Analyzing a Worm's Sleep

Caltech study identifies neuropeptides that collectively regulate C. elegans' sleep

Elephants, cats, flies, and even worms sleep. It is a natural part of many animals' lives. New research from Caltech takes a deeper look at sleep in the tiny roundworm Caenorhabditis elegans, or C. elegans, finding three chemicals that collectively work together to induce sleep. The study also shows that these chemicals—small proteins called neuropeptides that regulate neural activity—each control a different sleep behavior, such as the suppression of feeding or movement.

The results, accepted for publication in the journal Current Biology, suggest that other organisms, perhaps even humans, might similarly regulate individual components of sleep.

"The idea that multiple peptides work together to control sleep and its individual behaviors may translate to other animals," says Paul Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and a Howard Hughes Medical Institute investigator. "C. elegans is a simple animal model that allows us to isolate key molecular pathways. We start there and the information trickles up to other animal models."

C. elegans, which is about the size of the comma in this sentence, can be induced to sleep by certain stressors, such as heat. Researchers have used this behavior to get at the root of what regulates the worm's sleep. The transparent organisms, which live for only a few weeks, can be easily observed through microscopes. In 2007, a former Caltech postdoc, Cheryl Van Buskirk, now at California State University, Northridge, discovered that one cell alone, known as the ALA neuron, is responsible for inducing sleep in C. elegans; if you remove that cell, the worms cannot fall asleep.

In the new study, Sternberg and his team isolated genes that encode various neuropeptides that are produced at high levels within this one sleep-inducing neuron. They discovered that knocking out three of the peptides (nlp-8, flp-24, and flp-13) in the worms prevented them from being able to sleep.

"Removing just three neuropeptide genes had the same result as taking out the entire ALA cell," says graduate student Ravi Nath, lead author of the new study.

When the researchers added the neuropeptides back, one by one, into insomniac worms, they discovered that certain sleep behaviors were restored. The peptide flp-13 suppressed feeding patterns; flp-13 and nlp-8 inhibited defecation; and flp-24 blocked movement in the worms.

"The different neuropeptides may act in parallel to control the sleep state," says Nath. "You can think of the peptides as workers building a house—or the state of sleep. One is like an electrician and the other a plumber, for example, working together as a team."

The researchers say there are still many questions left to pursue. They want to investigate to what extent the roles of these neuropeptides overlap with each other, and if they also have other purposes outside of regulating sleep.

The Current Biology study, titled, "C. elegans Stress-Induced Sleep Emerges from the Collective Action of Multiple Neuropeptides," was funded by the Howard Hughes Medical Institute, the National Institutes of Health, and the Della Martin Postdoctoral Fellowship. Other authors include Elly Chow and Han Wang of Caltech, and Erich Schwarz of Cornell University.

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Analyzing a Worm's Sleep
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New research from Caltech finds three chemicals that collectively work together to induce sleep in the roundworm <em>C. elegans.</em>

Hushing the X Chromosome

New study reveals how changes in chromosome structure lead to gene silencing

Early in the development of female embryos, a crucial event occurs in all cells: An X chromosome is silenced. Whereas males have only one X chromosome, females have two—which means they can have twice as many proteins generated from their X chromosomes. Too much of certain proteins can be lethal, so nature has figured out a way to turn off one X chromosome.

A new paper published in the journal Science highlights an essential step in this process, showing for the first time that changes to the three-dimensional structure of DNA in the nucleus are required for X-chromosome silencing, also known as X inactivation. More specifically, the study demonstrates that a single molecule called Xist is responsible for the DNA remodeling, and that these changes lead to chromosome silencing.

"We had this chicken and egg problem, because we knew DNA structure changes occurred during the process of X inactivation, but we didn't know whether these changes caused silencing or were merely a result of silencing," says Caltech Assistant Professor of Biology Mitchell Guttman, the leader of the research. "Beyond the X chromosome, our results highlight the important role that dynamic arrangement of DNA plays in turning on and off different genes."

