Research Area: Cell Biology

The proteins that package DNA to fit inside cells have another role: tuning gene expression
Raleigh McElvery
May 19, 2021

The DNA inside a single human cell is several meters long — yet it must be condensed to fit inside a space one-tenth the diameter of a hair. That’s like stretching a string from Philadelphia, Pennsylvania to Washington, D.C., and then trying to stuff it into a soccer ball. Imagine then organizing all of this information for each of the body’s 3 trillion cells! The DNA is condensed by proteins called histones that create a spool around which the DNA can wrap itself. How tightly the DNA is wound determines whether it is accessible enough for other proteins to bind to and copy into RNA, toggling gene expression levels up or down.

One specialized type of histone, H2A.Z, is ubiquitous and essential among multicellular organisms. But there have been conflicting reports about how it affects gene expression, especially during embryonic development.

Several years ago, Laurie Boyer’s lab at MIT was the first to show that H2A.Z wraps the DNA located around the start sites of most genes, where the molecular machine RNA polymerase II (RNAPII) binds to copy the DNA into RNA. Boyer’s team demonstrated that removing H2A.Z prevented embryonic cells from turning on genes that are important for forming organs and tissues. But scientists still weren’t sure how H2A.Z exerted its effects.

Now, in a recent Nature Structural and Molecular Biology study, a team from the Boyer lab, led by former postdoc Constantine Mylonas, has revealed how H2A.Z regulates the ability of RNAPII to properly transcribe DNA into the messages that specify all cell types in the body. The researchers found that in embryonic stem cells, H2A.Z serves as a “yellow traffic light,” signaling RNAPII to slow the process of transcribing DNA into RNA. Although there are other proteins that also contribute to RNAPII pausing, H2A.Z establishes a second barrier to transcription that allows gene expression to be tuned in response to developmental signals.

“H2A.Z appears to regulate how fast RNAPII begins to transcribe DNA, and this allows the cell time to respond to important cues that ultimately direct a stem cell to become a brain or heart cell, for example,” says Boyer, a professor of biology and biological engineering. “This connection was a critical missing piece of the puzzle, and explains why H2A.Z is essential for development across all multicellular organisms.”

Illustration of molecules
As RNAPII starts to transcribe a gene, it encounters a cluster of eight histones (a “nucleosome”) including H2A.z, which slows its progression — allowing for tuning of gene expression in response to developmental signals.

According to Boyer, H2A.Z’s role in gene expression has been difficult to pin down because previous approaches only provided static snapshots of how proteins interact with DNA days after loss of the histone. Boyer’s team overcame this shortcoming by leveraging a system that allowed for targeted degradation of H2A.Z within hours. They combined this technique with high-resolution genomic approaches and live cell imaging of RNAPII dynamics using super-resolution microscopy. With help from Ibrahim Cissé’s lab, they were able to visualize RNAPII dynamics in real time at the single molecule level in embryonic stem cells. Upon loss of H2A.Z, they found a remarkable increase in RNAPII movement in the cells, consistent with their genomic results showing a faster release of RNAPII and an increase in transcription in the absence of H2A.Z.

Next, the researchers plan to determine precisely how H2A.Z is targeted to the start sites of genes and how it forms a barrier to RNAPII passage.

Boyer says pinpointing the way histone variants like H2A.Z control gene expression is fundamental to understanding how developmental decisions are made, and will help researchers understand why misregulation of H2A.Z has been linked to diseases such as cancer.

“Emerging evidence indicates that DNA ‘packaging proteins’ like histones directly participate in how RNAPII can read and transcribe DNA,” she explains, “and that crucial connection wasn’t clear before.”

Image credits: courtesy of Laurie Boyer
Top image: Live cell super-resolution imaging showing RNAPII dynamics at a single molecule level in embryonic stem cells. The bright and colored clusters represent RNAPII molecules.

Citation:
“A dual role for H2A. Z. 1 in modulating the dynamics of RNA Polymerase II initiation and elongation.”
Nature Structural & Molecular Biology, online May 10, 2021, DOI: 10.1038/s41594-021-00589-3
Constantine Mylonas, Choongman Lee, Alexander L. Auld, Ibrahim I. Cisse, and Laurie A. Boyer.

Childhood hobbies jump-start a research career

MIT Biology junior Eduardo Canto tinkered with science long before he started studying Treacher Collins syndrome in the Calo lab.

Saima Sidik | Department of Biology
May 19, 2021

In seventh grade, Eduardo Canto wanted a dog. His mom said no, though. She didn’t want to spend her days vacuuming fur. They reached a compromise: Canto was allowed to have pet fish. Soon Canto’s disappointment with his new pets turned to curiosity. While he couldn’t train the fish to sit or roll over, he decided that breeding the fish could be a fun pastime.

An internet search told Canto that some aquarists use dried Indian almond leaves, a traditional Asian herbal remedy, to stimulate fish breeding, although no one is quite sure how the leaves do this. However, finding Indian almond leaves presented a problem for a kid without an Amazon account living far from the tree’s native habitat. On a whim, Canto picked up some similar-looking leaves in a park near his house in Puerto Rico. He knew they weren’t from an Indian almond tree, but he put them in the tank anyhow, just to see what would happen. A few days later, he noticed a collection of eggs attached to the bottom of a leaf!

