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An eye for a mouth: How regenerating flatworms keep track of body parts

Graduate student Lauren Cote identifies genes directing regeneration

Justin Chen
November 16, 2017

An eye for a mouth: How regenerating flatworms keep track of body parts

Person with brown hair in pony tail sits in front of computer and microscope.

Graduate student Lauren Cote identifies genes directing regeneration

Justin Chen

 

Peering down through a microscope at a petri dish, Lauren Cote, a sixth-year graduate student, watches the tip of a worm’s tail. Alone in the petri dish, the brown globule of tissue is regenerating an entirely new digestive system, a brain, and a pair of eye spots. After just a few weeks, the animal — a quarter-inch-long ribbon of flesh capped by a triangular head — is complete again. Swimming through the dish, the worm’s grainy, mahogany body fades to a translucent gray-blue along the edges, stretching and contracting as if hinting at its malleability.

Many animals regenerate. Salamanders replace their tails while zebrafish regrow damaged heart muscle. Even humans can renew large parts of their livers. However, few creatures can regenerate like planarians, a class of flatworms found in fresh and salt water habitats around the world — and in the Reddien lab at the Whitehead Institute.

Because planarians are masters of regeneration, able to replace any body part and even create a new animal from small chunks of tissue, they have become a focus of intense study. By examining the flatworm species Schmidtea mediterranea, Cote and other members of the Reddien lab have uncovered the ways cells communicate after injury to coordinate regeneration. Their work provides insight into how the ability to regenerate evolved, and how the healing process works in a variety of animals, including humans.

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Although regeneration seems mysterious, researchers have simplified the feat into two steps. First, planarians create the raw material to make new body parts by stimulating a group of rapidly dividing cells, called stem cells, that are the source of all new tissue in the worm. Second, these new cells need instructions to know what kind of tissue to become. Cote’s goal is to demystify this second step by locating a grid of information, like latitude and longitude lines on a map, that helps planarians keep track of their body parts and sense what is missing.

Hands suctioning small, black dots from petri dish.
Few creatures can regenerate like planarians, a class of flatworms found in fresh and salt water habitats around the world.

“The animal could have lost just the tip of its head or entire left side of its body,” Cote says, “and somehow it regrows the precise anatomy needed to make a complete worm.”

Over the past few years, research in the Reddien lab has demonstrated that a network of muscle cells spread throughout the worm’s body guides regeneration. To accomplish this task, muscle cells rely on a group of genes called position control genes (PCGs) which, based on Cote’s model, are predicted to encode proteins involved in cell communication. Depending on what PCGs are activated or expressed, muscle cells would send out a unique combination of signaling molecules that determine which body parts, such as eyes, stomach, or tail, would form.

“We like to imagine that muscle cells function like satellites and beam down information,” Cote says. “This allows stem cells to know where they are and what new body part to become.”

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To systematically identify PCGs from the roughly 20,000 genes expressed in Schmidtea mediterranea, Cote worked in tandem with postdoctoral researcher Lucila Scimone in the Reddien lab to perform a two-part study. First they created maps of gene expression by examining individual muscle cells. After inventorying the genes each individual muscle cell expressed, they aggregated the data into a whole body map, showing gene activity across the entire worm. Some genes were expressed in all muscle cells, implying a general function such as controlling contraction and relaxation. In contrast, other genes were expressed in precise regions of the worm, like the head or midsection, suggesting that they could act as PCGs by defining the identity of each area.

In the second half of the study, Cote and Scimone used molecular techniques to disrupt the activity of potential PCGs. “We hypothesized that if a gene were needed to direct regeneration, the worm would still be able to renew itself without that gene’s activity,” Cote says, “but the animal would end up with an abnormal body.”

Indeed, Cote found that disrupting four genes in particular, encoding signaling molecules and receptor-like proteins, led to defective regeneration; worms either grew extra eyes on their head or grew extra feeding tubes sprouting out of their midsection like elongated suction cups.  Together these four genes, along with a few previously identified genes controlling head and tail regeneration, comprise a short but expanding list of PCGs controlling the location and identity of new tissues. As scientists begin to understand the molecular details of planarian regeneration, they will test whether similar genes are used by other animals and humans.

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Although a biologist now, Cote began her academic life focusing on mathematics. As an undergraduate math major at the University of Chicago, she studied branches of mathematics such as analysis, algebra, and algebraic topology, a discipline that describes the properties of multidimensional shapes. After a summer project, Cote realized that — while she enjoyed learning mathematics — she found the research far too abstract.

“I was having a mid-college crisis,” she recalls. “I wanted to study something more visual where you could actually see what is going on.” Following this urge, Cote began to work in a lab examining fly development during her junior year. “I remember watching sheets of cells on the outside of a fly embryo folding in on themselves and sliding under the surface away from view. It made me wonder how cells make decisions and choreograph their movements to build a body. That’s how I got interested in developmental biology.”

