Research Area: Stem Cell and Developmental Biology
New research from the Yamashita Lab indicates that in male fruit flies, asymmetrical division in germline stem cells is crucial for maintaining ribosomal DNA in the stem cell pool.
Greta Friar | Whitehead Institute
November 13, 2023
Germline stem cells are the pool of stem cells capable of becoming eggs or sperm. They divide asymmetrically, such that one of the cells resulting from a division is another stem cell and the other is a differentiated cell, which has progressed one step further down the path towards becoming an egg or sperm. Researchers have thought that this asymmetrical division served to replenish the pool of stem cells—making sperm or eggs, but also making more stem cells to produce future sperm or eggs. However, the germline has another way to replenish itself: cells that have differentiated only one or two steps down the path to becoming eggs or sperm are capable of reverting into stem cells. Why, then, do stem cells divide asymmetrically?
New research from Whitehead Institute Member Yukiko Yamashita, who is also a professor of biology at the Massachusetts Institute of Technology and an HHMI Investigator, and former postdoc in her lab Jonathan Nelson shows that asymmetrical division in germline stem cells serves a different but equally important purpose in male fruit flies (Drosophila melanogaster), a common model animal for germline research. The work, published in the journal Proceedings of the National Academy of Sciences (PNAS) on November 13, suggests that in flies, germline stem cells divide asymmetrically in order to unequally split a certain kind of DNA, called ribosomal DNA (rDNA), between the two dividing cells and then keep the cell with more rDNA in the stem cell pool. This is necessary in order to keep the germline viable over generations of cell divisions, and so to keep individual flies fertile and capable of reproduction. The researchers show that only germline stem cells, and not other types of germ cells, drive this process, and explain why stem cells’ asymmetric divisions make them uniquely suited to maintaining rDNA.
Cells must maintain rDNA to be viable and fertile
Ribosomal DNA is critical to maintain in the germline because it contains the instructions for making a major part of ribosomes, the cellular machines that build proteins from genetic instructions. Proteins are the main workhorses of the cell, and so cells need to make many ribosomes in order to build all of the proteins that they need. Consequently, rDNA exists as many copies repeated in a row of the code for components of the ribosome. All of these repeats make it easy for the cell to mass produce ribosomes, but they also come with a risk: repetitive DNA is prone to losing repeats during cell division. When the cell’s rDNA is copied, it’s easy for a few of the many identical repeats to get cut out, so that the resulting copy of the genome has fewer rDNA repeats than the original.
Most cells can afford to lose a few rDNA repeats without too many negative effects, but the germline cannot. Whereas other cells die with the body they are in, germ cells produce eggs and sperm that will form a new body, which produces new germ cells, and so on. The germ cell lineage is effectively immortal. Over the course of its endless cycle of cell division, the loss of rDNA repeats would add up until the cells became dysfunctional and then died. This would make the individual bearing those germ cells infertile, and so cause their lineage to go extinct.
A role for a “selfish” parasite in maintaining rDNA
Researchers have known that germ cells have some way to regain rDNA repeats when the number gets too low—if germ cells couldn’t do this, none of us would exist—but the details of how cells achieve this have been largely mysterious. One proposed model was that when a germ cell divides, sometimes it might divide up its rDNA unequally between the two resulting cells, so that one cell would gain rDNA repeats. Yamashita and Nelson have previously found evidence that this model is correct, and they discovered some of the specific mechanisms that enable it to happen. In a 2023 PNAS paper, the researchers showed that a retrotransposon, a “selfish” genetic element whose function is to make more copies of itself, actually helps germ cells maintain rDNA. During cell division, the retrotransposon R2 slices open one copy of the chromosome containing rDNA in its quest to insert extra copies of itself into the genome. The cell tries to repair the break using the copy on the other intact chromosome, but the tricky nature of repetitive DNA can cause the cell to lose its place, so that it stitches a stretch of rDNA repeats from one copy of the chromosome into the other copy instead.
Through this process, the germline can boost the level of rDNA in a cell—but only by as much as another cell loses. How does this win-lose exchange lead to an overall increase in rDNA levels across the germline cell population to compensate for lost rDNA? In this latest work, Yamashita and Nelson show through mathematical modeling that in cells that divide symmetrically, it would not. Gains and losses in rDNA through this form of exchange would occur essentially at random and cancel each other out over time.
In praise of asymmetry
Now consider an asymmetric division. After a germline stem cell divides, the cell that differentiates will go through a few more divisions and ultimately create a specific number of sperm cells–the number happens to be sixty-four. If this daughter cell gets the chromosome with more rDNA repeats, then that would lead to sixty-four sperm with more rDNA repeats—but that would be it, as the sperm have exited the pool of replicating germline stem cells.
However, the daughter cell that remains a germline stem cell will divide again to create a differentiated cell (which will become sixty-four sperm) and another stem cell, which will divide again, leading to another sixty-four sperm and another stem cell—and so on. All of these cells, including many sperm, would inherit the higher number of rDNA repeats. Furthermore, at each division, there would be an opportunity for another unequal split of rDNA. As long as the stem cell always gets the boost in rDNA, then the cumulative number of rDNA repeats would keep growing in the overall population over time—and Yamashita and colleagues’ past work shows that the germline can ensure this. A 2022 Science Advances paper from Yamashita and then-postdoc in her lab George Watase showed that when a germline stem cell divides, the DNA strand with more rDNA repeats is tagged with a protein that the researchers named Indra, which helps mark it to stay in the daughter cell that will become another stem cell. Yamashita and Nelson’s new paper includes mathematical modeling by second author Tomohiro Kumon, a postdoc in Yamashita’s lab, that proves that this is not only sufficient to restore the level of rDNA repeats over time, but that it is the most effective and efficient way for the germline to do so.
