Research Area: Cell Biology

Origin story

Junior Leah McKinney practiced kitchen microbiology on her ranch in Nevada before exploring the intricacies of DNA replication initiation in bacteria at MIT Biology.

Raleigh McElvery
February 6, 2019

Leah McKinney grew up on a 50,000-acre cattle ranch in Nevada — vaccinating sheep, roping calves, digging for fossils, and occasionally hauling home old bovine femurs. She saddled horses, treated sick lambs, and helped ewes struggling to give birth. One Christmas, she even asked Santa for a fetal pig. “He delivered,” McKinney, now a junior in Course 7, recalls with a laugh.

When she was 12 years old, she saved up enough birthday money to purchase a microscope. Even though she permanently dyed the kitchen sink a distinct shade of blue while making slides, her parents (who both hold degrees in animal science) didn’t mind. They even let her grow bacteria in the heater closet and tally them on the kitchen counter — all in the name of the elementary school science fair.

“They were always encouraging my weird scientific endeavors,” she says. “I think my love for science, and microbiology specifically, came out of my agricultural upbringing.”

She grew to appreciate basic science because it allowed her to study the fundamental mechanisms behind key biological processes. She arrived at MIT in 2016 determined to major in Biology, and hasn’t wavered in her decision. Although she relishes the subject matter, she initially feared the classes would be tedious and memorization-heavy.

“I was quite happy to learn that’s not the case here,” she says. “MIT Biology values problem-solving over rote memorization, and encourages you to take the information you’ve learned in class and apply it to interesting problems. And that mindset extends from the classroom into the lab.”

One of the things that drew McKinney to MIT was the institute’s Undergraduate Research Opportunities Program (UROP), which allows students to join labs and collaborate with faculty as early as their first year. She recalls that, while other universities touted similar opportunities, MIT placed theirs front and center.

“I’d heard that all you had to do was email a professor and ask to join the lab, but I didn’t believe it — that just seemed way too easy,” McKinney says. “But when I was looking for a UROP, I just emailed my current principal investigator to set up a time to talk, and now I’ve been in his lab for over a year.”

McKinney is part of Department Head Alan Grossman’s lab, which investigates the molecular mechanisms and regulation underlying basic cellular processes in bacteria. The entire group works with the rod-shaped Bacillus subtilis, but some members study horizontal gene transfer while others focus on DNA replication and gene expression. McKinney and her graduate student mentor Mary Anderson are in this second category, examining a protein called DnaA that is required to initiate DNA replication and also modulates the expression of several genes.

In order to successfully grow and reproduce, a bacterium must first replicate its single chromosome before dividing into two identical daughter cells. DnaA is responsible for beginning DNA replication in all bacteria. It binds to the origin of replication on the chromosome, unwinds some of the nearby DNA, and recruits the other proteins needed to copy the chromosome.

This operation is highly regulated to ensure that each daughter cell receives only a single chromosome. B. subtilis controls replication via several proteins, including YabA. When YabA binds to DnaA, it prevents replication from ever getting started.

Since DnaA also serves as a transcription factor — binding to other DNA sequences called promoters to increase or decrease expression of certain genes, including its own gene dnaA — YabA may also impact DnaA’s gene targets. McKinney hopes to eventually determine exactly how.

While McKinney discovers something new about her bacteria each time she conducts a successful experiment, she learns almost as much when her tests go awry. “I’ve had to practice a lot of troubleshooting,” she says, “and that’s not something you can learn in class. But everyone in the lab is incredibly friendly and always willing to answer questions or give advice.”

As a teaching assistant for the lab class 7.02 (Introduction to Experimental Biology and Communication), McKinney had the chance to help other students conduct experiments, answering their questions and grading their lab notebooks. She took 7.02 last spring, but says it’s been enlightening to experience the class through a different lens. She adds: “I definitely understand the material more deeply than I did before.”

In addition to TAing, McKinney teaches an SAT preparatory program run by MIT students. “At first, standing up and talking in front of a 20-person section was rather terrifying, but it’s become so much easier,” she says. “The experience has been really good for me.”

After she graduates, McKinney knows she wants to go to graduate school — likely for microbiology — but beyond that, nothing is concrete. She is sure of one thing, though: joining the Grossman lab was one of the best decisions she’s made at MIT.

She advises all current and prospective students to do a UROP. “Find something you’re really interested in,” she says. “It’s okay not to know a lot coming in; you’re going to learn so much, including topics and techniques you won’t learn in class. And don’t be too disappointed when things don’t work; that’s just part of the process. And when you finally get something to work that you’ve been troubleshooting for a while, the feeling is absolutely amazing.”

Posted 2.5.19
Biologist Adam Martin studies the mechanics of tissue folding

The dynamic process is critical to embryonic development and other cellular phenomena.

Anne Trafton | MIT News Office
February 1, 2019

Embryonic development is tightly regulated by genes that control how body parts form. One of the key responsibilities of these genes is to make sure that tissues fold into the correct shapes, forming structures that will become the spine, brain, and other body parts.

During the 1970s and ’80s, the field of embryonic development focused mainly on identifying the genes that control this process. More recently, many biologists have shifted toward investigating the physics behind the tissue movements that occur during development, and how those movements affect the shape of tissues, says Adam Martin, an MIT associate professor of biology.

Martin, who recently earned tenure, has made key discoveries in how tissue folding is controlled by the movement of cells’ internal scaffolding, known as the cytoskeleton. Such discoveries can not only shed light on how tissues form, including how birth defects such as spina bifida occur, but may also help guide scientists who are working on engineering artificial human tissues.