After graduating from the University of Chicago, Cote worked as a lab technician for two years. During this time, she realized that her background in math and ability to think logically was an asset. “Putting together a mathematical proof is similar to publishing a research paper,” she says. “In both cases you are piecing together smaller bits of evidence into a cohesive argument.”

A series of blobs with white, green, purple and yellow specs inside them.
Gene expression maps from the first half of Cote’s and Scimone’s study. The head of the worm faces the top of the screen while the tail of the worm faces the bottom of the screen. Each worm is marked by purple, yellow, and green dots indicating the expression of three different genes expressed in muscle cells. These colors show how genes are localized to different areas of the worm and could act as PCGs.  In the second half of the study, Cote and Scimone identified PCGs by using molecular techniques to disrupt gene activity and looking for worms that regenerated abnormal bodies.

Encouraged by her successful venture into biological research, Cote decided to pursue a PhD in biology. She learned about the Reddien lab while taking a genetics course during her first year at MIT. Like Cote, many members of this group have backgrounds in other areas of science — including computational biology, development, evolution, biochemistry, and immunology — which helps them examine planarian regeneration from many perspectives.

“They were beginning to put together a story linking muscle cells to regeneration that was really intriguing,” Cote says. “I also liked the challenge of working with planarians because they are a fairly new lab animal. We’re still developing a lot of research tools so there is room to be creative and ask fundamental questions.”

By following an initial strand of curiosity as an undergraduate and identifying PCGs as a graduate student, Cote has begun to decipher the molecular language of regeneration.  As scientists learn more about how planarians replace missing body parts, new areas of exploration open. One pressing question­ is how planarian regeneration compares to that of other animals. To pursue that mystery, Cote plans on studying another animal as a postdoctoral researcher and eventually starting her own laboratory.

“I still haven’t made up my mind, “she says, “but I’m considering a lot of possibilities such as crustaceans, sea squirts, zebrafish, and axolotls.” Regardless of her final choice, Cote will be investigating how cells — essentially fatty membranes encasing a slurry of water and proteins — manage to form complex and intricate structures. She will be pursuing the same questions that first captivated her as an undergraduate in Chicago. “How do cells make decisions? How do they know to become an eye or a stomach or a brain?” she asks. “There is a lot more that I want to understand.”

Photo credit: Raleigh McElvery
Biologists’ new peptide could fight many cancers

Drug that targets a key cancer protein could combat leukemia and other types of cancer.

Anne Trafton | MIT News Office
January 15, 2018

MIT biologists have designed a new peptide that can disrupt a key protein that many types of cancers, including some forms of lymphoma, leukemia, and breast cancer, need to survive.

The new peptide targets a protein called Mcl-1, which helps cancer cells avoid the cellular suicide that is usually induced by DNA damage. By blocking Mcl-1, the peptide can force cancer cells to undergo programmed cell death.

“Some cancer cells are very dependent on Mcl-1, which is the last line of defense keeping the cell from dying. It’s a very attractive target,” says Amy Keating, an MIT professor of biology and one of the senior authors of the study.

Peptides, or small protein fragments, are often too unstable to use as drugs, but in this study, the researchers also developed a way to stabilize the molecules and help them get into target cells.

Loren Walensky, a professor of pediatrics at Harvard Medical School and a physician at Dana-Farber Cancer Institute, is also a senior author of the study, which appears in the Proceedings of the National Academy of Sciences the week of Jan. 15. Researchers in the lab of Anthony Letai, an associate professor of medicine at Harvard Medical School and Dana-Farber, were also involved in the study, and the paper’s lead author is MIT postdoc Raheleh Rezaei Araghi.

A promising target

Mcl-1 belongs to a family of five proteins that play roles in controlling programmed cell death, or apoptosis. Each of these proteins has been found to be overactive in different types of cancer. These proteins form what is called an “apoptotic blockade,” meaning that cells cannot undergo apoptosis, even when they experience DNA damage that would normally trigger cell death. This allows cancer cells to survive and proliferate unchecked, and appears to be an important way that cells become resistant to chemotherapy drugs that damage DNA.

“Cancer cells have many strategies to stay alive, and Mcl-1 is an important factor for a lot of acute myeloid leukemias and lymphomas and some solid tissue cancers like breast cancers. Expression of Mcl-1 is upregulated in many cancers, and it was seen to be upregulated as a resistance factor to chemotherapies,” Keating says.

Many pharmaceutical companies have tried to develop drugs that target Mcl-1, but this has been difficult because the interaction between Mcl-1 and its target protein occurs in a long stretch of 20 to 25 amino acids, which is difficult to block with the small molecules typically used as drugs.

Peptide drugs, on the other hand, can be designed to bind tightly with Mcl-1, preventing it from interacting with its natural binding partner in the cell. Keating’s lab spent many years designing peptides that would bind to the section of Mcl-1 involved in this interaction — but not to other members of the protein family.

