Research Area: Genetics

Revising the textbook on introns

Whitehead Institute researchers uncover a group of introns in yeast that possess surprising stability and function.

Nicole Davis | Whitehead Institute
January 16, 2019

A research team from Whitehead Institute has uncovered a surprising and previously unrecognized role for introns, the parts of genes that lack the instructions for making proteins and are typically cut away and rapidly destroyed. Through studies of baker’s yeast, the researchers identified a highly unusual group of introns that linger and accumulate, in their fully intact form, long after they have been freed from their neighboring sequences, which are called exons. Importantly, these persistent introns play a role in regulating yeast growth, particularly under stressful conditions.

The researchers, whose work appears online in the journal Nature, suggest that some introns also might accumulate and carry out functions in other organisms.

“This is the first time anyone has found a biological role for full-length, excised introns,” says senior author David Bartel, a member of the Whitehead Institute. “Our findings challenge the view of these introns as simply byproducts of gene expression, destined for rapid degradation.”

Imagine the DNA that makes up your genes as the raw footage of a movie. The exons are the scenes used in the final cut, whereas the introns are the outtakes — shots that are removed, or spliced out, and therefore not represented in the finished product.

Despite their second-class status, introns are known to play a variety of important roles. Yet these activities are primarily confined to the period prior to splicing — that is, before introns are separated from their nearby exons. After splicing, some introns can be whittled down and retained for other uses — part of a group of so-called “non-coding RNAs.” But by and large, introns have been thought to be relegated to the genome’s cutting room floor.

Bartel and his Whitehead Institute colleagues, including world-renowned yeast expert Gerald Fink, now add an astonishing new dimension to this view: Full-length introns — that is, those that have been cut out but remain otherwise intact — can persist and carry out useful biological functions. As reported in their Nature paper, the team discovered that these extraordinary introns are regulated by and function within the essential TORC1 growth signaling network, forming a previously unknown branch of this network that controls cell growth during periods of stress.

“Our initial reaction was: ‘This is really weird,’” recalls first author Jeffrey Morgan, a former graduate student in Bartel’s lab who is now a postdoc in Jared Rutter’s lab at the University of Utah. “We came across genes where the introns were much more abundant than the exons, which is the exact opposite of what you’d expect.”

The researchers identified a total of 34 of these unusually stable introns, representing 11 percent of all introns in the yeast, also known as Saccharomyces cerevisiae. Surprisingly, there are very few criteria that determine which introns will become stable introns. For example, the genetic sequences of the introns or the regions that surround them are of no significance. The only defining — and necessary — feature, the team found, is a structural one, and involves the precise shape the introns adopt as they are being excised from their neighboring exons. Excised introns typically form a lasso-shaped structure, known as a lariat. The length of the lasso’s handle appears to dictate whether an intron will be stabilized or not.

Remarkably, both yeast and introns have been studied for several decades. Yet until now, these unique introns went undetected. One reason, Bartel and his colleagues believe, is the conditions under which yeast are typically grown. Often, researchers study yeast that are growing very rapidly — so-called log-phase growth. That is because abnormalities are often easiest to detect when cells are multiplying quickly.

“Biologists have focused heavily on log-phase for very good reasons, but in the wild, yeast are very rarely in that condition, whether it’s because of limited nutrients or other stresses,” says Bartel, who is also professor of biology at MIT and a Howard Hughes Medical Institute investigator.

He and his colleagues decided to grow yeast under more stressful circumstances, and that is what ultimately led them to their discovery. Although their experiments were confined to yeast, the researchers believe it is possible other organisms may harbor this long-overlooked class of introns — and that similar approaches using less-often-studied conditions could help illuminate them.

“Right now, we can say it is happening in yeast, but we’d be surprised if this is the only organism in which it is happening,” Bartel says.

The research was supported by the National Institutes of Health and the Howard Hughes Medical Institute.

A tough case cracked
Greta Friar | Whitehead Institute
December 17, 2018

CAMBRIDGE, Mass. — For hundreds of millions of years, plants thrived in the Earth’s oceans, safe from harsh conditions found on land, such as drought and UV radiation. Then, roughly 450 million years ago, plants found a way to make the move to land: They evolved spores—small reproductive cells—and eventually pollen grains with tough, protective outer walls that could withstand the harsh conditions in the terrestrial environment until they could germinate and grow into a plant or fertilize an ovule. A key component of the walls is sporopollenin, a durable polymer — a large molecule made up of many small subunits — that is absent in algae but remains ubiquitous in all land plants to this day.

Understanding the molecular composition of polymers found in nature is a fundamental pursuit of biology, with a long history tracing back to the early days of elucidating DNA and protein structures. However, the very toughness that makes sporopollenin so important for all land plants also makes it tough for researchers to study. It is extremely inert, resistant to reacting with other chemicals, including the ones researchers typically use to determine the structures of other plant biopolymers, such as polysaccharides, lignin, and natural rubber. Consequently, scientists have struggled for decades to figure out exactly what the sporopollenin polymer is made of. Now, in an article published in the journal Nature Plants on December 17, Whitehead Institute Member Jing-Ke Weng and first author and Weng lab postdoc Fu-Shuang Li, together with collaborators Professor Mei Hong and graduate student Pyae Phyo from the Massachusetts Institute of Technology (MIT) Department of Chemistry, have used innovative chemical degradation methods and state-of-the-art nuclear magnetic resonance (NMR) spectroscopy to determine the chemical structure of sporopollenin.

“Plants could not have colonized the land if they had not developed a way to withstand harsh environments,” says Weng, who is also an assistant professor of biology at MIT. “Sporopollenin helped make the terrestrial ecosystem as we know it possible.”

In addition to solving a longstanding puzzle in plant chemistry, identifying the structure of sporopollenin opens the door for its potential use in a host of other applications. Sporopollenin’s inertness is a desirable attribute to replicate in the development of, for example, medical implants such as stents, which prop open clogged arteries, to prevent negative interactions between the device and the body. It could also be a good model for durable paints and coatings, such as those used on boats, where its inertness would prevent reactions with compounds in the water and so protect the ship’s hull from environmental degradation.

