Research Area: Human Disease

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
Heart-healthy plant chemistry
Greta Friar | Whitehead Institute
October 29, 2018

Plants have been a rich source of medicines for thousands of years. Compounds such as artemisinin, for example, used to treat malaria, and morphine, a pain reliever, are mainstay therapeutics derived from plants. However, several roadblocks in plant chemistry research have prevented scientists from tapping into the full potential of plant-based medicinal compounds, thwarting drug discovery and development. Researchers typically screen for molecules of interest by breaking the plant into very small pieces, using biochemistry to test the activity of the pieces, and isolating the molecules responsible for the activity. It is often difficult, however, to pick the right compound responsible for a medicinal effect out of the plant mixture, or to identify the genes responsible for producing it.

The Chinese wolfberry plant (Lycium barbarum), also known as goji berry, has been used in traditional Chinese herbal medicine for millennia to treat symptoms such as high blood pressure. Researchers had identified small protein-like molecules called lyciumins, produced by the goji berry, as the source of its antihypertensive properties but little else was known about the molecules.

In research published online October 29 in the journal Proceedings of the National Academy of Sciences, Whitehead Institute Member Jing-Ke Weng and postdoctoral researcher Roland Kersten describe an approach to speed up the process of identifying plant chemistry that they used to investigate lyciumins. The approach capitalizes on the growing number of plants that have had their genomes sequenced. The wealth of genomic data available enabled Kersten to identify the gene that is associated with lyciumin production in goji berries by searching for a DNA sequence that matched the sequences of the lyciumins. Once Kersten found the matching precursor gene in goji berries he inserted it into a tobacco plant, which began producing lyciumins, confirming that he had found the right gene.

Kersten then hunted for lyciumin-producing genes in other plant genomes using a common feature of the genes that he had identified as a search query. He discovered more than one hundred unknown lyciumins in everything from potatoes to beets to soybeans.

Having sped up the gene discovery stage, Kersten used gene expression techniques to likewise speed up the molecule production stage. Being able to quickly produce large quantities of a drug candidate is necessary for testing and manufacturing the drug. Kersten edited the lyciumin precursor genes to make more copies of the molecule and then inserted the edited genes into the tobacco plant to mass produce lyciumins up to 40 times faster than the original plants. Kersten was also able to edit the lyciumins’ DNA sequences to alter the molecules’ structure, creating new varieties of lyciumins not found in nature. Together, these results allow for the future creation of a lyciumin library, a valuable repository for drug discovery research. Millions of different lyciumins can be grown in tobacco and tested for their efficacy as antihypertensive drugs or in other potential agrochemical and pharmaceutical applications.

Weng and Kersten’s approach leverages the recent explosion in plant genomics to uncover important medicinal compounds in plants and reveal the secrets of plants used in traditional global medicine for generations. For Kersten, the research was also an exciting demonstration of just how much undiscovered chemistry lies waiting to be tapped in even the best-studied crop plants.

This work was supported by grants from the Thome Foundation, the Pew Scholars Program in the Biomedical Sciences, the Searle Scholars Program, and the Family Larsson Rosenquist Foundation.

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Jing-Ke Weng’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:
“Gene-guided discovery and engineering of branched cyclic peptides in plants”
PNAS, online on October 29.
Roland D. Kersten (1), Jing-Ke Weng (1,2)
1. Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA, United States
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States
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).

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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.
How Many Evolutionary Events Can It Take To Screw in Nature’s Lightbulb?

Firefly genomics reveals independent evolution of bioluminescence in beetles

Lisa Girard | Whitehead Institute
October 16, 2018

Cambridge, MA — Researchers at Whitehead Institute and collaborators from fourteen other institutions around the world have shed light on the evolutionary origins of luciferase, the key enzyme behind the glow of fireflies and other bioluminescent beetles. By sequencing the genomes of two American and Japanese firefly species that diverged approximately 100 million years ago, along with a more evolutionarily distant bioluminescent Caribbean click beetle, the team discovered that luciferase appears to have arisen independently in fireflies and click beetles. Examining the genes flanking that encoding the luciferase gene, suggests an evolutionary path along which the luciferase gene arose from duplications and divergences of CoA ligase genes involved in fat metabolism. As described online October 16 in the journal eLife, these findings provide fundamental insights into how enzymes can evolve, potentially inform strategies to help protect bioluminescent beetles from a shifting climate and habitat, and could extend the utility of luciferase, which has also been harnessed for biomedical and agricultural research, as a laboratory tool.