“We’d like to understand the molecular mechanisms that tune how forces are generated by cells in a tissue, such that the tissue then gets into a proper shape,” Martin says. “It’s important that we understand fundamental mechanisms that are in play when tissues are getting sculpted in development, so that we can then harness that knowledge to engineer tissues outside of the body.”

Cellular forces

Martin grew up in Rochester, New York, where both of his parents were teachers. As a biology major at nearby Cornell University, he became interested in genetics and development. He went on to graduate school at the University of California at Berkeley, thinking he would study the genes that control embryonic development.

However, while in his PhD program, Martin became interested in a different phenomenon — the role of the cytoskeleton in a process called endocytosis. Cells use endocytosis to absorb many different kinds of molecules, such as hormones or growth factors.

“I was interested in what generates the force to promote this internalization,” Martin says.

He discovered that the force is generated by the assembly of arrays of actin filaments. These filaments tug on a section of the cell membrane, pulling it inward so that the membrane encloses the molecule being absorbed. He also found that myosin, a protein that can act as a motor and controls muscle contractions, helps to control the assembly of actin filaments.

After finishing his PhD, Martin hoped to find a way to combine his study of cytoskeleton mechanics with his interest in developmental biology. As a postdoc at Princeton University, he started to study the phenomenon of tissue folding in fruit fly embryonic development, which is now one of the main research areas of his lab at MIT. Tissue folding is a ubiquitous shape change in tissues to convert a planar sheet of cells into a three-dimensional structure, such as a tube.

In developing fruit fly embryos, tissue folding invaginates cells that will form internal structures in the fly. This folding process is similar to tissue folding events in vertebrates, such as neural tube formation. The neural tube, which is the precursor to the vertebrate spinal cord and brain, begins as a sheet of cells that must fold over and “zip” itself up along a seam to form a tube. Problems with this process can lead to spina bifida, a birth defect that results from an incomplete closing of the backbone.

When Martin began working in this area, scientists had already discovered many of the transcription factors (proteins that turn on networks of specific genes) that control the folding of the neural tube. However, little was known about the mechanics of this folding.

“We didn’t know what types of forces those transcription factors generate, or what the mechanisms were that generated the force,” he says.

He discovered that the accumulation of myosin helps cells lined up in a row to become bottle-shaped, causing the top layer of the tissue to pucker inward and create a fold in the tissue. More recently, he found that myosin is turned on and off in these cells in a dynamic way, by a protein called RhoA.

“What we found is there’s essentially an oscillator running in the cells, and you get a cycle of this signaling protein, RhoA, that’s being switched on and off in a cyclical manner,” Martin says. “When you don’t have the dynamics, the tissue still tries to contract, but it falls apart.”

He also found that the dynamics of this myosin activity can be disrupted by depleting genes that have been linked to spina bifida.

Breaking free

Another important cellular process that relies on tissue folding is the epithelial-mesenchymal transition (EMT). This occurs during embryonic development when cells gain the ability to break free and move to a new location. It is also believed to occur when cancer cells metastasize from tumors to seed new tumors in other parts of the body.

During embryonic development, cells lined up in a row need to orient themselves so that when they divide, both daughter cells remain in the row. Martin has shown that when the mechanism that enables the cells to align correctly is disrupted, one of the daughter cells will be squeezed out of the tissue.

“This has been proposed as one way you can get an epithelial-to-mesenchymal transition, where you have cells dissociate from native tissue,” Martin says.  He now plans to further study what happens to the cells that get squeezed out during the EMT.

In addition to these projects, he is also collaborating with Jörn Dunkel, an MIT associate professor of mathematics, to map the network connections between the myosin proteins that control tissue folding during development. “That project really highlights the benefits of getting people from diverse backgrounds to analyze a problem,” Martin says.

Study shows how specific gene variants may raise bipolar disorder risk

Findings could help inform new therapies, improve diagnosis.

David Orenstein | Picower Institute for Learning and Memory
January 18, 2019

A new study by researchers at the Picower Institute for Learning and Memory at MIT finds that the protein CPG2 is significantly less abundant in the brains of people with bipolar disorder (BD) and shows how specific mutations in the SYNE1 gene that encodes the protein undermine its expression and its function in neurons.

Led by Elly Nedivi, professor in MIT’s departments of Biology and Brain and Cognitive Sciences, and former postdoc Mette Rathje, the study goes beyond merely reporting associations between genetic variations and psychiatric disease. Instead, the team’s analysis and experiments show how a set of genetic differences in patients with bipolar disorder can lead to specific physiological dysfunction for neural circuit connections, or synapses, in the brain.

The mechanistic detail and specificity of the findings provide new and potentially important information for developing novel treatment strategies and for improving diagnostics, Nedivi says.

“It’s a rare situation where people have been able to link mutations genetically associated with increased risk of a mental health disorder to the underlying cellular dysfunction,” says Nedivi, senior author of the study online in Molecular Psychiatry. “For bipolar disorder this might be the one and only.”

The researchers are not suggesting that the CPG2-related variations in SYNE1 are “the cause” of bipolar disorder, but rather that they likely contribute significantly to susceptibility to the disease. Notably, they found that sometimes combinations of the variants, rather than single genetic differences, were required for significant dysfunction to become apparent in laboratory models.

“Our data fit a genetic architecture of BD, likely involving clusters of both regulatory and protein-coding variants, whose combined contribution to phenotype is an important piece of a puzzle containing other risk and protective factors influencing BD susceptibility,” the authors wrote.