Once they came up with some promising candidates, they encountered another obstacle, which is the difficulty of getting peptides to enter cells.

“We were exploring ways of developing peptides that bind selectively, and we were very successful at that, but then we confronted the problem that our short, 23-residue peptides are not promising therapeutic candidates primarily because they cannot get into cells,” Keating says.

To try to overcome this, she teamed up with Walensky’s lab, which had previously shown that “stapling” these small peptides can make them more stable and help them get into cells. These staples, which consist of hydrocarbons that form crosslinks within the peptides, can induce normally floppy proteins to assume a more stable helical structure.

Keating and colleagues created about 40 variants of their Mcl-1-blocking peptides, with staples in different positions. By testing all of these, they identified one location in the peptide where putting a staple not only improves the molecule’s stability and helps it get into cells, but also makes it bind even more tightly to Mcl-1.

“The original goal of the staple was to get the peptide into the cell, but it turns out the staple can also enhance the binding and enhance the specificity,” Keating says. “We weren’t expecting that.”

Killing cancer cells

The researchers tested their top two Mcl-1 inhibitors in cancer cells that are dependent on Mcl-1 for survival. They found that the inhibitors were able to kill these cancer cells on their own, without any additional drugs. They also found that the Mcl-1 inhibitors were very selective and did not kill cells that rely on other members of the protein family.

Keating says that more testing is needed to determine how effective the drugs might be in combating specific cancers, whether the drugs would be most effective in combination with others or on their own, and whether they should be used as first-line drugs or when cancers become resistant to other drugs.

“Our goal has been to do enough proof-of-principle that people will accept that stapled peptides can get into cells and act on important targets. The question now is whether there might be any animal studies done with our peptide that would provide further validation,” she says.

Joshua Kritzer, an associate professor of chemistry at Tufts University, says the study offers evidence that the stapled peptide approach is worth pursuing and could lead to new drugs that interfere with specific protein interactions.

“There have been a lot of biologists and biochemists studying essential interactions of proteins, with the justification that with more understanding of them, we would be able to develop drugs that inhibit them. This work now shows a direct line from biochemical and biophysical understanding of protein interactions to an inhibitor,” says Kritzer, who was not involved in the research.

Keating’s lab is also designing peptides that could interfere with other relatives of Mcl-1, including one called Bfl-1, which has been less studied than the other members of the family but is also involved in blocking apoptosis.

The research was funded by the Koch Institute Dana-Farber Bridge Project and the National Institutes of Health.

How the brain selectively remembers new places

Neuroscientists identify a circuit that helps the brain record memories of new locations.

Anne Trafton | MIT News Office
December 25, 2017

When you enter a room, your brain is bombarded with sensory information. If the room is a place you know well, most of this information is already stored in long-term memory. However, if the room is unfamiliar to you, your brain creates a new memory of it almost immediately.

MIT neuroscientists have now discovered how this occurs. A small region of the brainstem, known as the locus coeruleus, is activated in response to novel sensory stimuli, and this activity triggers the release of a flood of dopamine into a certain region of the hippocampus to store a memory of the new location.

“We have the remarkable ability to memorize some specific features of an experience in an entirely new environment, and such ability is crucial for our adaptation to the constantly changing world,” says Susumu Tonegawa, the Picower Professor of Biology and Neuroscience and director of the RIKEN-MIT Center for Neural Circuit Genetics at the Picower Institute for Learning and Memory.

“This study opens an exciting avenue of research into the circuit mechanism by which behaviorally relevant stimuli are specifically encoded into long-term memory, ensuring that important stimuli are stored preferentially over incidental ones,” adds Tonegawa, the senior author of the study.

Akiko Wagatsuma, a former MIT research scientist, is the lead author of the study, which appears in the Proceedings of the National Academy of Sciences the week of Dec. 25.

New places

In a study published about 15 years ago, Tonegawa’s lab found that a part of the hippocampus called the CA3 is responsible for forming memories of novel environments. They hypothesized that the CA3 receives a signal from another part of the brain when a novel place is encountered, stimulating memory formation.

They believed this signal to be carried by chemicals known as neuromodulators, which influence neuronal activity. The CA3 receives neuromodulators from both the locus coeruleus (LC) and a region called the ventral tegmental area (VTA), which is a key part of the brain’s reward circuitry. The researchers decided to focus on the LC because it has been shown to project to the CA3 extensively and to respond to novelty, among many other functions.

The LC responds to an array of sensory input, including visual information as well as sound and odor, then sends information on to other brain areas, including the CA3. To uncover the role of LC-CA3 communication, the researchers genetically engineered mice so that they could block the neuronal activity between those regions by shining light on neurons that form the connection.