Throughout much of the world, the silent flash of a firefly on a warm evening can only mean one thing-Summer has arrived. But fireflies don’t just signal summer, their glow serves as a mating signal to other fireflies, and is even a warning that they are chemically defended, having a noxious taste capable of repelling the boldest of predators.

Belying its grandeur, the chemistry of firefly bioluminescence is relatively straightforward. Their light is produced by a specialized firefly enzyme, luciferase, that breaks down a molecule called luciferin, producing light in the process. Luciferase has become a mainstay tool in the laboratory. Scientists can fuse their gene of interest to luciferase and assay for gene expression by measuring the intensity of the glow after luciferin is added.

Beyond fireflies, there are other bioluminescent beetles (despite their name, fireflies are actually beetles), including certain tropical click-beetles. Perplexingly, these diverse bioluminescent beetles use very similar luciferase enzymes and luciferin molecules, but have an unrelated anatomy of their light-producing organs (also known as lanterns), making it unclear if their bioluminescence evolved from a common luminous ancestor, or if their special glow evolved independently.

Since fireflies and bioluminescent click-beetles are not model organisms like mice or fruit flies for which there is a wealth of genetic information, Jing-Ke Weng, Whitehead Institute Member and assistant professor of biology at Massachusetts Institute of Technology (MIT), along with a  graduate student in Weng’s lab, Tim Fallon, and Cornell postdoctoral researcher, Sarah Lower, began their investigations by sequencing the genome of the American Big Dipper firefly, Photinus pyralis. Named for its distinctive swooping “J” flash , this common inhabitant of meadows and suburban lawns has been called the “All-American firefly”. Due to its abundance and ease of identification, it was also the firefly of choice for scientific study, and is the species from which luciferin and luciferase were first characterized. Wanting to start their work quickly and make their progress and data available to others in the firefly community, Whitehead Institute researchers and collaborators crowdsourced funds to sequence the Big Dipper firefly.

The Big Dipper genome sequence, they discovered, revealed interesting insights into the origin of the luciferase gene. Examining the genes flanking that encoding luciferase, they found a cluster, or tandem repeat, of fatty acid CoA ligase genes with the luciferase gene sitting in the middle of this cluster. Sequence similarity and proximity between the luciferase and fatty acid CoA ligase genes suggested an evolutionary path along which the luciferase gene was produced from tandem duplication and divergence of an ancestral fatty acid CoA ligase gene.

“When the luciferase gene was cloned, people knew it was similar to the fatty acid CoA ligase gene in sequence, and hypothesized that it must be related to that ancestry. But what we uncovered from the luciferase gene locus is a tandem repeat of five genes, four are still the fatty acid CoA ligases, but then luciferase evolved right in the middle we believe from divergence of one of these duplications,” says Weng.

The Big Dipper sequencing provided important insights into the origin of luciferase and additional factors involved in bioluminescence, but in order to gain additional insights into the evolution of bioluminescence, the researchers set out to sequence two additional species that they hoped would provide the additional context to help them triangulate on some answers.

The bioluminescent click beetle, Ignelater luminosus, is related to the firefly, but on another branch of the tree of life entirely. Instead of producing light at its tail, it has two lanterns behind its head.

“We thought that sequencing the click beetle would provide insights into the evolution of bioluminescence as well as perhaps into how these animals could acquire very similar traits in terms of their biochemistry, but not in terms of their development,” says Fallon.

The third species they selected to sequence was a Japanese aquatic firefly (Aquatica lateralis), known in Japan as the Heike firefly.  Heike and the Big Dipper diverged from one another over 100 million years ago (to give you a sense of how far this is, it is older than the evolutionary distance between humans and rodents).

