Placement: Promote to Homepage
Whitehead Institute scientists find chemical modification contributes to trafficking between non-membrane-bound compartments that control gene expression.
Nicole Davis | Whitehead Institute
August 9, 2019
Cells often create compartments to control important biological functions. The nucleus is a prime example; surrounded by a membrane, it houses the genome. Yet cells also harbor enclosures that are not membrane-bound and more transient, like oil droplets in water. Over the past two years, these droplets (called “condensates”) have become increasingly recognized as major players in controlling genes. Now, a team led by Whitehead Institute scientists helps expand this emerging picture with the discovery that condensates play a role in splicing, an essential activity that ensures the genetic code is prepared to be translated into protein. The researchers also reveal how a critical piece of cellular machinery moves between different condensates. The team’s findings appear in the Aug. 7 online issue of Nature.
“Condensates represent a real paradigm shift in the way molecular biologists think about gene control,” says senior author Richard Young, a member of the Whitehead Institute and professor of biology at MIT. “Now, we’ve added a critical new layer to this thinking that enhances our understanding of splicing as well as the major transcriptional apparatus RNA polymerase II.”
Young’s lab has been at the forefront of studying how and when condensates form as well as their functions in gene regulation. In the current study, Young and his colleagues, including first authors Eric Guo and John Manteiga, focused their efforts on a key transition that happens when genes undergo transcription — an early step in gene activation whereby an RNA copy is created from the genes’ DNA template. First, all of the molecular machinery needed to make RNA, including a large protein complex known as RNA polymerase II, assembles at a given gene. Then, specific chemical modifications to RNA polymerase II allow it to begin transcribing DNA into RNA. This shift from so-called transcription initiation to active transcription also involves another important molecular transition: As RNA molecules begin to grow, the splicing apparatus must also move in and carry out its job.
“We wanted to step back and ask, ‘Do condensates play an important role in this switch, and if so, what mechanism might be responsible?’” explains Young.
For roughly three decades, it has been recognized that the factors required for splicing are stored in compartments called speckles. Yet whether these speckles play an active role in splicing, or are simply storage vessels, has remained unclear.
Using confocal microscopy, the Whitehead team discovered condensates filled with components of the splicing machinery in the vicinity of highly active genes. Notably, these structures exhibited similar liquid-like characteristics to those condensates described in prior studies from Young’s lab that are involved in transcription initiation.
“These findings signaled to us that there are two types of condensates at work here: one involved in transcription initiation and the other in splicing and transcriptional elongation,” said Manteiga, a graduate student in Young’s lab.
With two different condensates at play, the researchers wondered: How does the critical transcriptional machinery, specifically RNA polymerase II, move from one condensate to the other?
Guo, Manteiga, and their colleagues found that chemical modification, specifically the addition of phosphate groups, serves as a kind of molecular switch that alters the protein complex’s affinity for a particular condensate. With fewer phosphate groups, it associates with the condensates for transcription initiation; when more phosphates are added, it enters the splicing condensates. Such phosphorylation occurs on one end of the protein complex, which contains a specialized region known as the C-terminal domain (CTD). Importantly, the CTD lacks a specific three-dimensional structure, and previous work has shown that such intrinsically disordered regions can influence how and when certain proteins are incorporated into condensates.
“It is well-documented that phosphorylation acts as a signal to help regulate the activity of RNA polymerase II,” says Guo, a postdoc in Young’s lab. “Now, we’ve shown that it also acts as a switch to alter the protein’s preference for different condensates.”
In light of their discoveries, the researchers propose a new view of splicing compartments, where speckles serve primarily as warehouses, storing the thousands of molecules required to support the splicing apparatus when they are not needed. But when splicing is active, the phosphorylated CTD of RNA Pol II serves as an attractant, drawing the necessary splicing materials toward the gene where they are needed and into the splicing condensate.
According to Young, this new outlook on gene control has emerged in part through a multidisciplinary approach, bringing together perspectives from biology and physics to learn how properties of matter predict some of the molecular behaviors he and his team have observed experimentally. “Working at the interface of these two fields is incredibly exciting,” says Young. “It is giving us a whole new way of looking at the world of regulatory biology.”
Support for this work was provided by the U.S. National Institutes of Health, National Science Foundation, Cancer Research Institute, Damon Runyon Cancer Research Foundation, Hope Funds for Cancer Research, Swedish Research Council, and German Research Foundation DFG.
Along the genome, proteins form liquid-like droplets that appear to boost the expression of particular genes.
Anne Trafton | MIT News Office
August 8, 2019
In recent years, MIT scientists have developed a new model for how key genes are controlled that suggests the cellular machinery that transcribes DNA into RNA forms specialized droplets called condensates. These droplets occur only at certain sites on the genome, helping to determine which genes are expressed in different types of cells.