“There was this problem with the unequal exchange model of rescuing rDNA, because every cell that gained rDNA did so at the expense of another that was losing it,” Nelson says. “What we show here is that the reason why there’s a bias towards gain in the germline is because this process is happening within these asymmetrically dividing germline stem cells that can gain and gain and gain, while the cells that lose rDNA exit the cycle and so have a limited effect.”
The researchers complemented their mathematical modeling with evidence that the process to increase rDNA repeats occurs primarily or solely in germline stem cells. They found that when the number of rDNA repeats got low enough, then expression of R2 and the presence of double-stranded DNA breaks both increased in germline stem cells, but not significantly in other germ cell types.
Yamashita and Nelson propose that the different cell types in the germline take on different functions to create a pipeline for maximizing the health of future sperm. Germ cells that are one or two steps down the path of differentiation from stem cells are essentially identical to them, to the point that they can be difficult to tell apart in testing, but they divide symmetrically. They are also much more sensitive to DNA damage; the researchers found that R2 exposure kills these cells.
Germline stem cells, with their asymmetrical division and ability to tolerate R2 expression, serve to restore rDNA levels when they get too low. Then the differentiated germ cells serve to weed out mutations—including those introduced during R2 expression in the earlier stem cell stage—by killing off cells with DNA damage. The different strengths of the different types of germ cells creates an effective pipeline to produce the largest number of sperm cells with high rDNA repeat number and low DNA damage.
Eventually, this new understanding of the details of how cells maintain their rDNA could lead to medical therapies. For example, cancer cells are, like germ cells, an essentially immortal cell line, and so must have a way to maintain their rDNA. If researchers could someday find a way to prevent them from doing so, that could be a good treatment strategy. The work also may have implications for research on aging, as rDNA decreases with age in other cell types. In the meantime, Yamashita and Nelson are excited to have solved several long-standing mysteries in their field, including how germ cells can restore rDNA at a population level when each division creates an equal loss and gain of rDNA, and why germline stem cells divide asymmetrically.
“Typically, when you publish a paper, you feel like you’ve fit two puzzle pieces together, but in this case, I feel like we fit a bunch of puzzle pieces together,” Yamashita says. “It’s been immensely satisfying to find answers to multiple questions and see how they all fit together to explain the mechanisms of this process that’s necessary for germline immortality.”
Omer Yilmaz’s work on how diet influences intestinal stem cells could lead to new ways to treat or prevent gastrointestinal cancers.
Anne Trafton | MIT News Office
May 25, 2023
Every three to five days, all of the cells lining the human intestine are replaced. That constant replenishment of cells helps the intestinal lining withstand the damage caused by food passing through the digestive tract.
This rapid turnover of cells relies on intestinal stem cells, which give rise to all of the other types of cells found in the intestine. Recent research has shown that those stem cells are heavily influenced by diet, which can help keep them healthy or stimulate them to become cancerous.
“Low-calorie diets such as fasting and caloric restriction can have antiaging effects and antitumor effects, and we want to understand why that is. On the other hand, diets that lead to obesity can promote diseases of aging, such as cancer,” says Omer Yilmaz, the Eisen and Chang Career Development Associate Professor of Biology at MIT.
For the past decade, Yilmaz has been studying how different diets and environmental conditions affect intestinal stem cells, and how those factors can increase the risk of cancer and other diseases. This work could help researchers develop new ways to improve gastrointestinal health, either through dietary interventions or drugs that mimic the beneficial effects of certain diets, he says.
“Our findings have raised the possibility that fasting interventions, or small molecules that mimic the effects of fasting, might have a role in improving intestinal regeneration,” says Yilmaz, who is also a member of MIT’s Koch Institute for Integrative Cancer Research.
A clinical approach
Yilmaz’s interest in disease and medicine arose at an early age. His father practiced internal medicine, and Yilmaz spent a great deal of time at his father’s office after school, or tagging along at the hospital where his father saw patients.
“I was very interested in medicines and how medicines were used to treat diseases,” Yilmaz recalls. “He’d ask me questions, and many times I wouldn’t know the answer, but he would encourage me to figure out the answers to his questions. That really stimulated my interest in biology and in wanting to become a doctor.”
Knowing that he wanted to go into medicine, Yilmaz applied and was accepted to an eight-year, combined bachelor’s and MD program at the University of Michigan. As an undergraduate, this gave him the freedom to explore areas of interest without worrying about applying to medical school. While majoring in biochemistry and physics, he did undergraduate research in the field of protein folding.
During his first year of medical school, Yilmaz realized that he missed doing research, so he decided to apply to the MD/PhD program at the University of Michigan. For his PhD research, he studied blood-forming stem cells and identified new markers that allowed such cells to be more easily isolated from the bone marrow.
“This was important because there’s a lot of interest in understanding what makes a stem cell a stem cell, and how much of it is an internal program versus signals from the microenvironment,” Yilmaz says.
After finishing his PhD and MD, he thought about going straight into research and skipping a medical residency, but ended up doing a residency in pathology at Massachusetts General Hospital. During that time, he decided to switch his research focus from blood-forming stem cells to stem cells found in the gastrointestinal tract.