Canto often took on little experiments like this, which caused his grandfather to predict early on that he would have a scientific career. Eight years after the breeding endeavor, Canto is fulfilling his grandfather’s prediction by studying Course 7 (Biology) at MIT, where he’s currently in his third year of a bachelor’s degree. Once again, fish have come into Canto’s life — he’s working in Eliezer Calo’s lab, where researchers use zebrafish to study a genetic disorder called Treacher Collins syndrome, which causes deformities in eyes, ears, cheekbones, and chins.

Throughout middle school and high school, Canto dipped his toes into many scientific disciplines. School science fairs motivated him to build a dry ice-powered trolley, a solar-powered water heater, and start a vegetable garden.

Sometimes, he admits, his motivation for joining science clubs wasn’t lofty. “I joined the math club because I got to miss a day of school every year for their annual competition,” he says with a laugh. But he also talks excitedly about his early experiments, particularly in biology. “I’ve always loved working with my hands,” he says.

Canto’s father, a medical doctor, encouraged his son’s interest by letting Canto shadow him at work. He also started a molecular biology summer program at Canto’s high school that taught students how to pipette and do simple experiments. By the time Canto applied to college, he was convinced he wanted to study biology, and MIT drew his attention because of its reputation as a top science school with excellent biology teachers. He knew it was the right choice for him when he attended Campus Preview Weekend, and found a large Puerto Rican community ready to welcome him. Even far from the island, he felt at home.

Canto has kept up with his roots since joining MIT by playing on a soccer team for Puerto Rican students. He’s also become part of a new community in a lab run by Eliezer Calo — who is a Puerto Rican himself. The lab is interested in ribosomes, the molecular machines that build proteins. Treacher Collins syndrome arises when cells can’t make ribosomes properly, and Canto wants to understand why that is.

Before Canto joined the Calo lab, the group had already started studying a protein called DDX21 that’s involved in making ribosomes in both humans and zebrafish. When genetic mutations in zebrafish cause DDX21 to go to the wrong part of the cell, the fish develop jaw deformations that mirror Treacher Collins syndrome. The Calo lab thinks cells with mislocalized DDX21 probably don’t produce ribosomes as well as normal cells, but they’re still testing this hypothesis.

Canto wants to probe the relationship between DDX21 and Treacher Collins syndrome further, but fish reproduce slowly, so they’re not ideal organisms for his research. Instead, he’s built a strain of Escherichia coli bacteria that carry DDX21 in place of the equivalent bacterial gene. DDX21 helps these bacteria survive the stress associated with cold temperatures, so without it, the bacteria will die in the cold. Canto hopes to take advantage of this trait by finding small molecules that stop the bacteria from growing at low temperatures — just like a DDX21 mutation would. Studying how these molecules bind DDX21 will help him understand which parts of this protein are important for its function.

The possibility that this work will one day reveal how Treacher Collins syndrome develops in patients is rewarding to Canto, and in fact he hopes helping patients will soon become his life’s focus. He wants to attend medical school, and eventually become a doctor. The human physiology class he took last semester was one of his favorites, even though it was over Zoom due to the COVID-19 pandemic. Becoming a doctor will let him help others while studying topics he finds fascinating. “Medicine is like biology on steroids!” he says.

And who knows — one day after he’s a doctor, maybe he’ll even get that pet he’s always wanted. But unlike Canto’s interest in biology, some of his interests have evolved over time. These days, he prefers cats over dogs.

Photo credit: Saima Sidik
Posted: 5.19.21
Kristin Knouse

Education

  • PhD, 2017, MIT; MD, 2018, Harvard Medical School
  • Undergraduate: BS, 2010, Biology, Duke University

Research Summary

We aim to understand how tissues sense and respond to damage with the goal of developing novel treatments for diverse human diseases. We focus on the mammalian liver, which has the unique ability to completely regenerate itself, in order to identify the molecular requirements for effective organ repair. To this end, we innovate genetic, molecular, and cellular tools that allow us to investigate and modulate organ injury and regeneration directly within living organisms.

Awards

  • NIH Director’s Early Independence Award, 2018
  • Henry Asbury Christian Award, 2018
Biologists discover a trigger for cell extrusion

Study suggests this process for eliminating unneeded cells may also protect against cancer.

Anne Trafton | MIT News Office
May 5, 2021

For all animals, eliminating some cells is a necessary part of embryonic development. Living cells are also naturally sloughed off in mature tissues; for example, the lining of the intestine turns over every few days.

One way that organisms get rid of unneeded cells is through a process called extrusion, which allows cells to be squeezed out of a layer of tissue without disrupting the layer of cells left behind. MIT biologists have now discovered that this process is triggered when cells are unable to replicate their DNA during cell division.

The researchers discovered this mechanism in the worm C. elegans, and they showed that the same process can be driven by mammalian cells; they believe extrusion may serve as a way for the body to eliminate cancerous or precancerous cells.

“Cell extrusion is a mechanism of cell elimination used by organisms as diverse as sponges, insects, and humans,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, a Howard Hughes Medical Institute investigator, and the senior author of the study. “The discovery that extrusion is driven by a failure in DNA replication was unexpected and offers a new way to think about and possibly intervene in certain diseases, particularly cancer.”