CPG2 in the bipolar brain

During years of fundamental studies of synapses, Nedivi discovered CPG2, a protein expressed in response to neural activity, that helps regulate the number of receptors for the neurotransmitter glutamate at excitatory synapses. Regulation of glutamate receptor numbers is a key mechanism for modulating the strength of connections in brain circuits. When genetic studies identified SYNE1 as a risk gene specific to bipolar disorder, Nedivi’s team recognized the opportunity to shed light into the cellular mechanisms of this devastating neuropsychiatric disorder typified by recurring episodes of mania and depression.

For the new study, Rathje led the charge to investigate how CPG2 may be different in people with the disease. To do that, she collected samples of postmortem brain tissue from six brain banks. The samples included tissue from people who had been diagnosed with bipolar disorder, people who had neuropsychiatric disorders with comorbid symptoms such as depression or schizophrenia, and people who did not have any of those illnesses. Only in samples from people with bipolar disorder was CPG2 significantly lower. Other key synaptic proteins were not uniquely lower in bipolar patients.

“Our findings show a specific correlation between low CPG2 levels and incidence of BD that is not shared with schizophrenia or major depression patients,” the authors wrote.

From there they used deep-sequencing techniques on the same brain samples to look for genetic variations in the SYNE1 regions of BD patients with reduced CPG2 levels. They specifically looked at ones located in regions of the gene that could regulate expression of CPG2 and therefore its abundance.

Meanwhile, they also combed through genomic databases to identify genetic variants in regions of the gene that code CPG2. Those mutations could adversely affect how the protein is built and functions.

Examining effects

The researchers then conducted a series of experiments to test the physiological consequences of both the regulatory and protein coding variants found in BD patients.

To test effects of non-coding variants on CPG2 expression, they cloned the CPG2 promoter regions from the human SYNE1 gene and attached them to a “reporter” that would measure how effective they were in directing protein expression in cultured neurons. They then compared these to the same regions cloned from BD patients that contained specific variants individually or in combination. Some did not affect the neurons’ ability to express CPG2 but some did profoundly. In two cases, pairs of variants (but neither of them individually), also reduced CPG2 expression.

Previously Nedivi’s lab showed that human CPG2 can be used to replace rat CPG2 in culture neurons, and that it works the same way to regulate glutamate receptor levels. Using this assay they tested which of the coding variants might cause problems with CPG2’s cellular function. They found specific culprits that either reduced the ability of CPG2 to locate in the “spines” that house excitatory synapses or that decreased the proper cycling of glutamate receptors within synapses.

The findings show how genetic variations associated with BD disrupt the levels and function of a protein crucial to synaptic activity and therefore the health of neural connections. It remains to be shown how these cellular deficits manifest as biopolar disorder.

Nedivi’s lab plans further studies including assessing behavioral implications of difference-making variants in lab animals. Another is to take a deeper look at how variants affect glutamate receptor cycling and whether there are ways to fix it. Finally, she said, she wants to continue investigating human samples to gain a more comprehensive view of how specific combinations of CPG2-affecting variants relate to disease risk and manifestation.

In addition to Rathje and Nedivi, the paper’s other authors are Hannah Waxman, Marc Benoit, Prasad Tammineni, Costin Leu, and Sven Loebrich.

The JPB Foundation, the Gail Steel Fund, the Carlsberg Foundation, the Lundbeck Foundation and the Danish Council for Independent Research funded the study.

Decoding patterns and meaning in biological data

Senior Anna Sappington found her perfect balance of “innovative computer science and innovative biology” as a member of the team mapping every cell in the human body.

Raleigh McElvery
December 5, 2018

When Anna Sappington was six years old, her parents gave her a black and white composition notebook. Together, they began jotting down observations to identify the patterns in their wooded backyard near the Chesapeake Bay. How would the harsh winters or the early springs affect the blooming trees? How many bluebirds nested each season and how many eggs would they lay? When would the cicada population cycle peak? Her father, the environmental scientist, taught her to sift through data to uncover the trends. Her mother, the journalist, gave her the words to describe her findings.

But it wasn’t until Sappington competed in the Intel International Science and Engineering Fair her junior year of high school that she probed one tiny niche of the natural world more keenly than she ever had before: the physiology of the water flea. Specifically, she investigated the developmental changes that these minute creatures experienced after being exposed to the antimicrobial compound triclosan, present in many soaps and toothpastes. She was surprised to learn that it required only a low concentration of triclosan (0.5 ppm) to cause developmental defects.

She’d been familiar with the concept of DNA since middle school, but her fellow science fair finalists were delving beyond their observations and into the letters of the genetic code. This gave her a new impetus: to understand how triclosan worked at the level of the genome and epigenome to engender the physical deformities she observed under the microscope. She just needed the proper tools, so she made some calls.

Environmental geneticist and water flea aficionado John Colbourne took an interest, and invited her to his lab at University of Birmingham in the U.K. the following summer so she could learn basic lab techniques. Although her friends and classmates didn’t quite get why she needed to travel to an entirely different country to study an organism they’d never heard of, as she puts it, she had burning scientific questions that needed answers.

“That was the experience that really turned me on to genomics,” says Sappington, now a senior and 6-7 (Computer Science and Molecular Biology) major. “I was finally getting the tools to dig through large amounts of data, using code to find patterns and meaning. I wanted to keep asking ‘why?’ and ‘how?’ all the way down to the molecular level.”

The summer before her freshman year of college, Sappington asked these questions in humans for the time as an intern at the National Human Genome Research Institute (NHGRI). There, she helped create a computational pipeline to identify the genomic changes associated with heightened risk of cardiovascular disease.