“The GI tract seemed very interesting because in contrast to the bone marrow, we knew very little about the identity of GI stem cells,” Yilmaz says. “I knew that once GI stem cells were identified, there’d be a lot of interesting questions about how they respond to diet and how they respond to other environmental stimuli.”
Dietary questions
To delve into those questions, Yilmaz did postdoctoral research at the Whitehead Institute, where he began investigating the connections between stem cells, metabolism, diet, and cancer.
Because intestinal stem cells are so long-lived, they are more likely to accumulate genetic mutations that make them susceptible to becoming cancerous. At the Whitehead Institute, Yilmaz began studying how different diets might influence this vulnerability to cancer, a topic that he carried into his lab at MIT when he joined the faculty in 2014.
One question his lab has been exploring is why low-calorie diets often have protective effects, including a boost in longevity — a phenomenon that has been seen in many studies in animals and humans.
In a 2018 study, his lab found that a 24-hour fast dramatically improves stem cells’ ability to regenerate. This effect was seen in both young and aged mice, suggesting that even in old age, fasting or drugs that mimic the effects of fasting could have a beneficial effect.
On the flip side, Yilmaz is also interested in why a high-fat diet appears to promote the development of cancer, especially colorectal cancer. In a 2016 study, he found that when mice consume a high-fat diet, it triggers a significant increase in the number of intestinal stem cells. Also, some non-stem-cell populations begin to resemble stem cells in their behavior. “The upshot of these changes is that both stem cells and non-stem-cells can give rise to tumors in a high-fat diet state,” Yilmaz says.
To help with these studies, Yilmaz’s lab has developed a way to use mouse or human intestinal stem cells to generate miniature intestines or colons in cell culture. These “organoids” can then be exposed to different nutrients in a very controlled setting, allowing researchers to analyze how different diets affect the system.
Recently, his lab adapted the system to allow them to expand their studies to include the role of immune cells, fibroblasts, and other supportive cells found in the microenvironment of stem cells. “It would be remiss of us to focus on just one cell type,” Yilmaz says. “We’re looking at how these different dietary interventions impact the entire stem cell neighborhood.”
While Yilmaz spends most of his time running his lab at MIT, he also devotes six to eight weeks per year to his work at MGH, where he is an associate pathologist focusing on gastrointestinal pathology.
“I enjoy my clinical work, and it always reminds me about the importance of the research we do,” he says. “Seeing colon cancer and other GI cancers under the microscope, and seeing their complexity, reminds me of the importance of our mission to figure out how we can prevent these cancers from forming.”
Biology Professor and Whitehead Institute Member Siniša Hrvatin has been named as one of the 15 researchers to be selected as 2023 Searle Scholars. The Searle Scholars Program supports the research of exceptional young faculty in the biomedical sciences and chemistry.
Merrill Meadow | Whitehead Institute
May 12, 2023
Whitehead Institute Member Siniša Hrvatin has been named as one of the 15 researchers to be selected as 2023 Searle Scholars. The Searle Scholars Program supports the research of exceptional young faculty in the biomedical sciences and chemistry.
Chosen by an advisory board of eminent scientists, Searle Scholars are considered among the most creative researchers pursuing careers in academic research. Their investigations address challenging research questions and can lead to new insights that fundamentally change their fields—and to opportunities for translating discoveries into new therapeutics and diagnostics.
“I am truly grateful for the support of the Searle Scholar Program as we embark on this ambitious project,” says Hrvatin, who joined the Institute in 2021 and is also an assistant professor of biology at Massachusetts Institute of Technology. The three-year grant accompanying the award will support his work developing a new animal model for the study of hibernation.
“The ability to maintain nearly constant body temperature is a defining feature of mammalian and avian evolution; but, when challenged by harsh environments, many species decrease body temperature and metabolic rate and initiate energy-conserving states of torpor and hibernation,” Hrvatin notes. “Science has not yet answered the fundamental questions of how mammals initiate, regulate, and survive these extraordinary hypometabolic and hypothermic states.
“However, those answers could have profound medical applications,” he explains. “For example, harnessing the mechanisms behind hibernation might provide new approaches to protect neurons from ischemic injury and to preserve tissues and organs for transplantation.”
In the Searle-supported study, Hrvatin aims to discover a control center in the brain that regulates distinct stages of hibernation in the Syrian hamster. His lab will start by identifying the brain regions active during the deep torpor stage of hibernation and, using molecular profiling techniques, will then identify the specific neuronal populations and molecular pathways involved. Finally, the team will develop new tools to determine specific activities in those neural populations that are necessary for natural hibernation—and that may be sufficient to induce a synthetic state of hibernation.
“Taken together,” Hrvatin says, “I believe that our discoveries and the tools we build will help establish the first controllable animal model of hibernation.”
Since 1981, 677 scientists have been named Searle Scholars and the Program has awarded more than $152 million in support for Scholars’ research. To date, 85 Searle Scholars have been inducted into the National Academy of Sciences, 20 have been recognized with a MacArthur Fellowship, and two have been awarded the Nobel Prize for Chemistry.
Developing a new neuroscience model is no small feat. New faculty member Brady Weissbourd has risen to the challenge in order to study nervous system evolution, development, regeneration, and function.