In a new study that supports that model, researchers at MIT and the Whitehead Institute for Biomedical Research have discovered physical interactions between proteins and with DNA that help explain why these droplets, which stimulate the transcription of nearby genes, tend to cluster along specific stretches of DNA known as super enhancers. These enhancer regions do not encode proteins but instead regulate other genes.
“This study provides a fundamentally important new approach to deciphering how the ‘dark matter’ in our genome functions in gene control,” says Richard Young, an MIT professor of biology and member of the Whitehead Institute.
Young is one of the senior authors of the paper, along with Phillip Sharp, an MIT Institute Professor and member of MIT’s Koch Institute for Integrative Cancer Research; and Arup K. Chakraborty, the Robert T. Haslam Professor in Chemical Engineering, a professor of physics and chemistry, and a member of MIT’s Institute for Medical Engineering and Science and the Ragon Institute of MGH, MIT, and Harvard.
Graduate student Krishna Shrinivas and postdoc Benjamin Sabari are the lead authors of the paper, which appears in Molecular Cell on Aug. 8.
“A biochemical factory”
Every cell in an organism has an identical genome, but cells such as neurons or heart cells express different subsets of those genes, allowing them to carry out their specialized functions. Previous research has shown that many of these genes are located near super enhancers, which bind to proteins called transcription factors that stimulate the copying of nearby genes into RNA.
About three years ago, Sharp, Young, and Chakraborty joined forces to try to model the interactions that occur at enhancers. In a 2017 Cell paper, based on computational studies, they hypothesized that in these regions, transcription factors form droplets called phase-separated condensates. Similar to droplets of oil suspended in salad dressing, these condensates are collections of molecules that form distinct cellular compartments but have no membrane separating them from the rest of the cell.
In a 2018 Science paper, the researchers showed that these dynamic droplets do form at super enhancer locations. Made of clusters of transcription factors and other molecules, these droplets attract enzymes such as RNA polymerases that are needed to copy DNA into messenger RNA, keeping gene transcription active at specific sites.
“We had demonstrated that the transcription machinery forms liquid-like droplets at certain regulatory regions on our genome, however we didn’t fully understand how or why these dewdrops of biological molecules only seemed to condense around specific points on our genome,” Shrinivas says.
As one possible explanation for that site specificity, the research team hypothesized that weak interactions between intrinsically disordered regions of transcription factors and other transcriptional molecules, along with specific interactions between transcription factors and particular DNA elements, might determine whether a condensate forms at a particular stretch of DNA. Biologists have traditionally focused on “lock-and-key” style interactions between rigidly structured protein segments to explain most cellular processes, but more recent evidence suggests that weak interactions between floppy protein regions also play an important role in cell activities.
In this study, computational modeling and experimentation revealed that the cumulative force of these weak interactions conspire together with transcription factor-DNA interactions to determine whether a condensate of transcription factors will form at a particular site on the genome. Different cell types produce different transcription factors, which bind to different enhancers. When many transcription factors cluster around the same enhancers, weak interactions between the proteins are more likely to occur. Once a critical threshold concentration is reached, condensates form.
“Creating these local high concentrations within the crowded environment of the cell enables the right material to be in the right place at the right time to carry out the multiple steps required to activate a gene,” Sabari says. “Our current study begins to tease apart how certain regions of the genome are capable of pulling off this trick.”
These droplets form on a timescale of seconds to minutes, and they blink in and out of existence depending on a cell’s needs.
“It’s an on-demand biochemical factory that cells can form and dissolve, as and when they need it,” Chakraborty says. “When certain signals happen at the right locus on a gene, the condensates form, which concentrates all of the transcription molecules. Transcription happens, and when the cells are done with that task, they get rid of them.”
A new view
Weak cooperative interactions between proteins may also play an important role in evolution, the researchers proposed in a 2018 Proceedings of the National Academy of Sciences paper. The sequences of intrinsically disordered regions of transcription factors need to change only a little to evolve new types of specific functionality. In contrast, evolving new specific functions via “lock-and-key” interactions requires much more significant changes.
“If you think about how biological systems have evolved, they have been able to respond to different conditions without creating new genes. We don’t have any more genes that a fruit fly, yet we’re much more complex in many of our functions,” Sharp says. “The incremental expanding and contracting of these intrinsically disordered domains could explain a large part of how that evolution happens.”
Similar condensates appear to play a variety of other roles in biological systems, offering a new way to look at how the interior of a cell is organized. Instead of floating through the cytoplasm and randomly bumping into other molecules, proteins involved in processes such as relaying molecular signals may transiently form droplets that help them interact with the right partners.
“This is a very exciting turn in the field of cell biology,” Sharp says. “It is a whole new way of looking at biological systems that is richer and more meaningful.”