The research is part of an international effort to understand better how DNA is packaged as a three-dimensional structure in the nucleus, and how changes to this structure lead to the silencing and activation of genes. Different genes—which are located on the DNA strands—are either turned on or off in any given cell; when they are on, they produce proteins. The shape and folding patterns of DNA control this process, but the details are unclear. A recently funded consortium established by the National Institutes of Health, called the 4D Nucleome program (with the fourth dimension being time), aims to solve this mystery of DNA architecture and its role in various human diseases such as cancer and premature aging diseases.

In the new study, the researchers focused on Xist, which was previously known to play a role in X-chromosome silencing. Xist belongs to a class a RNA molecules known as long non-coding RNA, or lncRNA (pronounced link RNA). Whereas traditional RNA molecules known as messenger RNAs act as templates that are made into proteins, lncRNAs such as Xist can directly bind to DNA to turn on and off genes. In the last decade, tens of thousands of lncRNAs have been discovered.

Previous studies have shown that Xist binds all along the DNA strands of silenced X chromosomes, but not to the active X chromosomes. Researchers also knew that changes to DNA's three-dimensional structure and position in the cell were associated with silencing. But how and if Xist played a role in the structural DNA changes was not known.

The new study outlines the process by which Xist orchestrates chromosome silencing and introduces a key player: a protein called the lamin B receptor, located in the envelope that surrounds a cell's nucleus.

"Our work demonstrates that Xist, through its direct interaction with the lamin B receptor, recruits the X chromosome to the nuclear envelope and through this leads to changes in the three-dimensional structure of DNA in the nucleus," says Chun-Kan Chen, a graduate student in Guttman's lab and lead author of the Science paper. "This process enables Xist to spread across the entire X chromosome to achieve chromosome-wide silencing—its essential role in embryonic development."

The X chromosome is a good model for studying the relationship between DNA structure and gene silencing, because the whole structure is inactivated by a single RNA molecule. However, other chromosomes experience more localized gene silencing. Guttman and his team are planning additional studies to probe the relationship between DNA's shifting shape and the regulation of other genes in cells.

The Science paper, entitled "The Xist lncRNA recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing," was funded by National Institutes of Health, the Heritage Research Institute for the Advancement of Medicine and Science at Caltech, the New York Stem Cell Foundation, Pew-Steward Foundation, the Edward Mallinckrodt Foundation, the Sontag Foundation, the Searle Scholars Program, and funds from Caltech. Other Caltech authors include postdoctoral scholars Mario Blanco and Noah Ollikainen; former research assistants Constanza Jackson, Erik Aznauryan, and Christine Surka; and senior research scientist Amy Chow.

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Thursday, August 11, 2016
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Team of Proteins Works Together to Turn on T Cells

The fates of various cells in our bodies—whether they become skin or another type of tissue—are controlled by genetic switches. In a new study, Caltech scientists investigate the switch for T cells, which are immune cells produced in the thymus that destroy virus-infected cells and cancers. The researchers wanted to know how cells make the choice to become T cells.

"We already know which genetic switch directs cells to commit to becoming T cells, but we wanted to figure out what enables that switch to be turned on," says Hao Yuan Kueh, a postdoctoral scholar at Caltech and lead author of a Nature Immunology report about the work, published on July 4.

The study found that a group of four proteins, specifically DNA-binding proteins known as transcription factors, work in a multi-tiered fashion to control the T-cell genetic switch in a series of steps. This was a surprise because transcription factors are widely assumed to work in a simultaneous, all-at-once fashion when collaborating to regulate genes.

The results may ultimately allow doctors to boost a person's T-cell population. This has potential applications in fighting various diseases, including AIDS, which infects mature T cells.

"In the past, combinatorial gene regulation was thought to involve all the transcription factors being required at the same time," says Kueh, who works in the lab of  Ellen Rothenberg, Caltech's Albert Billings Ruddock Professor of Biology. "This was particularly true in the case of the genetic switch for T-cell commitment, where it was thought that a quorum of the factors working simultaneously was needed to ensure that the gene would only be expressed in the right cell type."

