held by Harvard University related to NAD-based therapies on which Sinclair is an inventor are licensed to GlaxoSmithKline and Metrobiotech. The agreements between Harvard University and these entities are managed by the Harvard Office of Technology Development in accordance with Harvard Medical School’s conflict-of-interest policy.



Circuit Breaker

Genetic “toggle switch” reveals regulation of sociability in autism model

Harvard Medical School researchers at Beth Israel Deaconess Medical Center have gained new insight into the genetic and neuronal circuit mechanisms that may contribute to impaired sociability in some forms of autism spectrum disorder.

Led by Matthew Anderson, HMS associate professor of pathology and director of neuropathology at Beth Israel Deaconess, the scientists determined how a gene linked to one common form of autism works in a specific population of brain cells to impair sociability.

The research, published today in the journal Nature, reveals the neurobiological control of sociability and could represent important first steps toward interventions for patients with autism.

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Anderson and colleagues focused on the gene UBE3A, multiple copies of which cause a form of autism in humans (called isodicentric chromosome 15q syndrome). Conversely, the lack of this same gene in humans leads to a developmental disorder called Angelman syndrome, characterized by increased sociability.

In previous work, Anderson’s team demonstrated that mice engineered with extra copies of the UBE3A gene show impaired sociability, as well as heightened repetitive self grooming and reduced vocalizations with other mice.

“In this study, we wanted to determine where in the brain this social behavior deficit arises and where and how increases of the UBE3A gene repress it,” said Anderson, who is also director of the Autism BrainNET, Boston Node.

“We had tools in hand that we built ourselves. We not only introduced the gene into specific brain regions of the mouse, but we could also direct it to specific cell types to test which ones played a role in regulating sociability,” Anderson said. 

When Anderson and colleagues compared the brains of the mice engineered to model autism to those of normal—or wild type—mice, they observed that the increased UBE3A gene copies interacted with nearly 600 other genes.

After analyzing and comparing protein interactions between the UBE3A regulated gene and genes altered in human autism, the researchers noticed that increased doses of UBE3A repressed Cerebellin genes.

Cerebellin is a family of genes that physically interact with other autism genes to form glutamatergic synapses, the junctions where neurons communicate with each other via the neurotransmitter glutamate.

The researchers chose to focus on one of them, Cerebellin 1 (CBLN1), as the potential mediator of UBE3A’s effects. When they deleted CBLN1 in glutamate neurons, they recreated the same impaired sociability produced by increased UBE3A.

“Selecting Cerebellin 1 out of hundreds of other potential targets was something of a leap of faith,” Anderson said. “When we deleted the gene and were able to reconstitute the social deficits, that was the moment we realized we’d hit the right target. Cerebellin 1 was the gene repressed by UBE3A that seemed to mediate its effects,” he said. 

In another series of experiments, Anderson and colleagues demonstrated an even more definitive link between UBE3A and CBLN1. Seizures are a common symptom among people with autism including this genetic form.

Seizures themselves, when sufficiently severe, also impaired sociability.

Anderson’s team suspected this seizure-induced impairment of sociability was the result of repressing the Cerebellin genes. Indeed, the researchers found that deleting UBE3A, upstream from Cerebellin genes, prevented the seizure-induced social impairments and blocked seizures ability to repress CBLN1.

“If you take away UBE3A, seizures can’t repress sociability or Cerebellin,” said Anderson. “The flip side is, if you have just a little extra UBE3A—as a subset of people with autism do—and you combine that with less severe seizures, you can get a full-blown loss of social interactions.” 

The researchers next conducted a variety of brain-mapping experiments to locate where in the brain these crucial seizure-gene interactions take place.

“We mapped this seat of sociability to a surprising location,“ Anderson explained. Most scientists would have thought they take place in the cortex—the area of the brain where sensory processing and motor commands take place—but, in fact, these interactions take place in the brain stem, in the reward system.”

Then the researchers used their engineered mouse model to confirm the precise location as the ventral tegmental area, part of the midbrain that plays a role in the reward system and addiction.