The researchers analyzed genomic data from the Japanese aquatic firefly and saw a similar arrangement around the luciferase gene locus as they had in the Big Dipper genome, suggesting that luciferase arose from a common ancestral event in both firefly species. The structure around the luciferase locus in the click beetle, however, was entirely absent, suggesting that luciferase arose through a different event.  Taken together, by sequencing and analyzing data from the genomes of two firefly species that diverged approximately 100 million years ago, along with a more evolutionarily distant bioluminescent click beetle, the team discovered that luciferase appears to have evolved independently in both fireflies and click beetles.

“Having the genome allowed us to understand how the evolution of luciferase happened. Before sequencing, we knew there were five genes cloned, including luciferase, in firefly. By sequencing the genomes we actually uncovered those genomic loci where those initial gene duplication events occured,” says Lower.

In addition to the origins of luciferase, these findings also provided the researchers with insights into the evolution of the light organs.

“Since our findings suggest that luciferase originated independently in both lineages, we can infer that anything that came after luciferase, for example the light organs, or other things dependent on luciferase should also be independent,” says Fallon.

Discovering how bioluminescence arose, as well as other complex traits, can be studied now that genomic information is available. The information can also inform strategies to protect fireflies, whose populations in many parts of the world are diminishing. In addition to adding tools to help reveal a constituent parts list that could allow researchers to optimize bioluminescence as a tool, these findings reveal important insights into the evolution of bioluminescence as well as genomic evolution more broadly.

“Luciferase is a perfect example of how to build a new enzyme, duplication of a related progenitor gene followed by mutation and selection,” says Weng. “And one of the most exciting parts of this study was that by examining the evolutionary scars in the genomes we studied we could actually see it happen.”

* * *
Jing-Ke Weng’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:
“Firefly genomes illuminate parallel origins of bioluminescence in beetles”
eLife, online on October 9, 2018. doi: 10.7554/eLife.36495
Timothy R. Fallon (1,2,*), Sarah E. Lower (3,*), Ching-Ho Chang (4) , Manabu Bessho-Uehara (5,6), Gavin J. Martin (7), Adam J. Bewick (8) , Megan Behringer (9) , Humberto J. Debat (10), Isaac Wong (4) , John C. Day (11), Anton Suvorov (7) , Christian J. Silva (4,12), Kathrin F. Stanger-Hall1 (3), David W. Hall (8) , Robert J. Schmitz (8), David R. Nelson (14), Sara M. Lewis (15), Shuji Shigenobu (16), Seth M. Bybee (7) , Amanda M. Larracuente (4), Yuichi Oba (5), and Jing-Ke Weng (1,2)
1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
2.Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
3. Department of Molecular Biology & Genetics, Cornell University, Ithaca, New York 14850, USA.
4. Department of Biology, University of Rochester, Rochester, New York 14627, USA.
5. Department of Environmental Biology, Chubu University, Kasugai, Aichi 487-8501, Japan.
6. Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan.
7. Department of Biology, Brigham Young University, Provo, Utah 84602, USA.
8. Department of Genetics, University of Georgia, Athens, Georgia 30602, USA.
9. Biodesign Center for Mechanisms of Evolution, Arizona State University, Tempe, Arizona 85287, USA.
10. Center of Agronomic Research National Institute of Agricultural Technology, Córdoba, Argentina.
11. Centre for Ecology and Hydrology (CEH) Wallingford, Wallingford, Oxfordshire, UK.
12. Department of Plant Sciences, University of California Davis, Davis, California, USA.
13. Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA.
14. Department of Microbiology Immunology and Biochemistry, University of Tennessee  HSC, Memphis 38163, USA.
15. Department of Biology, Tufts University, Medford, Massachusetts 02155, USA.
16. NIBB Core Research Facilities, National Institute for Basic Biology, Okazaki 444-8585, Japan.
Only in Your Head
Greta Friar | Whitehead Institute
October 9, 2018

Cambridge, Mass. — Brain development is a delicately choreographed dance in which cell division and differentiation into mature cell types must be performed in the right balance for normal growth. In order to better understand factors affecting brain development, Whitehead Institute researchers investigated a genetic mutation that leads to a brain-specific developmental disorder in spite of the gene’s prevalent expression in other cell types.

