Research Area: Genetics

The molecules behind metastasis
Greta Friar | Whitehead Institute
January 4, 2023

Many cancer cells never leave their original tumors. Some cancer cells evolve the ability to migrate to other tissues, but once there cannot manage to form new tumors, and so remain dormant. The deadliest cancer cells are those that can not only migrate to, but also thrive and multiply in distant tissues. These metastatic cancer cells are responsible for most of the deaths associated with cancer. Understanding what enables some cancer cells to metastasize—to spread and form new tumors—is an important goal for researchers, as it will help them develop therapies to prevent or reverse those deadly occurrences.

Past research from Whitehead Institute Member Robert Weinberg and others suggests that cancer cells are best able to form metastatic tumors when the cells are in a particular state called the quasi-mesenchymal (qM) state. New research from Weinberg and Arthur Lambert, once a postdoc in Weinberg’s lab and now an associate director of translational medicine at AstraZeneca, has identified two gene-regulating molecules as important for keeping cancer cells in the qM state. The research, published in the journal Developmental Cell on December 19, shows that these molecules, ΔNp63 and p73, enable breast cancer cells to form new tumors in mice, and illuminates important aspects of how they do so.

Most potent in the middle

Cells enter the qM state by undergoing the epithelial-mesenchymal transition (EMT), a developmental process that can be co-opted by cancer cells. In the EMT, cells transition from an epithelial state through a spectrum of more mesenchymal states, which allows them to become more mobile and aggressive. Cells in the qM state have only transitioned partway through the EMT, becoming more, but not fully, mesenchymal. This middle ground is perfect for metastasis, whereas cells on either end of the spectrum—cells that are excessively epithelial or excessively mesenchymal—lose their metastatic abilities.

Lambert and colleagues wanted to understand more about how cancer stem cells, which can seed metastases and recurrent tumors, remain in a metastasis-prone qM state. They analyzed how gene activity was regulated in those cells and identified two transcription factors—molecules that influence the activity of target genes—as important. One of the transcription factors, ΔNp63, appeared to most directly control cancer stem cells’ ability to maintain a qM state. The other molecule, p73, seemed to have a similar role because it can activate ΔNp63. When either transcription factor was inactivated, the cancer stem cells transitioned to the far end of the EMT spectrum and so were unable to metastasize.

Next, the researchers looked at what genes ΔNp63 regulates in cancer stem cells. They expected to find a pattern of gene regulation resembling what they would see in healthy breast stem cells. Instead they found a pattern closely resembling what one would see in cells involved in wound healing and regeneration. Notably, ΔNp63 stimulates EGFR signaling, which is used in wound healing to promote rapid multiplication of cells.

“Although this is not what we expected to see, it makes a lot of sense because the process of metastasis requires active proliferation,” Lambert says. “Metastatic cancer cells need both the properties of stem cells—such as the ability to self-renew and differentiate into different cell types—and the ability to multiply their numbers to grow new tumors.”

This finding may help to explain why qM cells are so uniquely good at metastasizing. Only in the qM state can the cells strongly stimulate EGFR signaling and so promote their own proliferation.

“This work gives us some mechanistic understanding of what it is about the quasi-mesenchymal state that drives metastatic tumor growth,” says Weinberg, who is also the Daniel K. Ludwig Professor for Cancer Research at the Massachusetts Institute of Technology.

The researchers hope that these insights could eventually contribute to therapies that prevent metastasis. They also hope to pursue further research into the role of ΔNp63. For example, this work illuminated a possible connection between ΔNp63 and the activation of dormant cancer cells, the cells that travel to new tissues but then cannot proliferate after they arrive there. Such dormant cells are viewed as ticking time bombs, as at any point they may reawaken. Lambert hopes that further research may reveal new insights into what causes dormant cancer cells to eventually gain the ability to grow tumors, adding to our understanding of the mechanisms of metastatic cancer.

Notes

Arthur W. Lambert, Christopher Fiore, Yogesh Chutake, Elisha R. Verhaar, Patrick C. Strasser, Mei Wei Chen, Daneyal Farouq, Sunny Das, Xin Li, Elinor Ng Eaton, Yun Zhang, Joana Liu Donaher, Ian Engstrom, Ferenc Reinhardt, Bingbing Yuan, Sumeet Gupta, Bruce Wollison, Matthew Eaton, Brian Bierie, John Carulli, Eric R. Olson, Matthew G. Guenther, Robert A. Weinberg. “ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.” Developmental Cell, Volume 57, Issue 24,
2022, 2714-2730.e8, https://doi.org/10.1016/j.devcel.2022.11.015.

Scientists discover a new way of sharing genetic information in a common ocean microbe

Prochlorococcus, the world’s most abundant photosynthetic organism, reveals a gene-transfer mechanism that may be key to its abundance and diversity.

David L. Chandler | MIT News Office
January 5, 2023

From the tropics to the poles, from the sea surface to hundreds of feet below, the world’s oceans are teeming with one of the tiniest of organisms: a type of bacteria called Prochlorococcus, which despite their minute size are collectively responsible for a sizable portion of the oceans’ oxygen production. But the remarkable ability of these diminutive organisms to diversify and adapt to such profoundly different environments has remained something of a mystery.

Now, new research reveals that these tiny bacteria exchange genetic information with one another, even when widely separated, by a previously undocumented mechanism. This enables them to transmit whole blocks of genes, such as those conferring the ability to metabolize a particular kind of nutrient or to defend themselves from viruses, even in regions where their population in the water is relatively sparse.

The findings describe a new class of genetic agents involved in horizontal gene transfer, in which genetic information is passed directly between organisms — whether of the same or different species — through means other than lineal descent. The researchers have dubbed the agents that carry out this transfer “tycheposons,” which are sequences of DNA that can include several entire genes as well as surrounding sequences, and can spontaneously separate out from the surrounding DNA. Then, they can be transported to other organisms by one or another possible carrier system including tiny bubbles known as vesicles that cells can produce from their own membranes.