Lillian Eden | Department of Biology
April 26, 2023
How does animal behavior emerge from networks of connected neurons? How are these incredible nervous systems and behaviors actually generated by evolution? Are there principles shared by all nervous systems or is evolution constantly innovating? What did the first nervous system look like that gave rise to the incredible diversity of life that we see around us?
Combining the study of animal behavior with studies of nervous system form, function, and evolution, Brady Weissbourd, a new faculty member in the Department of Biology and investigator in The Picower Institute for Learning and Memory, uses the tiny, transparent jellyfish Clytia hemisphaerica, a new neuroscience model.
Q: In your work, you developed a new model organism for neuroscience research, the transparent jellyfish Clytia hemisphaerica. How do these jellyfish answer questions about neuroscience, the nervous system, and evolution in ways that other models cannot?
A: First, I believe in the importance of more broadly understanding the natural world and diversifying the organisms that we deeply study. One reason is to find experimentally tractable organisms to identify generalizable biological principles – for example, we understand the basis of how neurons “fire” from studies of the squid giant axon. Another reason is that transformative breakthroughs have come from identifying evolutionary innovations that already exist in nature – for example, green fluorescent protein (GFP, from jellyfish) or CRISPR (from bacteria). In both ways, this jellyfish is a valuable complement to existing models.
I have always been interested in the intersection of two types of problems: how nervous systems generate our behaviors; and how these incredible systems were actually created by evolution.
On the systems neuroscience side, ever since working on the serotonin system during my PhD I have been fascinated by the problem of how animals control all of their behaviors simultaneously in a flexible and context-dependent manner, and how behavioral choices depend not just on incoming stimuli but on how those stimuli interact with constantly changing states of the nervous system and body. These are extremely complex and difficult problems, with the particular challenge of interactions across scales, from chemical signaling and dynamic cell biology to neural networks and behavior.
To address these questions, I wanted to move into a model organism with exceptional experimental tractability.
There have been exciting breakthroughs in imaging techniques for neuroscience, including these incredible ways in which we can actually watch and manipulate neuronal activity in a living animal. So, the first thing I wanted was a small and transparent organism that would allow for this kind of optical approach. These jellyfish are a few millimeters in diameter and perfectly transparent, with interesting behaviors but relatively compact nervous systems. They have thousands of neurons where we have billions, which also puts them at a nice intermediate complexity compared to other transparent models that are widely used – for example, C. elegans have 302 neurons and larval zebrafish have something like 100,000 in the brain alone. These features will allow us to look at the activity of the whole nervous system in behaving animals to try to understand how that activity gives rise to behaviors and how that activity itself arises from networks of neurons.
On the evolution side of our work, we are interested in the origins of nervous systems, what the first nervous systems looked like, and broadly what the options are for how nervous systems are organized and functioning: to what extent there are principles versus interesting and potentially useful innovations, and if there are principles, whether those are optimal or somehow constrained by evolution. Our last common ancestor with jellyfish and their relatives (the cnidarians) was something similar to the first nervous system, so by comparing what we find in cnidarians with work in other models we can make inferences about the origins and early evolution of nervous systems. As we further explore these highly divergent animals, we are also finding exciting evolutionary innovations: specifically, they have incredible capabilities for regenerating their nervous systems. In the future, it will be exciting to better understand how these neural networks are organized to allow for such robustness.
Q: What work is required to develop a new organism as a model, and why did you choose this particular species of jellyfish?
A: If you’re choosing a new animal model, it’s not just about whether it has the right features for the questions you want to ask, but also whether it technically lets you do the right experiments. The model we’re using was first developed by a research group in France, who spent many years doing the really hard work of figuring out how to culture the whole life cycle in the lab, injecting eggs, and developing other key resources. For me, the big question was whether we’d be able to use the genetic tools that I was describing earlier for looking at neural activity. Working closely with collaborators in France, our first step was figuring out how to insert things into the jellyfish genome. If we couldn’t figure that out, I was going to switch back to working with mice. It took us about two years of troubleshooting, but now we can routinely generate genetically modified jellyfish in the lab.
Switching to a new animal model is tough – I have a mouse neuroscience background and joined a postdoc lab that used mice and flies; I was the only person working with jellyfish but had no experience. For example, building an aquaculture system and figuring out how to keep jellyfish healthy is not trivial, particularly now that we’re trying to do genetics. One of my goals is now to optimize and simplify this whole process so that when other labs want to start working with jellyfish we have a simple aquaculture platform to get them started, even if they have no experience.
In addition to the fact that these things are tiny and transparent, the main reason that we chose this particular species is because it has an amazing life cycle that makes it an exciting laboratory animal.
They have separate sexes that spawn daily with the fertilized eggs developing into larvae that then metamorphose into polyps. We grow these polyps on microscope slides, where they form colonies that are thought to be immortal. These colonies are then constantly releasing jellyfish, which are all genetically identical “clones” that can be used for experiments. That means that once you create a genetically modified strain, like a transgenic line or a knockout, you can keep it forever as a polyp colony – and since the animals are so small, we can culture them in large numbers in the lab.
There’s still a huge amount of foundational work to do, like characterizing their behavioral repertoire and nervous system organization. It’s shocking how little we know about the basics of jellyfish biology – particularly considering that they kill more people per year than sharks and stingrays combined – and the more we look into it the more questions there are.
Q: What drew you to a faculty position at MIT?