MIT postdoc Vivek Dwivedi is the lead author of the paper, which appears today in Nature. Other authors of the paper are King’s College London research fellow Carlos Pardo-Pastor, MIT research specialist Rita Droste, MIT postdoc Ji Na Kong, MIT graduate student Nolan Tucker, Novartis scientist and former MIT postdoc Daniel Denning, and King’s College London professor of biology Jody Rosenblatt.

Stuck in the cell cycle

In the 1980s, Horvitz was one of the first scientists to analyze a type of programmed cell suicide called apoptosis, which organisms use to eliminate cells that are no longer needed. He made his discoveries using C. elegans, a tiny nematode that contains exactly 959 cells. The developmental lineage of each cell is known, and embryonic development follows the same pattern every time. Throughout this developmental process, 1,090 cells are generated, and 131 cells undergo programmed cell suicide by apoptosis.

Horvitz’s lab later showed that if the worms were genetically mutated so that they could not eliminate cells by apoptosis, a few of those 131 cells would instead be eliminated by cell extrusion, which appears to be able to serve as a backup mechanism to apoptosis. How this extrusion process gets triggered, however, remained a mystery.

To unravel this mystery, Dwivedi performed a large-scale screen of more than 11,000 C. elegans genes. One by one, he and his colleagues knocked down the expression of each gene in worms that could not perform apoptosis. This screen allowed them to identify genes that are critical for turning on cell extrusion during development.

To the researchers’ surprise, many of the genes that turned up as necessary for extrusion were involved in the cell division cycle. These genes were primarily active during first steps of the cell cycle, which involve initiating the cell division cycle and copying the cell’s DNA.

Further experiments revealed that cells that are eventually extruded do initially enter the cell cycle and begin to replicate their DNA. However, they appear to get stuck in this phase, leading them to be extruded.

Most of the cells that end up getting extruded are unusually small, and are produced from an unequal cell division that results in one large daughter cell and one much smaller one. The researchers showed that if they interfered with the genes that control this process, so that the two daughter cells were closer to the same size, the cells that normally would have been extruded were able to successfully complete the cell cycle and were not extruded.

The researchers also showed that the failure of the very small cells to complete the cell cycle stems from a shortage of the proteins and DNA building blocks needed to copy DNA. Among other key proteins, the cells likely don’t have enough of an enzyme called LRR-1, which is critical for DNA replication. When DNA replication stalls, proteins that are responsible for detecting replication stress quickly halt cell division by inactivating a protein called CDK1. CDK1 also controls cell adhesion, so the researchers hypothesize that when CDK1 is turned off, cells lose their stickiness and detach, leading to extrusion.

Cancer protection

Horvitz’s lab then teamed up with researchers at King’s College London, led by Rosenblatt, to investigate whether the same mechanism might be used by mammalian cells. In mammals, cell extrusion plays an important role in replacing the lining of the intestines, lungs, and other organs.

The researchers used a chemical called hydroxyurea to induce DNA replication stress in canine kidney cells grown in cell culture. The treatment quadrupled the rate of extrusion, and the researchers found that the extruded cells made it into the phase of the cell cycle where DNA is replicated before being extruded. They also showed that in mammalian cells, the well-known cancer suppressor p53 is involved in initiating extrusion of cells experiencing replication stress.

That suggests that in addition to its other cancer-protective roles, p53 may help to eliminate cancerous or precancerous cells by forcing them to extrude, Dwivedi says.

“Replication stress is one of the characteristic features of cells that are precancerous or cancerous. And what this finding suggests is that the extrusion of cells that are experiencing replication stress is potentially a tumor suppressor mechanism,” he says.

The fact that cell extrusion is seen in so many animals, from sponges to mammals, led the researchers to hypothesize that it may have evolved as a very early form of cell elimination that was later supplanted by programmed cell suicide involving apoptosis.

“This cell elimination mechanism depends only on the cell cycle,” Dwivedi says. “It doesn’t require any specialized machinery like that needed for apoptosis to eliminate these cells, so what we’ve proposed is that this could be a primordial form of cell elimination. This means it may have been one of the first ways of cell elimination to come into existence, because it depends on the same process that an organism uses to generate many more cells.”

Dwivedi, who earned his PhD at MIT, was a Khorana scholar before entering MIT for graduate school. This research was supported by the Howard Hughes Medical Institute and the National Institutes of Health.

Two heads are better than one, but two disciplines are even better

How biologists and mathematicians reached across departmental lines to solve a long-standing problem in electron microscopy

Saima Sidik | Department of Biology
April 19, 2021

MIT’s Hockfield Court is bordered on the west by the ultra-modern Stata Center, with its reflective, silver alcoves that jut off at odd angles, and on the east by Building 68, which is a simple, window-lined, cement rectangle. At first glance, Bonnie Berger’s mathematics lab in the Stata Center and Joey Davis’s biology lab in Building 68 are as different as the buildings that house them. And yet, a recent collaboration between these two labs shows how their disciplines complement each other. The partnership started when Ellen Zhong, a graduate student from the Computational and Systems Biology (CSB) Program, decided to use a computational pattern-recognition tool called a neural network to study the shapes of molecular machines. Three years later, Zhong’s project is letting scientists see patterns that run beneath the surface of their data, and deepening their understanding of the molecules that shape life.