The authors report that a key to their finding was the ability to image live cells in real-time. They genetically engineered mouse cells so that a gene called Bcl11b—the key switch for T cells—would express a fluorescent protein in addition to its own Bcl11b protein. This caused the mouse cells to glow when the Bcl11b gene was turn on. By monitoring how different transcription factors, or proteins, affected the activation of this genetic switch in individual cells, the researchers were able to isolate the distinct roles of the proteins.

The results showed that four proteins work together in three distinct steps to flip the switch for T cells. Kueh says to think of the process as a team of people working together to get a light turned on. He says first two proteins in the chain (TCF1 and GATA3) open a door where the main light switch is housed, while the next protein (Notch) essentially switches the light on. A fourth protein (Runx1) controls the amplitude of the signal, like sliding a light dimmer.

"We identify the contributions of four regulators of Bcl11b, which are all needed for its activation but carry out surprisingly different functions in enabling the gene to be turned on," says Rothenberg. "It's interesting—the gene still needs the full quorum of transcription factors, but we now find that it also needs them to work in the right order. This makes the gene respond not only to the cell's current state, but also to the cell's recent developmental history."

Team member Kenneth Ng, a visiting student from California Polytechnic State University, says he was surprised by how much detail they could learn about gene regulation using live imaging of cells.

"I had read about this process in textbooks, but here in this study we could pinpoint what the proteins are really doing," he says.

The next step in the research is to get a closer look at precisely how the T cell genetic switch itself works. Kueh says he wants to "unscrew the panels" of the switch and understand what is physically going on in the chromosomal material around the Bcl11b gene.

The Nature Immunology paper, titled, "Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment," includes seven additional Caltech coauthors: Mary Yui, Shirley Pease, Jingli Zhang, Sagar Damle, George Freedman, Sharmayne Siu, and Michael Elowitz; as well as a collaborator at the Fred Hutchinson Cancer Research Center, Irwin Bernstein. The work at Caltech was funded by a CRI/Irvington Postdoctoral Fellowship, the National Institutes of Health, the California Institute for Regenerative Medicine, the Al Sherman Foundation, and the Louis A. Garfinkle Memorial Laboratory Fund.

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Scientists Transform Lower-Body Cells into Facial Cartilage

Caltech scientists have converted cells of the lower-body region into facial tissue that makes cartilage, in new experiments using bird embryos. The researchers discovered a "gene circuit," composed of just three genes, that can alter the fate of cells destined for the lower bodies of birds, turning them instead into cells that produce cartilage and bones in the head.

The results, published in the June 24 issue of the journal Science, could eventually lead to therapies for conditions where facial bone or cartilage is lost. For example, cartilage destroyed in the nose due to cancer is particularly hard to replace. Understanding the genetic pathways that lead to the development of facial cartilage may help in future stem-cell therapies, where a patient's own skin cells could be transformed and used to repair the nose.

"When facial cartilage and bone is lost, from cancer or an accident, it has been difficult to replace," says Marianne Bronner, the Albert Billings Ruddock Professor of Biology at Caltech, and senior author of the Science report. "Our long term hope is that uncovering this gene circuit may be useful in reprogramming a patient's own stem cells to make facial cartilage."

The bones below our necks, referred to by scientists as the "long" bones, originate from a different source of tissue than the bones in our head. As embryos, we are born with a type of early tissue called the neural crest that forms along the entire body, from the head to the end of the spinal cord. Those neural crest cells which originate in the head, called cranial neural crest, differentiate into the cartilage and bone of our faces, including the jaws and skull. In contrast, the so-called trunk neural crest cells, forming below the neck, do not make cartilage or bone but instead turn into nerve cells and pigment cells elsewhere in our bodies. Bronner and her colleagues want to understand what genes regulate the development of cranial neural crest cells and enable them to make cartilage and bones in the head.

To this end, they divided the trunk and cranial neural crest cells of bird embryos into separate groups, and looked for differences in gene activity. Fifteen genes were initially identified as being turned on in only the cranial cells. The researchers chose six of these genes for further study. All six code for transcription factors—molecules that bind to DNA to turn on and off the expression of other genes. After studying how these factors interact with each other, the scientists focused on three, called Sox8, Tfap2b and Ets, that are part of the cranial neural crest circuit.