A: I wanted to be in a department that does fundamental research, is enthusiastic about basic science, is open-minded, and is very diverse in what people work on and think about. My goal is also to be able to ultimately link mechanisms at the molecular and cellular level to organismal behavior, which is something that MIT Biology is particularly strong at doing. It’s been an exciting first few months! MIT Biology is such an amazing place to do science and it’s been wonderful how enthusiastic and supportive everyone in the department has been.
I was additionally drawn to MIT by the broader community and have already found it so easy to start collaborations with people in neuroscience, engineering, and math. I’m also thrilled to have recently become a member of The Picower Institute for Learning and Memory, which further enables these collaborations in a way that I believe will be transformational for the work in my lab.
It’s a new lab. It’s a new organism. There isn’t a huge, well-established field that is taking these approaches. There’s so much we don’t know, and so much that we have to establish from scratch. My goal is for my lab to have a sense of adventure and fun, and I’m really excited to be doing that here in MIT Biology.
The prestigious honor society announces more than 250 new members, including MIT Biology Professor Yukiko Yamashita.
MIT News Office
April 24, 2023
Eight MIT faculty members are among more than 250 leaders from academia, the arts, industry, public policy, and research elected to the American Academy of Arts and Sciences, the academy announced April 19.
One of the nation’s most prestigious honorary societies, the academy is also a leading center for independent policy research. Members contribute to academy publications, as well as studies of science and technology policy, energy and global security, social policy and American institutions, the humanities and culture, and education.
Those elected from MIT in 2023 are:
- Arnaud Costinot, professor of economics;
- James J. DiCarlo, the Peter de Florez Professor of Brain and Cognitive Sciences and director of the MIT Quest for Intelligence;
- Piotr Indyk, the Thomas D. and Virginia W. Cabot Professor of Electrical Engineering and Computer Science;
- Senthil Todadri, professor of physics;
- Evelyn N. Wang, the Ford Professor of Engineering (on leave) and director of the Department of Energy’s Advanced Research Projects Agency-Energy;
- Boleslaw Wyslouch, professor of physics and director of the Laboratory for Nuclear Science and Bates Research and Engineering Center;
- Yukiko Yamashita, professor of biology and core member of the Whitehead Institute; and
- Wei Zhang, professor of mathematics.
“With the election of these members, the academy is honoring excellence, innovation, and leadership and recognizing a broad array of stellar accomplishments. We hope every new member celebrates this achievement and joins our work advancing the common good,” says David W. Oxtoby, president of the academy.
Since its founding in 1780, the academy has elected leading thinkers from each generation, including George Washington and Benjamin Franklin in the 18th century, Maria Mitchell and Daniel Webster in the 19th century, and Toni Morrison and Albert Einstein in the 20th century. The current membership includes more than 250 Nobel and Pulitzer Prize winners.
Developing sperm cells swap out histones for proteins called protamines to coil DNA tightly enough to fit inside the hydrodynamic shape ideal for the task of swimming swiftly to an egg in order to fertilize it. If the balance of protamines in the sperm is wrong, however, the sperm may become misshapen and die, making the animal infertile. Whitehead Institute Member Yukiko Yamashita and former graduate student Jun Park have discovered why this imbalance causes infertility in the fruit fly.
Greta Friar | Whitehead Institute
April 10, 2023
Sperm must swim swiftly to an egg in order to fertilize it, and so they have evolved hydrodynamic shapes. Most of the space in the head of sperm cells is taken up by the DNA they carry, so the cells coil up their DNA super tightly to stay small and streamlined. In most cell types, DNA is coiled around proteins called histones. These do not package DNA tightly enough for sperm, so when a sperm cell is developing, it swaps out histones for another type of protein called protamines that coil DNA very tightly.
Many species, including humans, mice, and flies, have multiple types of protamines. If the balance between the different types is wrong, then the sperm’s DNA may not be packaged correctly and it may become misshapen and die, making the animal infertile. Whitehead Institute Member Yukiko Yamashita and former graduate student Jun Park have discovered why this imbalance causes infertility in the fruit fly (Drosophila melanogaster). The finding, published in the Proceedings of the National Academy of Sciences on April 10, showed a mechanism that balances different types of protamines to ensure male fertility.
Mst77F is a major fruit fly protamine. Yamashita and Park determined that the fruit fly protamine Mst77Y, which is related to Mst77F, can interfere with the function of Mst77F. Fruit flies usually make a lot of Mst77F and a little of Mst77Y. The researchers found that when expression of the Mst77Y gene is too high, especially when expression of Mst77F is low, it disrupts the process of DNA packaging, leading to infertility.
How does Mst77Y interfere with Mst77F? The researchers discovered that this is because the Mst77Y gene makes faulty protamines. There are multiple copies of Mst77Y on the fly’s Y chromosome. They likely evolved from a copy of Mst77F, which is not on a sex chromosome. However, the different versions of Mst77Y have lost or altered parts that they need in order to function, so unlike the Mst77F protamine, Mst77Y protamines likely cannot coil DNA tightly around themselves. In spite of the fact that the Mst77Y protamines do not work correctly, they are dominant: when they are present, the sperm cell will use them over the functional Mst77F protamines.
“Mst77Y is a half-broken tool,” says Yamashita, who is also a professor of biology at the Massachusetts Institute of Technology and a Howard Hughes Medical Institute investigator. “It is able to take the place of the working tool, Mst77F, but not to do its job, so when too much Mst77Y is present, the sperm cell does not have enough working tools in place to compact its DNA.”