Zhong’s work builds on a technique from the 1970s called cryo-electron microscopy (cryo-EM), which lets researchers take high-resolution images of frozen protein complexes. Over the last decade, better microscopes and cameras have led to a “resolution revolution” in cryo-EM that’s allowed scientists to see individual atoms within proteins. But, as good as these images are, they’re still only static snapshots. In reality, many of these molecular machines are constantly changing shape and composition as cells carry out their normal functions and adjust to new situations.

Along with former Berger lab member Tristan Belper, Zhong devised software called cryoDRGN. The tool uses neural nets to combine hundreds of thousands of cryo-EM images, and shows scientists the full range of three-dimensional conformations that protein complexes can take, letting them reconstruct the proteins’ motion as they carry out cellular functions. Understanding the range of shapes that protein complexes can take helps scientists develop drugs that block viruses from entering cells, study how pests kill crops, and even design custom proteins that can cure disease. COVID-19 vaccines, for example, work partly because they include a mutated version of the virus’s spike protein that’s stuck in its active conformation, so vaccinated people produce antibodies that block the virus from entering human cells. Scientists needed to understand the variety of shapes that spike proteins can take in order to figure out how to force spike into its active conformation.

Two women standing by rock wall
Graduate student Ellen Zhong (right), and her co-advisor, Professor of Mathematics Bonnie Berger (left)

Getting off the computer and into the lab

Zhong’s interest in computational biology goes back to 2011 when, as a chemical engineering undergrad at the University of Virginia, she worked with Professor Michael Shirts to simulate how proteins fold and unfold. After college, Zhong took her skills to a company called D. E. Shaw Research, where, as a Scientific Programmer, she took a computational approach to studying how proteins interact with small molecule drugs.

“The research was very exciting,” Zhong says, “but all based on computer simulations. To really understand biological systems, you need to do experiments.”

This goal of combining computation with experimentation motivated Zhong to join MIT’s CSB PhD program, where students often work with multiple supervisors to blend computational work with bench work. Zhong “rotated” in both the Davis and Berger labs, then decided to combine the Davis lab’s goal of understanding how protein complexes form with the Berger lab’s expertise in machine learning and algorithms. Davis was interested in building up the computational side of his lab, so he welcomed the opportunity to co-supervise a student with Berger, who has a long history of collaborating with biologists.

Davis himself holds a dual bachelor’s degree in computer science and biological engineering, so he’s long believed in the power of combining complementary disciplines. “There are a lot of things you can learn about biology by looking in a microscope,” he says. “But as we start to ask more complicated questions about entire systems, we’re going to require computation to manage the high-dimensional data that come back.”

Before rotating in the Davis lab, Zhong had never performed bench work before — or even touched a pipette. She was fascinated to find how streamlined some very powerful molecular biology techniques can be. Still, Zhong realized that physical limitations mean that biology is much slower when it’s done at the bench instead of on a computer. “With computational research, you can automate experiments and run them super quickly, whereas in the wet lab, you only have two hands, so you can only do one experiment at a time,” she says.

Zhong says that synergizing the two different cultures of the Davis and Berger labs is helping her become a well-rounded, adaptable scientist. Working around experimentalists in the Davis lab has shown her how much labor goes into experimental results, and also helped her to understand the hurdles that scientists face at the bench. In the Berger lab, she enjoys having coworkers who understand the challenges of computer programming.

“The key challenge in collaborating across disciplines is understanding each other’s ‘languages’,” Berger says. “Students like Ellen are fortunate to be learning both biology and computing dialects simultaneously.”

Bringing in the community

Man smiling outside
Zhong’s second co-advisor, Professor Joey Davis

Last spring revealed another reason for biologists to learn computational skills: these tools can be used anywhere there’s a computer and an internet connection. When the COVID-19 pandemic hit, Zhong’s colleagues in the Davis lab had to wind down their bench work for a few months, and many of them filled their time at home by using cryo-EM data that’s freely available online to help Zhong test her cryoDRGN software. The difficulty of understanding another discipline’s language quickly became apparent, and Zhong spent a lot of time teaching her colleagues to be programmers. Seeing the problems that non-programmers ran into when they used cryoDRGN was very informative, Zhong says, and helped her create a more user-friendly interface.

Although the paper announcing cryoDRGN was only recently published, the tool created a stir as soon as Zhong posted her code online, many months prior. The cryoDRGN team thinks this is because leveraging knowledge from two disciplines let them visualize the full range of structures that protein complexes can have, and that’s something researchers have wanted to do for a long time. For example, the cryoDRGN team recently collaborated with researchers from Harvard and Washington University to study locomotion of the single-celled organism Chlamydomonas reinhardtii. The mechanisms they uncovered could shed light on human health conditions, like male infertility, that arise when cells lose the ability to move. The team is also using cryoDRGN to study the structure of the SARS-CoV-2 spike protein, which could help scientists design treatments and vaccines to fight coronaviruses.

Zhong, Berger, and Davis say they’re excited to continue using neural nets to improve cryo-EM analysis, and to extend their computational work to other aspects of biology. Davis cited mass spectrometry as “a ripe area to apply computation.” This technique can complement cryo-EM by showing researchers the identities of proteins, how many of them are bound together, and how cells have modified them.