The research, which included studying hundreds of Prochlorococcus genomes from different ecosystems around the world, as well as lab-grown samples of different variants, and even evolutionary processes carried out and observed in the lab, is reported today in the journal Cell, in a paper by former MIT postdocs Thomas Hackl and Raphaël Laurenceau, visiting postdoc Markus Ankenbrand, Institute Professor Sallie “Penny” Chisholm, and 16 others at MIT and other institutions.

Chisholm, who played a role in the discovery of these ubiquitous organisms in 1988, says of the new findings, “We’re very excited about it because it’s a new horizontal gene-transfer agent for bacteria, and it explains a lot of the patterns that we see in Prochlorococcus in the wild, the incredible diversity.” Now thought to be the world’s most abundant photosynthetic organism, the tiny variants of what are known as cyanobacteria are also the smallest of all photosynthesizers.

Hackl, who is now at the University of Groningen in the Netherlands, says the work began by studying the 623 reported genome sequences of different species of Prochlorococcus from different regions, trying to figure out how they were able to so readily lose or gain particular functions despite their apparent lack of any of the known systems that promote/boost horizontal gene transfer, such as plasmids or viruses known as prophages.

What Hackl, Laurenceau, and Ankenbrand investigated were “islands” of genetic material that seemed to be hotspots of variability and often contained genes that were associated with known key survival processes such as the ability to    assimilate essential, and often limiting, nutrients such as iron, or nitrogen, or phosphates. These islands contained genes that varied enormously between different species, but they always occurred in the same parts of the genome and sometimes were nearly identical even in widely different species — a strong indicator of horizontal transfer.

But the genomes showed none of the usual features associated with what are known as mobile genetic elements, so initially this remained a puzzle. It gradually became apparent that this system of gene transfer and diversification was different from any of the several other mechanisms that have been observed in other organisms, including in humans.

Hackl describes what they found as being something like a genetic LEGO set, with chunks of DNA bundled together in ways that could almost instantly confer the ability to adapt to a particular environment. For example, a species limited by the availability of particular nutrients could acquire genes necessary to enhance the uptake of that nutrient.

The microbes appear to use a variety of mechanisms to transport these tycheposons (a name derived from the name of the Greek goddess Tyche, daughter of Oceanus). One is the use of membrane vesicles, little bubbles pouched off from the surface of a bacterial cell and released with tycheposons inside it. Another is by “hijacking” virus or phage infections and allowing them to carry the tycheposons along with their own infectious particles, called capsids. These are efficient solutions, Hackl says, “because in the open ocean, these cells rarely have cell-to-cell contacts, so it’s difficult for them to exchange genetic information without a vehicle.”

And sure enough, when capsids or vesicles collected from the open ocean were studied, “they’re actually quite enriched” in these genetic elements, Hackl says. The packets of useful genetic coding are “actually swimming around in these extracellular particles and potentially being able to be taken up by other cells.”

Chisholm says that “in the world of genomics, there’s a lot of different types of these elements” — sequences of DNA that are capable of being transferred from one genome to another. However, “this is a new type,” she says. Hackl adds that “it’s a distinct family of mobile genetic elements. It has similarities to others, but no really tight connections to any of them.”

While this study was specific to Prochlorococcus, Hackl says the team believes the phenomenon may be more generalized. They have already found similar genetic elements in other, unrelated marine bacteria, but have not yet analyzed these samples in detail. “Analogous elements have been described in other bacteria, and we now think that they may function similarly,” he says.

“It’s kind of a plug-and-play mechanism, where you can have pieces that you can play around with and make all these different combinations,” he says. “And with the enormous population size of Prochlorococcus, it can play around a lot, and try a lot of different combinations.”

Nathan Ahlgren, an assistant professor of biology at Clark University who was not associated with this research, says “The discovery of tycheposons is important and exciting because it provides a new mechanistic understanding of how Prochlorococcus are able to swap in and out new genes, and thus ecologically important traits. Tycheposons provide a new mechanistic explanation for how it’s done.” He says “they took a creative way to fish out and characterize these new genetic elements ‘hiding’ in the genomes of Prochlorococcus.”

He adds that genomic islands, the portions of the genome where these tycheposons were found, “are found in many bacteria, not just marine bacteria, so future work on tycheposons has wider implications for our understanding of the evolution of bacterial genomes.”

The team included researchers at MIT’s Department of Civil and Environmental Engineering, the University of Wuerzburg in Germany, the University of Hawaii at Manoa, Ohio State University, Oxford Nanopore Technologies in California, Bigelow Laboratory for Ocean Sciences in Maine, and Wellesley College. The work was supported by the Simons Foundation, the Gordon and Betty Moore Foundation, the U.S. Department of Energy, and the U.S. National Science Foundation.

Scientists unveil the functional landscape of essential genes

Researchers harness new pooled, image-based screening method to probe the functions of over 5,000 essential genes in human cells.

Nicole Davis | Whitehead Institute
November 21, 2022

A team of scientists at the Whitehead Institute for Biomedical Research and the Broad Institute of MIT and Harvard has systematically evaluated the functions of over 5,000 essential human genes using a novel, pooled, imaged-based screening method. Their analysis harnesses CRISPR-Cas9 to knock out gene activity and forms a first-of-its-kind resource for understanding and visualizing gene function in a wide range of cellular processes with both spatial and temporal resolution. The team’s findings span over 31 million individual cells and include quantitative data on hundreds of different parameters that enable predictions about how genes work and operate together. The new study appears in the Nov. 7 online issue of the journal Cell.

“For my entire career, I’ve wanted to see what happens in cells when the function of an essential gene is eliminated,” says MIT Professor Iain Cheeseman, who is a senior author of the study and a member of Whitehead Institute. “Now, we can do that, not just for one gene but for every single gene that matters for a human cell dividing in a dish, and it’s enormously powerful. The resource we’ve created will benefit not just our own lab, but labs around the world.”