To test the mice’s ability to form new memories, the researchers placed the mice in a large open space that they had never seen before. The next day, they placed them in the same space again. Mice whose LC-CA3 connections were not disrupted spent much less time exploring the space on the second day, because the environment was already familiar to them. However, when the researchers interfered with the LC-CA3 connection during the first exposure to the space, the mice explored the area on the second day just as much as they had on the first. This suggests that they were unable to form a memory of the new environment.

The LC appears to exert this effect by releasing the neuromodulator dopamine into the CA3 region, which was surprising because the LC is known to be a major source of norepinephrine to the hippocampus. The researchers believe that this influx of dopamine helps to boost CA3’s ability to strengthen synapses and form a memory of the new location.

They found that this mechanism was not required for other types of memory, such as memories of fearful events, but appears to be specific to memory of new environments. The connections between the LC and CA3 are necessary for long-term spatial memories to form in CA3.

“The selectivity of successful memory formation has long been a puzzle,” says Richard Morris, a professor of neuroscience at the University of Edinburgh, who was not involved in the research. “This study goes a long way toward identifying the brain mechanisms of this process. Activity in the pathway between the locus coeruleus and CA3 occurs most strongly during novelty, and it seems that activity fixes the representations of everyday experience, helping to register and retain what’s been happening and where we’ve been.”

Choosing to remember

This mechanism likely evolved as a way to help animals survive, allowing them to remember new environments without wasting brainpower on recording places that are already familiar, the researchers say.

“When we are exposed to sensory information, we unconsciously choose what to memorize. For an animal’s survival, certain things are necessary to be remembered, and other things, familiar things, probably can be forgotten,” Wagatsuma says.

Still unknown is how the LC recognizes that an environment is new. The researchers hypothesize that some part of the brain is able to compare new environments with stored memories or with expectations of the environment, but more studies are needed to explore how this might happen.

“That’s the next big question,” Tonegawa says. “Hopefully new technology will help to resolve that.”

The research was funded by the RIKEN Brain Science Institute, the Howard Hughes Medical Institute, and the JPB Foundation.

Pairing mismatch helps impaired fish RNA cleavage proceed swimmingly
December 21, 2017

Beyond tending to its multitudes of genetic, metabolic, and developmental processes, eukaryotic cells must additionally be vigilant against invasion by parasitic sequences such as viruses and transposons. RNA interference (RNAi) is a defense used by eukaryotic cells to protect themselves from such threats to their genomic harmony. Cellular RNAi components slice and destroy invading double-stranded RNA sequences and also help snip and process microRNAs, RNA sequences encoded by the genome that play key roles in gene regulation. An important process that occurs naturally in our cells, RNAi has also been harnessed by scientists as a tool to study gene function in common models such as worms, fruit flies, and mice. While many researchers have been using RNAi to tease apart gene function for over a decade, those using zebrafish, a powerful vertebrate model, have been forced to use other approaches because RNAi just did not seem to work well in these animals. Now, researchers at Whitehead Institute have uncovered how small changes in the fish Argonaute (Ago) protein, an RNA slicing protein, that happened in its lineage an estimated 300 million years ago greatly diminished the efficiency of RNAi in these animals, while another ancestral feature, in a critical pre-microRNA, was retained that enabled the microRNA to still be produced despite the fish’s impaired Ago protein.

In an article published December 21 in the journal Molecular Cell, graduate student Grace Chen, along with both Whitehead Member David Bartel, also a professor of biology at Massachusetts Institute of Technology (MIT) and investigator with the Howard Hughes Medical Institute, and Whitehead Member and MIT professor of biology Hazel Sive, describe their discovery of a roughly 300 million-year-old, two amino acid substitutions in the fish Ago protein. The substitution is present in the ancestor all teleost fish, the class of fish which includes not only zebrafish but also the vast majority of fish species spanning those populating the ocean, aquarium, and supermarket. These two changes reside in and near the protein’s catalytic site and greatly decrease the ability of the fish Ago to perform its RNA slicing function, offering an explanation for why RNAi has not been a useful tool in zebrafish.

Despite the zebrafish’s deficiencies in RNAi, it is still able to produce the microRNA miR-451, an important regulator of red blood cell maturation and the only microRNA processed by Ago (the rest are produced with another protein called Dicer). MicroRNAs are short stretches of RNA that can regulate gene expression by inhibiting translation of mRNA into a protein and directing the destruction of mRNA before it can be used to make more protein. Since Chen had discovered that zebrafish lack an efficient Ago protein, it was mysterious as to how are fish were able to produce Ago cleavage-dependent miR-451. The Ago protein must process miR-451 by slicing the sequence out of a longer strand of RNA that has folded up on itself, forming a hairpin structure. What they determined was that in the pre-miR-451 hairpin in zebrafish, at a critical position in the miRNA, they found a “G–G” pairing mismatch that actually appears to facilitate cleavage by the impaired zebrafish Ago. No mismatch, no efficient cleavage.