Finding the shape and composition of sporopollenin was not a simple task. The first challenge was getting enough of the material to study, as pollen amounts that can be collected from most plants are minute. However, pollen from the pitch pine, Pinus rigida, is sold in bulk in China as a topping for rice cakes, so Weng used an unconventional sample collection method: He asked his parents in China to ship him copious quantities of pitch pine pollen.

A common approach to determine a complex plant polymer’s structure is to dissolve it in solutions with specific chemical compounds that will break it apart into smaller and smaller pieces from which the complete structure can be deduced. But since sporopollenin is inert and does not react with the researchers’ usual cadre of chemicals, figuring out how to break down the molecule was a key challenge.

In order to crack this problem — and make the sporopollenin dissolve more easily — Li used a specially designed grinder known as the high-energy ball mill to physically shear the tiny pollen coat into even finer pieces. Then he began testing different chemical mixtures to find ones that could break apart the sporopollenin polymer into more accessible fragments. The big breakthrough came when he tried a chemical degradation process called thioacidolysis, an acid catalyzed reaction with a pinch of a special sulfur-containing compound. This allowed Li to consistently break down 50% of the total sporopollenin polymer into small pieces, with the structure of each of these pieces resolved one by one.

To help complete the puzzle, the researchers collaborated with Mei Hong’s group in MIT’s Department of Chemistry and used magic-angle-spinning solid-state NMR spectroscopy, which can determine the chemical structures of insoluble compounds by having them interact with magnetic fields. This investigation narrowed the possible structures for sporopollenin. Combined with more chemical degradation tests to verify certain possibilities and eliminate others, it ultimately led to the complete structure.

With the structure of sporopollenin in hand, the researchers were then able to identify aspects of this unique polymer that make it such a good protective wall for spores and pollen.

A key finding was that sporopollenin molecules contain two types of cross-linkages, esters and acetals, that act like chemical clips, binding the chains of the molecule together. Other known plant polymers have only one main type of cross-link, and this unique characteristic likely provides the extreme chemical inertness of sporopollenin. Ester bonds are resistant to mildly acidic conditions, while acetals are resistant to basic conditions, meaning the molecule won’t break down in either type of environment in the wild or in the lab.

Other components of sporopollenin that the researchers found include multiple molecules known to provide UV protection, as well as fatty acids, which are water resistant and may protect spores and pollen from drought or other changes in water availability.

The researchers are now looking for differences in sporopollenin between species. Pine is not a flowering plant, but the majority of plants of interest to agriculture and medicine are, so Weng and Li are investigating how sporopollenin may have changed with the evolution of the flowering plants.

“Since I was a student, inspired by the magnificent discovery of the structure of DNA, I have been driven to discover the fundamental forms of things in nature,” Weng says. “It has been so rewarding to illuminate the structure of this crucial biopolymer in plants.”

This work was supported by the Pew Scholar Program in the Biomedical Sciences and the Searle Scholars Program, and the U.S. Department of Energy (# DE-SC0001090).

***

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.

***

Full citation:
“The molecular structure of plant sporopollenin”
Nature Plants, December 17, 2018, DOI: 10.1038/s41477-018-0330-7
Fu-Shuang Li (1), Pyae Phyo (2), Joseph Jacobowitz (1,3), Mei Hong (2), Jing-Ke Weng (1,3)
1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, United States.
2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
Engineering “capture compounds” to probe cell growth

Researchers develop a method to investigate how bacteria respond to starvation and to identify which proteins bind to what they call the “magic spot” — ppGpp.

Raleigh McElvery | Department of Biology
December 17, 2018

In 1969, scientist Michael Cashel was analyzing the compounds produced by starved bacteria when he noticed two spots appearing on his chromatogram as if by magic. Today, we know one of these “magic spots,” as researchers call them, as guanosine tetraphosphate, or ppGpp for short. We also understand that it is a signaling molecule present in virtually all bacteria, helping tune cell growth and size based on nutrient availability.

And yet, despite decades of study, precisely how ppGpp regulates bacterial growth has remained rather mysterious. Delving further requires a more comprehensive list of the molecules that ppGpp binds to exert its effects.

Now, collaborators from MIT’s departments of Biology and Chemistry have developed a method to do just that, and used their new approach to pinpoint over 50 ppGpp targets in Escherichia coli — roughly half which had not been identified previously. Many of these targets are enzymes required to produce nucleotides, the building blocks of DNA and RNA. During times when the bacteria do not have enough nutrients to grow and divide normally, the researchers propose that ppGpp prevents these enzymes from creating new nucleotides from scratch, helping cells enter a dormant state.

“With small molecules or metabolites like ppGpp, it’s been difficult historically to determine which proteins they bind,” says Michael Laub, a professor of biology, a Howard Hughes Medical Institute investigator, and the senior author of the study. “This has been an intractable problem that’s held the field back for some time, but our new approach allows you to nail down the likely targets in a matter of weeks.”

Postdoc Boyuan Wang is the first author of the study, which appeares in Nature Chemical Biology on Dec. 17.

Since ppGpp was discovered nearly 50 years ago, it has been shown to suppress DNA replication, transcription, translation, and various metabolic pathways. It puts the brakes on cell growth and allows bacteria to persist in the face of starvation, stress, and antibiotics. Its influence over numerous regulatory processes has remained somewhat of a mystery, however — after all, it doesn’t just modulate a single pathway but coordinates multiple operations simultaneously to orchestrate a mass shutdown of the cell.

In order to discern which proteins ppGpp binds to effect such widespread change, the researchers built what they call “capture compounds” that contain ppGpp, allowing them to fish out its targets from bacterial extracts. These compounds included a photoreactive crosslinker that latched tightly onto the proteins of interest in the presence of light, and a biotin handle that helped the scientists pull out the proteins to identify them. Most importantly, they were joined to ppGpp in such a way that they wouldn’t interfere with its ability to bind to its targets. This method is more efficient and accurate compared to more traditional means of distinguishing ppGpp targets, which are far more arduous and lack sensitivity.