Systematically disrupting the function of essential genes is not a new concept, but conventional methods have been limited by various factors, including cost, feasibility, and the ability to fully eliminate the activity of essential genes. Cheeseman, who is the Herman and Margaret Sokol Professor of Biology at MIT, and his colleagues collaborated with MIT Associate Professor Paul Blainey and his team at the Broad Institute to define and realize this ambitious joint goal. The Broad Institute researchers have pioneered a new genetic screening technology that marries two approaches — large-scale, pooled, genetic screens using CRISPR-Cas9 and imaging of cells to reveal both quantitative and qualitative differences. Moreover, the method is inexpensive compared to other methods and is practiced using commercially available equipment.

“We are proud to show the incredible resolution of cellular processes that are accessible with low-cost imaging assays in partnership with Iain’s lab at the Whitehead Institute,” says Blainey, a senior author of the study, an associate professor in the Department of Biological Engineering at MIT, a member of the Koch Institute for Integrative Cancer Research at MIT, and a core institute member at the Broad Institute. “And it’s clear that this is just the tip of the iceberg for our approach. The ability to relate genetic perturbations based on even more detailed phenotypic readouts is imperative, and now accessible, for many areas of research going forward.”

Cheeseman adds, “The ability to do pooled cell biological screening just fundamentally changes the game. You have two cells sitting next to each other and so your ability to make statistically significant calculations about whether they are the same or not is just so much higher, and you can discern very small differences.”

Cheeseman, Blainey, lead authors Luke Funk and Kuan-Chung Su, and their colleagues evaluated the functions of 5,072 essential genes in a human cell line. They analyzed four markers across the cells in their screen — DNA; the DNA damage response, a key cellular pathway that detects and responds to damaged DNA; and two important structural proteins, actin and tubulin. In addition to their primary screen, the scientists also conducted a smaller, follow-up screen focused on some 200 genes involved in cell division (also called “mitosis”). The genes were identified in their initial screen as playing a clear role in mitosis but had not been previously associated with the process. These data, which are made available via a companion website, provide a resource for other scientists to investigate the functions of genes they are interested in.

“There’s a huge amount of information that we collected on these cells. For example, for the cells’ nucleus, it is not just how brightly stained it is, but how large is it, how round is it, are the edges smooth or bumpy?” says Cheeseman. “A computer really can extract a wealth of spatial information.”

Flowing from this rich, multi-dimensional data, the scientists’ work provides a kind of cell biological “fingerprint” for each gene analyzed in the screen. Using sophisticated computational clustering strategies, the researchers can compare these fingerprints to each other and construct potential regulatory relationships among genes. Because the team’s data confirms multiple relationships that are already known, it can be used to confidently make predictions about genes whose functions and/or interactions with other genes are unknown.

There are a multitude of notable discoveries to emerge from the researchers’ screening data, including a surprising one related to ion channels. Two genes, AQP7 and ATP1A1, were identified for their roles in mitosis, specifically the proper segregation of chromosomes. These genes encode membrane-bound proteins that transport ions into and out of the cell. “In all the years I’ve been working on mitosis, I never imagined ion channels were involved,” says Cheeseman.

He adds, “We’re really just scratching the surface of what can be unearthed from our data. We hope many others will not only benefit from — but also build upon — this resource.”

This work was supported by grants from the U.S. National Institutes of Health as well as support from the Gordon and Betty Moore Foundation, a National Defense Science and Engineering Graduate Fellowship, and a Natural Sciences and Engineering Research Council Fellowship.

Unusual Labmates: How C. elegans Wormed Its Way into Science Stardom
Greta Friar | Whitehead Institute
September 20, 2022

 

Introduction

Michael Stubna, a graduate student in Whitehead Institute Member David Bartel’s lab, peers into his microscope at the Petri dish full of agar gel below. He spots one of his research specimens, a millimeter-long nematode worm known as Caenorhabditis elegans (C. elegans), slithering across the coating of bacteria–the worm’s food source–on the surface of the gel. The worm leaves sinuous tracks in its wake like a skier slaloming down a slope.

 

New players in an essential pathway to destroy microRNAs

In a study from the lab of Whitehead Institute Member David Bartel, researchers have identified genetic sequences that can lead to the degradation of cellular regulators called microRNAs in the fruit fly Drosophila melanogaster.

Eva Frederick | Whitehead Institute
September 26, 2022

In a study from the lab of Whitehead Institute Member David Bartel, researchers have identified genetic sequences that can lead to the degradation of cellular regulators called microRNAs in the fruit fly Drosophila melanogaster. The findings were published September 22 in Molecular Cell.

“This is an exciting study that paves the way for a deeper understanding of the microRNA degradation pathway,” says Bartel, who is also a professor of biology at the Massachusetts Institute of Technology and investigator with the Howard Hughes Medical Institute. “Finding these ‘trigger’ sequences will allow us to more precisely probe the workings of this pathway in the lab, which is likely critical for flies — and possibly other species — to survive to adulthood.”

In order to produce new proteins, cells transcribe their DNA into messenger RNAs (or mRNAs), which provide information required to make the proteins . When a given mRNA has served its purpose, it’s degraded. The process of degradation is often led by tiny RNA sequences called microRNAs.

In previous work, researchers showed that certain mRNA or non coding RNA transcripts, rather than being degraded by microRNAs, can instead turn the tables on the microRNAs and lead to their destruction through a pathway called target-directed microRNA degradation, or TDMD. “This pathway leads to rapid turnover of certain microRNAs within the cell,” says former Bartel Lab graduate student Elena Kingston.

Kingston wanted to further understand the functions of the TDMD pathway in cells. “I wanted to get at the ‘why,’” she said. “Why are microRNAs regulated in this way, and why does it matter in an organism?”

Previous work on the TDMD pathway was primarily conducted in cultured cells. For the new study, the researchers decided to use the fruit fly Drosophila melanogaster.  A fly model could provide more insight into how the pathway worked in a live organism — including whether or not it had an effect on the organism’s fitness or was essential for survival.

The researchers created a model to study TDMD by using flies with mutations in an essential TDMD pathway gene called Dora (the equivalent human gene is called ZSWIM8, as detailed in this paper). Very few flies with mutations in Dora were seen to make it to adulthood. Most died early in development, suggesting the TDMD pathway was likely important for their embryonic viability.