The researchers also figured out how sperm cells keep expression of Mst77F high and Mst77Y low: with the help of a protein called Modulo. In order for an RNA read from a gene to be made into a protein, it needs to have a tail added to it made up of a string of adenines—one of the four building blocks that make up RNA. Modulo makes sure that the cell preferentially adds this tail to the RNA coding for Mst77F. Although Yamashita and Park did not determine the exact mechanism by which Modulo ensures this preferential treatment, they did observe that Modulo and the Mst77F RNA group together in the same part of the cell, the nucleolus, whereas Mst77Y does not.
Altogether, these findings explain why and how fruit fly sperm cells carefully balance the levels of these two protamines. However, the research raises the question, what are sperm cells using the non-functional Mst77Y protamines for? Yamashita and Park can only speculate, but the answer may have to do with their observation that high levels of Mst77Y killed off more X-chromosome bearing sperm than Y-chromosome bearing sperm. Past research has suggested that protamines may be involved in a process called meiotic drive, which animals can use to skew the sex ratio of their offspring. This new work is not only consistent with that hypothesis, but provides a possible mechanism to explain how protamines contribute. The researchers note that they did not see a strong effect on the sex ratio of offspring in this experiment, but hope that this work could set the stage to understand the role of non-functional protamines in meiotic drive.
“At the cell level, we were able to show that there’s some basis for this protamine to be involved in biasing whether X or Y chromosome bearing sperm survive,” Park says. “An interesting next question would be to see if there are certain conditions in which this mechanism more clearly acts as a driver at the level of offspring’s sex ratio.”
Notes
Park, Jun I., George W. Bell, and Yukiko M. Yamashita. 2023. “Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster,” PNAS 120 (16). https://www.pnas.org/doi/10.1073/pnas.2220576120
Whitehead Institute Member David Page has spent his career understanding how the differences between X and Y contribute to these sex differences, but a recent project is taking his lab in a new direction: understanding how the differences between X chromosomes contribute to sex differences.
Greta Friar | Whitehead Institute
February 7, 2023
Nearly every cell in our body contains pairs of each of our chromosomes, and these pairs are identical in all but one case: that of our sex chromosomes. Males typically have one X and one Y sex chromosome, while females typically have two X chromosomes. In recent years, research has suggested that these different chromosomes can influence far more than sex determination. Gene expression from the sex chromosomes appears to contribute to sex differences in health and disease, which males and females experience in everything from the incidence of getting certain diseases, to the symptoms of diseases, to responses to drugs, and more. For example, women are more likely to develop autoimmune disorders, while men are more likely to develop heart conditions.
Whitehead Institute Member David Page has spent his career understanding how the differences between X and Y contribute to these sex differences, but a recent project is taking his lab in a new direction: understanding how the differences between X chromosomes contribute to sex differences. Although females’ pair of X chromosomes contain the same genes, they have different patterns of gene expression. New research from Page and postdoc Adrianna San Roman reveals just how different the two types of X chromosomes are. The findings, published in the journal Cell Genomics on February 8, show that one type of X chromosome, known as the inactive X chromosome, can modulate the gene expression of the other type of X chromosome, known as the active X chromosome. Their work indicates that inactive X chromosomes have underappreciated roles in gene regulation and, most likely, in sex differences in health and disease.
Difference rooted in history
Females’ two X chromosomes have different gene expression activity because of the sex chromosomes’ evolutionary history. The X and Y sex chromosomes evolved from a pair of identical non-sex chromosomes. Because of this ancestry, the sex chromosomes still contain genes that are important outside of regulating sex differences, such as genes that contribute to our immune system or regulate gene expression throughout the body. However, over time the Y chromosome shrank and lost most of its genes. Researchers think that in order to make up for the loss of necessary genes on the Y, expression of the corresponding genes on the X chromosome increased. This ensured that males still had the necessary levels of gene expression from their sex chromosomes, but now females, with two copies of X both working overtime, had levels of gene expression that were too high. To solve this problem, our bodies developed a process called X chromosome inactivation, by which the majority of genes on all but one copy of the X chromosome in each cell are silenced, or turned off. This means that everyone, male and female alike, has one copy of the X chromosome working at full strength–the active X chromosome. In males, the active X chromosome is paired with a Y chromosome, and in females, it is paired with a so-called inactive X chromosome, on which most of the genes are turned off.
In spite of the evolution of X chromosome inactivation, some percentage of genes on the inactive X chromosome are still expressed, such as genes that have an active counterpart on the Y chromosome. Previous research has indicated that about a quarter of the genes on the inactive X are, in fact, active, so researchers have long been aware that the chromosome is not completely silent. However, it’s still often painted as a passive copy playing backup for its more active partner. San Roman’s work shows that the inactive X chromosome’s gene expression is much more potent and complex than that.
A spectrum of sex chromosomes
In order to understand the inactive X chromosome’s contributions to gene expression, San Roman and colleagues in the Page lab collected blood and skin samples from people born with unusual combinations of sex chromosomes—everything from X0 (one X chromosome) to XXXXY. People with these different sets of chromosomes often have health issues; for example, X0 females have Turner syndrome, which can cause heart defects, hearing impairment, and more; and XXY males have Klinefelter syndrome, which can cause infertility, weak muscles, and more. Page and San Roman hope their research could provide useful insights into these health issues as well as into sex differences between XY males and XX females.