“Our approach solves these problems because you’re no longer required to do such labor-intensive protocols in order to identify ppGpp targets — and it works even in bacteria beyond E. coli,” says Wang. “Although ppGpp is common among many bacterial species, it seems to exert its effects through different mechanisms, which complicates things. Our capture compounds provide a way to unravel this diversity, and in short order.”

Although the 56 ppGpp targets Wang identified in his screen control a myriad of cellular processes, he homed in on the enzyme PurF — which initiates the biosynthesis of purine nucleotides bearing adenine and guanine bases, also known as A and G.

When bacteria are stressed or starved, they enter a dormant state to survive. But simply curbing translation and transcription is not enough; nucleotides are still being generated and will build up if their synthesis is not put on pause. Cells can build nucleotides in one of two ways: either by salvaging existing materials or starting completely from scratch. PurF kicks off the first step in the latter process leading to the A and G nucleotides. However, when ppGpp binds to PurF, it causes the enzyme to change its shape, which prevents it from doing its job, thus reducing nucleotide production in the cell.

“This is the first time that an enzyme involved in that specific pathway or function has been identified as a ppGpp target,” Wang says. “If you limit the consumption of nucleotides but not their production, the nucleotide pool is going to explode, which isn’t good for the cell. So we’ve shown that ppGpp actually addresses this problem as well.”

In addition to PurF and other enzymes required for nucleotide production, the researchers noticed that ppGpp also binds to many GTPase enzymes involved in translation. This could indicate a failsafe mechanism slowing down translation by striking multiple, similar enzymes in an almost redundant manner in the face of starvation.

As Wang continues to refine his method, he aims to increase its specificity and ensure his capture compounds bind to the exact same proteins they would inside a live cell. He also hopes to screen for ppGpp binding proteins in other bacteria, including pathogens that rely on ppGpp to survive within their hosts and propagate conditions like tuberculosis.

“This is an exciting chemical approach to better understand the function of a long-studied conserved signaling molecule in bacteria,” says Jue Wang, professor of bacteriology at the University of Wisconsin at Madison, who was not involved with the study. “Their findings and techniques are highly relevant to many other bacteria, and will greatly improve knowledge of how bacteria use this critical signaling molecule to mediate everything from surviving in the human gut to causing disease.”

Adds Laub: “We are still discovering new nucleotide-based signaling molecules in bacteria even today, and every single one of them could eventually be derivatized in a similar way to identify their binding partners.”

This research was supported by a fellowship from the Jane Coffin Childs Memorial Fund for Medical Research and a grant from the National Institutes of Health.

Uncovering the “must-haves” of tissue regeneration
Nicole Davis | Whitehead Institute
November 27, 2018

Cambridge, MA.  – The ability to regrow missing or damaged body parts is one of the great marvels of modern biology. In an effort to lay bare the biological underpinnings of this phenomenon, scientists at Whitehead Institute have begun to define the core features that are required for regeneration in flatworms. Their research, which appears online November 27 in Cell Reports, reveals that a set of cellular and molecular responses — previously thought to be essential for regeneration following amputations and other major injuries — is in fact dispensable.

“This is a real surprise,” said senior author Peter Reddien, a Member of Whitehead Institute, professor of biology at Massachusetts Institute of Technology, and investigator with the Howard Hughes Medical Institute. “These responses are broad, prominent attributes of tissue regeneration and repair and, a reasonable bet was that they function to bring about regeneration.”

About eight years ago, Reddien and his team described a set of biological activities that are triggered by injuries that remove tissue. Whereas a cut or a scrape removes little if any tissue, more damaging injuries, like amputations, cause significant tissue loss. That missing tissue must be regenerated to ensure the organism retains its proper anatomical proportions.

A series of cellular and molecular activities — known collectively as the missing tissue response — were believed to enable this regeneration to occur. They include the sustained action of genes that respond to injury, a period of intense cell division in areas surrounding the wound, and a general increase in cell death throughout the body. “This happens prominently, not only in planarians but also in other organisms capable of regeneration, so we suspected that the missing tissue response must play a very fundamental role in regeneration,” recalled Reddien.

What types of injuries require the missing tissue response for repair, and what is the function of the missing tissue response in regeneration? Graduate student and first author Aneesha Tewari, Reddien and colleagues, including Sarah Stern and Isaac Oderberg, set out to uncover the answers. This work forms the basis of their latest Cell Reports study.

The researchers harnessed an earlier discovery that a gene known as follistatin is required for the missing tissue response in flatworms (known as planarians). By using molecular tools to inhibit this gene, they could block the missing tissue response and observe what happens under various wound conditions, ranging from minimal (the removal of an eye, for example) to moderate (the removal of the pharynx or part of the head) to significant tissue loss (the removal of a complete side of the body). Remarkably, in every case, the missing tissue was regenerated, albeit much more slowly than it would be otherwise.

“These results tell us that what the missing tissue response is really doing is simply pushing the foot down on the gas pedal — basically accelerating the process of regeneration,” explained Reddien. “If you can’t accelerate, you’ll still get there, it just takes longer.”

Tewari, Reddien, and their colleagues also cracked a thorny mystery surrounding the missing tissue response. Although their results show that it is not required across a wide range of injuries, there is one lingering instance in which regeneration failed to occur when they blocked the missing tissue response: head amputation.

“This was a big puzzle,” said Tewari. “It left us wondering whether or not we could generalize our findings to all types of wounds — is there something special about the head that makes it uniquely dependent on the missing tissue response?”

The answer, it turns out, is no. When follistatin is blocked, a key signaling protein, called Wnt1, kicks into overdrive. And when that happens, the tissue destined to form the head does not receive the positional cues it needs to properly regrow, which means regeneration fails to proceed. But, when both the missing tissue response and Wnt1 are blocked, the head does indeed regenerate, the team uncovered.

Taken together, the researchers’ findings begin to clarify what is essential for regeneration to take place and what is not. “Our study greatly simplifies the picture of what it takes to regenerate,” said Reddien. “And that’s an important step along the path towards dissecting the central elements of regeneration in animals that do regenerate well, like flatworms, and then applying that knowledge to understand what the limits might be in those animals that don’t regenerate as well, like humans.”