Putting a finger on the triggers of the TDMD pathway

While microRNAs don’t need many complementary base pairs to bind and regulate their mRNA targets, the opposite is true in the TDMD pathway. In order to work properly, the TDMD pathway needs a highly specific trigger, which can either be a mRNA that codes for proteins, or a non-coding RNA. “What’s unique about a trigger is it has a site that the microRNA can bind to that has a lot of complementarity to the microRNA,” Kingston said.

During the isolation of the early Covid-19 pandemic, Kingston set out to write a program that could pick out probable triggers of microRNA degradation in Drosophila based on their sequences. The program returned thousands of hits, and the researchers set to work narrowing down which sites were the best candidates to test in flies.

“As soon as we were able to get back into lab [after the lockdown], I took our top 10 or so candidates and tried perturbing them in flies,” she said. “Fortunately for me, about half of them ended up working out.”

These six new triggers more than double the list of known RNA sequences that can direct degradation of microRNAs. To take this finding a step further, the researchers conducted an analysis of what happened to the flies when a trigger was disrupted.

The researchers found that one of the triggers — a long non-coding RNA — plays a role in proper development of the cuticle, or the waterproof outer shell of a fly embryo. “We noticed that when we perturbed this trigger, the cuticles of fly embryos had altered elasticity,” Kingston said. “When we popped the embryos out of their egg shells, we could see these cuticles expand up and bloat.”

Because of the bloated phenotype, Kingston decided to name the long non-coding RNA marge after Aunt Marge, a character in the Harry Potter series. In “Harry Potter and the Prisoner of Azkaban”, Aunt Marge’s taunts lead Harry to accidentally perform magic on her, causing her to inflate and float away.

In the future, Kingston, who has since graduated and begun a career in the biotech industry, hopes researchers will pick up the torch on learning the roles of other TDMD triggers. “We still have several other triggers [from this paper] where there’s no known biological role for them in the fly,” she said. “I think this opens up the field for others to go in and to ask the questions, ‘Where are these triggers acting? What are they doing? And what’s the phenotype when you lose them?’”

Notes

Elena Kingston, Lianne Blodgett and David Bartel. “Endogenous transcripts direct microRNA degradation in Drosophila, and this targeted degradation is required for proper embryonic development.” Molecular Cell, September 22, 2022. DOI: https://doi.org/10.1016/j.molcel.2022.08.029

Biologists glean insight into repetitive protein sequences

A computational analysis reveals that many repetitive sequences are shared across proteins and are similar in species from bacteria to humans.

Anne Trafton | MIT News Office
September 13, 2022

About 70 percent of all human proteins include at least one sequence consisting of a single amino acid repeated many times, with a few other amino acids sprinkled in. These “low-complexity regions” are also found in most other organisms.

The proteins that contain these sequences have many different functions, but MIT biologists have now come up with a way to identify and study them as a unified group. Their technique allows them to analyze similarities and differences between LCRs from different species, and helps them to determine the functions of these sequences and the proteins in which they are found.

Using their technique, the researchers have analyzed all of the proteins found in eight different species, from bacteria to humans. They found that while LCRs can vary between proteins and species, they often share a similar role — helping the protein in which they’re found to join a larger-scale assembly such as the nucleolus, an organelle found in nearly all human cells.

“Instead of looking at specific LCRs and their functions, which might seem separate because they’re involved in different processes, our broader approach allows us to see similarities between their properties, suggesting that maybe the functions of LCRs aren’t so disparate after all,” says Byron Lee, an MIT graduate student.

The researchers also found some differences between LCRs of different species and showed that these species-specific LCR sequences correspond to species-specific functions, such as forming plant cell walls.

Lee and graduate student Nima Jaberi-Lashkari are the lead authors of the study, which appears today in eLife. Eliezer Calo, an assistant professor of biology at MIT, is the senior author of the paper.

Large-scale study

Previous research has revealed that LCRs are involved in a variety of cellular processes, including cell adhesion and DNA binding. These LCRs are often rich in a single amino acid such as alanine, lysine, or glutamic acid.

Finding these sequences and then studying their functions individually is a time-consuming process, so the MIT team decided to use bioinformatics — an approach that uses computational methods to analyze large sets of biological data — to evaluate them as a larger group.

“What we wanted to do is take a step back and instead of looking at individual LCRs, to try to take a look at all of them and to see if we could observe some patterns on a larger scale that might help us figure out what the ones that have assigned functions are doing, and also help us learn a bit about what the ones that don’t have assigned functions are doing,” Jaberi-Lashkari says.

To do that, the researchers used a technique called dotplot matrix, which is a way to visually represent amino acid sequences, to generate images of each protein under study. They then used computational image processing methods to compare thousands of these matrices at the same time.

Using this technique, the researchers were able to categorize LCRs based on which amino acids were most frequently repeated in the LCR. They also grouped LCR-containing proteins by the number of copies of each LCR type found in the protein. Analyzing these traits helped the researchers to learn more about the functions of these LCRs.

As one demonstration, the researchers picked out a human protein, known as RPA43, that has three lysine-rich LCRs. This protein is one of many subunits that make up an enzyme called RNA polymerase 1, which synthesizes ribosomal RNA. The researchers found that the copy number of lysine-rich LCRs is important for helping the protein integrate into the nucleolus, the organelle responsible for synthesizing ribosomes.

Biological assemblies

In a comparison of the proteins found in eight different species, the researchers found that some LCR types are highly conserved between species, meaning that the sequences have changed very little over evolutionary timescales. These sequences tend to be found in proteins and cell structures that are also highly conserved, such as the nucleolus.

“These sequences seem to be important for the assembly of certain parts of the nucleolus,” Lee says. “Some of the principles that are known to be important for higher order assembly seem to be at play because the copy number, which might control how many interactions a protein can make, is important for the protein to integrate into that compartment.”

The researchers also found differences between LCRs seen in two different types of proteins that are involved in nucleolus assembly. They discovered that a nucleolar protein known as TCOF contains many glutamine-rich LCRs that can help scaffold the formation of assemblies, while nucleolar proteins with only a few of these glutamic acid-rich LCRs could be recruited as clients (proteins that interact with the scaffold).