Anderson and colleagues used chemogenetics—an approach that makes use of modified receptors introduced into neurons that respond to drugs but not to naturally occurring neurotransmitters—to switch this specific group of neurons on or off.

Turning these neurons on could magnify sociability and rescue seizure and UBE3A-induced sociability deficits.

“We were able to abolish sociability by inhibiting these neurons, and we could magnify and prolong sociability by turning them on,” said Anderson. “So we have a toggle switch for sociability. It has a therapeutic flavor; someday, we might be able to translate this into a treatment that will help patients.”

The researchers thank Oriana DiStefano, Greg Salimando and Rebecca Broadhurst for colony work and the HMS Neurobiology Imaging Facility (NINDS P30 Core Center Grant #NS07203).

This work was supported an American Academy of Neurology Research Training Fellowship, the National Institutes of Health (grants 1R25NS070682, 1R01NS08916, 1R21MH100868 and 1R21HD079249), the Nancy Lurie Marks Family Foundation, the Landreth Family Foundation, the Simons Foundation, Autism Speaks/National Alliance for Autism Research and the Klarman Family Foundation.

Adapted from a Beth Israel Deaconess news release.


Honoring the CRISPR Revolution

Genetics pioneers to be recognized at 28th annual Warren Alpert Foundation Prize Symposium

All five scientists sharing the 2016 Warren Alpert Foundation Prize for elucidating the CRISPR bacterial defense system and recognizing its utility for gene editing will tell the story of their discoveries at the 28th annual Warren Alpert Foundation Prize Symposium at Harvard Medical School on Thursday, Oct. 6.

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The symposium will also feature invited speakers Austin Burt, professor of evolutionary genetics at Imperial College London, who will discuss developing CRISPR-based gene drive for malaria control, and Luhan Yang, HMS research fellow in genetics and co-founder and chief scientific officer of eGenesis, who will discuss rewriting the pig genome to transform xenotransplantation.

The 2016 Warren Alpert Foundation Prize recipients are:

  • Rodolphe Barrangou, associate professor in the Department of Food, Bioprocessing and Nutrition Sciences and the Todd R. Klaenhammer Distinguished Scholar in Probiotics Research at North Carolina State University
  • Philippe Horvath, senior scientist at DuPont in Dangé-Saint-Romain, France
  • Jennifer Doudna, the Li Ka Shing Chancellor’s Chair in Biomedical and Health Sciences and professor of molecular and cell biology and of chemistry at the University of California, Berkeley
  • Emmanuelle Charpentier, scientific member and director at the Max Planck Institute for Infection Biology in Berlin and professor at Umeå University in Sweden
  • Virginijus Siksnys, professor, chief scientist and department head at the Institute of Biotechnology at Vilnius University in Lithuania

​Barrangou and Horvath established that bacteria protect themselves from being killed by pathogens, such as viruses, using a system called CRISPR (short for clustered regularly interspaced short palindromic repeats) to cut up specific segments of the invading viruses’ DNA.

Building on their findings, Doudna, Charpentier and Siksnys realized that the system could be programmed to zero in on any desired genetic sequence in a broader array of organisms, including humans, and that this purposeful cutting could be used to alter or replace the targeted DNA at will.

Together, these discoveries, which were further refined and expanded by the prize recipients and other researchers, have generated a powerful tool for rapidly determining gene function and have democratized the ability to pursue clinical advances, such as correcting genetic defects and designing better drugs by making gene editing faster, easier and cheaper than the technologies available previously.

“The game-changing insights achieved by these five scientists led to a technique that has been swiftly embraced across the globe, altering the way we study and understand eukaryotic genetics and offering enormous potential for developing new gene- and cell-based therapies, including treatment strategies for previously intractable genetic diseases,” said Jeffrey S. Flier, former dean of the faculty of medicine at Harvard Medical School and chair of the Warren Alpert Foundation Prize Scientific Advisory Committee, when the prize winners were announced in March 2016.