This research was supported by the NIH (R01GM080639), the National Science Foundation, the Eleanor Schwartz Charitable Foundation, and the Howard Hughes Medical Institute.

***
Peter Reddien’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 Howard Hughes Medical Institute Investigator and a professor of biology at Massachusetts Institute of Technology.
***
Full citation:
Cell Reports,  Vol. 25, Is. 9, P2577-2590.E3, November 27, 2018, DOI:https://doi.org/10.1016/j.celrep.2018.11.004
“Cellular and molecular responses unique to major injury are dispensable for planarian regeneration”
Aneesha G. Tewari (1,2), Sarah R. Stern (1,2), Isaac M. Oderberg (1,2,4), and Peter W. Reddien (1,2,3)
 1.Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
4. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Plant characteristics shaped by parental conflict
Greta Friar | Whitehead Institute
November 19, 2018

CAMBRIDGE, Mass. – Different subpopulations of a plant species can have distinct traits, varying in size, seed count, coloration, and so on. The primary source of this variation is genes: different versions of a gene can lead to different traits. However, genes are not the only determinant of such traits, and researchers are learning more about another contributor: epigenetics. Epigenetic factors are things that regulate genes, altering their expression, and like genes they can be inherited from generation to generation, even though they are independent of the actual DNA sequences of the genes.

One epigenetic mechanism is DNA methylation, in which the addition of chemical tags called methyl groups can turn genes on or off. Genes that share the identical DNA sequence but have different patterns of methylation are called epialleles. Several studies have shown that epialleles, like different versions of genes, can cause differences in traits between plant subpopulations, or strains, but whether genetic factors are also at play can be difficult to determine.

The lab of Whitehead Member Mary Gehring, who is also an associate professor at Massachusetts Institute of Technology, has described evidence that epialleles alone can lead to different heritable traits in plants. In research published online November 5 in the journal PLoS Genetics, Gehring, along with co-first authors and former lab members Daniela Pignatta and Katherine Novitzky, showed that altering the methylation state of the gene HDG3 in different strains of the plant Arabidopsis thaliana was enough to cause changes in seed weight and in the timing of certain aspects of seed development.

In plants, methylation states of genes change most frequently during seed development, when genes are switched on or off to progress development of the organism. This period is also when a conflict of interest arises in the genome of each seed between the parts inherited from its mother and father. The mother plants produce seeds fertilized by different fathers at the same time. It’s in the mother’s interest to give an equal share of nutrients to each seed—to have many smaller seeds. But it’s in the father’s interest for its seed to get the most nutrients and grow larger. This conflict plays out through an epigenetic mechanism called imprinting, in which, through differential methylation between the father’s and mother’s copies of a gene, one parent’s copy is silenced in the offspring so that only the other parent’s version of the gene is expressed.

The gene HDG3 is imprinted in one strain of Arabidopsis so that only the father’s copy is expressed. Gehring and her team found that when the strain loses its paternal imprinting, the timing of seed development is affected and the plant ends up with smaller seeds. This is consistent with the theory of imprinting: When the father’s genes have the advantage, the seeds are larger than when both parents’ genes are equally expressed.

Other experiments tested the effect of either activating or silencing HDG3 by methylation in a variety of scenarios, both in a separate strain of Arabidopsis in which the gene starts off silenced, as well as in crosses between the two strains. The researchers found that altering the methylation state of the gene was sufficient to affect seed size and the timing of seed development. In the crosses, these traits depended on whether the paternal copy of the gene came from the strain in which HDG3 was normally silenced or the strain in which it was normally activated.

Altogether these experiments demonstrate a link between changes in methylation state and differences in seed development and size. This suggests that epialleles in natural populations function much like variations in genes, creating heritable traits that differ within the larger population.

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

***

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 variably imprinted epiallele impacts seed development”
PLoS Genetics, online November 5 2018, https://doi.org/10.1371/journal.pgen.1007469
Daniela Pignatta (1,3), Katherine Novitzky (1,3), P. R. V. Satyaki (1), Mary Gehring (1,2)
1. Whitehead Institute for Biomedical Research, Cambridge, MA, United States of America
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States of America
3. These authors contributed equally to this work.
Heart-healthy plant chemistry
Greta Friar | Whitehead Institute
October 29, 2018

Plants have been a rich source of medicines for thousands of years. Compounds such as artemisinin, for example, used to treat malaria, and morphine, a pain reliever, are mainstay therapeutics derived from plants. However, several roadblocks in plant chemistry research have prevented scientists from tapping into the full potential of plant-based medicinal compounds, thwarting drug discovery and development. Researchers typically screen for molecules of interest by breaking the plant into very small pieces, using biochemistry to test the activity of the pieces, and isolating the molecules responsible for the activity. It is often difficult, however, to pick the right compound responsible for a medicinal effect out of the plant mixture, or to identify the genes responsible for producing it.

The Chinese wolfberry plant (Lycium barbarum), also known as goji berry, has been used in traditional Chinese herbal medicine for millennia to treat symptoms such as high blood pressure. Researchers had identified small protein-like molecules called lyciumins, produced by the goji berry, as the source of its antihypertensive properties but little else was known about the molecules.

In research published online October 29 in the journal Proceedings of the National Academy of Sciences, Whitehead Institute Member Jing-Ke Weng and postdoctoral researcher Roland Kersten describe an approach to speed up the process of identifying plant chemistry that they used to investigate lyciumins. The approach capitalizes on the growing number of plants that have had their genomes sequenced. The wealth of genomic data available enabled Kersten to identify the gene that is associated with lyciumin production in goji berries by searching for a DNA sequence that matched the sequences of the lyciumins. Once Kersten found the matching precursor gene in goji berries he inserted it into a tobacco plant, which began producing lyciumins, confirming that he had found the right gene.

Kersten then hunted for lyciumin-producing genes in other plant genomes using a common feature of the genes that he had identified as a search query. He discovered more than one hundred unknown lyciumins in everything from potatoes to beets to soybeans.