She enrolled at MIT the following fall, because she wanted to be around people from every scientific subfield imaginable. When she arrived, the joint major in computer science and biology was still relatively new.

“While a few of the required classes did meld the two, many of them offered training in each separately,” she says. “That approach really appealed to me because I was hoping to develop both skill sets independently. I wanted to learn code and write algorithms that could be applied to any field, and I also loved understanding the biological mechanisms behind different diseases and viruses.”

Before she’d even officially declared her major, Sappington was already running experiments in Sangeeta Bhatia’s lab. There, at the Koch Institute, she studied the effects of HPV infection on gene expression in liver cells. Sappington’s main role was data analysis, striving to determine which genes were amplified in response to disease.Despite their obvious differences, Sappington found the two areas to be more similar than she had initially anticipated. In her Introduction to Algorithms class, she leveraged an arsenal of algorithms with certain outputs, conditions, and run times to decode her problem sets. In Organic Chemistry, she deployed a list of foundational reactions to solve synthesis questions on her exams. “In each case, you have to combine your understanding of these fundamental rules and come up with a creative solution to decipher an unknown,” she says.

One year later, Sappington moved to Aviv Regev’s lab at the Broad Institute. There, she learned computational techniques for decoding protein interaction networks. After a year, she began working on an international project called the Human Cell Atlas as a member of the Regev and the Sanes lab collaboration.

“The overarching mission is to create a reference map of all human cells,” Sappington explains. “We want to add a layer of functional understanding on top of what we know about the genome, to understand how different cell types differ and how they interact to impact disease. This kind of endeavor has never been undertaken on such a large scale before, so it’s incredibly exciting.”

Even within a single cell type — say, retinal cells — there are about six main cell categories, each of which splinter into as many as 40 subtypes with distinct molecular profiles and roles.

Beyond the biological challenges that go along with trying to distinguish all these cell types, there are numerous computational hurdles as well. Sappington enjoys these the most — grappling with how best to analyze the gene expression of a single cell separated from its tissue of origin.

“Since you’re only working with single cells rather than entire groups of cells from a tissue, the data that you get are much more sparse,” she says. “You have to sequence a lot of individual cells and build up lots of statistical power before you can be confident that a given cell is expressing specific genes. Coming up with models to determine what constitutes a cell type — and map cell types between time points or between species — are broad problems in computer science that we’re now applying to this very specific type of data.”

Although she’s been at the Broad since her sophomore year, Sappington has supplemented her MIT research experiences with summer studies elsewhere: another stint at the NHGRI and an Amgen Scholars fellowship in Japan. She’s especially excited because her first co-authored paper will soon be published. As she puts it, she’s finally found her ideal balance of “innovative computer science and innovative biology.”

But Sappington’s time at MIT has been defined by more than just lab work. She is the co-president of the Biology Undergraduate Student Association, which serves as a liaison between the Department of Biology and the wider community. She’s also a member of MedLinks, a volunteer at the Massachusetts General Hospital Department of Radiology, former managing director of TechX, and a performer for several campus dance troupes. In 2018, Sappington earned the prestigious Barry Goldwater Scholarship Award, alongside fellow 6-7 major Meena Chakraborty.

She was recently awarded the Marshall Scholarship, which will fund her master’s degrees in machine learning at University College London and oncology at the University of Cambridge beginning in the fall of 2019. After two years, she plans to start her MD-PhD. That way, she can become a practicing physician without having to give up her computer science research.

Her advice to prospective students: “When you get to MIT, just explore. Try different academic disciplines, different extracurriculars, and talk to as many people as you can. The campus is full of passionate individuals in every field imaginable, whether that’s computer science or political science.”

Posted 12.5.18
The long and short of CDK12

A new study linking RNA processing to DNA repair may open new avenues to cancer therapy.

Bendta Schroeder | Koch Institute
December 3, 2018

Mutations in the BRCA1 and BRCA2 genes pose a serious risk for breast and ovarian cancer because they endanger the genomic stability of a cell by interfering with homologous recombination repair (HR), a key mechanism for accurately repairing harmful double-stranded breaks in DNA. Without the ability to use HR to fix double-stranded breaks, the cell is forced to resort to more error-prone — and thus more cancer-prone — forms of DNA repair.

The BRCA1 and BRCA2 genes are not the only genes whose mutations foster tumorigenesis by causing an inability to repair DNA double strand breaks by HR. Mutations in twenty-two genes are known to disrupt HR, giving rise to tumors with what researchers call “BRCAness” characteristics. All but one of these BRCAness genes are known to be directly involved in the HR pathway.

The one exception, CDK12, is thought to facilitate a set of different processes altogether, involving how RNA transcripts are elongated, spliced and cleaved into their mature forms. While the connection between this RNA-modulating gene to DNA repair remained poorly understood, the identification of CDK12 as a BRCAness gene piqued significant clinical interest.

The researchers who pinpointed this connection, Sara Dubbury and Paul Boutz, both work in the laboratory of Phillip Sharp, Institute Professor, professor of biology, and member of the Koch Institute for Integrative Cancer Research. In a study appearing online in Nature on Nov. 28, they describe how they discovered a previously unknown mechanism by which CDK12 enables the production of full-length RNA transcripts and that this mechanism was especially critical to maintain functional expression of the other BRCAness genes.

When the researchers knocked out expression of CDK12, mouse stem cells showed many signs of accumulating DNA damage that prevented DNA replication from going forward, classic indications of a BRCAness phenotype. To identify what roles CDK12 may play in regulating gene expression, the researchers turned to RNA sequencing to determine which genes had increased or decreased their overall expression.