Some of the MIT researchers, led by Young, have helped form a company called Dewpoint Therapeutics to develop potential treatments for a wide variety of diseases by exploiting cellular condensates. There is emerging evidence that cancer cells use condensates to control sets of genes that promote cancer, and condensates have also been linked to neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease.
The research was funded by the National Science Foundation, the National Institutes of Health, and the Koch Institute Support (core) Grant from the National Cancer Institute.
Novel study shows protein CPG15 acts as a molecular proxy of experience to mark synapses for stabilization.
David Orenstein | Picower Institute for Learning and Memory
August 7, 2019
Brain cells, or neurons, constantly tinker with their circuit connections, a crucial feature that allows the brain to store and process information. While neurons frequently test out new potential partners through transient contacts, only a fraction of fledging junctions, called synapses, are selected to become permanent.
The major criterion for excitatory synapse selection is based on how well they engage in response to experience-driven neural activity, but how such selection is implemented at the molecular level has been unclear. In a new study, MIT neuroscientists have identified the gene and protein, CPG15, that allows experience to tap a synapse as a keeper.
In a series of novel experiments described in Cell Reports, the team at MIT’s Picower Institute for Learning and Memory used multi-spectral, high-resolution two-photon microscopy to literally watch potential synapses come and go in the visual cortex of mice — both in the light, or normal visual experience, and in the darkness, where there is no visual input. By comparing observations made in normal mice and ones engineered to lack CPG15, they were able to show that the protein is required in order for visual experience to facilitate the transition of nascent excitatory synapses to permanence.
Mice engineered to lack CPG15 only exhibit one behavioral deficiency: They learn much more slowly than normal mice, says senior author Elly Nedivi, the William R. (1964) and Linda R. Young Professor of Neuroscience in the Picower Institute and a professor of brain and cognitive sciences at MIT. They need more trials and repetitions to learn associations that other mice can learn quickly. The new study suggests that’s because without CPG15, they must rely on circuits where synapses simply happened to take hold, rather than on a circuit architecture that has been refined by experience for optimal efficiency.
“Learning and memory are really specific manifestations of our brain’s ability in general to constantly adapt and change in response to our environment,” Nedivi says. “It’s not that the circuits aren’t there in mice lacking CPG15, they just don’t have that feature — which is really important — of being optimized through use.”
Watching in light and darkness
The first experiment reported in the paper, led by former MIT postdoc Jaichandar Subramanian, who is now an assistant professor at the University of Kansas, is a contribution to neuroscience in and of itself, Nedivi says. The novel labeling and imaging technologies implemented in the study, she says, allowed tracking key events in synapse formation with unprecedented spatial and temporal resolution. The study resolved the emergence of “dendritic spines,” which are the structural protrusions on which excitatory synapses are formed, and the recruitment of the synaptic scaffold, PSD95, that signals that a synapse is there to stay.
The team tracked specially labeled neurons in the visual cortex of mice after normal visual experience, and after two weeks in darkness. To their surprise, they saw that spines would routinely arise and then typically disappear again at the same rate regardless of whether the mice were in light or darkness. This careful scrutiny of spines confirmed that experience doesn’t matter for spine formation, Nedivi said. That upends a common assumption in the field, which held that experience was necessary for spines to even emerge.
By keeping track of the presence of PSD95 they could confirm that the synapses that became stabilized during normal visual experience were the ones that had accumulated that protein. But the question remained: How does experience drive PSD95 to the synapse? The team hypothesized that CPG15, which is activity dependent and associated with synapse stabilization, does that job.
CPG15 represents experience
To investigate that, they repeated the same light-versus-dark experiences, but this time in mice engineered to lack CPG15. In the normal mice, there was much more PSD95 recruitment during the light phase than during the dark, but in the mice without CPG15, the experience of seeing in the light never made a difference. It was as if CPG15-less mice in the light were like normal mice in the dark.
Later they tried another experiment testing whether the low PSD95 recruitment seen when normal mice were in the dark could be rescued by exogenous expression of CPG15. Indeed, PSD95 recruitment shot up, as if the animals were exposed to visual experience. This showed that CPG15 not only carries the message of experience in the light, it can actually substitute for it in the dark, essentially “tricking” PSD95 into acting as if experience had called upon it.
“This is a very exciting result, because it shows that CPG15 is not just required for experience-dependent synapse selection, but it’s also sufficient,” says Nedivi, “That’s unique in relation to all other molecules that are involved in synaptic plasticity.”
A new model and method
In all, the paper’s data allowed Nedivi to propose a new model of experience-dependent synapse stabilization: Regardless of neural activity or experience, spines emerge with fledgling excitatory synapses and the receptors needed for further development. If activity and experience send CPG15 their way, that draws in PSD95 and the synapse stabilizes. If experience doesn’t involve the synapse, it gets no CPG15, very likely no PSD95, and the spine withers away.