Another structure that appears to have many conserved LCRs is the nuclear speckle, which is found inside the cell nucleus. The researchers also found many similarities between LCRs that are involved in forming larger-scale assemblies such as the extracellular matrix, a network of molecules that provides structural support to cells in plants and animals.

The research team also found examples of structures with LCRs that seem to have diverged between species. For example, plants have distinctive LCR sequences in the proteins that they use to scaffold their cell walls, and these LCRs are not seen in other types of organisms.

The researchers now plan to expand their LCR analysis to additional species.

“There’s so much to explore, because we can expand this map to essentially any species,” Lee says. “That gives us the opportunity and the framework to identify new biological assemblies.”

The research was funded by the National Institute of General Medical Sciences, National Cancer Institute, the Ludwig Center at MIT, a National Institutes of Health Pre-Doctoral Training Grant, and the Pew Charitable Trusts.

Scientists identify a plant molecule that sops up iron-rich heme

The peptide is used by legumes to control nitrogen-fixing bacteria; it may also offer leads for treating patients with too much heme in their blood.

Anne Trafton | MIT News Office
August 11, 2022

Symbiotic relationships between legumes and the bacteria that grow in their roots are critical for plant survival. Without those bacteria, the plants would have no source of nitrogen, an element that is essential for building proteins and other biomolecules, and they would be dependent on nitrogen fertilizer in the soil.

To establish that symbiosis, some legume plants produce hundreds of peptides that help bacteria live within structures known as nodules within their roots. A new study from MIT reveals that one of these peptides has an unexpected function: It sops up all available heme, an iron-containing molecule. This sends the bacteria into an iron-starvation mode that ramps up their production of ammonia, the form of nitrogen that is usable for plants.

“This is the first of the 700 peptides in this system for which a really detailed molecular mechanism has been worked out,” says Graham Walker, the American Cancer Society Research Professor of Biology at MIT, a Howard Hughes Medical Institute Professor, and the senior author of the study.

This heme-sequestering peptide could have beneficial uses in treating a variety of human diseases, the researchers say. Removing free heme from the blood could help to treat diseases caused by bacteria or parasites that need heme to survive, such as P. gingivalis (periodontal disease) or toxoplasmosis, or diseases such as sickle cell disease or sepsis that release too much heme into the bloodstream.

“This study demonstrates that basic research in plant-microbe interactions also has potential to be translated to therapeutic applications,” says Siva Sankari, an MIT research scientist and the lead author of the study, which appears today in Nature Microbiology.

Other authors of the paper include Vignesh Babu, an MIT research scientist; Kevin Bian and Mary Andorfer, both MIT postdocs; Areej Alhhazmi, a former KACST-MIT Ibn Khaldun Fellowship for Saudi Arabian Women scholar; Kwan Yoon and Dante Avalos, MIT graduate students; Tyler Smith, an MIT instructor in biology; Catherine Drennan, an MIT professor of chemistry and biology and a Howard Hughes Medical Institute investigator; Michael Yaffe, a David H. Koch Professor of Science and a member of MIT’s Koch Institute for Integrative Cancer Research; and Sebastian Lourido, the Latham Family Career Development Professor of Biology at MIT and a member of the Whitehead Institute for Biomedical Research.

Iron control

For nearly 40 years, Walker’s lab has been studying the symbiosis between legumes and rhizobia, a type of nitrogen-fixing bacteria. These bacteria convert nitrogen gas to ammonia, a critical step of the Earth’s nitrogen cycle that makes the element available to plants (and to animals that eat the plants).

Most of Walker’s work has focused on a clover-like plant called Medicago truncatula. Nitrogen-fixing bacteria elicit the formation of nodules on the roots of these plants and eventually end up inside the plant cells, where they convert to their symbiotic form called bacteroids.

Several years ago, plant biologists discovered that Medicago truncatula produces about 700 peptides that contribute to the formation of these bacteroids. These peptides are generated in waves that help the bacteria make the transition from living freely to becoming embedded into plant cells where they act as nitrogen-fixing machines.

Walker and his students picked one of these peptides, known as NCR247, to dig into more deeply. Initial studies revealed that when nitrogen-fixing bacteria were exposed to this peptide, 15 percent of their genes were affected. Many of the genes that became more active were involved in importing iron.

The researchers then found that when they fused NCR247 to a larger protein, the hybrid protein was unexpectedly reddish in color. This serendipitous observation led to the discovery that NCR247 binds heme, an organic ring-shaped iron-containing molecule that is an important component of hemoglobin, the protein that red blood cells use to carry oxygen.

Further studies revealed that when NCR247 is released into bacterial cells, it sequesters most of the heme in the cell, sending the cells into an iron-starvation mode that triggers them to begin importing more iron from the external environment.

“Usually bacteria fine-tune their iron metabolism, and they don’t take up more iron when there is already enough,” Sankari says. “What’s cool about this peptide is that it overrides that mechanism and indirectly regulates the iron content of the bacteria.”

Nitrogenase, the main enzyme that bacteria use to fix nitrogen, requires 24 to 32 atoms of iron per enzyme molecule, so the influx of extra iron likely helps those enzymes to become more active, the researchers say. This influx is timed to coincide with nitrogen fixation, they found.

“These peptides are produced in a wave in the nodules, and the production of this particular peptide is higher when the bacteria are preparing to fix nitrogen. If this peptide was secreted throughout the whole process, then the cell would have too much iron all the time, which is bad for the cell,” Sankari says.

Without the NCR247 peptide, Medicago truncatula and rhizobium cannot form an effective nitrogen-fixing symbiosis, the researchers showed.

“Many possible directions”

The peptide that the researchers studied in this work may have potential therapeutic uses. When heme is incorporated into hemoglobin, it performs a critical function in the body, but when it’s loose in the bloodstream, it can kill cells and promote inflammation. Free heme can accumulate in stored blood, so having a way to filter out the heme before the blood is transfused into a patient could be potentially useful.