The Warren Alpert Foundation Prize recognizes scientists whose research has led to the prevention, cure or treatment of human diseases or disorders and constitutes a seminal scientific finding that holds great promise for ultimately changing our understanding of, or ability to treat, disease.

The late Warren Alpert, a philanthropist dedicated to advancing biomedical research, established the prize in 1987. To date, the foundation has awarded more than $3 million to 54 individuals. Eight honorees have also received a Nobel Prize.

Click here for more information or to register for the Symposium.

The Warren Alpert Foundation

Each year the Warren Alpert Foundation receives 30 to 50 nominations for the Alpert Prize from scientific leaders worldwide. Prize recipients are selected by the foundation’s scientific advisory board, composed of distinguished biomedical scientists and chaired by the dean of Harvard Medical School.

Warren Alpert (1920-2007), a native of Chelsea, Massachusetts, established the Warren Alpert Foundation Prize in 1987 after reading about the development of a vaccine for hepatitis B. Alpert decided on the spot that he would like to reward such breakthroughs, so he picked up the phone and told the vaccine’s creator, Kenneth Murray of the University of Edinburgh, that he had won a prize. Alpert then set about creating the foundation.

To award subsequent prizes, Alpert asked Daniel Tosteson (1925-2009), then dean of Harvard Medical School, to convene a panel of experts to identify scientists from around the world whose research has had a direct impact on the treatment of disease.

The Warren Alpert Foundation does not solicit funds. It is a private philanthropic organization funded solely by the Warren Alpert Estate.


A Diversity of Genomes

New DNA from understudied groups reveals modern genetic variation, ancient population shifts

A study of hundreds of new genomes from across the globe has yielded insights into modern human genetic diversity and ancient population dynamics, including compelling evidence that essentially all non-Africans today descend from a single migration out of Africa.

The multinational research effort, led by Harvard Medical School geneticists and published Sept. 21 in Nature, also suggests that no single genetic change or small group of changes is likely to explain the substantial transformations in human culture and cognition seen in the last 50,000 years.

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The study represents the largest data set yet of high-quality genome sequences from understudied populations, adding nearly 6 million DNA base pairs to the “canonical” human genome sequence published in 2001. The data identify millions of previously unknown mutations that may help scientists develop precision-targeted diagnostics and treatments and improve health care for the world’s underserved populations.

Most genome-wide population sequencing studies to date have focused on a handful of populations of large census size. The HMS-led study, by comparison, sequenced samples from 142 smaller populations, most of which were previously unstudied.

“As humans, we are not just the people who live in industrialized countries, and we are not just the people who live in numerically large groups,” said David Reich, professor of genetics at HMS and senior author of the study. “If we want to understand who we really are, we have to realize that some of the most interesting aspects of human variation are only present in underrepresented, small populations.”

“We wanted to go out into the world and pull together as many of the ethnically, linguistically and anthropologically diverse samples as we possibly could,” said Swapan Mallick, bioinformatic systems director in the Reich lab and first author of the study.

The team’s analyses are already answering questions about various populations’ genetic origins, but, the researchers note, these insights are only a milestone on a longer journey.

“Of course, there are thousands of ethnically distinct populations in the world, and much more work needs to be done,” said Mallick.

Clarifying history

Reich, Mallick and their international team of colleagues began by selecting two genomes each from 51 populations represented in a collection called the Human Genome Diversity Project. Next, they assembled samples from members of 91 other groups, including diverse Native American, South Asian and African populations not previously included in genome-wide studies, and sent the DNA for sequencing. In all, the project analyzed the genomes of 300 people.

A key conclusion—that the vast majority of modern human ancestry in non-Africans derives from a single population that migrated out of Africa—is also supported by two other whole-genome sequencing studies appearing simultaneously in Nature. One, led by an Estonian group, focused on 379 whole genome sequences; the other, led by a Danish group, analyzed 108 Australians and New Guineans.