Exploring the effects of a seed sequence mismatch on Ago-catalyzed cleavage kinetics further, they then tested its ability to slice other bound transcripts. The researchers discovered that while, as might be expected, a G–G mismatch slows Ago binding, it significantly enhances both slicing efficiency as well as the release of the bound product, more than off-setting the slower binding reaction kinetics and suggesting that non- “Watson–Crick” base pairing creates an exceptionally favorable geometry for the cleavage and release parts of the reaction.

These findings offer interesting insights into how animals can survive and thrive without an efficient RNAi system and suggest how the Ago protein could be “repaired” in order to allow zebrafish researchers to use RNAi in their experiments. Restoring a function that a lineage hasn’t had for 300 million years might also fuel additional findings into how the teleost class has diverged over time.

Written by Lisa Girard
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David Bartel’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology and investigator with the Howard Hughes Medical Institute.
***
Paper cited:
Chen GR, Sive H, and Bartel DP. A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially Rescues Severely Impaired Cleavage Found in Fish. Molecular Cell, Dec 21 2017 DOI: 10.1016/j.molcel.2017.11.032.
Harnessing nature’s riches
December 19, 2017

Cambridge, MA – Researchers at Whitehead Institute have reconstructed the full suite of biochemical steps required to make salidroside, a plant-derived compound widely used in traditional medicine to combat depression and fatigue and boost immunity and memory. Their new study, which appears online this week in the journal Molecular Plant, resolves some long-standing questions about how this compound is manufactured by a type of high-altitude plant, known commonly as golden root. This work not only paves a path toward large-scale synthetic efforts—thereby protecting plants already in danger of extinction—but also provides a model for dissecting the biochemical synthesis of a host of natural products, which represent a treasure trove for modern medical discoveries.

“By cracking open the natural synthesis of this compound, known as salidroside, we have helped eliminate a major bottleneck in the broader development of plant-derived natural products into pharmaceuticals,” says Jing-Ke Weng, the senior author of the paper, a Member of Whitehead Institute, and an assistant professor of biology at Massachusetts Institute of Technology. “We simply can’t rely on the native plants as the sole sources of these biologically important molecules.”

Golden root, also called Tibetan ginseng, typically grows in high-altitude, arctic environments, such as Tibet. It is well known in Eastern cultures for its medicinal properties and produces a variety of chemical substances, particularly salidroside, which have garnered interest in the biomedical research community for their potential therapeutic effects.

“People have tried to farm golden root, but the medicinal value is much lower because the plants make much less salidroside when cultivated outside of their normal habitat,” says Weng.

That means collecting enough salidroside to fuel scientific studies is largely impossible, without risking the viability of these plants and their surroundings. So Weng and his team, including first author Michael Torrens-Spence, set out to find a better way. “If we can figure out how plants make these high-value natural products, then we can devise sustainable engineering approaches to recreate such molecules—we won’t have to destroy nature in order to harness its riches,” says Torrens-Spence, a postdoctoral researcher in Weng’s laboratory.

Torrens-Spence and his colleagues used a systematic multi-omics approach to characterize various tissues from a three-month-old, greenhouse-grown golden root plant. By correlating the active genes with the abundance of key metabolites between various tissue types, the researchers created a massive biochemical catalog of the plant’s tissues.

The researchers then mined these data and matched the likely biochemical precursors of salidroside with the candidate genes (and their corresponding enzymes) responsible for those compounds’ synthesis. This approach allowed Weng and his team to create a kind of draft blueprint of how salidroside is made in nature.

To test the validity of this draft blueprint—and the molecular players from the golden root plant that comprise it—the scientists turned to two well-studied laboratory organisms: the baker’s yeast Saccharomyces cerevisiae and the tobacco plant Nicotiana benthamiana. Normally, these organisms do not make salidroside. But if the researchers’ model was correct, by inserting the candidate genes involved in salidroside synthesis Weng and his colleagues should be able to bestow that special property upon them.

That is precisely what the researchers did. Using three key enzymes they identified through their “-omics” approach, including 4HPAAS (4-hydroxyphenylacetaldehyde synthase), 4HPAR (4-hydroxyphenylacetaldehyde reductase), and T8GT (tyrosol:UDP-glucose 8-O-glucosyltransferase), they engineered yeast and tobacco plants with the capacity to make salidroside. Notably, this biochemical pathway for synthesizing salidroside involves three enzymes, rather than four, as had previously been proposed.

“This is an exciting proof-of-principle for how we can systematically unlock the biochemistry behind a range of intriguing plant-derived natural products,” says Weng. “With this capability, we can accelerate biomedical studies of these unique compounds as well as their potential therapeutic development.”

