Research Area: Genetics

CRISPR-based approach reveals Achilles’ heels of a common herpesvirus
Eva Frederick | Whitehead Institute
October 25, 2021

Many people — around half of the adult population — are infected with a type of herpesvirus called human cytomegalovirus, or HCMV. Though mostly asymptomatic, the virus can be dangerous for immunocompromised people and unborn babies. Because HCMV is so widespread, the chance of a baby becoming infected in utero is around one in 200, and that infection can lead to problems with the baby’s brain, lungs and growth.

In a new paper from Whitehead Institute Member Jonathan Weissman published on October 25 in Nature Biotechnology, Weissman and colleagues turn cutting-edge CRISPR and single cell sequencing technologies on this virus, providing the most detailed picture yet on how viral and human genes interact to create an HCMV infection — and revealing new ways to potentially derail the virus’ progression through manipulating viral and host genes.

The research could provide an important road map for future studies of host-pathogen interactions, as well as inform antiviral drug design. Over the course of the project, the researchers generated a list of both viral and host genes that were either essential for the virus to replicate, or could potentially be manipulated to confer some immunity to the host cell. “Now that we have this list, we have a list of potential targets that one might now go ahead and develop drugs against,” said Marco Hein, the first author and a former postdoctoral researcher in the Weissman Lab.

Seeing both sides 

Millions of years of evolution have created a complex web of interactions between virus and host. For example, viruses have their own set of genes, but they also depend on some human genes, called host factors. Hijacking these host factors allows the viruses to invade cells in the body and replicate their own genetic material.

Hein, who is now a researcher at the Chan Zuckerberg Biohub in San Francisco, and Whitehead Institute Member Jonathan Weissman, who is also a professor of biology at the Massachusetts Institute of Technology and Koch Institute and an investigator of the Howard Hughes Medical Institute, sought to gain a more thorough understanding of the web of host-viral interactions that arises throughout the course of an infection. “[We wanted to know] what actually happens when we [knock out or weaken] those critical factors,” Hein said. “Can we prevent infection? If so, what ‘goes wrong’ from the perspective of the virus?”

They chose HCMV as a test subject because, for one thing, the virus has a double-stranded DNA genome like humans. That means that CRISPR technologies that work by snipping DNA could theoretically work for both the virus and the host. “And because CMV is an important human pathogen and it’s such a complex and intricate virus, we thought we would have a chance to really discover something new,” said Weissman.

A series of screens 

The researchers first set out, using a molecular technique called CRISPR screening, to determine whether any regions of the viral or host genomes in particular had an impact on the fate of infected host cells. By systematically knocking out individual genes in a large population of viruses and host cells, the researchers could then assess how essential each gene was to the infection.

The project took on a new dimension in 2016 with the development of accessible, large-scale single cell sequencing. “We had this idea to put together the CRISPR screening and the single cell sequencing, and [a screening method called PerturbSeq],” Hein said. “Basically, you perturb genes in a cell population and then you read out what happens to the cells, not just by measuring survival, but by actually looking at the pattern of gene expression in those cells over time.”

Combining these methods generated a huge set of data, which provided the researchers with a clearer view of which genes were important and when. “The single cell sequencing lets us watch the steps in the viral life cycle with much higher precision, and then the perturbation lets us understand how host and viral factors allow the virus to manipulate the host and complete its life cycle,” Weissman said.

The resultant data showed how the virus’ typical trajectory — from the initial waves of viral gene expression, to replication of the full viral genome, to the final step of budding off into newly-formed virions —  could be derailed by altering specific viral genes. It also clued the researchers in to which host genes the virus depended on at what stages for a ‘successful’ infection.

“The course of infection is pre-programmed into the viral genome,” Hein said. “If you want to interfere with the course of infection you can do that by targeting a viral factor, or you can do it indirectly by targeting the host factor. And the outcomes are conceptually different. If you target a virus factor you derail the program that the virus would normally follow. If you target a host factor, the program itself is unchanged, but you change how far the virus gets in executing the program.”