To their surprise, only genes activated by p53 and early differentiation (side effects of accumulating unrepaired DNA damage and BRCAness in mouse stem cells) accounted for the lion’s share of changes to RNA transcription. However, when the researchers instead focused on the types of RNAs transcribed, they found that many genes produced unusually short transcripts when CDK12 was absent.

Not every stretch of DNA in a gene makes it into the final RNA transcript. The initial RNA from a gene often includes sections, which researchers call “introns,” that are cut out of transcript, the discovery that earned Sharp the 1993 Nobel Prize in Physiology or Medicine and the remaining sections. “Exons,” are spliced together to form a mature transcript (mRNA). Alternately, an intronic polyadenylation (IPA) site may be activated to cleave away the RNA sequence that follows it preventing intron removal and generating a prematurely shortened transcript. These processes allow the same gene to produce alternate forms of messenger RNA (mRNA), and thus be translated into different protein sequences.

Surprisingly CDK12 knockout cells produced significantly more IPA-truncated transcripts genome-wide, while full-length transcripts for the same genes were reduced. These shortened mRNAs can vary greatly in their stability, their ability to be translated into protein, and their protein function. Thus, even while a gene may be actively transcribed, its translation into functional proteins can be radically altered or depleted by IPA activation.

While this observation began to illuminate CDK12’s role in regulating mRNA processing, what remained puzzling was why CDK12 loss affected the HR pathway so disproportionately. In investigating this question, Dubbury and Boutz found that BRCAness genes were overrepresented as a group among those genes that have increased IPA activity upon CDK12 loss.

Additionally, while CDK12 suppresses IPA activity genome-wide, 13 of the other 21 BRCAness genes were found to be particularly vulnerable to CDK12 loss, in part, because they possess multiple high-sensitivity IPA sites, which have a compound effect in decreasing the total amount of full-length transcripts. Moreover, because multiple CDK12-senstive BRCAness genes operate in the same HR pathway, the researchers believe that the disruption to HR repair of double-stranded DNA breaks is amplified.

CDK12 mutations are found recurrently in prostate and ovarian cancer patients, making them an attractive diagnostic and therapeutic target for cancer. However, not enough is known about CDK12 to distinguish between true loss-of-function mutations and so-called “passenger mutations” with no functional consequence.

“The ability to identify patients with true loss-of-function mutations in CDK12 would enable clinicians to label a new cohort of patients with bona fide BRCAness tumors that could benefit from certain highly effective and targeted chemotherapeutics against BRCAness, such as PARP1 inhibitors,” says Dubbury, a former David H. Koch Fellow.

Dubbury and Boutz were able to confirm that IPA sites in key BRCAness genes were also used more frequently upon CDK12 loss in human tumor cells using RNA sequencing data from prostate and ovarian tumor patients with CDK12 mutations and by treating human prostate adenocarcinoma and ovarian carcinoma cells with a CDK12 inhibitor. This result suggests that the CDK12 mechanism observed in mouse cell lines is conserved in humans and that CDK12 mutations in human ovarian and prostate tumors may promote tumorigenesis by increasing IPA activity and thus functionally attenuating HR repair.

“These results not only give us a better understanding how CDK12 contributes to BRCAness, they also may have exciting potential impact in the clinic,” Dubbury says. “Currently available diagnostic techniques could be used to probe the usage of IPA sites found in this study to rapidly screen for patients with true loss-of-function CDK12 mutations, who would respond to BRCAness-targeted treatments.”

Paul Boutz, a research scientist in the Sharp Lab, is co-first author of the study, and has plans to follow-up many of these implications for ovarian and prostate cancer his lab at the University of Rochester School of Medicine and Dentistry.

“CDK12 provides a remarkable example of how factors that control the processing of RNA molecules can function as master regulators of gene networks, and thereby profoundly affect the physiology of both normal and cancerous cells,” he says.

Phil Sharp, the senior author on the work, says “Sara’s and Paul’s surprising discovery that CDK12 suppresses intronic polyadenylation has implications for fundamental new insights into gene structure as well as for control of cancer.”

Activating a new understanding of gene regulation
Greta Friar | Whitehead Institute
November 15, 2018

CAMBRIDGE, Mass. – Regulation of gene expression — turning genes on or off, increasing or decreasing their expression — is critical for defining cell identity during development and coordinating cellular activity throughout the cell’s lifetime. The common model of gene regulation imagines the nucleus of the cell as a large space in which molecules involved in DNA transcription float around seemingly at random until they stumble across a DNA sequence or other transcriptional machinery to which they can bind — a haphazard approach. However, this paradigm is being upended as over the last few years researchers have discovered that rather than being amorphous spaces dependent upon fortuitous collisions, cells actually compartmentalize their processes into discrete membraneless structures in order to congregate relevant molecules, thereby better coordinating their interactions. Research from the lab of Whitehead Member Richard Young and others earlier this year reported that such compartmentalization is a crucial, previously unobserved aspect of gene regulation.[1]

The latest research from Young’s lab, published online November 15 in the journal Cell, delves further into how such compartmentalization helps orchestrate transcriptional regulation by revealing the role of the activation domain, a part of transcription factors previously shrouded in mystery. One side of transcription factors, containing the DNA binding domain, binds to a region of DNA near a gene. The other end, called the activation domain, then captures molecules that impact gene expression, anchoring that transcriptional machinery near the gene.