Written by Nicole Davis
* * *
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 assistant professor of biology at Massachusetts Institute of Technology.
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Full citation:
“Complete pathway elucidation and heterologous reconstitution of Rhodiola salidroside biosynthesis”
Molecular Plant, online December 19, 2017. DOI: 10.1016/j.molp.2017.12.007
Michael P. Torrens-Spence (1), Tomáš Pluskal (1), Fu-Shuang Li (1), Valentina Carballo (1) and Jing-Ke Weng (1,2).
1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
Small RNA mediates genetic parental conflict in seed endosperm
December 19, 2017

CAMBRIDGE, MA–When it comes to gene expression in the endosperm of seeds, gene provenance matters. In this specialized tissue, plants actively strive to keep the expression of genes inherited from the mother versus the father in balance, according to Whitehead Institute scientists.

The endosperm, the starchy part of a seed that envelopes and nourishes the developing embryo, comprises two-thirds of the calories in a typical human diet. It is the meat of a coconut and the sweet part of the corn on the cob we eat.  In a paper published online December 19 in the journal Cell Reports, Whitehead Member Mary Gehring, first author and former Gehring graduate student Robert Erdmann, and colleagues reveal that the endosperm is also the site where the plant must actively orchestrate a delicate balance between expression of genes inherited from the mother and those of the father.  If this critical balance errs toward one parent or the other, seeds can be too small or even abort.

Unlike most plant cells, which have two copies of the genome, cells within the endosperm have three copies: one inherited from the father, and two inherited from the mother. This ratio is established when a sperm cell in the fertilizing pollen grain fuses with the central cell associated with the egg cell in a flower’s ovule. Unlike most cells, the central cell has two nuclei, so when the sperm’s nucleus merges with the central cell, the resulting endosperm is triploid.

 The 2-to-1 ratio of maternal to paternal gene expression is crucial, and deviation can have dire consequences:  If maternal gene expression is too high, the seeds are too small; if paternal gene expression is too high, the seeds abort. Although plant biologists have known the importance of this ratio for seed viability, the balance was assumed to be passively maintained for the majority of genes.  Previously, Gehring determined that a subset of genes expressed in the endosperm are imprinted—their expression is inherited from their parent. But what about the remaining majority of the genome?

Now Gehring and colleagues have discovered a role for small RNAs—snippets of RNA that interfere with and can reduce gene expression—in actively maintaining this 2-to-1 balance in those genes that are not imprinted.  This the first time scientists have documented small RNAs maintaining such a ratio. Using Arabadopsis thaliana and Arabadopsis lyrata plants, Gehring and her lab determined that these small RNAs tamp down the expression of maternally inherited genes. When the enzyme that creates the small RNAs is mutated, fewer small RNAs are produced, and the plant’s carefully balanced gene expression is thrown off. The resulting seeds have excessive maternal gene expression. To understand the significance of this elevated maternal gene expression, Satyaki Rajavasireddy, a postdoctoral researcher in Gehring’s lab and an author of the Cell Reports paper, turned to plants with seeds that abort  because they have additional copies of paternal genes. When these plants with extra paternal DNA had their small-RNA-producing enzyme mutated, the outcome was striking: The seeds were rescued and developed to maturity.

Although the research analyzed this phenomenon in A. thaliana and A. lyrata, Gehring expects it to be a widespread manifestation of the tug-of-war between maternal and paternal genetic contributions.

“Maintaining this maternal/paternal balance is crucial for seed development, including in crop plants,” says Gehring, who is also an associate professor of biology at Massachusetts Institute of Technology.  “We’ve looked at two species that are separated by 10 million years of evolution, and I anticipate we will find this mechanism in other species as well.”

This work was supported by the National Science Foundation (NSF CAREER grant 1453459).

Written by Nicole Giese Rura
* * *
Mary Gehring’s primary affiliation is with Whitehead Institute for Biomedical Research, where her laboratory is located and all her research is conducted. She is also an associate professor of biology at Massachusetts Institute of Technology.
* * *
Full Citation:
“A small RNA pathway mediates allelic dosage in endosperm”
Cell Reports, online December 19, 2017.
Robert M. Erdmann (1,2), P.R. V. Satyaki (1), Maja Klosinska (1), Mary Gehring (1,2).
1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142 USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA
The need to know

Driven by curiosity, former auto mechanic Ryan Kohn now pursues a PhD in biology.

Bridget E. Begg | Office of Graduate Education
December 18, 2017

The name of Ryan Kohn’s son, Jayden, is tattooed in Hindi on his left outer forearm. Other tattoos on his inner arms declare “Respect” and “Loyalty.” A Latin phrase balances the tableau on his right outer forearm: “Many fear their reputation. Few their conscience.”

Kohn may stand out in the corporate milieu of Kendall Square, but he feels home at MIT. “No one has ever judged me,” he says. “For as rigorous scientifically and academically as MIT is, it can be such a laid-back place. I’ve always felt included, if I wanted to be.”

Kohn, now a PhD candidate in the Jacks Lab at MIT’s Koch Institute for Integrative Cancer Research, has overcome a challenging adolescence, colored by economic difficulties and punctuated by personal loss. These hardships developed in him a resilient curiosity that made an unexpected cultural match between MIT and Kohn, a father and former mechanic from Boyertown, Pennsylvania.