These findings will be useful tools for the development of drugs that can be used as part of an antiviral “cocktail.” Because viruses and other pathogens are living creatures that can mutate and adapt to changing conditions, a common thread among antiviral treatments involves combining several drugs with different viral targets. This ensures the most complete eradication of viruses possible, reducing the chance that some will survive and create a new resistant population.

While the researchers’ list of essential viral genes provide parts of HCMV to target with drug cocktails, the list of contributing human genes could open the door for a more indirect therapy. “If you target a host factor to affect the virus, it’s much more difficult for the virus to escape because it can’t just mutate so the drug doesn’t bind anymore — it would have to mutate away from dependency on a host protein, which is much more complicated,” Hein said.

Of course, there are drawbacks to potentially targeting a human gene or protein to treat an infection, and much more work would need to be done for a viable treatment to emerge via this avenue of research. “If you target a host factor, you’re by definition targeting a protein that’s in our body, doing its normal job, so the risk of side effects is much higher,” Hein said.

Few drugs like this have made it past clinical trials; one famous example is hydroxychloroquine, which has been used successfully to treat malaria, and unsuccessfully to treat COVID-19.

In the future, Hein and Weissman hope to turn their multi-level approach for studying infection toward other viruses such as SARS-CoV-2. Although the novel coronavirus does not have double-stranded DNA that can be altered via CRISPR, the researchers can still investigate which host genes are essential at what stage of infection, and use their methods-driven approach to hopefully glean unexpected findings from a well-studied virus.

“I’m always driven by what technology can do,” Hein said. “I like to run a study in a systems-wide manner and then come up with some findings that you would have not found if you had only looked at one gene or protein at a time or looked at things more in the conventional way. This kind of high-level conclusion is what I personally always find the most exciting.”

New CRISPR-based screening method improves gene editors
Eva Frederick | Whitehead Institute
October 24, 2021

Gene editing methods often involve breaking a strand of DNA in order to make specific changes to the sequence. They then rely on the cell’s DNA repair pathways to mend the break. These cellular repair pathways, however, are not completely understood, and introduce an element of chance to gene editing; for example, a repair mechanism may patch up the edited strand, but also leave behind an unwanted mutation.

In a paper published online in Cell on October 20, 2021, a collaborative team of researchers in the lab of Whitehead Institute Member, Massachusetts Institute of Technology (MIT) biology professor and Howard Hughes Medical Institute Investigator Jonathan Weissman, the lab of Britt Adamson at Princeton University, and Cecilia Cotta-Ramusino, then a research scientist at Editas Medicine (now at Tessera Therapeutics), present a new experimental method that could help bridge this gap in our understanding.

The method, called Repair-seq, allows researchers to find out which genes and genetic pathways are involved in DNA repair mechanisms. It provides a useful tool for fundamental research on gene repair, as well as a way to test the action of new genome editing methods as they are developed, says Weissman Lab postdoc and first author Jeffrey Hussmann. A companion paper published concurrently in Cell in collaboration with researchers at the Broad Institute of MIT and Harvard provides a glimpse into the utility of Repair-seq when applied to new gene editing technologies.

“The field of gene editing has moved so quickly and people have been so creative developing new methods that our ability to apply them has dramatically outpaced our understanding of exactly how they work,” Hussmann said. “We think that Repair-seq will be a valuable tool going forward so that as new editing methods are developed, we can quickly do a better job of characterizing how they interact with different repair mechanisms.”

Studying repair mechanisms in one fell swoop

Cells have several different methods they use to repair breaks in DNA strands, and the path to any one method depends on a tree of decisions based on the circumstances. “Over decades, a huge number of people have worked out parts of these pathways  through focused experiments,” said Adamson, a senior author on both papers and an assistant professor at Princeton University.

Together, the team of researchers saw an opportunity to harness existing CRISPR-based methods to take a broad look at repair pathways in the cell. The method they created combines several CRISPR-based technologies. First, the researchers used a method they previously developed called CRISPRi to inactivate hundreds of genes known to be involved in DNA repair across a cell population. Then, they induced double strand breaks in the cells’ DNA at specific places that the cell would need to heal.