A variety of human diseases lead to free heme circulating in the bloodstream, including sickle cell anemia, sepsis, and malaria. Additionally, some infectious parasites and bacteria depend on heme for their survival but cannot produce it, so they scavenge it from their environment. Treating such infections with a protein that takes up all available heme could help prevent the parasitic or bacterial cells from being able to grow and reproduce.

In this study, Lourido and members of his lab showed that treating the parasite Toxoplasma gondii with NCR427 prevented the parasite from forming plaques on human cells.

The researchers are now pursuing collaborations with other labs at MIT to explore some of these potential applications, with funding from a Professor Amar G. Bose Research Grant.

“There are many possible directions, but they’re all at a very early stage,” Walker says. “The number of potential clinical applications is very broad. You can place more than one bet in this game, which is an intriguing thing.”

Currently, the human protein hemopexin, which also binds to heme, is being explored as a possible treatment for sickle cell anemia. The NCR247 peptide could provide an easier to deploy alternative, the researchers say, because it is much smaller and could be easier to manufacture and deliver into the body.

The research was funded in part by the MIT Center for Environmental Health Sciences, the National Science Foundation, and the National Institutes of Health.

Yiyin Erin Chen and Sam Chunte Peng named as core members of Broad Institute and MIT
Broad Communications
July 12, 2022

Broad Institute of MIT and Harvard has named Erin Chen, a dermatologist and microbiologist, and Sam Peng, a biophysicist and physical chemist with expertise in single-molecule imaging, as core institute members.

Chen will join in January 2023 and will also serve as an assistant professor in the Department of Biology at MIT and an attending dermatologist at Massachusetts General Hospital. Peng joined in July 2022 and will serve as an assistant professor in the Department of Chemistry at MIT.

Chen’s lab will study the communication between the immune system and the diverse microbes that colonize every surface of the human body, with a focus on the human body’s largest organ, the skin.

Peng’s lab will develop novel probes and microscopy techniques to visualize the dynamics of individual molecules in living cells, which will improve the understanding of molecular mechanisms underlying human diseases.

“We are delighted to welcome Sam and Erin to the Broad community,” said Todd Golub, director of the Broad. “These creative scientists are each taking inventive approaches to understand the molecular signals and interactions that underlie biological processes in health and disease. These insights will help further the Broad’s mission of advancing the understanding and treatment of human disease.”

Erin Chen.
Erin Chen

Erin Chen earned her BA in biology from the University of Chicago, her PhD from MIT, and her MD from Harvard Medical School. Prior to joining the Broad, Chen was a Howard Hughes Medical Institute Hanna Gray Postdoctoral Fellow at Stanford University, in the lab of Michael Fischbach. She was also an attending dermatologist at the University of California San Francisco and at the San Francisco VA Medical Center. During her postdoctoral research, Chen developed genetic methods to study harmless commensal skin bacteria. She engineered these bacteria to generate anti-tumor immunity, pioneering a novel approach to vaccination and cancer immunotherapy.

At the Broad, members of the Chen lab will continue to employ microbial genetics, immunologic approaches, and mouse models to dissect the molecular signals used by commensal microbes to educate the immune system. Ultimately, Chen aims to harness these microbe-host interactions to engineer novel therapeutics for human disease.

“I’m excited to join the collaborative scientific community at the Broad and MIT, including those who have pioneered novel tools for examining biological mechanisms at higher spatial resolution,” said Chen. “The biology I study is quite basic, but I’m motivated by the potential impact it could have on patients. Figuring out how commensal skin bacteria are captured by the immune system could unlock a whole new therapeutic toolbox.”

Sam Peng
Sam Peng

Sam Peng earned his BS in chemistry from the University of California, Berkeley, and his PhD from MIT in physical chemistry. He completed his postdoctoral research at Stanford University as an NIH K99 Pathway to Independence scholar in the lab of Steve Chu. During his postdoctoral research, he developed long-term single molecule imaging in live cells using a novel class of nanoprobes. He applied this new technique to study axonal transport in neurons and the molecular dynamics of dynein — a motor protein involved in transporting cargo in cells.

At the Broad, the Peng group will aim to elucidate the molecular mechanisms underlying human diseases. Lab members will develop and integrate a diverse toolbox spanning single-molecule microscopy, super-resolution microscopy, spectroscopy, nanomaterial engineering, biophysics, chemical biology, and quantitative modeling to uncover previously unexplored biological processes. With bright and photostable probes, lab members will have unprecedented capability to record ultra-long-term “molecular movies” in living systems with high spatiotemporal resolutions and to reveal molecular interactions that drive biological functions. Peng’s group will focus on studying molecular dynamics, protein-protein interactions, and cellular heterogeneity involved in neurobiology and cancer biology. Their long-term goal is to translate these mechanistic insights into drug discovery.

“Because my research is so multi-disciplinary, joining the Broad and MIT communities allows us to integrate a range of experimental tools and to collaborate with colleagues and students from diverse backgrounds,” said Peng. “I’m excited to see how our techniques can enable discoveries for a variety of cellular processes, including those underlying complex brain functions and dysfunctions. Many problems that previously seemed inaccessible now appear to be within reach in the foreseeable future.”

New CRISPR-based map ties every human gene to its function

Jonathan Weissman and collaborators used their single-cell sequencing tool Perturb-seq on every expressed gene in the human genome, linking each to its job in the cell.

Eva Frederick | Whitehead Institute
June 9, 2022

The Human Genome Project was an ambitious initiative to sequence every piece of human DNA. The project drew together collaborators from research institutions around the world, including MIT’s Whitehead Institute for Biomedical Research, and was finally completed in 2003. Now, over two decades later, MIT Professor Jonathan Weissman and colleagues have gone beyond the sequence to present the first comprehensive functional map of genes that are expressed in human cells. The data from this project, published online June 9 in Cell, ties each gene to its job in the cell, and is the culmination of years of collaboration on the single-cell sequencing method Perturb-seq.

The data are available for other scientists to use. “It’s a big resource in the way the human genome is a big resource, in that you can go in and do discovery-based research,” says Weissman, who is also a member of the Whitehead Institute and an investigator with the Howard Hughes Medical Institute. “Rather than defining ahead of time what biology you’re going to be looking at, you have this map of the genotype-phenotype relationships and you can go in and screen the database without having to do any experiments.”