The paper potentially has significance beyond the findings about experience-dependent synapse stabilization, Nedivi says. The method it describes of closely monitoring the growth or withering of spines and synapses amid a manipulation (like knocking out or modifying a gene) allows for a whole raft of studies in which examining how a gene, or a drug, or other factors affect synapses.
“You can apply this to any disease model and use this very sensitive tool for seeing what might be wrong at the synapse,” she says.
In addition to Nedivi and Subramanian, the paper’s other authors are Katrin Michel and Marc Benoit.
The National Institutes of Health and the JPB Foundation provided support for the research.
Whitehead Institute team finds drugs that activate a key brain gene; initial tests in cells and mice show promise for rare, untreatable neurodevelopmental disorder.
Nicole Davis
August 2, 2019
A research team led by Whitehead Institute scientists has identified 30 distinct chemical compounds — 20 of which are drugs undergoing clinical trial or have already been approved by the FDA — that boost the protein production activity of a critical gene in the brain and improve symptoms of Rett syndrome, a rare neurodevelopmental condition that often provokes autism-like behaviors in patients. The new study, conducted in human cells and mice, helps illuminate the biology of an important gene, called KCC2, which is implicated in a variety of brain diseases, including autism, epilepsy, schizophrenia, and depression. The researchers’ findings, published in the July 31 online issue of Science Translational Medicine, could help spur the development of new treatments for a host of devastating brain disorders.
“There’s increasing evidence that KCC2 plays important roles in several different disorders of the brain, suggesting that it may act as a common driver of neurological dysfunction,” says senior author Rudolf Jaenisch, a founding member of Whitehead Institute and professor of biology at MIT. “These drugs we’ve identified may help speed up the development of much-needed treatments.”
KCC2 works exclusively in the brain and spinal cord, carrying ions in and out of specialized cells known as neurons. This shuttling of electrically charged molecules helps maintain the cells’ electrochemical makeup, enabling neurons to fire when they need to and to remain idle when they don’t. If this delicate balance is upset, brain function and development go awry.
Disruptions in KCC2 function have been linked to several human brain disorders, including Rett syndrome (RTT), a progressive and often debilitating disorder that typically emerges early in life in girls and can involve disordered movement, seizures, and communication difficulties. Currently, there is no effective treatment for RTT.
Jaenisch and his colleagues, led by first author Xin Tang, devised a high-throughput screen assay to uncover drugs that increase KCC2 gene activity. Using CRISPR/Cas9 genome editing and stem cell technologies, they engineered human neurons to provide rapid readouts of the amount of KCC2 protein produced. The researchers created these so-called reporter cells from both healthy human neurons as well as RTT neurons that carry disease-causing mutations in the MECP2 gene. These reporter neurons were then fed into a drug-screening pipeline to find chemical compounds that can enhance KCC2 gene activity.
Tang and his colleagues screened over 900 chemical compounds, focusing on those that have been FDA-approved for use in other conditions, such as cancer, or have undergone at least some level of clinical testing. “The beauty of this approach is that many of these drugs have been studied in the context of non-brain diseases, so the mechanisms of action are known,” says Tang. “Such molecular insights enable us to learn how the KCC2 gene is regulated in neurons, while also identifying compounds with potential therapeutic value.”
The Whitehead Institute team identified a total of 30 drugs with KCC2-enhancing activity. These compounds, referred to as KEECs (short for KCC2 expression-enhancing compounds), work in a variety of ways. Some block a molecular pathway, called FLT3, which is found to be overactive in some forms of leukemia. Others inhibit the GSK3b pathway that has been implicated in several brain diseases. Another KEEC acts on SIRT1, which plays a key role in a variety of biological processes, including aging.
In followup experiments, the researchers exposed RTT neurons and mouse models to KEEC treatment and found that some compounds can reverse certain defects associated with the disease, including abnormalities in neuronal signaling, breathing, and movement. These efforts were made possible by a collaboration with Mriganka Sur’s group at the Picower Institute for Learning and Memory, in which Keji Li and colleagues led the behavioral experiments in mice that were essential for revealing the drugs’ potency.
“Our findings illustrate the power of an unbiased approach for discovering drugs that could significantly improve the treatment of neurological disease,” says Jaenisch. “And because we are starting with known drugs, the path to clinical translation is likely to be much shorter.”
In addition to speeding up drug development for Rett syndrome, the researchers’ unique drug-screening strategy, which harnesses an engineered gene-specific reporter to unearth promising drugs, can also be applied to other important disease-related genes in the brain. “Many seemingly distinct brain diseases share common root causes of abnormal gene expression or disrupted signaling pathways,” says Tang. “We believe our method has broad applicability and could help catalyze therapeutic discovery for a wide range of neurological conditions.”