As the cells mended the breaks, the researchers used targeted sequencing to examine the ‘repair outcomes’ — mutations or the lack thereof — in the DNA strand resulting from different methods of repair. Finally, they were able to extrapolate which genes were essential to various repair mechanisms and how they were involved in producing or preventing each type of resulting mutation. They also posted their data online in an interactive format so others can use it to investigate DNA repair genes and pathways.

“This combination of different CRISPR-based technologies has made it possible to, in one fell swoop, recapitulate a lot of the work that was done painstakingly over the past decades to study each repair pathway one at a time,” Hussmann said. “The high-level view of repair that our method produces shows us many of the things that people saw before, and at the same time reveals unexpected connections that we only get by having the comprehensive picture.”

These unforeseen relationships between repair genes may help fundamental researchers refine the decision tree of double strand break repair in the future, said Weissman. “One of the big themes that’s come out of this is that outcomes that superficially look similar can actually have very different mechanisms,” Weissman said.

A ‘prime’ example of Repair-seq’s utility 

As the team was developing their Repair-seq methodology, Broad Institute of MIT and Harvard Core Member David Liu’s lab was working on prime editing, a gene editing method that promises more precise control over genetic outcomes than traditional CRISPR methods. Instead of snipping both strands of DNA’s double helix, prime editing makes a ‘nick’ in only one of the strands and introduces a short sequence template containing the desired genetic change.

“When Liu’s group came out with prime editing, our Repair-seq team realized that we had the perfect tool for quickly trying to understand exactly how it was working,” Hussmann said.

The three labs collaborated to use Repair-seq to identify which pathways were at play during the installation of mutations by prime editing, and identified one in particular, called the DNA mismatch repair pathway, that seemed to be interfering with the efficiency and accuracy of the method. When the researchers inhibited this pathway, the performance of Liu’s prime editing technique greatly improved.

“Working with Britt, Jonathan, and their labs has been a beautiful integration of basic science, tool application, and technology development—a real testament to the power of multidisciplinary collaboration” said Liu, also an Investigator of the Howard Hughes Medical Institute.

The researchers also applied Repair-seq to a base editor — a tool to swap specific bases in a DNA sequence — and were able to illuminate the DNA repair genes involved in swapping in particular base.

In the future, the researchers plan to continue adapting the method to new sequencing methods and applying it to new editors as they are developed. “We think Repair-seq is a really practical way of making better genome editors,” Weissman said.

“It has been rewarding to see the efforts of our collaboration come together,” said Adamson. “We hope the insights from our study and tools that those insights have led to will be widely useful to the research community.”

So-called “junk” DNA plays a key role in speciation
Eva Frederick | Whitehead Institute
August 23, 2021

More than 10 percent of our genome is made up of repetitive, seemingly nonsensical stretches of genetic material called satellite DNA that do not code for any proteins. In the past, some scientists have referred to this DNA as “genomic junk.”

Over a series of papers spanning several years, however, Whitehead Institute Member Yukiko Yamashita and colleagues have made the case that satellite DNA is not junk, but instead has an essential role in the cell: it works with cellular proteins to keep all of a cell’s individual chromosomes together in a single nucleus.

Now, in the latest installment of their work, published online July 24 in the journal Molecular Biology and Evolution, Yamashita and former postdoctoral fellow Madhav Jagannathan, currently an assistant professor at ETH Zurich, Switzerland, take these studies a step further, proposing that the system of chromosomal organization made possible by satellite DNA is one reason that organisms from different species cannot produce viable offspring.

“Seven or eight years ago when we decided we wanted to study satellite DNA, we had zero plans to study evolution,” said Yamashita, who is also a professor of biology at the Massachusetts Institute of Technology and an investigator with the Howard Hughes Medical Institute. “This is one very fun part of doing science: when you don’t have a preconceived idea, and you just follow the lead until you bump into something completely unexpected.”

The origin of species: DNA edition 

Researchers have known for years that satellite DNA is highly variable between species. “If you look at the chimpanzee genome and the human genome, the protein coding regions are, like, 98 percent, 99 percent identical,” she says. “But the junk DNA part is very, very different.”