“Collaborations between disciplines are the future,” Berger says. “Researchers focused on a single discipline can take it only so far with existing techniques. Shining a different lens on the problem is how advances can be made.”

Zhong says it’s not a bad way to spend a PhD, either. Asked what she’d say to incoming graduate students considering interdisciplinary projects, she says: “definitely do it.”

An on-off switch for gene editing
Eva Frederick | Whitehead Institute
April 9, 2021

Now, in a paper published online in Cell on April 9, researchers describe a new gene editing technology called CRISPRoff that allows researchers to control gene expression with high specificity while leaving the sequence of the DNA unchanged. Designed by Whitehead Institute Member Jonathan Weissman, University of California San Francisco assistant professor Luke Gilbert, Weissman lab postdoc James Nuñez and collaborators, the method is stable enough to be inherited through hundreds of cell divisions, and is also fully reversible.

“The big story here is we now have a simple tool that can silence the vast majority of genes,” says Weissman, who is also a professor of biology at MIT and an investigator with the Howard Hughes Medical Institute. “We can do this for multiple genes at the same time without any DNA damage, with great deal of homogeneity, and in a way that can be reversed. It’s a great tool for controlling gene expression.”

The project was partially funded by a 2017 grant from the Defense Advanced Research Projects Agency to create a reversible gene editor. “Fast forward four years [from the initial grant], and CRISPRoff finally works as envisioned in a science fiction way,” says co-senior author Gilbert. “It’s exciting to see it work so well in practice.”

Genetic engineering 2.0

The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9 found in bacterial immune systems. The system can be targeted to specific genes in human cells using a single guide RNA, where the Cas9 proteins create tiny breaks in the DNA strand. Then the cell’s existing repair machinery patches up the holes.

Because these methods alter the underlying DNA sequence, they are permanent. Plus, their reliance on “in-house” cellular repair mechanisms means it is hard to limit the outcome to a single desired change. “As beautiful as CRISPR-Cas9 is, it hands off the repair to natural cellular processes, which are complex and multifaceted,” Weissman says. “It’s very hard to control the outcomes.”

That’s where the researchers saw an opportunity for a different kind of gene editor — one that didn’t alter the DNA sequences themselves, but changed the way they were read in the cell.

This sort of modification is what scientists call “epigenetic” — genes may be silenced or activated based on chemical changes to the DNA strand. Problems with a cell’s epigenetics are responsible for many human diseases such as Fragile X syndrome and various cancers, and can be passed down through generations.

Epigenetic gene silencing often works through methylation — the addition of chemical tags to to certain places in the DNA strand — which causes the DNA to become inaccessible to RNA polymerase, the enzyme which reads the genetic information in the DNA sequence into messenger RNA transcripts, which can ultimately be the blueprints for proteins.

Weissman and collaborators had previously created two other epigenetic editors called CRISPRi and CRISPRa — but both of these came with a caveat. In order for them to work in cells, the cells had to be continually expressing artificial proteins to maintain the changes.

“With this new CRISPRoff technology, you can [express a protein briefly] to write a program that’s remembered and carried out indefinitely by the cell,” says Gilbert. “It changes the game so now you’re basically writing a change that is passed down through cell divisions — in some ways we can learn to create a version 2.0 of CRISPR-Cas9 that is safer and just as effective, and can do all these other things as well.”

Building the switch

To build an epigenetic editor that could mimic natural DNA methylation, the researchers created a tiny protein machine that, guided by small RNAs, can tack methyl groups onto specific spots on the strand. These methylated genes are then “silenced,” or turned off, hence the name CRISPRoff.

Because the method does not alter the sequence of the DNA strand, the researchers can reverse the silencing effect using enzymes that remove methyl groups, a method they called CRISPRon.

As they tested CRISPRoff in different conditions, the researchers discovered a few interesting features of the new system. For one thing, they could target the method to the vast majority of genes in the human genome — and it worked not just for the genes themselves, but also for other regions of DNA that control gene expression but do not code for proteins. “That was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes,” says first author Nuñez.

Also, surprisingly to the researchers, CRISPRoff was even able to silence genes that did not have large methylated regions called CpG islands, which had previously been thought necessary to any DNA methylation mechanism.

“What was thought before this work was that the 30 percent of genes that do not have a CpG island were not controlled by DNA methylation,” Gilbert says. “But our work clearly shows that you don’t require a CpG island to turn genes off by methylation. That, to me, was a major surprise.”

CRISPRoff in research and therapy

To investigate the potential of CRISPRoff for practical applications, the scientists tested the method in induced pluripotent stem cells. These are cells that can turn into countless cell types in the body depending on the cocktail of molecules they are exposed to, and thus are powerful models for studying the development and function of particular cell types.

The researchers chose a gene to silence in the stem cells, and then induced them to turn into nerve cells called neurons. When they looked for the same gene in the neurons, they discovered that it had remained silenced in 90 percent of the cells, revealing that cells retain a memory of epigenetic modifications made by the CRISPRoff system even as they change cell type.