Support for this work was provided by the National Institutes of Health, the Simons Foundation Autism Research Initiative, the Simons Center for the Social Brain at MIT, the Rett Syndrome Research Trust, the International Rett Syndrome Foundation, the Damon Runyon Cancer Foundation, and the National Cancer Institute.
July 26, 2019
Researchers from MIT will work with teams in the U.K. and Europe to use nanoparticles to carry multiple drug therapies to treat glioblastoma.
Koch Institute
July 30, 2019
Tiny “Russian doll-like” particles that deliver multiple drugs to brain tumors, developed by researchers at MIT and funded by Cancer Research UK, are at the center of a new international collaboration.
Professor Paula Hammond from the Department of Chemical Engineering developed the nanoparticle technology, which will be used in an effort to treat glioblastoma — the most aggressive and deadly type of brain tumor.
Hammond will be working with Professor Michael Yaffe from the Department of Biological Engineering to determine the combinations of drugs placed within the particles, and the order and timing in which the drugs are released.
The nanoparticles — 1,000 times smaller than a human hair — are coated in a protein called transferrin, which helps them cross the blood-brain barrier. This is a membrane that keeps a tight check on anything trying to get in to the brain, including drugs.
Not only are the nanoparticles able to access hard-to-reach areas of the brain, they have also been designed to carry multiple cancer drugs at once by holding them inside layers, similarly to the way Russian dolls fit inside one another.
To make the nanoparticles even more effective, they will carry signals on their surface so that they are only taken up by brain tumor cells. This means that healthy cells should be left untouched, which will minimize the side effects of treatment.
The researchers, who are based at the Koch Institute for Integrative Cancer Research, are also working with Professor Forest White from the Department of Biological Engineering. The group are one of three international teams to have been given Cancer Research UK Brain Tumor Awards — in partnership with The Brain Tumour Charity — receiving $7.9 million of funding. The awards are designed to accelerate the pace of brain tumor research. Altogether, teams were awarded a total of $23 million.
Just last year, around 24,200 people in the United States were diagnosed with brain tumors. With around 17,500 deaths from brain tumors in the same year, survival remains tragically low.
Brain tumors represent one of the hardest types of cancer to treat because not enough is known about what starts and drives the disease, and current treatments are not effective enough.
The researchers from MIT will now work with teams in the U.K. and Europe to use the nanoparticles to carry multiple drug therapies to treat glioblastoma.
Early research carried out in the lab has already shown that nanoparticles loaded with two different drugs were able to shrink glioblastomas in mice. The team has also demonstrated that the nanoparticles can kill lymphoma cells grown in the lab, and they are also exploring their use in ovarian cancer.
The Cancer Research UK Brain Tumor Award will now allow the researchers and their collaborators to use different drug combinations to find the best parameters to tackle glioblastomas.
Drugs that have already been approved, as well as experimental drugs that have passed initial safety testing in people, will be used. Because of this, if an effective drug combination is found, the team won’t have to navigate the initial regulatory hurdles needed to get them into clinical testing, which could help get promising treatments to patients faster.
“Glioblastoma is particularly challenging because we want to get highly effective but toxic drug combinations safely across the blood-brain barrier, but also want our nanoparticles to avoid healthy brain cells and only target the cancer cells,” Hammond says. “We are very excited about this alliance between the MIT Koch Institute and our colleagues in Edinburgh to address these critical challenges.”
An algorithm developed to study the structure of galaxies helps explain a key feature of embryonic development.
Anne Trafton | MIT News Office
July 25, 2019
As embryos develop, they follow predetermined patterns of tissue folding, so that individuals of the same species end up with nearly identically shaped organs and very similar body shapes.
MIT scientists have now discovered a key feature of embryonic tissue that helps explain how this process is carried out so faithfully each time. In a study of fruit flies, they found that the reproducibility of tissue folding is generated by a network of proteins that connect like a fishing net, creating many alternative pathways that tissues can use to fold the right way.
“What we found is that there’s a lot of redundancy in the network,” says Adam Martin, an MIT associate professor of biology and the senior author of the study. “The cells are interacting and connecting with each other mechanically, but you don’t see individual cells taking on an all-important role. This means that if one cell gets damaged, other cells can still connect to disparate parts of the tissue.”
To uncover these network features, Martin worked with Jörn Dunkel, an MIT associate professor of physical applied mathematics and an author of the paper, to apply an algorithm normally used by astronomers to study the structure of galaxies.
Hannah Yevick, an MIT postdoc, is the lead author of the study, which appears today in Developmental Cell. Graduate student Pearson Miller is also an author of the paper.
A safety net
During embryonic development, tissues change their shape through a process known as morphogenesis. One important way tissues change shape is to fold, which allows flat sheets of embryonic cells to become tubes and other important shapes for organs and other body parts. Previous studies in fruit flies have shown that even when some of these embryonic cells are damaged, sheets can still fold into their correct shapes.