Kinetochore null protein 1 (KNL1) acts throughout the body during cell division to help ensure the accurate segregation of chromosomes into each daughter cell. A mutation in the KNL1 gene caused by a single change in its DNA sequence leads to microcephaly, a condition in which the brain fails to properly develop, causing babies to be born with small heads, often accompanied by intellectual disabilities and other health problems. In an article published online October 9 in the journal Cell Reports, Whitehead Institute Founding Member Rudolf Jaenisch and colleagues investigated how this KNL1 mutation can lead to microcephaly without affecting other cell types, providing important insights into the underlying basis of microcephaly and the role that KNL1 normally plays in brain development.

“The key question we were interested in was why, if the gene is ubiquitously expressed, is there a brain-specific phenotype,” says Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology.

Jaenisch lab graduate student Attya Omer Javed, a co-first author on the paper along with past lab members Yun Li and Julien Muffat, used CRISPR-Cas9 to recreate the mutation—a point mutation, or one-letter change in the DNA sequence of the KNL1 gene—in several different cell types derived from human stem cells in the lab. Of the three cell types tested, they found that only the neural progenitor cells, early stage cells that become brain cells, appeared to be affected.

As the brain develops, each neural progenitor can either keep dividing to increase the overall number of cells in the brain, or it can mature into a differentiated brain cell, at which point it is no longer able to divide. For a healthy brain to develop, there needs to be a careful balance between these two processes of proliferation and differentiation. If the progenitors take too long to differentiate, the developing brain won’t have the specific cells it needs to assemble. But if all of the cells differentiate too quickly, before they can divide, there will be a shortage of cells and the brain will be too small.

“Neural progenitors are going through many cell cycles, dividing quickly during brain development. Even a small defect could accumulate to have a huge impact,” Omer Javed says.

The researchers discovered that neural progenitors with the KNL1 mutation differentiated prematurely at the cost of proliferation, resulting in the small brain size that characterizes microcephaly. The brain cells with the mutation also were at a greater risk of cell death, disruption of the cell cycle, ending up with the wrong number of chromosomes, and malfunctions during attempted cell division.

KNL1’s role is in the kinetochore, an assembly of proteins that operate during mitosis to attach chromosomes to the machinery that will pull them apart into the daughter cells. This is why the KNL1 mutation negatively affects cell division. Co-author and Whitehead Member Iain Cheesemanhelped identify KNL1’s role in the kinetochore as a postdoctoral researcher years ago, and his expertise provided an opportunity for collaboration between his lab and Jaenisch’s.

“I have always found it interesting that inherited mutations to the kinetochore seem to lead to microcephaly,” Cheeseman says. “Investigating KNL1 together was an exciting chance to combine our labs’ diverse scientific knowledge.”

In order for the researchers to study the cells in an environment that more closely mimicked a human brain, they used a 3D cell culture technique to grow organoids made up of neural progenitors. Omer Javed found that the neural progenitors were extremely sensitive, as the organoids with the mutation expressed the microcephaly phenotype after as little as two weeks of growth.

Omer Javed then looked for differences between neural progenitors and the other cell types that would explain the brain-specific effects of the mutation. Even with the mutation, the KNL1 gene appeared able to make a functioning protein, explaining its lack of effect on the other cell types. So Omer Javed turned her attention to factors involved in regulating gene expression. For many of our genes to be expressed, first sections called introns must be removed, or spliced out, in order for the correct DNA sequence to then be read into RNA and then translated into a functional protein.

Omer Javed found that the KNL1 mutation created a site for splicing inhibitors to bind and silence the KNL1 gene by preventing it from being read into RNA. She also found a disparity in the level of a protein involved in this process between the cell types: the inhibitory splicing protein hnRNPA1 was much more prevalent in neural progenitors than elsewhere. When hnRNPA1 came across the site caused by the mutation, it prevented the gene from being expressed. The high quantity of hnRNPA1 in neural progenitors appears to be the main factor mediating the brain-specific effects of the mutation.