Together, the three studies put to rest a lingering question about whether indigenous peoples of Australia, New Guinea and the Andaman Islands descend in large part from a second group that left Africa earlier and skirted the coast of the Indian Ocean. They do not, the HMS researchers say.

“Our best estimate for the proportion of ancestry from an early-exit population is zero,” said Reich, who is also an investigator of the Howard Hughes Medical Institute and associate member of the Broad Institute. “Taken together, all three studies leave wiggle room for, at most, around two percent.”

The HMS-led study further revealed that the common ancestors of modern humans began to differentiate at least 200,000 years ago, long before the out-of-Africa dispersal occurred.

“It had been unclear whether the group that expanded out of Africa represented a large subset of the populations within Africa,” said Mallick. “This really shows that there was a lot of substructure prior to the expansion.”

The additional discovery that genetics alone can’t account for the acceleration of cultural, economic and intellectual progress in the last 50,000 years runs contrary to a popular hypothesis in the field.

“There does not seem to have been one or a few enabling mutations that suddenly appeared among our ancestors and allowed them to think in profoundly different ways,” said Reich.

Instead, the researchers say, a constellation of factors, including environment, lifestyle and possibly genetics, likely drove the great changes that occurred.

“Geneticists often search for examples where genetics is the explanation. Here, paradoxically, genetic data are showing that there will be no clear genetic answers,” Reich said.

Overcoming hurdles

Mallick and colleagues overcame significant logistical hurdles posed by sharing and processing an enormous amount of data.

Often, in studies of this size, data are collected in many laboratories that use different sequencing machines and different experimental protocols. This can create so-called batch effects that make it difficult to distinguish true differences among samples. The current study minimized batch effects by sending all of the samples to a single center to be sequenced at the same time.

The team made much of the data set publicly available in 2014; multiple research groups have already used it for their studies.

In a way, the authors say, the findings reported thus far are just the tip of the iceberg.

“It’s impossible for our group to analyze even a tiny fraction of what the data represents,” said Mallick. “Our goal is to push the data out and let people use it to consider their own questions.”

“A project of this magnitude is a combination of considerable efforts from multiple groups and creates an important resource for researchers worldwide,” Mallick added. “Such work is only possible in the environment and with the remarkable support from administration and computing groups here at HMS.”

Primary funding for the study, called the Simons Genome Diversity Project, was provided by the Simons Foundation (SFARI 280376) and the National Science Foundation (BCS-1032255).


Insights into Protein Recycling

Findings in roundworms may apply to treatment of cancers, neurodegenerative disorders

Maintaining appropriate levels of proteins within cells largely relies on a cellular component called the proteasome, which degrades unneeded or defective proteins to recycle the components for the eventual assembly of new proteins.

Deficient proteasome function can lead to a buildup of unneeded and potentially toxic proteins, so cells usually respond to proteasome dysfunction by increasing production of its component parts.

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Now two Harvard Medical School investigators at Massachusetts General Hospital have identified key molecules in the pathway by which cells in the C. elegans roundworm sense proteasome dysfunction. Their findings may have applications for the treatment of several human diseases.

“Proteasome inhibitors are currently being used to treat some cancers, including multiple myeloma, so future drugs targeting this pathway could enhance their activity,” said Gary Ruvkun, professor of genetics at HMS, an investigator at Mass General and corresponding author of the report in the journal eLife. 

Lead author Nicolas Lehrbach, HMS postdoctoral fellow in Ruvkun’s laboratory, added, “Boosting proteasome activity might help treat some neurodegenerative disorders–including Alzheimer’s and Parkinson’s disease, both of which are characterized by abnormal protein deposits in the brain that exceed the ability of the proteasome to respond.”

While it has been known that a transcription factor called SKN-1 was essential for the response to proteasome dysfunction in C. elegans, exactly how SKN-1 is coupled to a sensor of proteasome activity to induce expression of proteasome subunits was unknown. Lehrbach conducted a comprehensive genetic screen of C. elegans mutants that were unable to activate SKN-1 in order to identify the mutated genes.