Having sped up the gene discovery stage, Kersten used gene expression techniques to likewise speed up the molecule production stage. Being able to quickly produce large quantities of a drug candidate is necessary for testing and manufacturing the drug. Kersten edited the lyciumin precursor genes to make more copies of the molecule and then inserted the edited genes into the tobacco plant to mass produce lyciumins up to 40 times faster than the original plants. Kersten was also able to edit the lyciumins’ DNA sequences to alter the molecules’ structure, creating new varieties of lyciumins not found in nature. Together, these results allow for the future creation of a lyciumin library, a valuable repository for drug discovery research. Millions of different lyciumins can be grown in tobacco and tested for their efficacy as antihypertensive drugs or in other potential agrochemical and pharmaceutical applications.

Weng and Kersten’s approach leverages the recent explosion in plant genomics to uncover important medicinal compounds in plants and reveal the secrets of plants used in traditional global medicine for generations. For Kersten, the research was also an exciting demonstration of just how much undiscovered chemistry lies waiting to be tapped in even the best-studied crop plants.

This work was supported by grants from the Thome Foundation, the Pew Scholars Program in the Biomedical Sciences, the Searle Scholars Program, and the Family Larsson Rosenquist Foundation.

***
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.
***
Full citation:
“Gene-guided discovery and engineering of branched cyclic peptides in plants”
PNAS, online on October 29.
Roland D. Kersten (1), Jing-Ke Weng (1,2)
1. Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA, United States
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States
How Many Evolutionary Events Can It Take To Screw in Nature’s Lightbulb?

Firefly genomics reveals independent evolution of bioluminescence in beetles

Lisa Girard | Whitehead Institute
October 16, 2018

Cambridge, MA — Researchers at Whitehead Institute and collaborators from fourteen other institutions around the world have shed light on the evolutionary origins of luciferase, the key enzyme behind the glow of fireflies and other bioluminescent beetles. By sequencing the genomes of two American and Japanese firefly species that diverged approximately 100 million years ago, along with a more evolutionarily distant bioluminescent Caribbean click beetle, the team discovered that luciferase appears to have arisen independently in fireflies and click beetles. Examining the genes flanking that encoding the luciferase gene, suggests an evolutionary path along which the luciferase gene arose from duplications and divergences of CoA ligase genes involved in fat metabolism. As described online October 16 in the journal eLife, these findings provide fundamental insights into how enzymes can evolve, potentially inform strategies to help protect bioluminescent beetles from a shifting climate and habitat, and could extend the utility of luciferase, which has also been harnessed for biomedical and agricultural research, as a laboratory tool.

Throughout much of the world, the silent flash of a firefly on a warm evening can only mean one thing-Summer has arrived. But fireflies don’t just signal summer, their glow serves as a mating signal to other fireflies, and is even a warning that they are chemically defended, having a noxious taste capable of repelling the boldest of predators.

Belying its grandeur, the chemistry of firefly bioluminescence is relatively straightforward. Their light is produced by a specialized firefly enzyme, luciferase, that breaks down a molecule called luciferin, producing light in the process. Luciferase has become a mainstay tool in the laboratory. Scientists can fuse their gene of interest to luciferase and assay for gene expression by measuring the intensity of the glow after luciferin is added.

Beyond fireflies, there are other bioluminescent beetles (despite their name, fireflies are actually beetles), including certain tropical click-beetles. Perplexingly, these diverse bioluminescent beetles use very similar luciferase enzymes and luciferin molecules, but have an unrelated anatomy of their light-producing organs (also known as lanterns), making it unclear if their bioluminescence evolved from a common luminous ancestor, or if their special glow evolved independently.

Since fireflies and bioluminescent click-beetles are not model organisms like mice or fruit flies for which there is a wealth of genetic information, Jing-Ke Weng, Whitehead Institute Member and assistant professor of biology at Massachusetts Institute of Technology (MIT), along with a  graduate student in Weng’s lab, Tim Fallon, and Cornell postdoctoral researcher, Sarah Lower, began their investigations by sequencing the genome of the American Big Dipper firefly, Photinus pyralis. Named for its distinctive swooping “J” flash , this common inhabitant of meadows and suburban lawns has been called the “All-American firefly”. Due to its abundance and ease of identification, it was also the firefly of choice for scientific study, and is the species from which luciferin and luciferase were first characterized. Wanting to start their work quickly and make their progress and data available to others in the firefly community, Whitehead Institute researchers and collaborators crowdsourced funds to sequence the Big Dipper firefly.

The Big Dipper genome sequence, they discovered, revealed interesting insights into the origin of the luciferase gene. Examining the genes flanking that encoding luciferase, they found a cluster, or tandem repeat, of fatty acid CoA ligase genes with the luciferase gene sitting in the middle of this cluster. Sequence similarity and proximity between the luciferase and fatty acid CoA ligase genes suggested an evolutionary path along which the luciferase gene was produced from tandem duplication and divergence of an ancestral fatty acid CoA ligase gene.

“When the luciferase gene was cloned, people knew it was similar to the fatty acid CoA ligase gene in sequence, and hypothesized that it must be related to that ancestry. But what we uncovered from the luciferase gene locus is a tandem repeat of five genes, four are still the fatty acid CoA ligases, but then luciferase evolved right in the middle we believe from divergence of one of these duplications,” says Weng.

The Big Dipper sequencing provided important insights into the origin of luciferase and additional factors involved in bioluminescence, but in order to gain additional insights into the evolution of bioluminescence, the researchers set out to sequence two additional species that they hoped would provide the additional context to help them triangulate on some answers.

The bioluminescent click beetle, Ignelater luminosus, is related to the firefly, but on another branch of the tree of life entirely. Instead of producing light at its tail, it has two lanterns behind its head.

“We thought that sequencing the click beetle would provide insights into the evolution of bioluminescence as well as perhaps into how these animals could acquire very similar traits in terms of their biochemistry, but not in terms of their development,” says Fallon.

The third species they selected to sequence was a Japanese aquatic firefly (Aquatica lateralis), known in Japan as the Heike firefly.  Heike and the Big Dipper diverged from one another over 100 million years ago (to give you a sense of how far this is, it is older than the evolutionary distance between humans and rodents).