The work complements and extends previous investigations by the researchers into how neural progenitor proliferation may have contributed to the evolution of large human brains, as well as studies investigating why neural progenitors are so vulnerable to the Zika virus, which has been associated with microcephaly. Given their work suggesting that KNL1 could be a regulator of brain size, Omer Javed hopes that future research will reveal its role in the evolution of the human brain.

 

This research was supported by Boehringer Ingelheim Fonds, the Simons Foundation, the International Rett Syndrome Foundation, Brain & Behavior Research Foundation, the European Leukodystrophy Association, the National Institutes of Health (NIH grants HD 045022, R37-CA084198 and 1U19AI131135-01). Jaenisch is a cofounder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics.

 

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Rudolf Jaenisch’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.
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Full citation:
“Microcephaly modeling of kinetochore mutation reveals a brain-specific phenotype”
Cell Reports, online October 9, 2018
Attya Omer (1,2,8), Yun Li (2,3,4,8), Julien Muffat (2,4,5,8), Kuan-Chung Su (2), Malkiel A. Cohen (2), Tenzin Lungjangwa (2), Patrick Aubourg (1,6), Iain M. Cheeseman (2,7), and Rudolf Jaenisch (2,7).
1. Université Paris-Saclay, ED 569, 5 Rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France
2.  Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA
3. Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M4G 0A4, Canada
4. Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada
5. Program in Neurosciences and Mental Health, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
6. INSERM U1169, CHU Bicêtre Paris Sud, Le Kremlin-Bicêtre, France.
7. Department of Biology, MIT, 31 Ames Street, Cambridge, MA 02139, USA
8. These authors contributed equally
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.

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

Ankur Jain

Education

  • PhD, 2013, University of Illinois, Urbana-Champaign
  • BTech, 2007,  Biotechnology and Biochemical Engineering, Indian Institute of Technology Kharagpur

Research Summary

We study how biomolecules in a cell self-organize. In particular, we are interested in understanding how membrane-free cellular compartments such as RNA granules form and function. Our lab develops new biochemical and biophysical techniques to investigate these compartments and to understand their dysfunction in human disease.

Awards

  • Young Alumni Achiever’s Award, Indian Institute of Technology Kharagpur, 2019
  • NIH K99/R00 Pathway to Independence Award, 2017
  • Pew Scholar in the Biomedical Sciences, 2022
Study suggests glaucoma may be an autoimmune disease

Unexpected findings show that the body’s own immune system destroys retinal cells.

Anne Trafton | MIT News Office
August 11, 2018

Glaucoma, a disease that afflicts nearly 70 million people worldwide, is something of a mystery despite its prevalence. Little is known about the origins of the disease, which damages the retina and optic nerve and can lead to blindness.

A new study from MIT and Massachusetts Eye and Ear has found that glaucoma may in fact be an autoimmune disorder. In a study of mice, the researchers showed that the body’s own T cells are responsible for the progressive retinal degeneration seen in glaucoma. Furthermore, these T cells appear to be primed to attack retinal neurons as the result of previous interactions with bacteria that normally live in our body.

The discovery suggests that it could be possible to develop new treatments for glaucoma by blocking this autoimmune activity, the researchers say.

“This opens a new approach to prevent and treat glaucoma,” says Jianzhu Chen, an MIT professor of biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and one of the senior authors of the study, which appears in Nature Communications on Aug. 10.

Dong Feng Chen, an associate professor of ophthalmology at Harvard Medical School and the Schepens Eye Research Institute of Massachusetts Eye and Ear, is also a senior author of the study. The paper’s lead authors are Massachusetts Eye and Ear researchers Huihui Chen, Kin-Sang Cho, and T.H. Khanh Vu.

Genesis of glaucoma

One of the biggest risk factors for glaucoma is elevated pressure in the eye, which often occurs as people age and the ducts that allow fluid to drain from the eye become blocked. The disease often goes undetected at first; patients may not realize they have the disease until half of their retinal ganglion cells have been lost.