“These are about the most rapidly evolving sequences in the genome, but the prior perspective has been, ‘Well, these are junk sequences, who cares if your junk is different from mine?’” said Jagannathan.

But as they were investigating the importance of satellite DNA for fertility and survival in pure species, Yamashita and Jagannathan had their first hint that these repetitive sequences might play a role in speciation.

When the researchers deleted a protein called Prod that binds to a specific satellite DNA sequence in the fruit fly Drosophila melanogaster, the flies’ chromosomes scattered outside of the nucleus into tiny globs of cellular material called micronuclei, and the flies died. “But we realized at this point that this [piece of] satellite DNA that was bound by the Prod protein was completely missing in the nearest relatives of Drosophila melanogaster,” Jagannathan said. “It completely doesn’t exist. So that’s an interesting little problem.”

If that piece of satellite DNA was essential for survival in one species but missing from another, it could imply that the two species of flies had evolved different satellite DNA sequences for the same role over time.  And since satellite DNA played a role in keeping all the chromosomes together, Yamashita and Jagannathan wondered whether these evolved differences could be one reason different species are reproductively incompatible.

“After we realized the function [of satellite DNA in the cell], the fact that satellite DNA is quite different between species really hit like lightning,” Yamashita said. “All of a sudden, it became a completely different investigation.”

A tale of two fruit fly species

To study how satellite DNA differences might underlie reproductive incompatibility, the researchers decided to focus on two branches of the fruit fly family tree: the classic lab model Drosophila melanogaster, and its closest relative, Drosophila simulans. These two species diverged from each other two to three million years ago.

Researchers can breed a Drosophila melanogaster female to a Drosophila simulans male, “but [the cross] generates very unhappy offspring,” Yamashita said. “Either they’re sterile or they die.”

Yamashita and Jagannathan bred the flies together, then studied the tissues of the offspring to see what was leading these “unhappy” hybrids to drop like flies. Right away they noticed something interesting: “When we looked at those hybrid tissues, it was very clear that their phenotype was exactly the same as if you had disrupted the satellite DNA [-mediated chromosomal organization] of a pure species,” Yamashita said. “The chromosomes were scattered, and not encapsulated in a single nucleus.”

Furthermore, the researchers could create a healthy hybrid fly by mutating certain genes in the parent flies called “hybrid incompatibility genes,” which have been shown to localize to satellite DNA in the cells of pure species.  Via these experiments, the researchers were able to demonstrate how these genes affect chromosomal packaging in hybrids, and pinpoint the cellular phenotypes associated with them for the first time. “I think for me, that is probably the most critical part of this paper,” Jagannathan said.

Taken together, these findings suggest that because satellite DNA mutates relatively frequently, the proteins that bind the satellite DNA and keep chromosomes together must evolve to keep up, leading each species to develop their own “strategy” for working with the satellite DNA. When two organisms with different strategies interbreed, a clash occurs, leading the chromosomes to scatter outside of the nucleus.

In future studies, Yamashita and Jagannathan hope to put their model to the ultimate test: if they can design a protein that can bind the satellite DNA of two different species and hold the chromosomes together, they could theoretically ‘rescue’ a doomed hybrid, allowing it to survive and produce viable offspring.

This feat of bioengineering is likely years off. “Right now it’s just a pure conceptual thing,” Yamashita said. “In doing this tinkering, there’s probably a lot of specifics that will have to be solved.”

For now, the researchers plan to continue investigating the roles of satellite DNA in the cell, armed with their new knowledge of the part it plays in speciation. “To me, the surprising part of this paper is that our hypothesis was correct,” Jagannathan said. “I mean, in retrospect, there are so many ways things could have been inconsistent with what we hypothesized, so it’s kind of amazing that we’ve sort of been able to chart a clear path from start to finish.”