“This is a process that’s fairly reproducible, and so we wanted to know what makes it so robust,” Martin says.
In this study, the researchers focused on the process of gastrulation, during which the embryo is reorganized from a single-layered sphere to a more complex structure with multiple layers. This process, and other morphogenetic processes similar to fruit fly tissue folding, also occur in human embryos. The embryonic cells involved in gastrulation contain in their cytoplasm proteins called myosin and actin, which form cables and connect at junctions between cells to form a network across the tissue. Martin and Yevick had hypothesized that the network of cell connectivity might play a role in the robustness of the tissue folding, but until now, there was no good way to trace the connections of the network.
To achieve that, Martin’s lab joined forces with Dunkel, who studies the physics of soft surfaces and flowing matter — for example, wrinkle formation and patterns of bacterial streaming. For this study, Dunkel had the idea to apply a mathematical procedure that can identify topological features of a three-dimensional structure, analogous to ridges and valleys in a landscape. Astronomers use this algorithm to identify galaxies, and in this case, the researchers used it to trace the actomyosin networks across and between the cells in a sheet of tissue.
“Once you have the network, you can apply standard methods from network analysis — the same kind of analysis that you would apply to streets or other transport networks, or the blood circulation network, or any other form of network,” Dunkel says.
Among other things, this kind of analysis can reveal the structure of the network and how efficiently information flows along it. One important question is how well a network adapts if part of it gets damaged or blocked. The MIT team found that the actomyosin network contains a great deal of redundancy — that is, most of the “nodes” of the network are connected to many other nodes.
This built-in redundancy is analogous to a good public transit system, where if one bus or train line goes down, you can still get to your destination. Because cells can generate mechanical tension along many different pathways, they can fold the right way even if many of the cells in the network are damaged.
“If you and I are holding a single rope, and then we cut it in the middle, it would come apart. But if you have a net, and cut it in some places, it still stays globally connected and can transmit forces, as long as you don’t cut all of it,” Dunkel says.
Folding framework
The researchers also found that the connections between cells preferentially organize themselves to run in the same direction as the furrow that forms in the early stages of folding.
“We think this is setting up a frame around which the tissue will adopt its shape,” Martin says. “If you prevent the directionality of the connections, then what happens is you can still get folding but it will fold along the wrong axis.”
Although this study was done in fruit flies, similar folding occurs in vertebrates (including humans) during the formation of the neural tube, which is the precursor to the brain and spinal cord. Martin now plans to apply the techniques he used in fruit flies to see if the actomyosin network is organized the same way in the neural tube of mice. Defects in the closure of the neural tube can lead to birth defects such as spina bifida.
“We would like to understand how it goes wrong,” Martin says. “It’s still not clear whether it’s the sealing up of the tube that’s problematic or whether there are defects in the folding process.”
The research was funded by the National Institute of General Medical Sciences and the James S. McDonnell Foundation.
Greta Friar | Whitehead Institute
July 22, 2019
Cambridge, MA — Kava (Piper methysticum) is a plant native to the Polynesian islands that people there have used in a calming drink of the same name in religious and cultural rituals for thousands of years. The tradition of cultivating kava and drinking it during important gatherings is a cultural cornerstone shared throughout much of Polynesia, though the specific customs — and the strains of kava — vary from island to island. Over the last few decades, kava has been gaining interest outside of the islands for its pain relief and anti-anxiety properties as a potentially attractive alternative to drugs like opioids and benzodiazepines because kavalactones, the molecules of medicinal interest in kava, use slightly different mechanisms to affect the central nervous system and appear to be non-addictive. Kava bars have been springing up around the United States, kava supplements and teas lining the shelves at stores like Walmart, and sports figures including former and current NFL players in need of safe pain relief are touting its benefits.
This growing usage suggests that there would be a sizeable market for kavalactone based medical therapies, but there are roadblocks to development: for one, kava is hard to cultivate, especially outside of the tropics. Kava takes years to reach maturity, and as a domesticated species that no longer produces seeds it can only be propagated using cuttings. This can make it difficult for researchers to get a large enough quantity of kavalactones for investigations or clinical trials. New research from Whitehead Institute Member and associate professor of biology at MIT Jing-Ke Weng and postdoctoral researcher Tomáš Pluskal, published online in Nature Plants on July 22, describes a way to solve that problem, as well as to create kavalactone variants not found in nature that may be more effective or safe as therapeutics.
“We’re combining historical knowledge of this plant’s medicinal properties, established through centuries of traditional usage, with modern research tools in order to potentially develop new drugs,” Pluskal says.