Compelled to seek answers

After being placed in an alternative high school outside of Philadelphia for insubordination, Kohn graduated with a 1.8 GPA. His son was born three years later, while Kohn worked for six and a half years as a mechanic and manager at a Dodge dealership. After losing his job during the Great Recession, he decided to go back to school, attending his local community college on a premed track before transferring to Kutztown University after two years.

Kohn attributes some of his troubled youth to early tragedy. His older sister, Nicole, died from sepsis when she was a senior in college, just 10 days after 9/11; on the morning of her funeral, Kohn’s grandfather passed away from colon cancer. Kohn felt compelled to understand why and how these illnesses happened to his loved ones, and found himself spending his time googling the immune system, the inflammatory response, and cancer.

This habit remained with him. Kohn recalls scouring the internet again and again to understand illness when it arose near him, from his own son’s immunoglobulin A deficiency to the early-onset multiple sclerosis of a friend. Though he admits he did not yet have the core scientific knowledge to fully grasp what he read at the time, Kohn says he needed, deeply, to try.

At Kutztown University, Kohn met his undergraduate mentor Angelika Antoni, a professor who taught both oncology and immunology. According to Kohn, Antoni constantly encouraged him to pursue his curiosity despite the college’s lack of laboratory resources. In fact, Antoni paid for laboratory reagents with her own credit card, while Kohn wrote his own grants and subscribed to well-known biology journals out of his own pocket because journal access was not available through Kutztown.

These challenges shaped Kohn as an experimental biologist, requiring him to precisely understand the mechanisms of experimental techniques in order to reconstruct them in the most creative and inexpensive ways possible. Perhaps most importantly, this small-college experience cultivated Kohn’s persistent curiosity.

Diving into cancer research

In his current position at the Jacks Lab, Kohn studies cancer immunotherapy, the use of a cancer patient’s own immune system to fight cancer cells. To do this, Kohn uses a mouse model of lung cancer that mimics the natural development of human cancer: Mutations identical to those found in many human cancers are triggered in the mouse, causing a tumor to arise that originates from the mouse’s own cells. These mice, like human cancer patients, have an immune system that can recognize the cancer as aberrant. Kohn’s work focuses modifying mouse immune cells to identify and attack a tumor.

Kohn is excited by the translational potential of his work, but also eagerly defends basic research at MIT when he encounters skepticism about its practicality in his conservative hometown.

Kohn often draws on metaphors in these types of conversations. He may leverage car talk, for example, to explain why there will never be a single cure for cancer: “So your ‘check engine’ light always presents the same way … but there’s literally a multitude of different things that can [cause] it. It could be a loose gas cap for the evaporative emissions system that set it off, it could be a misfire because of a bad spark plug, it could be a catalytic converter.”

Likewise, cancer can be caused by many possible biological errors that lead to an overgrowth of cells, Kohn explains. “So just like there will never be a cure for ‘check engine light,’ there will never be a [single] cure for cancer.”

Perhaps unsurprisingly, Kohn embraces the scientific freedom of the research in his lab. His advisor, Tyler Jacks, director of the Koch Institute, an HHMI investigator, and a David H. Koch Professor of Biology at MIT, is frequently in high demand, but Kohn says he has felt fully supported in his work — including in the bold ideas and unconventional projects he undertakes in his free time.

Jacks remains accessible despite his busy schedule, according to Kohn, and his emphasis on mentorship has inspired the postdocs in the lab to mentor the graduate students. The Jacks Lab also enjoys a thriving social environment. Kohn regularly attends casual weekend parties held by his labmates, and every other year Jacks organizes a cross-campus themed scavenger hunt for which the whole lab dresses in elaborate costumes.

“Real conversations about ideas”

Outside of lab, Kohn calls himself a homebody and prefers to relax after a full day, often with a beer and a movie. He spends much of this down time with his partner Ruthlyn, whether they are exploring the Boston area or talking with friends and colleagues at local pubs.

Kohn speaks about these conversations with genuine excitement: “You meet so many different people, every religion, every gender identity, every country, every language, and you just meet these people and you get to have these cool conversations … these real conversations about ideas. Because that’s really what you want, right?”

He enthusiastically notes that, in contrast to his largely homogenous hometown, more than 200 countries are represented at MIT. Kohn says the diversity and ideals of MIT reflect his own worldview.

Despite his deep sense of belonging on campus, leaving home did lay an exceptional burden on Kohn: Twelve-year-old Jayden remains in Pennsylvania with his mother, over 300 miles away.

Kohn speaks about his son with immense pride, describing Jayden as not only an extremely talented baseball player, but as a positive, energetic, and deeply mature young person. Kohn recounts with admiration, and a trace of relief, that despite the difficulty of the distance, Jayden said his father’s coming to MIT was the right thing to do.