Rewiring cell division to make eggs and sperm
Whitehead Institute
July 30, 2021

To create eggs and sperm, cells must rewire the process of cell division. Mitosis, the common type of cell division that our bodies use to grow everything from organs to fingernails and to replace aging cells, produces two daughter cells with the same number of chromosomes and approximately the same DNA sequence as the original cell. Meiosis, the specialized cell division that makes egg and sperm in two rounds of cell division, creates four granddaughter cells with new variations in their DNA sequence and half as many chromosomes in each cell. Meiosis uses most of the same cellular machinery as mitosis to achieve this very different outcome; only a few key molecular players prompt the rewiring from one type of division to another. One such key player is the protein Meikin, which is found exclusively in cells undergoing meiosis.

New research from Whitehead Institute Member Iain Cheeseman, graduate student Nolan Maier and collaborators Professor Michael Lampson and senior research scientist Jun Ma at the University of Pennsylvania demonstrates how Meikin is elegantly controlled, and sheds light on how the protein acts to serve multiple roles over different stages of meiosis. The findings, which appear in Developmental Cell on July 30, reveal that Meikin is precisely cut in half midway through meiosis. Instead of this destroying the protein, one half of the molecule, known as C-Meikin, goes on to play a critical role as a previously hidden protein actor in meiosis.

“Cells have this fundamental process, mitosis, during which they have to divide chromosomes evenly or it will cause serious problems like cancer, so the system has to be very robust,” Maier says. “What’s incredible is that you can add one or two unique meiotic proteins like Meikin and dramatically change the whole system very quickly.”

Helping chromosomes stick together

During both mitosis and meiosis, sister chromatids — copies of the same chromosome — pair up to form the familiar “X” shape that we recognize as a chromosome. In mitosis, each chromatid—each half of the X — is connected to a sort of cellular fishing line and these lines reel the chromatids to opposite ends of the cell, where the two new cells are formed around them. However, in the first round of division in meiosis, the sister chromatids stick together, and one whole “X” is reeled into each new cell. Meikin helps to achieve this different outcome by ensuring that, while the chromosomes are being unstuck from each other in preparation for being pulled apart, each pair of sister chromatids stays glued together in the right place. Meikin also helps ensure that certain cellular machinery on the sister chromatids is fused so that they will connect to the same line and be reeled together to the same side of the cell.

More specifically, when chromosomes are first paired up, they are glued together by adhesive molecules in three regions: the centromere, or center of the X, where Meikin localizes; the region around the center; and the arms of the X. In the first round of meiosis, Meikin helps to keep the glue in the region around the center intact, so the sister chromatids will stick together. Simultaneously, Meikin helps to prime the center region to be unglued, while a separate process unglues the arms. This ungluing allows the chromosomes to separate and be prepared for later stages of meiosis.

Cheeseman and Maier initially predicted that Meikin’s role ended after meiosis I, the first round of meiotic cell division. In meiosis II, the second round of cell division, the cells being created should end up with only one sister chromatid each, and so the chromatids must not be kept glued together. Maier found that near the end of meiosis I, Meikin is cleaved in two by an enzyme called Separase, the same molecule that cleaves the adhesive molecules gluing together the chromosomes. At first, this cleavage seemed like the end of Meikin and the end of this story.
A hidden role for a hidden proteinHowever, unexpectedly, the researchers found that cells lacking Meikin during the second half of meiosis do not divide properly, prompting them to take another look at what happens to Meikin after it gets cleaved. They found that Separase cleaves Meikin at a specific point — carving it with the precision of a surgeon’s scalpel — to create C-Meikin, a previously unknown protein that turns out to be necessary for meiosis II. C-Meikin has many of the same properties as the intact Meikin molecule, but it is just different enough to take on a different role: helping to make sure that the chromosomes align properly before their final division.

“There’s a lot of protein diversity in cells that you would never see if you don’t go looking for it, if you only look at the DNA or RNA. In this case, Separase is creating a completely different protein variant of Meikin than can function differently in meiosis II,” says Cheeseman, who is also a professor of biology at Massachusetts Institute of Technology. “I’m very excited to see what we might discover about other hidden protein forms in cell division.”

Recombining ideas

Answering the question of Meikin’s role and regulation throughout meiosis required a close collaboration and partnership between Maier and Lampson lab researcher Ma – the Lampson lab being experts on studying meiosis using mouse models. Working with mouse oocytes (immature egg cells), Ma was able to reveal the behaviors and critical contributions of Meikin cleavage in meiotic cells in mice. Both labs credit the close exchange with helping them to get a deeper understanding of how cells rewire for meiosis.