They also selected one gene to use as an example of how CRISPRoff might be applied to therapeutics: the gene that codes for Tau protein, which is implicated in Alzheimer’s disease. After testing the method in neurons, they were able to show that using CRISPRoff could be used to turn Tau expression down, although not entirely off.  “What we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed,” Weissman says. “The question is, then, how do you deliver this to an adult? And would it really be enough to impact Alzheimer’s? Those are big open questions, especially the latter.”

Even if CRISPRoff does not lead to Alzheimer’s therapies, there are many other conditions it could potentially be applied to. And while delivery to specific tissues remains a challenge for gene editing technologies such as CRISPRoff, “we showed that you can deliver it transiently as a DNA or as an RNA, the same technology that’s the basis of the Moderna and BioNTech coronavirus vaccine,” Weissman says.

Weissman, Gilbert, and collaborators are enthusiastic about the potential of CRISPRoff for research as well.  “Since we now can sort of silence any part of the genome that we want, it’s a great tool for exploring the function of the genome,” Weissman says.

Plus, having a reliable system to alter a cell’s epigenetics could help researchers learn the mechanisms by which epigenetic modifications are passed down through cell divisions. “I think our tool really allows us to begin to study the mechanism of heritability, especially epigenetic heritability, which is a huge question in the biomedical sciences,” Nuñez says.

“Selfish” DNA helps bacteria cheat and grow in densely-packed microbial communities
Raleigh McElvery
March 12, 2021

Scientists have a term for genes that spread themselves throughout a population at any cost: “selfish” DNA. One way that these genes transmit through bacterial communities is via a type of bacterial sex called conjugation. When one bacterium makes contact with another, DNA from the host cell can be injected into a recipient cell.

Alan Grossman’s lab at the MIT Department of Biology studies a small but selfish chunk of DNA called ICEBs1. His group has identified several ways in which this so-called mobile genetic element actually benefits its host bacterium as it fights to spread. Building off this body of work, Grossman’s lab collaborated with colleagues at Tel Aviv University on a new study recently published in eLife. The international team found that ICEBs1 contains one gene in particular, which allows the host cell to continue dividing in densely-packed microbial communities. This helps the host to grow in conditions where nutrients are scarce, while also potentially helping ICEBs1 to propagate.

“Mobile genetic elements like ICEBs1 are found in the chromosomes of many different types of bacteria,” says Grossman, department head and co-senior author on the study. “Studying these elements — how they spread and how they affect their host cells — is critical for understanding the evolution of bacteria, engineering some types of bacteria to do useful things, and possibly preventing the deleterious effects caused by harmful bacteria.”

Like many DNA segments on the move, ICEBs1 includes genes that encode the molecular machinery required to transfer itself from one cell to the next. But mobile genetic elements can also contain “cargo” genes that bestow the host bacterium with new traits, such as antibiotic resistance. However, in many cases, the properties a cargo gene will endow are hard to predict.

“The host cell can get a lot of new genes in a hurry through mobile genetic elements like ICEBs1, and there’s a lot we still don’t know about the types of phenotypes cargo genes confer,” says the study’s first author, Joshua Jones PhD ’20. “The array of possible traits is probably a lot more diverse than we currently appreciate.”

To investigate the changes that ICEBs1 triggers in the host cell, Jones and colleagues examined large microbial communities called biofilms. These form when many bacteria aggregate on a surface and secrete a slimy “glue” made of sugar, proteins, and DNA that encases the population. Common examples of biofilms include dental plaque, the sludge that coats the inside of pipes, or the deleterious infections that form on surgical implants in patients’ bodies.

Because there are so many bacteria in close contact, biofilms are hot spots for exchanging mobile genetic elements like ICEBs1. However, secreting the materials needed to produce the slimy glue can rapidly deplete resources. As a result, bacteria in a biofilm do not always have the capacity to grow, divide, and potentially spread ICEBs1. Instead, certain types of rod-shaped bacteria begin to produce spores that are analogous to plant seeds. This process, called sporulation, enables these bacteria to become dormant and survive extreme conditions.

Jones found that Bacillus subtilis bacteria containing ICEBs1 were delayed in contributing to the biofilm glue, and also delayed in producing dormant spores. As a result, these bacteria could continue dividing for longer than bacteria without ICEBs1 — increasing the number of bacteria with ICEBs1 and the likelihood that ICEBs1 would spread. The researchers were able to pinpoint one ICEBs1 cargo gene in particular, called Development Inhibitor (devI), that triggered this delay in both biofilm development and sporulation.

“In a way, the cells with ICEBs1 are ‘cheating’ by delaying sporulation and not contributing to the greater good of the biofilm community,” Jones says. But, he explains, they can get away with it because the devI pathway only initiates when ICEBs1-containing cells are the minority in a microbial population. In order to spread as widely as possible, it’s best for ICEBs1 to transfer to new cells that don’t already contain existing copies. Furthermore, accumulating duplicate copies can have detrimental effects on ICEBs1 itself.

“It’s a very clever system for assessing the situation around the cell, and deciding whether it’s worthwhile for ICEBs1 to attempt to transfer,” Jones adds.