Most treatments focus on lowering pressure in the eye (also known as intraocular pressure). However, in many patients, the disease worsens even after intraocular pressure returns to normal. In studies in mice, Dong Feng Chen found the same effect.

“That led us to the thought that this pressure change must be triggering something progressive, and the first thing that came to mind is that it has to be an immune response,” she says.

To test that hypothesis, the researchers looked for immune cells in the retinas of these mice and found that indeed, T cells were there. This is unusual because T cells are normally blocked from entering the retina, by a tight layer of cells called the blood-retina barrier, to suppress inflammation of the eye. The researchers found that when intraocular pressure goes up, T cells are somehow able to get through this barrier and into the retina.

The Mass Eye and Ear team then enlisted Jianzhu Chen, an immunologist, to further investigate what role these T cells might be playing in glaucoma. The researchers generated high intraocular pressure in mice that lack T cells and found that while this pressure induced only a small amount of damage to the retina, the disease did not progress any further after eye pressure returned to normal.

Further studies revealed that the glaucoma-linked T cells target proteins called heat shock proteins, which help cells respond to stress or injury. Normally, T cells should not target proteins produced by the host, but the researchers suspected that these T cells had been previously exposed to bacterial heat shock proteins. Because heat shock proteins from different species are very similar, the resulting T cells can cross-react with mouse and human heat shock proteins.

To test this hypothesis, the team brought in James Fox, a professor in MIT’s Department of Biological Engineering and Division of Comparative Medicine, whose team maintains mice with no bacteria. The researchers found that when they tried to induce glaucoma in these germ-free mice, the mice did not develop the disease.

Human connection

The researchers then turned to human patients with glaucoma and found that these patients had five times the normal level of T cells specific to heat shock proteins, suggesting that the same phenomenon may also contribute to the disease in humans. The researchers’ studies thus far suggest that the effect is not specific to a particular strain of bacteria; rather, exposure to a combination of bacteria can generate T cells that target heat shock proteins.

One question the researchers plan to study further is whether other components of the immune system may be involved in the autoimmune process that gives rise to glaucoma. They are also investigating the possibility that this phenomenon may underlie other neurodegenerative disorders, and looking for ways to treat such disorders by blocking the autoimmune response.

“What we learn from the eye can be applied to the brain diseases, and may eventually help develop new methods of treatment and diagnosis,” Dong Feng Chen says.

The research was funded by the National Institutes of Health, the Lion’s Foundation, the Miriam and Sheldon Adelson Medical Research Foundation, the National Nature Science Foundation of China, the Ivan R. Cottrell Professorship and Research Fund, the Koch Institute Support (core) Grant from the National Cancer Institute, and the National Eye Institute Core Grant for Vision Research.

Researchers discover new type of lung cell, critical insights for cystic fibrosis

A comprehensive single-cell analysis of airway cells in mice, validated in human tissue, reveals molecular details critical to understanding lung disease.

Karen Zusi
August 1, 2018

Researchers have identified a rare cell type in airway tissue, previously uncharacterized in the scientific literature, that appears to play a key role in the biology of cystic fibrosis. Using new technologies that enable scientists to study gene expression in thousands of individual cells, the team comprehensively analyzed the airway in mice and validated the results in human tissue.

Led by researchers from the Broad Institute of MIT and Harvard and Massachusetts General Hospital (MGH), the molecular survey also characterized gene expression patterns for other new cell subtypes. The work expands scientific and clinical understanding of lung biology, with broad implications for all diseases of the airway — including asthma, chronic obstructive pulmonary disease, and bronchitis.

Jayaraj Rajagopal, a physician in the Pulmonary and Critical Care Unit at MGH, associate member at the Broad Institute, and a Howard Hughes Medical Institute (HHMI) faculty scholar, and Broad core institute member Aviv Regev, director of the Klarman Cell Observatory at the Broad Institute, professor of biology at MIT, and an HHMI investigator, supervised the research. Daniel Montoro, a graduate student in Rajagopal’s lab, and postdoctoral fellows Adam Haber and Moshe Biton in the Regev lab are co-first authors on the paper published today in Nature.