“The genetic analysis that Nic did was extremely comprehensive, isolating across the genome of C. elegans nearly 100 new mutations that render these animals unable to sense and respond to decreased proteasome activity,” Ruvkun said. “These new mutations were mapped by determining the full genome sequence of all of the mutant strains.”

Among many mutated genes that were isolated was a protease enzyme called DDI-1, present in many different species but not previously associated with protein damage signaling. Another essential element of the pathway is a protein called PNG-1, which removes sugar modifications from other proteins.

In order for SKN-1 to be activated, DDI-1 must cleave or clip the protein, and PNG-1 must remove a sugar molecule. The mammalian versions of SKN-1 are the transcription factors Nrf1 and Nrf2, and blocking Nrf1 has been proposed as a way to improve protease inhibitor treatment of cancer.

In another paper published simultaneously in eLifeinvestigators from the University of Tokyo led by Shigeo Murata independently discovered that the human version of C. elegans DDI-1, called DDI-2, is needed to couple proteasome deficiency to the upregulation of proteasome genes in human cells. Taken together, these two papers show that the DDI-1/2 protease is essential to sensing proteasome stress in animals from roundworms to humans, making the enzyme an ideal target for drug development.

“We know from the successful development of protease inhibitors to treat HIV and hepatitis C infection that protease enzymes are highly amenable to the development of small-molecule inhibitors,” Ruvkun said. “In addition to potentially being powerful enhancers of the proteasome inhibitors being used to treat multiple myeloma, DDI-1/2 inhibitors may be more generally deployed against conditions in which proteasome deregulation has been implicated.”

The variety of diseases caused by aberrant protein aggregation include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and prion diseases, Ruvkun said.

Inactivating mutations in the human version of PNG-1, a protein called NGLY1, have recently been identified as the cause of a rare genetic disease, Ruvkun said. The new findings strongly suggest this condition could be treated by reactivation of proteasome genes.

The team’s discovery that PNG-1 acts with DDI-1 and SKN-1 in the proteasome pathway now places PNG-1/NGLY1 in a genetic pathway that is better understood. 

“We have the genetic tools now to ask in C. elegans, how can we cure NGLY1 deficiency?” said Ruvkun. “We are doing these screens right now and are anxiously awaiting what the genome sequence of the mutants will teach us.”

The research was supported by National Institutes of Health grant R01 AG016636.

Adapted from a Mass General news release.


Imprecise Diagnoses

Genetic tests for potentially fatal heart anomaly can misdiagnose condition in black Americans

Genetic testing has greatly improved physicians’ ability to detect potentially lethal heart anomalies among asymptomatic family members of people who suffer cardiac arrest or sudden cardiac death.

But a study from Harvard Medical School published in the Aug. 18 issue of The New England Journal of Medicine shows that over the last decade these lifesaving tools may have disproportionately misdiagnosed one cardiac condition — hypertrophic cardiomyopathy (HCM) – in black Americans.

HCM, which affects one in 500 people, is an often-asymptomatic thickening of the heart muscle that can spark fatal arrhythmias in seemingly healthy young adults.

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The notion that genetic tests could misread benign genetic alterations as disease-causing mutations is not entirely new, but this study is believed to be the first one to trace the root of the problem to racially biased methodologies in early studies that defined certain common genetic variants as causes of HCM.

Indeed, the analysis reveals that in the case of HCM, the false positive diagnoses stemmed from inadequately designed clinical studies that used predominantly white populations as control groups.

White Americans harbor far fewer benign mutations on several genes implicated in HCM than black Americans. The higher rate of benign alterations in the latter group can cause test results to be misread as abnormal, the researchers say.

Using statistical simulations, the HMS team demonstrated that including even small numbers of black participants in the original studies would have improved test accuracy and, consequently, helped avert some of the false-positive diagnoses.

The findings, the researchers say, highlight the importance of interpreting genetic test results against diverse control populations to ensure that normal variations of genetic markers common in one racial or ethnic group do not get misclassified as disease-causing in another.