Next, the Grossman lab plans to determine precisely how devI exerts its effects on biofilm formation and sporulation. They suspect that other ICEBs1-like elements may also use genes analogous to devI to execute similar propagation strategies. Probing such “cheating” tactics orchestrated by selfish genes will help scientists better understand microbial evolution and, eventually, perhaps even inspire drugs to disrupt harmful biofilms, like those that form around surgical implants.

Members of MIT Biology came together with alumni, industry representatives, and supporters to review the department’s challenges and accomplishments.

March 9, 2021
Study reveals how egg cells get so big

Oocyte growth relies on physical phenomena that drive smaller cells to dump their contents into a larger cell.

Anne Trafton | MIT News Office
March 10, 2021

Egg cells are by far the largest cells produced by most organisms. In humans, they are several times larger than a typical body cell and about 10,000 times larger than sperm cells.

There’s a reason why egg cells, or oocytes, are so big: They need to accumulate enough nutrients to support a growing embryo after fertilization, plus mitochondria to power all of that growth. However, biologists don’t yet understand the full picture of how egg cells become so large.

A new study in fruit flies, by a team of MIT biologists and mathematicians, reveals that the process through which the oocyte grows significantly and rapidly before fertilization relies on physical phenomena analogous to the exchange of gases between balloons of different sizes. Specifically, the researchers showed that “nurse cells” surrounding the much larger oocyte dump their contents into the larger cell, just as air flows from a smaller balloon into a larger one when they are connected by small tubes in an experimental setup.

“The study shows how physics and biology come together, and how nature can use physical processes to create this robust mechanism,” says Jörn Dunkel, an MIT associate professor of physical applied mathematics. “If you want to develop as an embryo, one of the goals is to make things very reproducible, and physics provides a very robust way of achieving certain transport processes.”

Dunkel and Adam Martin, an MIT associate professor of biology, are the senior authors of the paper, which appears this week in the Proceedings of the National Academy of Sciences. The study’s lead authors are postdoc Jasmin Imran Alsous and graduate student Nicolas Romeo. Jonathan Jackson, a Harvard University graduate student, and Frank Mason, a research assistant professor at Vanderbilt University School of Medicine, are also authors of the paper.

A physical process

In female fruit flies, eggs develop within cell clusters known as cysts. An immature oocyte undergoes four cycles of cell division to produce one egg cell and 15 nurse cells. However, the cell separation is incomplete, and each cell remains connected to the others by narrow channels that act as valves that allow material to pass between cells.

Members of Martin’s lab began studying this process because of their longstanding interest in myosin, a class of proteins that can act as motors and help muscle cells contract. Imran Alsous performed high-resolution, live imaging of egg formation in fruit flies and found that myosin does indeed play a role, but only in the second phase of the transport process. During the earliest phase, the researchers were puzzled to see that the cells did not appear to be increasing their contractility at all, suggesting that a mechanism other than “squeezing” was initiating the transport.

“The two phases are strikingly obvious,” Martin says. “After we saw this, we were mystified, because there’s really not a change in myosin associated with the onset of this process, which is what we were expecting to see.”

cluster of cells

Martin and his lab then joined forces with Dunkel, who studies the physics of soft surfaces and flowing matter. Dunkel and Romeo wondered if the cells might be behaving the same way that balloons of different sizes behave when they are connected. While one might expect that the larger balloon would leak air to the smaller until they are the same size, what actually happens is that air flows from the smaller to the larger.

This happens because the smaller balloon, which has greater curvature, experiences more surface tension, and therefore higher pressure, than the larger balloon. Air is therefore forced out of the smaller balloon and into the larger one. “It’s counterintuitive, but it’s a very robust process,” Dunkel says.

Adapting mathematical equations that had already been derived to explain this “two-balloon effect,” the researchers came up with a model that describes how cell contents are transferred from the 15 small nurse cells to the large oocyte, based on their sizes and their connections to each other. The nurse cells in the layer closest to the oocyte transfer their contents first, followed by the cells in more distant layers.

“After I spent some time building a more complicated model to explain the 16-cell problem, we realized that the simulation of the simpler 16-balloon system looked very much like the 16-cell network. It is surprising to see that such counterintuitive but mathematically simple ideas describe the process so well,” Romeo says.

The first phase of nurse cell dumping appears to coincide with when the channels connecting the cells become large enough for cytoplasm to move through them. Once the nurse cells shrink to about 25 percent of their original size, leaving them only slightly larger than their nuclei, the second phase of the process is triggered and myosin contractions force the remaining contents of the nurse cells into the egg cell.

“In the first part of the process, there’s very little squeezing going on, and the cells just shrink uniformly. Then this second process kicks in toward the end where you start to get more active squeezing, or peristalsis-like deformations of the cell, that complete the dumping process,” Martin says.

Cell cooperation

The findings demonstrate how cells can coordinate their behavior, using both biological and physical mechanisms, to bring about tissue-level behavior, Imran Alsous says.

“Here, you have several nurse cells whose job it is to nurse the future egg cell, and to do so, these cells appear to transport their contents in a coordinated and directional manner to the oocyte,” she says.

Oocyte and early embryonic development in fruit flies and other invertebrates bears some similarities to those of mammals, but it’s unknown if the same mechanism of egg cell growth might be seen in humans or other mammals, the researchers say.