“It was a pleasure working together to understand how some of the specialized meiotic functions that are necessary for making healthy eggs and sperm are controlled,” Lampson says.

Finally, once cells have completed these specialized meiotic divisions, the researchers found that it was critical for oocytes to fully eliminate Meikin. The researchers determined that, after meiosis two, C-Meikin is degraded by another molecule (the anaphase-promoting complex or APC/C)—this time for good. With Meikin gone and the rewiring of cell division reversed, eggs and sperm are ready for mitosis; should they fuse and form an embryo, that is the next cell division they will undergo. The researchers note that the way Meikin is regulated by being broken down — first into C-Meikin and then completely — may help cells to organize their timing during meiosis. Breaking apart a protein is an irreversible step that creates a clear demarcation between before and after in a multi-step process.The researchers hope that by uncovering the intricacies of meiosis, they may shed light on what happens when the creation of eggs and sperm goes wrong, and so perhaps contribute to our understanding of infertility. Cheeseman also hopes that by studying how mitotic processes are rewired for meiosis, his lab can gain new insights into the original wiring of mitosis.

A “tail” of two RNA regulatory systems
Greta Friar | Whitehead Institute
July 12, 2021

In new research, published in eLife on July 2, Bartel, who is also a professor of biology at Massachusetts Institute of Technology and an investigator with the Howard Hughes Medical Institute, and Bartel lab member Kehui Xiang, a CRI Irvington Postoctoral Fellow, have now discovered how cells establish this early gene regulatory regime and what conditions prompt a switch as the embryos mature. The researchers have observed the same regulatory switch in fish, frogs, and flies, and because the switch occurs across the animal kingdom, they would expect to see that the mechanism applies in other species including mammals.

“When I joined the lab, they had discovered that egg cells and early embryos had this different regulatory regime, and I wanted to know why,” Xiang says. “There must be fundamental changes to the cell, or to the molecules in the cell, that define this.”

The difference in how mRNAs are regulated during and after early development has to do with the length of their tails. mRNAs have tails made up of strings of adenines, one of the building blocks of RNA. Tail length varies between mRNAs from different genes and even between mRNAs from the same gene. Usually, the length of this “poly(A)-tail” corresponds to how long an mRNA lasts before getting degraded. An mRNA with a long tail is more stable, and will generally last longer. However, researchers had also observed that in some cases mRNA tail length corresponds to how readily an mRNA is used to make protein. Bartel’s earlier research had helped define when each of these connections occurs: mRNA tail length affects translational efficiency only in immature eggs and early embryos, and in other stages, it affects mRNA stability or lifespan.

In their new research, Xiang and Bartel uncovered three conditions that are required for the mRNA regulatory regime that exists in early development.

A competitive environment

The first condition is that there has to be a limited availability of a protein that binds to mRNA tails called cytoplasmic poly(A)-binding protein (PABPC). PABPC is known to help activate the translation of mRNA into protein. It binds to the mRNA tail and—in embryos—helps to increase translational efficiency; the researchers propose that it may do this by promoting a more favorable structure for translation. When PABPC is in limited supply, as it is in early embryos, then short-tailed mRNAs are less likely to bind any of the protein, as they will be outcompeted by long-tailed mRNAs, explaining the correlation between tail length and translational efficiency. Later in development, PABPC is in such ready supply that all of the mRNAs are able to bind at least one, decreasing the competitive edge of long-tailed mRNAs.

Early durability

However, the researchers observed that reducing the amount of PABPC in adult cells so that it becomes limiting in these cells did not cause mRNAs with longer tails to be translated more efficiently, which showed that other conditions must also contribute to early embryos’ unique regulation. The second condition that Xiang identified is that mRNAs must be relatively stable in spite of their inability to compete for PABPC. In adult cells, RNAs without PABPC bound to their tails are very unstable, and so are likely to degrade. If the same were true in early embryos, then the short-tailed mRNAs would degrade quickly because they are outcompeted for binding PABPC, and so one would again see a link between tail length and stability, rather than between tail length and translational efficiency—short-tailed mRNAs would be eliminated rather than poorly translated. However, the processes that would normally degrade mRNAs without PABPC have not yet started occurring in early embryos, allowing the short-tailed mRNAs to survive.