The researchers analyzed genomic data from the Japanese aquatic firefly and saw a similar arrangement around the luciferase gene locus as they had in the Big Dipper genome, suggesting that luciferase arose from a common ancestral event in both firefly species. The structure around the luciferase locus in the click beetle, however, was entirely absent, suggesting that luciferase arose through a different event.  Taken together, by sequencing and analyzing data from the genomes of two firefly species that diverged approximately 100 million years ago, along with a more evolutionarily distant bioluminescent click beetle, the team discovered that luciferase appears to have evolved independently in both fireflies and click beetles.

“Having the genome allowed us to understand how the evolution of luciferase happened. Before sequencing, we knew there were five genes cloned, including luciferase, in firefly. By sequencing the genomes we actually uncovered those genomic loci where those initial gene duplication events occured,” says Lower.

In addition to the origins of luciferase, these findings also provided the researchers with insights into the evolution of the light organs.

“Since our findings suggest that luciferase originated independently in both lineages, we can infer that anything that came after luciferase, for example the light organs, or other things dependent on luciferase should also be independent,” says Fallon.

Discovering how bioluminescence arose, as well as other complex traits, can be studied now that genomic information is available. The information can also inform strategies to protect fireflies, whose populations in many parts of the world are diminishing. In addition to adding tools to help reveal a constituent parts list that could allow researchers to optimize bioluminescence as a tool, these findings reveal important insights into the evolution of bioluminescence as well as genomic evolution more broadly.

“Luciferase is a perfect example of how to build a new enzyme, duplication of a related progenitor gene followed by mutation and selection,” says Weng. “And one of the most exciting parts of this study was that by examining the evolutionary scars in the genomes we studied we could actually see it happen.”

* * *
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.
* * *
Full citation:
“Firefly genomes illuminate parallel origins of bioluminescence in beetles”
eLife, online on October 9, 2018. doi: 10.7554/eLife.36495
Timothy R. Fallon (1,2,*), Sarah E. Lower (3,*), Ching-Ho Chang (4) , Manabu Bessho-Uehara (5,6), Gavin J. Martin (7), Adam J. Bewick (8) , Megan Behringer (9) , Humberto J. Debat (10), Isaac Wong (4) , John C. Day (11), Anton Suvorov (7) , Christian J. Silva (4,12), Kathrin F. Stanger-Hall1 (3), David W. Hall (8) , Robert J. Schmitz (8), David R. Nelson (14), Sara M. Lewis (15), Shuji Shigenobu (16), Seth M. Bybee (7) , Amanda M. Larracuente (4), Yuichi Oba (5), and Jing-Ke Weng (1,2)
1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
2.Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
3. Department of Molecular Biology & Genetics, Cornell University, Ithaca, New York 14850, USA.
4. Department of Biology, University of Rochester, Rochester, New York 14627, USA.
5. Department of Environmental Biology, Chubu University, Kasugai, Aichi 487-8501, Japan.
6. Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan.
7. Department of Biology, Brigham Young University, Provo, Utah 84602, USA.
8. Department of Genetics, University of Georgia, Athens, Georgia 30602, USA.
9. Biodesign Center for Mechanisms of Evolution, Arizona State University, Tempe, Arizona 85287, USA.
10. Center of Agronomic Research National Institute of Agricultural Technology, Córdoba, Argentina.
11. Centre for Ecology and Hydrology (CEH) Wallingford, Wallingford, Oxfordshire, UK.
12. Department of Plant Sciences, University of California Davis, Davis, California, USA.
13. Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA.
14. Department of Microbiology Immunology and Biochemistry, University of Tennessee  HSC, Memphis 38163, USA.
15. Department of Biology, Tufts University, Medford, Massachusetts 02155, USA.
16. NIBB Core Research Facilities, National Institute for Basic Biology, Okazaki 444-8585, Japan.
Detangling DNA replication

Researchers identify an essential protein that helps enzymes relax overtwisted DNA so each strand can be copied during cell division.

Raleigh McElvery | Department of Biology
September 18, 2018

DNA is a lengthy molecule — approximately 1,000-fold longer than the cell in which it resides — so it can’t be jammed in haphazardly. Rather, it must be neatly organized so proteins involved in critical processes can access the information contained in its nucleotide bases. Think of the double helix like a pair of shoe laces twisted together, coiled upon themselves again and again to make the molecule even more compact.

However, when it comes time for cell division, this supercoiled nature makes it difficult for proteins involved in DNA replication to access the strands, separate them, and copy them so one DNA molecule can become two.

Replication begins at specific regions of the chromosome where specialized proteins separate the two strands, pulling apart the double helix as you would the two shoe laces. However, this local separation actually tangles the rest of the molecule further, and without intervention creates a buildup of tension, stalling replication. Enter the enzymes known as topoisomerases, which travel ahead of the strands as they are being peeled apart, snipping them, untwisting them, and then rejoining them to relieve the tension that arises from supercoiling.

These topoisomerases are generally thought to be sufficient to allow replication to proceed. However, a team of researchers from MIT and the Duke University School of Medicine suggests the enzymes may require guidance from additional proteins, which recognize the shape characteristic of overtwisted DNA.

“We’ve known for a long time that topoisomerases are necessary for replication, but it’s never been clear if they were sufficient on their own,” says Michael Laub, an MIT professor of biology, Howard Hughes Medical Institute Investigator, and senior author of the study. “This is the first paper to identify a protein in bacteria, or eukaryotes, that is required to localize topoisomerases ahead of replication forks and to help them do what they need to do there.”

Postdoc Monica Guo ’07 and former graduate student Diane Haakonsen PhD ’16 are co-first authors of the study, which appeared online in the journal Cell on Sept. 13.

Necessary but not sufficient

Although it’s well established that topoisomerases are crucial to DNA replication, it has now becoming clear that we know relatively little about the mechanisms regulating their activity, including where and when they act to relieve supercoiling.