This most recent work reveals that activation domains do their job by meshing with other transcription proteins to form liquid droplets near the genes they regulate. The process by which the molecules form a distinct liquid compartment within the environment of the cell — like oil refusing to mix with vinegar in a salad dressing — is called phase separation.

Such an evolved understanding of gene regulation has enormous implications for medicine and drug discovery, as errors in gene regulation are key components of many diseases, including cancers. The new model could help illuminate how diseases coopt regulatory mechanisms and how therapeutic interventions might remedy such dysregulation. Transcription factors have traditionally been hard to target therapeutically, and the incomplete understanding of their structure and function may have been part of the reason.

“Transcriptional regulation is important for every human function, from cell differentiation to development to cell maintenance,” says Ann Boija, co-first author and postdoctoral researcher in Young’s lab. “Despite that fact the structure and function of the activation domain on the transcription factors have been poorly understood.”

Most proteins settle into defined three-dimensional structures and can only bind with other molecules that fit them perfectly, in a specific orientation, like a key in a lock. The activation domains of transcription factor proteins, however, contain what are known as intrinsically disordered regions, which behave more like strands of cooked spaghetti, tangling at random into flexible shapes. This disorder allows the molecules to bind at many points, creating a dynamic network of loose connections that appears to precipitate phase separation.

“I have taught regulatory biology for decades using inspiration from lock and key structures. They are elegant, and easy to visualize and model, but they don’t tell the whole story. Phase separation was the missing piece,” says Young, who is also a professor of biology at MIT.

In experiments with a variety of transcription factors, Boija and co-first author Isaac Klein, a postdoctoral researcher in Young’s lab and medical oncology fellow at the Dana-Farber Cancer Institute, found that the transcription factors meshed with Mediator, a molecule that helps activate genes, and phase separated into droplets, and that this process was associated with gene activation. The transcription factors they investigated included OCT4, which is important for maintaining the state of embryonic stem cells; the estrogen receptor (ER), which plays a role in breast cancer; and GCN4, a well-studied model transcription factor in yeast.

The discovery has implications for many diseases, such as cancer, in which cancer genes may use phase separated droplets to help ramp up their expression. New therapeutic approaches could focus on dissolving the droplets, and drug discovery can incorporate testing of how the drug — or target molecule — behaves inside versus outside of the droplets. This new model of how transcription factors function is not only rewriting the understanding of transcriptional regulation, it is opening up new paths for drug discovery and therapeutic approaches.“We found a link between gene activation and phase separation across a broad spectrum of contexts,” Klein says, suggesting that this mechanism is a common feature of transcriptional regulation.

The work was supported by the National Institutes of Health (NIH grants GM123511, GM117370, T32CA009172, T32GM08759), the National Science Foundation (NSF grant PHY1743900), Swedish Research Council (VR 2017-00372), Damon Runyon Cancer Research Foundation (2309-17), Hope Funds for Cancer Research, Cancer Research Institute, and Netherlands Organisation for Scientific Research (NWO).

***
Richard Young’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.
***
Full citation:
“Transcription factors activate genes through the phase separation capacity of their activation domains”
Cell, online November 15, DOI: 10.1016/j.cell.2018.10.042
Ann Boija (1,7), Isaac A. Klein (1,2,7), Benjamin R. Sabari (1), Alessandra Dall’Agnese (1), Eliot L. Coffey (1,3), Alicia V. Zamudio (1,3), Charles H. Li (1), Krishna Shrinivas (4,5), John C. Manteiga (1,3), Nancy M. Hannett (1), Brian J. Abraham (1), Lena K. Afeyan (1,3), Yang E. Guo (1), Jenna K. Rimel (6), Charli B. Fant (6), Jurian Schuijers (1), Tong Ihn Lee (1), Dylan J. Taatjes (6), and Richard A. Young (1,3)
  1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA
  3. Department of Biology
  4. Department of Chemical Engineering
  5. Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
  6. Department of Biochemistry, University of Colorado, Boulder, CO 80303, USA
  7. These authors contributed equally
[1] Sabari et al., “Coactivator condensation at super-enhancers links phase separation and gene control,”
Science, June 21, 2018; Cho et al., “Mediator and RNA polymerase II clusters associate in transcription-dependent condensates,” Science, June 21 2018.
Immune cell variations contribute to malaria severity

Natural killer cells’ failure to respond to infection may explain why the disease is more grave in some patients.

Anne Trafton | MIT News Office
October 4, 2018

At least 250 million people are infected with malaria every year, and about half a million of those die from the disease. A new study from MIT offers a possible explanation for why some people are more likely to experience a more severe, and potentially fatal, form of the disease.

The researchers found that in some patients, immune cells called natural killer cells (NK cells) fail to turn on the genes necessary to effectively destroy malaria-infected red blood cells.

The researchers also showed that they could stimulate NK cells to do a better job of killing infected red blood cells grown in a lab dish. This suggests a possible approach for developing treatments that could help reduce the severity of malaria infections in some people, especially children, says Jianzhu Chen, one of the study’s senior authors.

“This is one approach to that problem,” says Chen, an MIT professor of biology and a member of MIT’s Koch Institute for Integrative Cancer Research. “Most of the malaria patients who die are children under the age of 5, and their immune system has not completely formed yet.”

Peter Preiser, a professor at Nanyang Technical University (NTU) in Singapore, is also a senior author of the study, which appears in the journal PLOS Pathogens on Oct. 4. The paper’s lead authors are NTU and Singapore-MIT Alliance for Research and Technology (SMART) graduate students Weijian Ye and Marvin Chew.