The screen allowed the researchers to delve into diverse biological questions. They used it to explore the cellular effects of genes with unknown functions, to investigate the response of mitochondria to stress, and to screen for genes that cause chromosomes to be lost or gained, a phenotype that has proved difficult to study in the past. “I think this dataset is going to enable all sorts of analyses that we haven’t even thought up yet by people who come from other parts of biology, and suddenly they just have this available to draw on,” says former Weissman Lab postdoc Tom Norman, a co-senior author of the paper.

Pioneering Perturb-seq

The project takes advantage of the Perturb-seq approach that makes it possible to follow the impact of turning on or off genes with unprecedented depth. This method was first published in 2016 by a group of researchers including Weissman and fellow MIT professor Aviv Regev, but could only be used on small sets of genes and at great expense.

The massive Perturb-seq map was made possible by foundational work from Joseph Replogle, an MD-PhD student in Weissman’s lab and co-first author of the present paper. Replogle, in collaboration with Norman, who now leads a lab at Memorial Sloan Kettering Cancer Center; Britt Adamson, an assistant professor in the Department of Molecular Biology at Princeton University; and a group at 10x Genomics, set out to create a new version of Perturb-seq that could be scaled up. The researchers published a proof-of-concept paper in Nature Biotechnology in 2020.

The Perturb-seq method uses CRISPR-Cas9 genome editing to introduce genetic changes into cells, and then uses single-cell RNA sequencing to capture information about the RNAs that are expressed resulting from a given genetic change. Because RNAs control all aspects of how cells behave, this method can help decode the many cellular effects of genetic changes.

Since their initial proof-of-concept paper, Weissman, Regev, and others have used this sequencing method on smaller scales. For example, the researchers used Perturb-seq in 2021 to explore how human and viral genes interact over the course of an infection with HCMV, a common herpesvirus.

In the new study, Replogle and collaborators including Reuben Saunders, a graduate student in Weissman’s lab and co-first author of the paper, scaled up the method to the entire genome. Using human blood cancer cell lines as well noncancerous cells derived from the retina, he performed Perturb-seq across more than 2.5 million cells, and used the data to build a comprehensive map tying genotypes to phenotypes.

Delving into the data

Upon completing the screen, the researchers decided to put their new dataset to use and examine a few biological questions. “The advantage of Perturb-seq is it lets you get a big dataset in an unbiased way,” says Tom Norman. “No one knows entirely what the limits are of what you can get out of that kind of dataset. Now, the question is, what do you actually do with it?”

The first, most obvious application was to look into genes with unknown functions. Because the screen also read out phenotypes of many known genes, the researchers could use the data to compare unknown genes to known ones and look for similar transcriptional outcomes, which could suggest the gene products worked together as part of a larger complex.

The mutation of one gene called C7orf26 in particular stood out. Researchers noticed that genes whose removal led to a similar phenotype were part of a protein complex called Integrator that played a role in creating small nuclear RNAs. The Integrator complex is made up of many smaller subunits — previous studies had suggested 14 individual proteins — and the researchers were able to confirm that C7orf26 made up a 15th component of the complex.

They also discovered that the 15 subunits worked together in smaller modules to perform specific functions within the Integrator complex. “Absent this thousand-foot-high view of the situation, it was not so clear that these different modules were so functionally distinct,” says Saunders.

Another perk of Perturb-seq is that because the assay focuses on single cells, the researchers could use the data to look at more complex phenotypes that become muddied when they are studied together with data from other cells. “We often take all the cells where ‘gene X’ is knocked down and average them together to look at how they changed,” Weissman says. “But sometimes when you knock down a gene, different cells that are losing that same gene behave differently, and that behavior may be missed by the average.”

The researchers found that a subset of genes whose removal led to different outcomes from cell to cell were responsible for chromosome segregation. Their removal was causing cells to lose a chromosome or pick up an extra one, a condition known as aneuploidy. “You couldn’t predict what the transcriptional response to losing this gene was because it depended on the secondary effect of what chromosome you gained or lost,” Weissman says. “We realized we could then turn this around and create this composite phenotype looking for signatures of chromosomes being gained and lost. In this way, we’ve done the first genome-wide screen for factors that are required for the correct segregation of DNA.”

“I think the aneuploidy study is the most interesting application of this data so far,” Norman says. “It captures a phenotype that you can only get using a single-cell readout. You can’t go after it any other way.”

The researchers also used their dataset to study how mitochondria responded to stress. Mitochondria, which evolved from free-living bacteria, carry 13 genes in their genomes. Within the nuclear DNA, around 1,000 genes are somehow related to mitochondrial function. “People have been interested for a long time in how nuclear and mitochondrial DNA are coordinated and regulated in different cellular conditions, especially when a cell is stressed,” Replogle says.

The researchers found that when they perturbed different mitochondria-related genes, the nuclear genome responded similarly to many different genetic changes. However, the mitochondrial genome responses were much more variable.

“There’s still an open question of why mitochondria still have their own DNA,” said Replogle. “A big-picture takeaway from our work is that one benefit of having a separate mitochondrial genome might be having localized or very specific genetic regulation in response to different stressors.”

“If you have one mitochondria that’s broken, and another one that is broken in a different way, those mitochondria could be responding differentially,” Weissman says.

In the future, the researchers hope to use Perturb-seq on different types of cells besides the cancer cell line they started in. They also hope to continue to explore their map of gene functions, and hope others will do the same. “This really is the culmination of many years of work by the authors and other collaborators, and I’m really pleased to see it continue to succeed and expand,” says Norman.

A heart-racing deadline for a heartfelt collaboration

In a whirlwind team project, undergraduates Aniket Dehadrai SB ’22 and Brindha Rathinasabapathi SB ’24 of the Boyer lab pioneered a new method to study how hearts are built.

Celina Zhao
May 23, 2022

Can’t miss a beat

The lab was bustling with activity, with everyone working together on a team project comprised of many moving parts. Once one person finished a step of the experiment, it was whisked off to the next person. There was no time to lose.