“There’s evidence in mice that the oocyte develops as a cyst with other interconnected cells, and that there is some transport between them, but we don’t know if the mechanisms that we’re seeing here operate in mammals,” Martin says.

The researchers are now studying what triggers the second, myosin-powered phase of the dumping process to start. They are also investigating how changes to the original sizes of the nurse cells might affect egg formation.

The research was funded by the National Institute of General Medical Sciences, a Complex Systems Scholar Award from the James S. McDonnell Foundation, and the Robert E. Collins Distinguished Scholarship Fund.

Cells are known by the company they keep
Eva Frederick
March 2, 2021

In the paper, published online March 1 in the journal Cell Metabolism, researchers at Whitehead Institute and the Morgridge Institute for Research performed CRISPR-based genetic screens of cells cultured in either traditional media or a new physiologic medium previously designed in the Sabatini Lab at Whitehead Institute designed to more closely reflect the nutrient composition of human blood. The screen revealed that different genes became essential for survival and reproduction in the various conditions.

“This work underscores the importance of using more human-like, physiologically relevant media for culturing human cancer cell lines,” said Whitehead Institute Member and co-senior author David Sabatini, who is also a professor of biology at the Massachusetts Institute of Technology and an investigator of the Howard Hughes Medical Institute. “The information we can learn from screens in human plasma-like media — or media designed to mimic other fluids throughout the body — may inform new therapeutic methods down the line.”

The widespread use of a human plasma-like medium could open the door for many researchers to conduct experiments in the lab that could have more relevance to human disease, but without complicated methods or prohibitive costs.

“Medium composition is both relatively accessible and quite flexible,” said co-senior author Jason Cantor, an Investigator at the Morgridge Institute for Research and an assistant professor of biochemistry at the University of Wisconsin-Madison, and a former postdoc in Sabatini’s lab. “Not all researchers have access to specialized tissue culture incubators, nor can everyone easily pursue some of the more complex 3D and co-culture methods, but most can get their hands on a bottle of media.”

The big screen

The idea that different environmental conditions may lead to different genes being essential is not a new one. “People have done this in microorganisms, and shown that if you throw [bacteria] into different growth conditions — the contributions of different genes to cell fitness can change,” Cantor said.

With this reasoning in mind — that medium composition could affect which genes become necessary for human cells to grow — the researchers set up screens to identify essential genes in a single leukemia cell line in different kinds of culture media. One batch was grown in a traditional medium, and another cultured in the lab’s new medium called Human Plasma-Like Medium, or HPLM, which has a metabolic composition more reflective of that in human blood.

The approach they used — called a CRISPR screen —  takes advantage of CRISPR-Cas9 gene editing technology to systematically snip and knock out genes across the genome, with the goal of creating a population of cells in which every possible gene knockout is represented. The expression of some genes is essential to survival, and cells grow substantially slower or die when those genes are deleted. Other cells may have trouble functioning, and some may grow even faster. Once the pooled cells have had a chance to grow and proliferate, researchers sequence the genetic material of the entire population to determine which genes were critical for survival within the given screen.

Once they completed the initial screens, the researchers identified hundreds of genes that were conditionally essential — that is, necessary for cell growth in one medium versus another. Interestingly, these medium-dependent essential genes collectively had roles in a number of different biological processes.

To determine how much these genes were dependent on the type of cells studied, the researchers then ran similar screens across a panel of human blood cancer cell lines, and then pursued follow-up work to understand why certain genes were identified as conditionally essential.

Ultimately, they uncovered the underlying gene-nutrient interactions, and specifically for these hit genes, traced the effects to availability of certain metabolites — the nutrients and small molecules necessary for metabolism — that are uniquely defined in HPLM versus the traditional media.

The next steps

CRISPR screens can help scientists identify potential drug targets and map out important cellular interactions to inform cancer therapies. “There are so many ways that people use CRISPR screens right now,” said Cantor. “What this study is showing is that the availability metabolites can have a major impact on which genes are important for cell growth, and so I think there are a lot of implications here in terms of how these types of screens could be performed in the future in order to potentially increase the fidelity of what we see in the lab and what might happen in the body.”

The research also establishes more nuanced relationships between cells’ genes and their environment. “What this allows us to do down the line, theoretically, is to tune how important a gene is — how important the encoded protein is — by manipulating metabolite levels in the blood,” said Cantor. “That’s one of our bigger-picture ideas.”

In the future, these relationships could inform cancer treatments. For example, if scientists could “tune” the importance of a specific gene for cancer cell growth, then the protein encoded by that gene could become a more promising drug target — in effect, tricking cancer cells into revealing possible context-dependent vulnerabilities. “The idea of targeting metabolites to treat cancer isn’t itself new — in fact, it [underlies] a well-established anti-cancer therapeutic enzyme still in use today — but I think our work maybe enables us to look for ways to couple this type of approach with other targeted therapies.”

“At our core, we are a basic cell biology lab,” added Nicholas Rossiter, a technician in Cantor’s lab and the first author of the study. “But whenever you’re studying basic cell biology, there’s the potential to translate it into therapeutic strategy. Our plan is just to keep on chugging along in our lab and studying how exactly cell biology can be influenced by these environmental factors. We do the basics, and then there will hopefully be some auspicious findings that can be carried on into therapeutics.”