First line of defense

In 2010, Chen and his colleagues engineered strains of mice that produce several types of human immune cells and red blood cells. These “humanized” mice can be used to study the human immune response to pathogens that don’t normally infect mice, such as Plasmodium falciparum, the parasite that causes malaria.

A few years later, the researchers used those mice to investigate the roles of NK cells and macrophages in malaria infection. These two cell types are key players in the innate immune system, a nonspecific response that acts as the first line of defense against many microbes. Chen and his colleagues found that when they removed human NK cells from the mice and infected them with malaria, the quantity of parasites in the blood was much greater than in mice with NK cells. This did not happen when they removed human macrophages, suggesting that NK cells are the most important first-line defenders against malaria.

A natural killer (NK) cell binds to a malaria-infected red blood cell and destroys it. Credit: Weijian Ye

In that study, the researchers also found that in about 25 percent of the human blood samples they used, the NK cells did not respond to malaria at all. In the new paper, they set out to try to find out why that was the case. To do that, they sequenced the RNA of NK cells before and after they encountered malaria-infected red blood cells. This allowed the researchers to identify a small number of genes that get turned on in malaria-responsive NK cells but not in nonresponsive cells.

Among these genes was one that codes for a protein called MDA5, which was already known to be involved in helping immune cells such as NK cells and macrophages recognize foreign RNA. Further studies revealed that malaria-infected red blood cells secrete tiny bubbles called microvesicles that carry pieces of RNA from the malaria parasite. The studies also showed that NK cells absorb these microvesicles. If MDA5 is present, the NK cell is activated to kill the infected blood cell.

Nonresponsive NK cells, which have lower levels of MDA5, fail to recognize and kill the infected cells. NK cells are also responsible for secreting cytokines that summon T cells and other immune cells, so their failure to activate also hinders other elements of the immune response.

Boosting immunity

Chen and his colleagues also showed that they could activate the nonresponsive NK cells by treating them with a synthetic molecule called poly I:C, which is structurally similar to double-stranded RNA. For poly I:C to be effective, the researchers had to package it into tiny spheres called liposomes, which allow it to enter cells just like the RNA-carrying microvesicles do.

The researchers also found a correlation between the levels of MDA5 in the NK cells and the disease severity experienced by the patients who donated the blood samples. Next, they hope to take cells from human patients and use them to further examine this correlation in humanized mice, and also to explore whether treating the mice with poly I:C would have the same beneficial effect they saw in cells grown in a lab dish.

The research was funded by the National Research Foundation of Singapore through the SMART Interdisciplinary Research Group in Infectious Disease Research Program.

A Summer of Protein Degradation and the Beauty of Basic Science

MSRP-Bio student Elizabeth Bond worked in the Baker Lab, investigating the macromolecular machines that roam the cell and gobble up unneeded proteins.

Raleigh McElvery
September 25, 2018

Elizabeth Bond’s greatest summer accomplishment is proudly displayed as the background image on her phone. To the casual observer, it looks like columns of black blobs, but to Bond this stained protein gel signifies that, after two long weeks, she successfully isolated her protein of interest. The snapshot also underscores that she’s found her “people” — the kind who, as she describes, “will freak out with you over a great looking gel.”

A rising senior at UMass Amherst, Bond joined 18 fellow MIT Summer Research Program in Biology (MSRP-Bio) students — collaborating in labs across the biology department and various MIT-affiliated institutes for 10 weeks. Together, they attended seminars, lectures, Q&A sessions, meals, and field trips while living in dorms and bonding over science and life in general.

“You’re with a group of other college students looking towards the future, and you’re all stressing out about what comes next,” she says. “That’s amazing because you’re able to talk about your different insecurities and anxieties. You have a built-in support system that you might not get by staying at your home institution over the summer.”

Bond grew up not too far from MIT in the quiet town of Boxford, Massachusetts. Before setting foot on campus, she expected MIT to be cutthroat and competitive. Instead, she found “a bunch of nerds who are willing to help other nerds learn, make mistakes, and be human beings.” The researchers she met were supportive and eager to share their insights, scientific or otherwise. “In addition to being really interesting, these conversations helped me feel that I fit in with a group of very intelligent scientists,” she says.

As a biochemistry major, Bond appreciates basic science because it allows her to probe biological phenomena with no immediate goal other than to understand the underlying mechanisms. “Maybe 10 years down the line my research will help someone’s translational work, but right now I can pursue knowledge for its own sake,” she says. “The beauty of basic science is that you’re able to study things because they’re cool, while also contributing to the body of work your lab family began before you.”

At UMass Amherst, she serves as an undergraduate research assistant, investigating AAA+ proteases — the same protein degradation machines she studied all summer in Tania Baker’s lab, mentored by graduate student Kristin Zuromski. Back home, Bond examines these proteases in bacteria, using a combination of microbiology and computational biology. As a member of the Baker lab, she studied these macromolecular machines leveraging biochemical approaches.

She likens AAA+ proteases to Pac-Men from the classic arcade game, roaming the cell and gobbling up misfolded, excess, or unneeded proteins. One of the AAA+ proteases studied in the Baker lab is ClpAP, which is comprised of the AAA+ unfoldase ClpA and its partner peptidase ClpP. Bond’s protein of interest this summer was ClpA, a hexameric protein depicted as a cylinder with a central channel. ClpA unfolds and threads protein substrates through its channel, which contains pore loop structures that protrude from the chamber and play important roles in the function of ClpA. From there, the proteins enter ClpP, where they are degraded into small peptide fragments.