In people with more than one X chromosome, every X but one is an inactive X. The researchers graphed sex chromosome gene expression, measuring the change in expression level of each gene with the addition of each inactive X, for people with anything from zero to three inactive X chromosomes, as well as different numbers of Y chromosomes. They also looked at the relative contribution to overall expression from the active versus inactive X chromosomes. One might expect the graphs they made to be relatively straightforward: for genes that are turned off on the inactive X chromosome, the gene expression level would not change at all as the number of copies of the inactive X increased. For genes that are turned on, the gene expression level would double with two X chromosomes, triple with three X chromosomes, and so on. When the researchers looked at chromosomes other than X with extra copies—namely, Y and chromosome 21—this is essentially the pattern they observed. Gene expression from additional X chromosomes, however, was not so straightforward.
Each additional inactive X chromosome changes gene expression by the same amount. However, the researchers found a surprising diversity in expression levels across X chromosome genes. The presence of each additional inactive X might increase one gene’s expression by 20 percent and another’s by 70 percent. Then the results grew more surprising: for some genes, the addition of an inactive X decreased their expression. For some genes that are only expressed on the active X chromosome, and completely silent on the inactive X, additional inactive X chromosomes nonetheless changed their expression level.
These discrepancies led the researchers to a startling finding. The X chromosomes do not function independently of each other. Instead, the inactive X chromosome can modulate expression of genes on the active X chromosome. In other words, some genes on the inactive X chromosome regulate genes on the active X chromosome, dialing their expression up or down. Altogether, the researchers found that 38% of the X chromosome genes in the two cell types that they tested are affected by the presence of inactive X chromosomes, either because the genes are expressed on the inactive X, or because the inactive X regulates their expression on the active X, or through some combination of the two mechanisms.
These findings show that the inactive X plays a much more active role in gene expression and regulation than was previously appreciated. Rather than just playing second fiddle to the active X chromosome, the inactive X is sometimes harmonizing with and sometimes even conducting its partner.
Rethinking the role of the inactive X in health and disease
Page and San Roman hope that their findings will help refocus research into sex differences. Previous research into the mechanisms behind these differences has focused on the effects of having X versus Y chromosomes. Page and San Roman’s work show that researchers also need to consider how the presence (in females) or absence (in males) of an inactive X chromosome contributes to sex differences.
“Everybody on the planet carries one active X chromosome, so that first X chromosome really does not contribute, we think, to differences between males and females,” says Page, who is also a professor of biology at the Massachusetts Institute of Technology and Investigator with the Howard Hughes Medical Institute. “If we transition from saying that females are XX and males are XY, to saying that females are Xi [have an inactive X] and males are Y, that really focuses the question.”
Page lab researchers have already begun using their findings to identify X chromosome genes that are likely to be important for sex differences in health and disease. From their list of genes that change in expression based on the presence of an inactive X, the researchers narrowed in on a top ten list of genes that need to maintain a specific expression level or else there will be severe negative consequences. These genes are also likely to be responsible for causing the health issues associated with different atypical sex chromosome compositions, because changes in their expression level are most likely to have strong effects on cells.
“This is a new way of thinking about how the X chromosome is expressed and how it might be impacting our biology,” San Roman says. “This top ten list will be really interesting to consider in the future in terms of how the level of expression of these genes affects cells and tissues in very fundamental ways.”
Notes
Citation:
Adrianna K. San Roman, Alexander K. Godfrey, Helen Skaletsky, Daniel W. Bellott, Abigail F. Groff, Hannah L. Harris, Laura V. Blanton, Jennifer F. Hughes, Laura Brown, Sidaly Phou, Ashley Buscetta, Paul Kruszka, Nicole Banks, Amalia Dutra, Evgenia Pak, Patricia C. Lasutschinkow, Colleen Keen, Shanlee M. Davis, Nicole R. Tartaglia, Carole Samango-Sprouse, Maximilian Muenke, and David C. Page. (2023). The human inactive X chromosome modulates expression of the active X chromosome. Cell Genomics. https://doi.org/10.1016/j.xgen.2023.100259
A new technique for studying liver cells within an organism could shed light on the genes required for regeneration.
Anne Trafton | MIT News Office
November 15, 2022
The liver’s ability to regenerate itself is legendary. Even if more than 70 percent of the organ is removed, the remaining tissue can regrow an entire new liver.
Kristin Knouse, an MIT assistant professor of biology, wants to find out how the liver is able to achieve this kind of regeneration, in hopes of learning how to induce other organs to do the same thing. To that end, her lab has developed a new way to perform genome-wide studies of the liver in mice, using the gene-editing system CRISPR.
With this new technique, researchers can study how each of the genes in the mouse genome affects a particular disease or behavior. In a paper describing the technique, the researchers uncovered several genes important for liver cell survival and proliferation that had not been seen before in studies of cells grown in a lab dish.
“If we really want to understand mammalian physiology and disease, we should study these processes in the living organism wherever possible, as that’s where we can investigate the biology in its most native context,” says Knouse, who is also a member of MIT’s Koch Institute for Integrative Cancer Research.
Knouse is the senior author of the new paper, which appears today in Cell Genomics. Heather Keys, director of the Functional Genomics Platform at the Whitehead Institute, is a co-author on the study.
Extracellular context
As a graduate student at MIT, Knouse used regenerating liver tissue as a model to study an aspect of cell division called chromosome segregation. During this study, she observed that cells dividing in the liver did not behave the same way as liver cells dividing in a lab dish.