Weng’s lab has shown that if researchers figure out the genes behind a desirable natural molecule—in this case, kavalactones—they can clone those genes, insert them into species like yeast or bacteria that grow quickly and are easier to maintain in a variety of environments than a temperamental tropical plant, and then get these microbial bio-factories to mass produce the molecule. In order to achieve this, first Weng and Pluskal had to solve a complicated puzzle: how does kava produce kavalactones? There is no direct kavalactone gene; complex metabolites like kavalactones are created through a series of steps using intermediate molecules. Cells can combine these intermediates, snip out parts of them, and add bits onto them to create the final molecule—most of which is done with the help of enzymes, cells’ chemical reaction catalysts. So, in order to recreate kavalactone production, the researchers had to identify the complete pathway plants use to synthesize it, including the genes for all of the enzymes involved.
The researchers could not use genetic sequencing or common gene editing tools to identify the enzymes because the kava genome is huge; it has 130 chromosomes compared to humans’ 46. Instead they turned to other methods, including sequencing the plant’s RNA to survey the genes expressed, to identify the biosynthetic pathway for kavalactones.
“It’s like you have a lot of LEGO pieces scattered on the floor,” Weng says, “and you have to find the ones that fit together to build a certain object.”
Weng and Pluskal had a good starting point: they recognized that kavalactones had a similar structural backbone to chalcones, metabolites shared by all land plants. They hypothesized that one of the enzymes involved in producing kavalactones must be related to the one involved in producing chalcones, chalcone synthase (CHS). They looked for genes encoding similar enzymes and found two synthases that had evolved from an older CHS gene. These synthases, which they call PmSPS1 and PmSPS2, help to shape the basic scaffolding of kavalactones molecules.
Then, with some trial and error, Pluskal found the genes encoding a number of the tailoring enzymes that modify and add to the molecules’ backbone to create a variety of specific kavalactones. In order to test that he had identified the right enzymes, Pluskal cloned the relevant genes and confirmed that the enzymes they encode produced the expected molecules. The team also identified key enzymes in the biosynthetic pathway of flavokavains, molecules in kava that are structurally related to kavalactones and have been shown in studies to have anti-cancer properties.
Once the researchers had their kavalactone genes, they inserted them into bacteria and yeast to begin producing the molecules. This proof of concept for their microbial bio-factory model demonstrated that using microbes could provide a more efficient and scalable production vehicle for kavalactones. The model could also allow for the production of novel molecules engineered by combining kava genes with other genes so the microbes would produce modified kavalactones. This could allow researchers to optimize the molecules for efficiency and safety as therapeutics.
“There’s a very urgent need for therapies to treat mental disorders, and for safer pain relief options,” Weng says. “Our model eliminates several of the bottlenecks in drug development from plants by increasing access to natural medicinal molecules and allowing for the creation of new-to-nature molecules.”
Kava is only one of many plants around the world containing unique molecules that could be of great medicinal value. Weng and Pluskal hope that their model—combining the use of drug discovery from plants used in traditional medicine, genomics, synthetic biology, and microbial mass production—will be used to better harness the great diversity of plant chemistry around the world in order to help patients in need.
This work was supported by grants from the Smith Family Foundation, Edward N. and Della L. Thome Memorial Foundation, the Family Larsson-Rosenquist Foundation, and the National Science Foundation (CHE-1709616). T.P. is a Simons Foundation Fellow of the Helen Hay Whitney Foundation. J.K.W is supported by the Beckman Young Investigator Program, Pew Scholars Program in the Biomedical Sciences (grant number 27345), and the Searle Scholars Program (grant number 15-SSP-162).
Written by Greta Friar
***
Jing-Ke Weng’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an associate professor of biology at Massachusetts Institute of Technology.
***
Full citation:
“The biosynthetic origin of psychoactive kavalactones in kava”
Nature Plants, online July 22, 2019, doi: 10.1038/s41477-019-0474-0
Tomáš Pluskal (1), Michael P. Torrens-Spence (1), Timothy R. Fallon (1,2), Andrea De Abreu (1,2), Cindy H. Shi (1,2), and Jing-Ke Weng (1,2)
1. Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142 USA.
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.
Differences in male and female gene expression, including those contributing to height differences, found throughout the body in humans and other mammals.
Greta Friar | Whitehead Institute
July 19, 2019
Throughout the animal kingdom, males and females frequently exhibit sexual dimorphism: differences in characteristic traits that often make it easy to tell them apart. In mammals, one of the most common sex-biased traits is size, with males typically being larger than females. This is true in humans: Men are, on average, taller than women. However, biological differences among males and females aren’t limited to physical traits like height. They’re also common in disease. For example, women are much more likely to develop autoimmune diseases, while men are more likely to develop cardiovascular diseases.