These enzymes fall into two groups, type I and type II, depending on how many strands of DNA they cut. The researchers focused on type II topoisomerases found in a common species of freshwater bacteria, Caulobacter crescentus. Type II topoisomerases in bacteria are of particular interest because a number of antibiotics target them in order to prevent DNA replication, treating a wide variety of microbial infections, including tuberculosis. Without topoisomerases, the bacteria can’t grow. Since these bacterial enzymes are unique, poisons directed at them won’t harm human topoisomerases.

For a long time, type II topoisomerases were generally assumed adequate on their own to manage the overtwisted supercoils that arise during replication. Although researchers working in E. coli and other, higher organisms have pinpointed additional proteins that can activate or repress these enzymes, none of these proteins were required for replication.

Such findings hinted that there might be similar interactions taking place in other organisms. In order to understand the protein factors involved in compacting Caulobacter DNA — regulating topoisomerase activity specifically — the researchers screened their bacteria for proteins that bound tightly to supercoiled DNA. From there, they honed in on one protein, GapR, which they observed was essential for DNA replication. In bacteria missing GapR, the DNA became overtwisted, replication slowed, and the bacteria eventually died.

Surprisingly, the researchers found that GapR recognized the structure of overtwisted DNA rather than specific nucleotide sequences.

“The vast majority of DNA-binding proteins localize to specific locations of the genome by recognizing a specific set of bases,” Laub says. “But GapR basically pays no attention to the actual underlying sequence — just the shape of overtwisted DNA, which uniquely arises in front of replication forks and transcription machinery.”

The crystal structure of the protein bound to DNA, solved by Duke’s Maria Schumacher, revealed that GapR recognizes the backbone of DNA (rather than the bases), forming a snug clamp that encircles the overtwisted DNA. However, when the DNA is relaxed in its standard form, it no longer fits inside the clamp. This might signify that GapR sits on DNA only at positions where topoisomerase is needed.

An exciting milestone

Although GapR appears to be required for DNA replication, it’s still not clear precisely how this protein promotes topoisomerase function to relieve supercoiling.

“In the absence of any other proteins, GapR is able to help type II topoisomerases remove positive supercoils faster, but we still don’t quite know how,” Guo says. “One idea is that GapR interacts with topoisomerases, recognizing the overtwisted DNA and recruiting the topoisomerases. Another possibility is that GapR is essentially grabbing onto the DNA and limiting the movement of the positive supercoils, so topoisomerases can target and eliminate them more quickly.”

Anthony Maxwell, a professor of biological chemistry at the John Innes Centre who was not involved with the study, says the buildup of DNA supercoils is a key problem in both bacterial replication and transcription.

“Identifying GapR and its potential role in controlling supercoiling in vivo is an exciting milestone in understanding the control of DNA topology in bacteria,” he says. “Further work will be required to show how exactly these proteins cooperate to maintain bacterial genomic integrity.”

According to Guo, the study provides insight into a fundamental process — DNA replication — and the ways topoisomerases are regulated, which could extend to eukaryotes.

“This was the first demonstration that a topoisomerase activator is required for DNA replication,” she says. “Although there’s no GapR homolog in higher organisms, there could be similar proteins that recognize the shape of the DNA and aid or position topoisomerases.”

This could open up a new field of drug research, she says, targeting activators like GapR to increase the efficacy of existing topoisomerase poisons to treat conditions like respiratory and urinary tract infections. After all, many topoisomerase inhibitors have become less effective due to antibiotic resistance. But only time will tell; there is still much to learn in order to untangle the complex process of DNA replication, along with its many twists and turns.

The research was funded by NIH grants, the HHMI International Predoctoral Fellowship, and the Jane Coffin Childs Memorial Fellowship.

Jarrett Smith receives Hanna Gray Fellowship from HHMI
Greta Friar | Whitehead Institute
September 12, 2018

Cambridge, Mass — Jarrett Smith, postdoctoral researcher in David Bartel’s lab at the Whitehead Institute, has been announced as a recipient of the Howard Hughes Medical Institute (HHMI)’s 2018 Hanna Gray fellowship. The fellowship supports outstanding early career scientists from groups underrepresented in the life sciences. Each of this year’s fifteen awardees will be given up to $1.4 million dollars in funding over the course of their postdoctoral program and beginning of a tenure-track faculty position.

“This program will help us retain the most diverse talent in science,” said HHMI President Erin O’Shea. “We feel it’s critically important in academia to have exceptional people from all walks of life, all cultures, and all backgrounds – people who can inspire the next generation of scientists.”

For Smith, who began his postdoctoral training in the Bartel lab in January, finding out he got the fellowship was a defining moment.

“I’m grateful for the support that the fellowship will provide during the formative years of my career,” Smith says. “This kind of opportunity gives you the confidence to set ambitious research goals and find out what you can accomplish.”

In the Bartel lab, Smith studies how cells respond to stress. When a cell is exposed to environmental stressors such as heat, UV radiation, or viral infection, proteins and RNAs in the cell may clump together into dense aggregates called stress granules. Several diseases are associated with altered stress granule formation, but the exact function of stress granules and their potential role in disease are unknown. Smith is investigating changes in the cell linked to their formation. His findings could shed light on a potential role for stress granules in cancer, viral infection, and neurodegenerative disease.

Growing up, Smith was always interested in science but no one in his family had ever received a PhD, making biology research feel like an unlikely career path for him. Nevertheless, he followed his passion, which led him to a PhD program at the Johns Hopkins University School of Medicine. Despite his strong academic performance, Smith began graduate school with doubts about his ability to become a scientist. His mentors were incredible teachers but their self-assuredness could be intimidating.

“They were absolutely my role models, but I didn’t think of them as having gone through what I was going through. In the first few years, I felt like I had a lot of catching up to do,” Smith said.

Smith says he was frequently inspired and guided by his graduate school mentor, Geraldine Seydoux. Under her tutorship he became more confident in his abilities.

“I try to pick mentors who are the kind of scientist I aspire to be,” Smith said.