During MIT’s Independent Activities Period (IAP) in January of 2022, several members of the Boyer Lab were hard at work — among them, Aniket Dehadrai, a junior studying Course 5-7 (Chemistry and Biology), and Brindha Rathinasabapathi, a sophomore studying Course 7 (Biology). Fueled with coffee every morning from the lab’s handy Keurig, the team was on a time crunch.

Working alongside Dehadrai and Rathinasabapathi were research scientist Vera Koledova, lab manager Kirsten Schneider, and fellow undergraduate researcher Caroline Zhang. They had a hard deadline at the end of the month to finish the project: studying how the absence of a certain protein affects the growth of cardiomyocytes, the cells responsible for pumping blood around the heart.

The Boyer lab — headed by Professor Laurie Boyer, the “Queen of Hearts” — specializes in heart cells. The lab is particularly interested in one intriguing question: Is it possible to heal the heart? Injuries like heart attacks often cause permanent damage that can eventually lead to heart failure. Scientists have found that at birth, injured heart cells are able to repair or replace themselves after such an event. However, that ability shuts off just a few days post-birth. Afterwards, heart cells, once damaged, are unfixable.

But what if adult cardiomyocytes could regain the ability to repair themselves, and thus repair trauma in heart tissue? The Boyer lab is intrigued by this possibility. But in order to answer that question, they must start from ground zero: learning how cardiomyocytes themselves develop.

The operation

Dehadrai, Rathinasabapathi, and the rest of the team were studying one part of that puzzle — the role histones play in cardiomyocyte growth. Histones are proteins that act as spools for DNA to wind around. DNA is extremely long, so histones help fit all this genetic information into the tiny space of a nucleus.

There are many types of histones (called “variants”), each of which has a unique effect on how DNA is wrapped. The tighter the DNA is packed, the more difficult it is for proteins to access the DNA — all of which affects how genes are expressed. As a result, each variant has a unique effect on how certain genes are regulated.

For the IAP project, the Boyer lab’s team focused on one histone variant called H2AZ.1. Prior studies have shown that H2AZ.1 is essential in most organisms, particularly when it comes to gene expression in stem cells. Stem cells are cells that essentially begin as blank slates, with the ability to form the many different cell types in the body. But through a differentiation process, they develop specific identities: skin, brain, or heart, to name a few.

By the end of the four weeks, the team planned to create and streamline a completely new process to “knock out,” or entirely remove, H2AZ.1 by degrading it during cardiomyocyte differentiation — the process where stem cells become specialized heart cells. Building this procedure to remove H2AZ.1 could later help identify what role H2AZ.1 plays in cardiomyocyte differentiation, a key step in both heart development and regeneration.

Microscopy image of heart muscle cells
The histone variant H2A.Z.1 (red) is located in the nucleus (blue) of cardiac muscle cells. Actin, a component of the sarcomere, is shown in green. The striated structure of the muscle cells gives them strength to beat throughout our entire lives. Credit: Boyer lab

To begin creating the knockout procedure, the team started by culturing stem cells from a cell line specifically developed by the Boyer lab to study the H2AZ.1 histone. The goal was to see if removing H2AZ.1 would have a visible effect on how stem cells eventually become mature cardiomyocytes.

The amount of careful planning and execution to do in just one month — simply running through one full differentiation cycle took 15 days at a time — meant working together as a team was critical. “There was one late night with all five people in the lab, doing this giant experiment as well as we could without mixing up the different variables in play,” Rathinasabapathi says. “It was really critical for us to look over each other’s shoulders and double check each other.”

In all, the team tested out 10 different variations of a method to optimize the experimental procedure. Despite the time crunch, they succeeded in pioneering a procedure to efficiently remove H2AZ.1 during cardiac differentiation. It turns out that H2AZ.1 does, in fact, have a functional impact on heart cells.

Without H2AZ.1, the beating rate of mature cardiomyocytes was notably different, changing from rhythmic to arrhythmic. The research team also found varying levels of maturity in the cells, suggesting that the progression through the differentiation process was also changed.

All of this suggests that H2AZ.1 has a significant influence in gene regulation, which they plan to continue studying in greater detail in the future.

“We’re breaking new ground,” Dehadrai says. “And importantly, it’s a great framework for future work in this field.”

With the procedure the team developed, the lab is now able to ask and answer more questions. For one, they can zoom in on certain parts of cardiomyocyte differentiation to see when H2AZ.1 has the greatest impact on gene expression. They can also use this procedure as a model to study how other histone variants affect heart cell growth. Ultimately, they can begin piecing together how histones, their effect on gene regulation, and cardiomyocyte development unite to build the heart.

“The better we can understand how heart cell development works, the better we can understand heart development, injury, and response — all of which have a lot of different implications in the medical field,” Rathinasabapathi says.

Following their hearts

The two credit the cohesiveness of the team as a big part of their success. “Brindha is really responsible, helpful, and willing to put in the hours,” Dehadrai says . “You can’t take stuff like that for granted.”

“Ani is just as dependable, and I’ve learned a lot from him as a senior with a lot of experience in the lab,” Rathinasabapathi says.

Another strength of the team was their ability to draw upon many different academic areas: a hallmark of the Boyer lab, which is known for its interdisciplinary approach to heart research. Members come from all sorts of backgrounds: biology, chemistry, biological engineering, mechanical engineering, and more. Research in the lab also spans a wide expanse, from uncovering the secrets of heart regeneration to building better microscopy techniques to study the heart. In fact, that was one of the reasons why Dehadrai initially chose to join the lab. “Here, there’s people who pretty much know how to do everything,” he says.

Although the IAP project has concluded, both Dehadrai and Rathinasabapathi are committed to continuing their passion for medical research. Dehadrai, who is graduating in the spring, is planning to take a gap year to work on clinical research projects before applying to medical school.

Rathinasabapathi, on the other hand, still has two years at MIT. She plans to stay in the Boyer Lab and is eager to take more advanced courses in the Department of Biology. “I’m impatient — I wish I already had the solid foundation to attack the research at different angles and come up with more cool new things,” she says. “There’s just so much more that I want to know.”