In spite of the widespread nature of these sex biases, and their significant implications for medical research and treatment, little is known about the underlying biology that causes sex differences in characteristic traits or disease. In order to address this gap in understanding, Whitehead Institute Director David Page has transformed the focus of his lab in recent years from studying the X and Y sex chromosomes to working to understand the broader biology of sex differences throughout the body. In a paper published in Science, Page, a professor of biology at MIT and a Howard Hughes Medical Institute investigator; Sahin Naqvi, first author and former MIT graduate student (now a postdoc at Stanford University); and colleagues present the results of a wide-ranging investigation into sex biases in gene expression, revealing differences in the levels at which particular genes are expressed in males versus females.
The researchers’ findings span 12 tissue types in five species of mammals, including humans, and led to the discovery that a combination of sex-biased genes accounts for approximately 12 percent of the average height difference between men and women. This finding demonstrates a functional role for sex-biased gene expression in contributing to sex differences. The researchers also found that the majority of sex biases in gene expression are not shared between mammalian species, suggesting that — in some cases — sex-biased gene expression that can contribute to disease may differ between humans and the animals used as models in medical research.
Having the same gene expressed at different levels in each sex is one way to perpetuate sex differences in traits in spite of the genetic similarity of males and females within a species — since with the exception of the 46th chromosome (the Y in males or the second X in females), the sexes share the same pool of genes. For example, if a tall parent passes on a gene associated with an increase in height to both a son and a daughter, but the gene has male-biased expression, then that gene will be more highly expressed in the son, and so may contribute more height to the son than the daughter.
The researchers searched for sex-biased genes in tissues across the body in humans, macaques, mice, rats, and dogs, and they found hundreds of examples in every tissue. They used height for their first demonstration of the contribution of sex-biased gene expression to sex differences in traits because height is an easy-to-measure and heavily studied trait in quantitative genetics.
“Discovering contributions of sex-biased gene expression to height is exciting because identifying the determinants of height is a classic, century-old problem, and yet by looking at sex differences in this new way we were able to provide new insights,” Page says. “My hope is that we and other researchers can repeat this model to similarly gain new insights into diseases that show sex bias.”
Because height is so well studied, the researchers had access to public data on the identity of hundreds of genes that affect height. Naqvi decided to see how many of those height genes appeared in the researchers’ new dataset of sex-biased genes, and whether the genes’ sex biases corresponded to the expected effects on height. He found that sex-biased gene expression contributed approximately 1.6 centimeters to the average height difference between men and women, or 12 percent of the overall observed difference.
The scope of the researchers’ findings goes beyond height, however. Their database contains thousands of sex-biased genes. Slightly less than a quarter of the sex-biased genes that they catalogued appear to have evolved that sex bias in an early mammalian ancestor, and to have maintained that sex bias today in at least four of the five species studied. The majority of the genes appear to have evolved their sex biases more recently, and are specific to either one species or a certain lineage, such as rodents or primates.
Whether or not a sex-biased gene is shared across species is a particularly important consideration for medical and pharmaceutical research using animal models. For example, previous research identified certain genetic variants that increase the risk of Type 2 diabetes specifically in women; however, the same variants increase the risk of Type 2 diabetes indiscriminately in male and female mice. Therefore, mice would not be a good model to study the genetic basis of this sex difference in humans. Even when the animal appears to have the same sex difference in disease as humans, the specific sex-biased genes involved might be different. Based on their finding that most sex bias is not shared between species, Page and colleagues urge researchers to use caution when picking an animal model to study sex differences at the level of gene expression.
“We’re not saying to avoid animal models in sex-differences research, only not to take for granted that the sex-biased gene expression behind a trait or disease observed in an animal will be the same as that in humans. Now that researchers have species and tissue-specific data available to them, we hope they will use it to inform their interpretation of results from animal models,” Naqvi says.
The researchers have also begun to explore what exactly causes sex-biased expression of genes not found on the sex chromosomes. Naqvi discovered a mechanism by which sex-biased expression may be enabled: through sex-biased transcription factors, proteins that help to regulate gene expression. Transcription factors bind to specific DNA sequences called motifs, and he found that certain sex-biased genes had the motif for a sex-biased transcription factor in their promoter regions, the sections of DNA that turn on gene expression. This means that, for example, a male-biased transcription factor was selectively binding to the promoter region for, and so increasing the expression of, male-biased genes — and likewise for female-biased transcription factors and female-biased genes. The question of what regulates the transcription factors remains for further study — but all sex differences are ultimately controlled by either the sex chromosomes or sex hormones.
The researchers see the collective findings of this paper as a foundation for future sex-differences research.
“We’re beginning to build the infrastructure for a systematic understanding of sex biases throughout the body,” Page says. “We hope these datasets are used for further research, and we hope this work gives people a greater appreciation of the need for, and value of, research into the molecular differences in male and female biology.”
This work was supported by Biogen, Whitehead Institute, National Institutes of Health, Howard Hughes Medical Institute, and generous gifts from Brit and Alexander d’Arbeloff and Arthur W. and Carol Tobin Brill.