The team says their findings point to a pressing need to reevaluate decades-old genetic studies by using new racially diverse sequencing data.

“We believe that what we’re seeing in the case of hypertrophic cardiomyopathy may be the tip of the iceberg of a larger problem that transcends a single genetic disease,” said study first author Arjun Manrai, a research fellow in the Department of Biomedical Informatics at Harvard Medical School. “We hope our study motivates a systematic review of this issue across other genetic conditions.

“Ensuring that genomic medicine benefits all people and all populations equally is nothing short of a moral imperative, not only for scientists and clinicians but for political and health policy powers that be,” —Zak Kohane

Aside from the emotional toll that a genetic misdiagnosis can take on individuals and families, the researchers say their findings represent a cautionary tale with a broad relevance to geneticists, clinicians and policy-makers alike.

“Our study powerfully illustrates the importance of racial and ethnic diversity in research,” says Zak Kohane, senior investigator on the study and chair of the Department of Biomedical Informatics at Harvard Medical School. “Racial and ethnical inclusiveness improves the validity and accuracy of clinical trials and, in doing so, can better guide clinical decision-making and choice of optimal therapy. This is the essence of precision medicine.”

In the current study, the team analyzed more than 8,000 DNA samples stored in three national databases — the National Institutes of Health’s Mendelian Exome Sequencing Project, the 1000 Genomes Project and the Human Genome Diversity Project.

Five genetic variants — each of them benign — accounted for 75 percent of all genetic variation across populations. However, the team found, these five mutations occurred disproportionately in black Americans.

Between 2.9 and 27 percent of black Americans harbored one or more such variants, compared with 0.02 to 2.9 percent of white Americans.

Next, researchers examined records of more than 2,000 patients and family members tested at a leading genetic laboratory between 2004 and 2014. Seven patients received reports indicating they harbored disease-causing mutations that were subsequently reclassified as benign. Five of the seven patients were black, and two were of unspecified ancestry.

Researchers say it remains unclear how many of the seven patients had been re-contacted to communicate the change in test results.
The investigators caution that test results from a single genetic lab are not necessarily representative of the scope of the problem nationally, but say their findings likely point to a discrepancy that goes beyond a single laboratory and a single condition.

To trace the root of the misclassifications, researchers reviewed the five original studies that shaped early understanding of genetic variants and their role in the development of hypertrophic cardiomyopathy. All of them, the researchers found, analyzed small population sizes and none indicated that black people were included in the control groups.

But, the investigators add, even small studies can avert misclassification of genetic variants as long they include racially diverse populations.

Using statistical simulation, the team showed that a sample of 200 people that included 20 black participants would have only 50 percent chance of correctly ruling out a harmful mutation. The same sample would have more than an 80 percent accuracy if a third of patients were black and more than 90 percent accuracy if half of them were black.

Investigators say the newly created Exome Aggregation Consortium — a compilation of data from various large-scale sequencing projects that includes DNA from more than 60,000 individuals — is well-powered to discern between harmful and benign mutations even for relatively rare genetic variants and should help in the reanalysis of decades-old data.

The latest clinical guidelines urge physicians to interpret genetic test results by cross-referencing them against racially matched controls. However, with expanding efforts to sequence DNA from various ethnic and racial groups, researchers say more genetic variants will be reclassified in the next decade. Interpreting the meaning of test results within the context of such rapidly evolving knowledge will pose a serious challenge for clinicians.

One way to address the problem, the HMS team says, could be the use of point-of-care risk calculators to help clinicians and genetic counselors more precisely gauge the significance of a given genetic variant. Such risk calculators would use algorithms that incorporate statistical probability, race, ethnicity and family history to help sift variant noise from truly pathogenic mutations.

“Ensuring that genomic medicine benefits all people and all populations equally is nothing short of a moral imperative, not only for scientists and clinicians but for political and health policy powers that be,” Kohane said.