With that tenet in mind, he set his sights on David Bartel’s lab for his postdoctoral research. He had heard that Bartel was a great mentor and knew the Bartel lab had expertise in all of the research techniques he wanted to learn. Since arriving at Whitehead Institute, Smith says he has experienced support not only from Bartel, but from the entire lab as well.

“Jarrett’s graduate experience with P granules in nematodes brings much appreciated expertise to our lab, and we are all excited about what he will discover here on stress-granule function,” Bartel says. “Receiving this fellowship is a well-deserved honor, and I am very happy for him.”

Smith noted that he is deeply grateful for the community he’s found at Whitehead Institute. However, he also noted that throughout his scientific career he has typically been the only black person in the room. One of the joys of applying for the fellowship was meeting the rest of the candidates, a diverse and impressive group of scientists, he says. He looks forward to seeing the other fellows again at meetings hosted by the HHMI.

“I’ve never really had a scientific role model that shared those experiences or that I could identify with in that way,” Smith says, but he hopes that future aspiring scientists won’t have to go through the same experience. His brother-in-law recently began an undergraduate major in biology. Smith enjoys being there to answer his questions about school work or life as a researcher.

“I’d never ask him if he thinks of me as a role model,” Smith says, laughing. “But I’m glad that I have the chance to help people who—like I did—might question whether they could be successful in the sciences.” With the support of the fellowship and his lab, and an exciting research question he is eager to tackle, Smith has never been more certain that he belongs right where he is.

Tissue architecture affects chromosome segregation

Biologists discover that the environment surrounding a cell plays an integral role in its ability to accurately segregate its chromosomes.

Ashley Junger | Koch Institute
August 24, 2018

All growth and reproduction relies on a cell’s ability to replicate its chromosomes and produce accurate copies of itself. Every step of this process takes place within that cell.

Based on this observation, scientists have studied the replication and segregation of chromosomes as a phenomenon exclusively internal to the cell. They traditionally rely on warm nutritional cultures that promote growth but bear little resemblance to the cell’s external surroundings while in its natural environment.

New research by a group of MIT biologists reveals that this long-held assumption is incorrect. In a paper published this week, they describe how some types of cells rely on signals from surrounding tissue in order to maintain chromosome stability and segregate accurately.

Kristin Knouse, a fellow at the Whitehead Institute, is the lead author of the paper, which was published online in the journal Cell on Aug. 23. Angelika Amon, the Kathleen and Curtis Marble Professor in Cancer Research in the Department of Biology and a member of the Koch Institute for Integrative Cancer Research, is the senior author.

“The main takeaway from this paper is that we must study cells in their native tissues to really understand their biology,” Amon says. “Results obtained from cell lines that have evolved to divide on plastic dishes do not paint the whole picture.”

When cells replicate, the newly duplicated chromosomes line up within the cell and cellular structures pull one copy to each side. The cell then divides down the middle, separating one copy of each chromosome into each new daughter cell.

At least, that’s how it’s supposed to work. In reality, there are sometimes errors in the process of separating chromosomes into daughter cells, known as chromosome mis-segregation. Some errors simply result in damage to the DNA. Other errors can result in the chromosomes being unevenly divided between daughter cells, a condition called aneuploidy.

These errors are almost always harmful to cell development and can be fatal. In developing embryos, aneuploidy can cause miscarriages or developmental disorders such as Down syndrome. In adults, chromosome instability is seen in a large number of cancers.

To study these errors, scientists have historically removed cells from their surrounding tissue and placed them into easily controlled plastic cultures.

“Chromosome segregation has been studied in a dish for decades,” Knouse says. “I think the assumption was … a cell would segregate chromosomes the same way in a dish as it would in a tissue because everything was happening inside the cell.”

However, in previous work, Knouse had found that reported rates for aneuploidy in cells grown in cultures was much higher than the rates she found in cells that had grown within their native tissue. This prompted her and her colleagues to investigate whether the surroundings of a cell influence the accuracy with which that cell divided.

To answer this question, they compared mis-segregation rates between five different cell types in native and non-native environments.

But not all cells’ native environments are the same. Some cells, like those that form skin, grow in a very structured context, where they always have neighbors and defined directions for growth. Other cells, however, like cells in the blood, have greater independence, with little interaction with the surrounding tissue.

In the new study, the researchers observed that cells that grew in structured environments in their native tissues divided accurately within those tissues. But once they were placed into a dish, the frequency of chromosome mis-segregation drastically increased. The cells that were less tied to structures in their tissue were not affected by the lack of architecture in culture dishes.

The researchers found that maintaining the architectural conditions of the cell’s native environment is essential for chromosome stability. Cells removed from the context of their tissue don’t always faithfully represent natural processes.

The researchers determined that architecture didn’t have an obvious effect on the expression of known genes involved in segregation. The disruption in tissue architecture likely causes mechanical changes that disrupt segregation, in a manner that is independent of mutations or gene expression changes.

“It was surprising to us that for something so intrinsic to the cell — something that’s happening entirely within the cell and so fundamental to the cell’s existence — where that cell is sitting actually matters quite a bit,” Knouse says.

Through the Cancer Genome Project, scientists learned that despite high rates of chromosome mis-segregation, many cancers lack any mutations to the cellular machinery that controls chromosome partitioning. This left scientists searching for the cause of the increase of these division errors. This study suggests that tissue architecture could be the culprit.

Cancer development often involves disruption of tissue architecture, whether during tumor growth or metastasis. This disruption of the extracellular environment could trigger chromosome segregation errors in the cells within the tumor.

“I think [this paper] really could be the explanation for why certain kinds of cancers become chromosomally unstable,” says Iain Cheeseman, a professor of biology at MIT and a member of the Whitehead Institute, who was not involved in the study.

The results point not only to a new understanding of the cellular mechanical triggers and effects of cancers, but also to a new understanding of how cell biology must be studied.

“Clearly a two-dimensional culture system does not faithfully recapitulate even the most fundamental processes, like chromosome segregation,” Knouse says. “As cell biologists we really must start recognizing that context matters.”

This work was supported by the National Institutes of Health, the Kathy and Curt Marble Cancer Research Fund, and the Koch Institute Support (core) Grant from the National Cancer Institute.