“What I internalized from that research was the extent to which something as intrinsic to the cell as cell division, something we have long assumed to be independent of anything beyond the cell, is clearly influenced by the extracellular environment,” she says. “When we study cells in culture, we lose the impact of that extracellular context.”
However, many types of studies, including genome-wide screens that use technologies such as CRISPR, are more difficult to deploy at the scale of an entire organism. The CRISPR gene-editing system consists of an enzyme called Cas9 that cuts DNA in a given location, directed by a strand of RNA called a guide RNA. This allows researchers to knock out one gene per cell, in a huge population of cells.
While this approach can reveal genes and proteins involved in specific cellular processes, it has proven difficult to deliver CRISPR components efficiently to enough cells in the body to make it useful for animal studies. In some studies, researchers have used CRISPR to knock out about 100 genes of interest, which is useful if they know which genes they want to study, but this limited approach doesn’t reveal new genes linked to a particular function or disease.
A few research groups have used CRISPR to do genome-wide screens in the brain and in skin cells, but these studies required large numbers of mice to uncover significant hits.
“For us, and I think many other researchers, the limited experimental tractability of mouse models has long hindered our capacity to dive into questions of mammalian physiology and disease in an unbiased and comprehensive manner,” Knouse says. “That’s what I really wanted to change, to bring the experimental tractability that was once restricted to cell culture into the organism, so that we are no longer limited in our ability to explore fundamental principles of physiology and disease in their native context.”
To get guide RNA strands into hepatocytes, the predominant cell type in the liver, Knouse decided to use lentivirus, an engineered nonpathogenic virus that is commonly used to insert genetic material into the genome of cells. She injected the guide RNAs into newborn mice, such that once the guide RNA was integrated into the genome, it would be passed on to future generations of liver cells as the mice grew. After months of effort in the lab, she was able to get guide RNAs consistently expressed in tens of millions of hepatocytes, which is enough to do a genome-wide screen in just a single animal.
Cellular fitness
To test the system, the researchers decided to look for genes that influence hepatocyte fitness — the ability of hepatocytes to survive and proliferate. To do that, they delivered a library of more than 70,000 guide RNAs, targeting more than 13,000 genes, and then determined the effect of each knockout on cell fitness.
The mice used for the study were engineered so that Cas9 can be turned on at any point in their lifetime. Using a group of four mice — two male and two female — the researchers turned on expression of Cas9 when the mice were five days old. Three weeks later, the researchers screened their liver cells and measured how much of each guide RNA was present. If a particular guide RNA is abundant, that means the gene it targets can be knocked out without fatally damaging the cells. If a guide RNA doesn’t show up in the screen, it means that knocking out that gene was fatal to the cells.
This screen yielded hundreds of genes linked to hepatocyte fitness, and the results were very consistent across the four mice. The researchers also compared the genes they identified to genes that have been linked to human liver disease. They found that genes mutated in neonatal liver failure syndromes also caused hepatocyte death in their screen.
The screen also revealed critical fitness genes that had not been identified in studies of liver cells grown in a lab dish. Many of these genes are involved in interactions with immune cells or with molecules in the extracellular matrix that surrounds cells. These pathways likely did not turn up in screens done in cultured cells because they involve cellular interactions with their external environment, Knouse says.
By comparing the results from the male and female mice, the researchers also identified several genes that had sex-specific effects on fitness, which would not have been possible to pick up by studying cells alone.
Renew and regenerate
Knouse now plans to use this system to identify genes that are critical for liver regeneration.
“Many tissues such as the heart are unable to regenerate because they lack stem cells and the differentiated cells are unable to divide. However, the liver is also a highly differentiated tissue that lacks stem cells, yet it retains this amazing capacity to regenerate itself after injury,” she says. “Importantly, the genome of the liver cells is no different from the genome of the heart cells. All of these cells have the same instruction manual in their nucleus, but the liver cells are clearly reading different sentences in this manual in order to regenerate. What we don’t know is, what are those sentences? What are those genes? If we can identify those genes, perhaps someday we can instruct the heart to regenerate.”
This new screening technique could also be used to study conditions such as fatty liver disease and cirrhosis. Knouse’s lab is also working on expanding this approach to organs other than the liver.
“We need to find ways to get guide RNAs into other tissues at high efficiency,” she says. “In overcoming that technical barrier, then we can establish the same experimental tractability that we now have in the liver in the heart or other issues.”
The research was funded by the National Institutes of Health NIH Director’s Early Independence Award, the Koch Institute Support (core) Grant from the National Cancer Institute, and the Scott Cook and Signe Ostby Fund.
MIT Koch Institute
October 25, 2022
Cheers to the inaugural winners of the Koch Institute’s Angelika Amon Young Scientist Award, Alejandro Aguilera and Melanie de Almeida. The new award recognizes graduate students in the life sciences or biomedical research from institutions outside the U.S. who embody Dr. Amon’s infectious enthusiasm for discovery science.
Aguilera, a student at the Weizmann Institute of Science in Israel, has developed a platform for studying mammalian embryogenesis. De Almeida, who recently completed her doctoral work at the Research Institute of Molecular Pathology in Austria, develops CRISPR screens to explore cancer vulnerabilities and gene regulatory networks.
Aguilera and de Almeida will visit the Koch Institute in November to deliver scientific presentations to the MIT community and Amon Lab alumni.