“We have the framework now for a new cellular narrative of lung disease,” said Rajagopal, who is also a professor at Harvard Medical School and a principal faculty member at the Harvard Stem Cell Institute. “We’ve uncovered a whole distribution of cell types that seem to be functionally relevant. What’s more, genes associated with complex lung diseases can now be linked to specific cells that we’ve characterized. The data are starting to change the way we think about lung diseases like cystic fibrosis and asthma.”

“With single-cell sequencing technology, and dedicated efforts to map cell types in different tissues, we’re making new discoveries — new cells that we didn’t know existed, cell subtypes that are rare or haven’t been noticed before, even in systems that have been studied for decades,” said Regev, who is also co-chair of the international Human Cell Atlas consortium. “And for some of these, understanding and characterizing them sheds new light immediately on what’s happening inside the tissue.”

Using single-cell RNA sequencing, the researchers analyzed tens of thousands of cells from the mouse airway, mapping the physical locations of cell types and creating a cellular “atlas” of the tissue. They also developed a new method called pulse-seq to monitor development of cell types from their progenitors in the mouse airway. The findings were validated in human tissue.

One extremely rare cell type, making up roughly one percent of the cell population in mice and humans, appeared radically different from other known cells in the dataset. The team dubbed this cell the “pulmonary ionocyte” because its gene expression pattern was similar to ionocytes — specialized cells that regulate ion transport and hydration in fish gills and frog skin.

Strikingly, at levels higher than any other cell type, these ionocytes expressed the gene CFTR — which, when mutated, causes cystic fibrosis in humans. CFTR is critical for airway function, and for decades researchers and clinicians have assumed that it is frequently expressed at low levels in ciliated cells, a common cell type spread throughout the entire airway.

But according to the new data, the majority of CFTR expression occurs in only a few cells, which researchers didn’t even know existed until now.

When the researchers disrupted a critical molecular process in pulmonary ionocytes in mice, they observed the onset of key features associated with cystic fibrosis — most notably, the formation of dense mucus. This finding underscores how important these cells are to airway-surface regulation.

“Cystic fibrosis is an amazingly well-studied disease, and we’re still discovering completely new biology that may alter the way we approach it,” said Rajagopal. “At first, we couldn’t believe that the majority of CFTR expression was located in these rare cells, but the graduate students and postdocs on this project really brought us along with their data.”

The results may also have implications for developing targeted cystic fibrosis therapies, according to the team. For example, a gene therapy that corrects for a mutation in CFTRwould need to be delivered to the right cells, and a cell atlas of the tissue could provide a reference map to guide that process.

The study further highlighted where other disease-associated genes are expressed in the airway. For example, asthma development has been previously linked with a gene that encodes a sensor for rhinoviruses, and the data now indicate that this gene is expressed by ciliated cells. Another gene linked with asthma is expressed in tuft cells, which separated into at least two groups — one that senses chemicals in the airway and one that produces inflammation. The results suggest that a whole ensemble of cells may be responsible for different aspects of asthma.

Using the pulse-seq assay, the researchers tracked how the newly characterized cells and subtypes in the mouse airway develop. They demonstrated that mature cells in the airway arise from a common progenitor: the basal cells. The team also discovered a previously undescribed cellular structure in the tissue. These structures, which the researchers called “hillocks,” are unique zones of rapid cell turnover, and their function is not yet understood.

“The atlas that we’ve created is already starting to drastically re-shape our understanding of airway and lung biology,” said Regev. “And, for this and other organ systems being studied at the single-cell level, we’ll have to drape everything we know on top of this new cellular diversity to understand human health and disease.”

Funding for this study was provided in part by the Klarman Cell Observatory at the Broad Institute, Manton Foundation, HHMI, New York Stem Cell Foundation, Harvard Stem Cell Institute, Human Frontiers Science Program, and National Institutes of Health.

Paper(s) cited:

Montoro DT, Haber AL, Biton M et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature. Online August 1, 2018. DOI: 10.1038/s41586-018-0393-7