The work was funded by the National Human Genome Research Institute under grant 5T32HG002295-9, by the National Institute of Mental Health under grant P50MH094267 and by the National Centers for Biomedical Computing under grant 5U54-LM-008748.

Other investigators on the research included Birgit Funke, Ph.D., Heidi Rehm, Ph.D., Morten Olesen, Ph.D., Bradley Maron, M.D., Peter Szolovits, Ph.D., 
David Margulies, M.D., Joseph Loscalzo, M.D., Ph.D.


Predictive Genomics

Two new technologies define the consequences of genetic variation on a proteome-wide scale

Combining two emerging, large-scale technologies for the first time—multiplexed mass spectrometry and a mouse population with a high level of natural genetic diversity—researchers at Harvard Medical School and The Jackson Laboratory can now crack an outstanding question in biology and medicine: How do genetic variants affect protein levels?

Proteins are chains of amino acids that comprise the structural and functional “parts list” of all cells and organisms. Understanding the regulation of protein expression is therefore critical to understanding normal development and disease.

“We can now uncover relationships among genes, transcripts and proteins not previously known.”—Steven Gygi 

The central dogma of molecular biology describes this transfer of genetic information from DNA to RNA to protein. The DNA sequence is first transcribed into messenger RNA, or mRNA, and then the cell’s protein-building machinery translates the mRNA sequences into the amino-acid sequence of the protein.

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Given this direct relationship between RNA and proteins, it was widely assumed that protein expression would track closely with mRNA expression. Yet several studies comparing cellular mRNA levels and protein levels have shown a surprisingly high level of discordance between the two, suggesting that one or more mechanisms act to buffer protein levels from genetic variants that affect mRNA levels.

Previous experiments in mice and human cell lines aimed at identifying these mechanisms have been inconclusive.

To address this puzzle, Gary Churchill,  Jackson Lab professor and Karl Gunnar Johansson Chair and a pioneer in developing the Collaborative Cross and Diversity Outbred mouse populations, joined forces with Steven Gygi, HMS professor of cell biology, a leader in the rapidly advancing field of quantitative proteomics, which is the study of an organism’s entire complement of proteins.

"This makes an entirely new scale of analysis possible.”—Gary Churchill

Diversity Outbred mice, bred from eight founder strains, contain extensive genetic variation.

Our mouse populations have more than 50 million SNPs,” or single nucleotide polymorphisms, which are variations in individual DNA building blocks, said Churchill.  “Steve can measure the levels of thousands of proteins instead of dozens. This makes an entirely new scale of analysis possible.”

Gygi and Churchill are co-senior authors of a paper in Nature in which they compared mRNA and protein levels in the livers of 192 Diversity Outbred mice.

The researchers identified 2,866 genetic markers that correlate with differences in protein levels across mice (protein quantitative trait loci, or pQTL) and observed two striking patterns. Most proteins with “local” pQTL—where the genetic variant influencing protein abundance is located close to the DNA sequence that encodes that protein—showed strong evidence of transcriptional regulation where protein levels tracked closely with mRNA levels.

In stark contrast, proteins with “distant” pQTL—where the genetic variant influencing protein abundance is located far away from the DNA sequence that encodes the protein—appeared completely uncoupled from their corresponding mRNA’s abundance. By applying a novel statistical approach, they showed that the post-transcriptional effects of many distant pQTL could be attributed to a second protein, revealing an extensive network of direct protein–protein interactions and tightly regulated cellular pathways.

The researchers confirmed their findings in Collaborative Cross mice.

“We can now uncover relationships among genes, transcripts and proteins not previously known,” Gygi said. “Our findings suggest a new predictive genomics framework, combining quantitative proteomics and transcriptomics to infer the proteome-wide effects of a specific genetic variant.”

Within this framework, Gygi said, researchers can explore and fine-tune pathways associated with the physical process, disease or characteristic of interest.

The study was supported by Harvard Medical School, The Jackson Laboratory and the National Institutes of Health (grants P50GM076468, F32HD074299, GM67945 and U41HG006673).

Adapted from a Jackson Lab news release.