As for his own parents, Kohn finally feels that all the headaches he has given them over the years have been worthwhile. His intense desire for knowledge has driven him through many obstacles, connected him with like minds from all over the world, and still shows no signs of waning.

Kohn has a reputation in his lab for asking questions, big and small. Asked if he’s ever afraid to admit what he doesn’t know, he says no: “I want to know … and that’s really what it comes down to.”

Epigenetic rheostat helps uncover how gene regulation is inherited and maintained
December 14, 2017

While our genome contains a vast repertoire of genes that are responsible for virtually all of the cellular and developmental processes life requires, it is the complex dance of regulating their expression that is vital for genetic programs to be executed successfully. Genes must be turned on and off at appropriate times or, in some cases, never turned on or off at all.

Methylation—the addition of chemical tags to DNA—typically reduces the expression of methylated genes. In many cases, DNA methylation can be thought of as roadblocks on a gene. The more methylated a gene is, the less likely it is that it will be active. Such genetic demarcations are critical to ensure that genes involved in particular stages of development are active at the right time, for example. Methylation is essential for proper cellular function, and its dysregulation is associated with diseases, such as cancer in humans. Despite its importance, little is known about how critical methylation patterns are inherited or maintained. Whitehead Institute Member Mary Gehring and her lab have identified a mechanism important for maintaining methylation, that when disrupted, results in the demethylation of large sections of the Arabidopsis plant’s genome. Their work is described this week in the journal Nature Communications.

Using an unusual gene in the plant Arabidopsis, Gehring is teasing apart the mechanisms that underpin methylation. By breaking this unique gene’s “circuit,” Gehring and Ben Williams, a postdoctoral researcher in her lab, have gained important insights into how methylation is maintained, including a surprising finding that previously erased methylation can be restored under certain circumstances.

In order to better understand methylation’s heritability, Gehring and Williams looked closely at an anomaly, the ROS1 gene in Arabidopsis plants, which encodes a protein that removes methylation from its own gene as well as others. Previously, Gehring and Williams had determined that ROS1 methylation actually functions in the complete opposite way from the existing paradigm—unlike most genes, when a short section of this gene is methylated, the gene is actually activated instead of inactivated. Conversely, if it is methylated, the gene is turned on. As a result, ROS1 can act as a rheostat for the Arabidopsis genome: As methylation increases, ROS1 turns on and begins removing methyl groups, and as methylation decreases, ROS1 shuts off and reduces its demethylating activity.

In the current research, Williams altered methylation at ROS1 so that its activity was uncoupled from methylation levels in the genome, in order to see what effects such a change would have on methylation throughout the entire genome. When he analyzed the plants’ methylation, it was haywire. Methylation was lost throughout the genome and progressively decreased in subsequent generations, except in a particular part of the genome called the heterochromatin—genomic areas that are strongly repressed. Interestingly, Williams found that, despite the alteration of the ROS1regulatory circuit, these heterochromatic sections of the genome actually regain their methylation and approach full methylation by the fourth generation— the same time point by which the rest of the genome has lost much of its methylation .

The researchers determined that the ROS1 circuit they uncovered is important for methylation homeostasis because it causes heritable loss of methylation when disrupted.  And yet methylation returns at some locations, albeit not immediately, suggesting that Arabidopsis enlists multiple mechanisms to maintain methylation homeostasis. Gehring and Williams are intrigued by that delay in remethylation and are working to identify its cause as well as other mechanisms that may also be at work regulating this critical process.

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (R01GM112851).

Written by Nicole Giese Rura
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Mary Gehring’s primary affiliation is with Whitehead Institute for Biomedical Research, where her laboratory is located and all her research is conducted. She is also an associate professor of biology at Massachusetts Institute of Technology.
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Full Citation:
“Stable transgenerational epigenetic inheritance requires a DNA methylation-sensing circuit”
Nature Communications, December 14, 2017.
Ben P. Williams (1) and Mary Gehring (1,2).
1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
Jacqueline Lees

Education

  • PhD, 1990, University of London
  • BSc, 1986, Biochemistry, University of York

Research Summary

We identify the proteins and pathways involved in tumorigenicity — establishing their mechanism of action in both normal and tumor cells. To do so, we use a combination of molecular and cellular analyses, mutant mouse models and genetic screens in zebrafish.

Michael T. Hemann

Education

  • PhD, 2001, Johns Hopkins University
  • BS, 1993, Molecular Biology and Biochemistry, Wesleyan University

Research Summary

Many human cancers do not respond to chemotherapy, and often times those that initially respond eventually acquire drug resistance. Our lab uses high-throughput screening technology — combined with murine stem reconstitution and tumor transplantation systems — to investigate the genetic basis for this resistance. Our goal is to identify novel cancer drug targets, as well as strategies for tailoring existing cancer therapies to target the vulnerabilities associated with specific malignancies.