There are two types of pore loops in ClpA, D1 and D2, but their respective roles in the recognition, unfolding, and movement of proteins for degradation are not fully characterized. Bond hoped to discern their roles relative to one another.

She introduced a mutation into the gene that encodes ClpA, switching one amino acid for another in the D2 pore loop, in a region thought to be critical for recognizing proteins targeted for degradation. This mutation would, in theory, lead to a variant of ClpA where the D1 pore loop retained normal activity, but the D2 pore loop was unable to function. She used chemical crosslinking to generate a ClpA dimer variant that was half wildtype and half mutant in the D2 pore loops, and monitored the ability of the assembled hexameric AAA+ protease to function.

By observing the degradation activity of this crosslinked ClpA variant containing three active and three inactive D2 pore loops in an alternating order, she hoped to get a better sense of the role the D2 pore loops play in ClpAP protease function.

Although there is still more to be done to answer this particular question, reflecting on the summer Bond feels her project went “surprisingly well,” despite being more challenging than she initially anticipated — primarily due to multi-week protein purification experiments and performing many procedures simultaneously. She arrived with the sole intention of bolstering her biochemistry knowledge, and left with a greater appreciation for the breadth of scientific fields she could pursue.

“MSRP-Bio gave me the chance to talk with students and faculty members working in multiple branches of science,” she says. “I study bacteria, but I can learn a lot from someone researching roundworms or cancer cells, or using computational approaches to biology. Those conversations prompted me to think more critically about my own research.”

Besides feeling integrated into the tightly-knit Baker lab, her favorite aspect of the summer was the bond she formed within her MSRP-Bio cohort. In addition to freaking out over protein gels, they started their own journal club, and they discussed personal struggles, family, where they came from, and where they want to go.

“A lot of students of color who come from underrepresented groups in science, like I do, have this anxiety about not being smart enough or not fitting in,” she says. “The program allows you to bond over these shared feelings and that is part of what makes it really amazing for students who are trying to do great things, but do not often feel fully represented.”

At the beginning of the summer, Bond hadn’t fully admitted to herself that she wanted to apply to grad school. “It was easier for me to be ambiguous about what I wanted to do, because it was scary to admit that grad school was something I might actually want,” she recalls. After a summer at MIT, she’s gained the confidence to apply and state her ambitions out loud.

“My project’s been amazing and great, but now I want to have my own body of work,” she says. “It’s something I have this great urge to do — and, because of MSRP-Bio, I’m ready for it.”

Photo credit: Raleigh McElvery
Parasite’s riff on essential enzyme highlights unique biology
Nicole Giese Rura | Whitehead Institute
September 18, 2018

Cambridge, Mass. — The primary currency of energy in cells—adenosine triphosphate (ATP)—is essential for their survival and without it, cellular processes would seize. In the apicomplexan Toxoplasma gondii (T. gondii), a parasite that Whitehead Member Sebastian Lourido studies, key components of the ATP synthase—the enzyme responsible for ATP production—have remained elusive. While investigating indispensable proteins with unknown functions, Lourido and Diego Huet, a postdoctoral researcher in Lourido’s lab, identified a critical component of the enzyme. While highly conserved from yeast to humans, it proved to be considerably different in T. gondii. The findings, published online September 11 in the journal eLife, underscore the unique biology of these parasites and highlight differences between them and their human hosts.

More closely related to plants than to animals, the single-celled apicomplexans are among the most common and deadly human pathogens. According to the World Health Organization, every year these diseases sicken hundreds of millions, kill hundreds of thousands—primarily children—and cost billions of dollars to treat. Species of apicomplexans cause malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.), and toxoplasmosis (T. gondii).

Using a CRISPR-based genetic screen that they had adapted to T. gondii, Lourido and Huet had previously identified about 200 genes in T. gondii that are fitness-conferring and specific to apicomplexans. Of that cadre, a few were localized to the mitochondria, where cells manufacture ATP, the cellular currency of energy. Because those genes have not been annotated previously, and the proteins encoded by them have no known function, Huet ran their protein sequences through a database that compared them to protein sequences with known structures.

One of the proteins came back with an interesting hit: it shares structural similarity, but not sequence similarity, with an integral part of the ATP synthase. Most of the protein subunits that compose the apicomplexan ATP synthase have been identified, but key components of the stator—a portion of the enzyme essential for its function—was not yet known.

When Huet experimentally removed the function of the stator subunit in T. gondii, the parasites’ growth stalled, their mitochondria were misshapen and shrunken, and energy production halted—all traits typical of interrupted ATP synthase function.

Because the apicomplexan ATP synthase varies so much from its hosts’ version, those differences, like the unusual stator, could serve as future drug targets. But for Lourido, who is also an assistant professor of biology at Massachusetts Institute of Technology (MIT), the unique stator protein emphasizes how unique and extraordinary apicomplexan organisms are compared to us and their other hosts.

This work was supported by the National Institutes of Health (NIH grants 1DP5OD017892, R21AI123746, and K99AI137218).

* * *

Sebastian Lourido’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an assistant professor of biology at Massachusetts Institute of Technology.

* * *

Full Citation:

“Identification of cryptic subunits from an apicomplexan ATP synthase”

eLife, online September 11, 2018.  DOI: 10.7554/eLife.38097

Diego Huet (1) , Esther Rajendran (2) , Giel G van Dooren (2) , Sebastian Lourido (1,3*).

1. Whitehead Institute for Biomedical Research, Cambridge, United States

2. Research School of Biology, Australian National University, Canberra, Australia

3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States