Big fish in a small pond

Finally, Xiang discovered that in order for tail length and translational efficiency to be linked, PABPC has to be able to affect translational efficiency. He found that in adult cells PABPC does not appear to boost translational efficiency the way it does in embryos. The researchers hypothesize that this is because the process of translating mRNAs in adult cells is already so efficient that the small boost from binding PABPC does not make a significant difference. However, in early embryos PABPC is more of a big fish in a small pond. The cells do not have all of the machinery to maximize translational efficiency, so every bit of improvement, such as the benefit of binding PABPC, makes a noticeable difference.

Together, these three conditions enable early eggs and embryos to regulate their mRNA in a unique fashion that can control how much protein is made from each gene without destroying the limited pool of mRNA available. In the future, the researchers hope to recreate the three conditions in non-embryonic cells to confirm that the conditions Xiang identified are not only necessary but also sufficient to cause the switch in regulatory regimes.

“Knowing which function the poly(A)-tail is performing in a specific cell or scenario—providing mRNA stability or translational efficiency—is really critical for understanding how genes are regulated in the different cells,” Bartel says. “And understanding that is important for answering all kinds of questions about cells, from their functions to what can go wrong with them in diseases.”

Siniša Hrvatin

Education

  • PhD, 2013, Harvard University
  • A.B., 2007, Biochemical Sciences, Harvard University

Research Summary

To survive extreme environments, many animals have evolved the ability to profoundly decrease metabolic rate and body temperature and enter states of dormancy, such as torpor and hibernation. Our laboratory studies the mysteries of how animals and their cells initiate, regulate, and survive these adaptations. Specifically, we focus on investigating: 1) how the brain regulates torpor and hibernation, 2) how cells adapt to these states, and 3) whether inducing these states can slow down tissue damage, disease progression, and even aging. Our long-term goal is to explore potential applications of inducing similar states of “suspended animation” in humans.

Awards

  • Warren Alpert Distinguished Scholar, Warren Albert Foundation, 2019
  • NIH Director’s New Innovator Award, 2022
  • Searle Scholar, 2023
  • Pew Scholar, 2023
  • McKnight Scholar, 2024
Francisco J. Sánchez-Rivera

Education

  • PhD, 2016, Biology, MIT
  • BS, 2008, Microbiology, University of Puerto Rico at Mayagüez

Research Summary

The overarching goal of the Sánchez-Rivera laboratory is to elucidate the cellular and molecular mechanisms by which genetic variation shapes normal physiology and disease, particularly in the context of cancer. To do so, we develop and apply genome engineering technologies, genetically-engineered mouse models (GEMMs), and single cell lineage tracing and omics approaches to obtain comprehensive biological pictures of disease evolution at single cell resolution. By doing so, we hope to produce actionable discoveries that could pave the way for better therapeutic strategies to treat cancer and other diseases.

Awards

  • V Foundation Award, 2022
  • Hanna H. Gray Fellowship, Howard Hughes Medical Institute, 2018-2026
  • GMTEC Postdoctoral Researcher Innovation Grant, Memorial Sloan Kettering Cancer Center, 2020-2022
  • 100 inspiring Hispanic/Latinx scientists in America, Cell Mentor/Cell Press, 2020
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.

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
Olivia Corradin

Education

  • PhD, 2015, Case Western Reserve University
  • BS, 2010, Biochemistry, Marquette University

Research Summary

Our lab studies genetic and epigenetic variation that contributes to human disease by disrupting gene expression programs. We utilize biological insights into the mechanisms of gene regulation in order to determine the impact of disease-associated variants on cellular function. We aim to identify actionable insights into disease pathogenesis by studying the confluence of genetic and epigenetic risk factors of human diseases, including multiple sclerosis and opioid use disorder.

Awards

  • NIH Director’s Pioneer Award Program Avenir Award, 2017