These three genes were then inserted into the bodies of developing bird embryos, in particular the trunk neural crest, using a technique called electroporation. In this method, electric current is applied to cells to open up pores through which molecules such as DNA may pass. Next, the researchers transplanted the altered trunk cells to the cranial region of the embryos. Five days later, the trunk cells were doing something entirely new: producing cartilage.

"Normally, these trunk cells will not make cartilage," says Bronner. "Introducing just three genes into these cells reprogrammed them to acquire the ability to do so."

Bronner said that she hopes other researchers will use this information for experiments in cell culture. By adding the new-found gene circuit, perhaps with other known factors, to skin cells in a petri dish it may be possible to turn them into cartilage-producing cells—a key next step in creating future therapies for facial bone and cartilage loss.

The first author of the Science paper, titled, "Reprogramming of avian neural crest axial identity and cell fate," is Marcos Simoes-Costa of Caltech. The research is funded by the National Institutes of Health and the Pew Fellows Program in Biomedical Sciences.

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Creating Facial Cartilage in the Lab
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Wednesday, August 24, 2016
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Dietary Fiber and Microbes Change the Gel That Lines Our Gut

In the ongoing hustle and bustle of our intestines, where bacteria and food regularly intermingle, there is another substance that, to the surprise of researchers, has been found to rapidly change: the gel that lines the gut. A new Caltech study is the first to show how the structure of this gut gel, or mucus, can change in the presence of certain substances, such as bacteria and polymers—a class of long-chained molecules that includes dietary fiber.

The work, to be published online the week of June 13 in the Proceedings of the National Academy of Sciences, could lead to the development of new drugs or diets for intestinal conditions such as irritable bowel disease.

Our intestinal tracts are lined with a mucus gel that acts as a protective barrier between the insides of our bodies and the outside world. The gel lets in nutrients and largely blocks out bacteria, preventing infections. It also regulates how some drugs are delivered elsewhere in our bodies.

Researchers had previously studied how the gel can be damaged, for instance when bacteria feed on the gut's lining. The Caltech study is the first to look at the structure of the gel and how it morphs in the presence of other substances naturally found in the gut.

Performing their experiments in mice, the team tested the effects of polymers, which include dietary fiber as well as therapeutics such as medicines for constipation. The researchers fed some mice a diet rich in polymers and others (the controls) a polymer-free diet. Using a technique called confocal reflectance microscopy they measured the thickness of the gut gel and the degree to which the gel was compressed as a result of the consumed polymers. Mice given a high-polymer diet, they found, had a more compressed gel layer.

"The gel is like a sponge with holes that let material through," says the paper's lead author, Sujit Datta, a postdoctoral scholar in the laboratory of Rustem Ismagilov, Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering. "We are seeing that polymers, including dietary fiber, can compress the gel, potentially making the holes smaller, and we think that this might offer protective benefits," Datta adds.

In addition, the researchers applied different kinds of polymers—including dietary fibers like pectin, found in apples—directly to the gel lining to test its response. All of the polymers tested compressed the gel layer.

"It's too early to draw any conclusions, but it may be that eating an apple a day will affect the shape of the lining in your gut," says Asher Preska Steinberg, a Caltech graduate student and coauthor of the study.

The researchers also found that dietary fiber and gut bacteria—which are part of a community of microorganisms collectively known as gut microbiota—can work together to influence how the gut gel changes shape. They performed the same polymer/fiber experiments in germ-free mice, which are mice carefully raised to not have any bacteria in their gut. The results showed that the polymers compressed the gut gels of these germ-free mice to a greater degree. This implies that species of bacteria in our gut that are known to break down polymers can weaken the compressing effect.

"We previously thought of the gel as a static structure, so it was unexpected to find an interplay between diet and gut microbiota that rapidly and dynamically changes the biological structures that protect a host," says Ismagilov.

Both dietary fiber and certain gut microbes have been linked to good health. Fiber has been shown to lower cholesterol and regulate blood sugar levels—factors in heart disease and diabetes, respectively. Meanwhile, some bacteria, including the good "probiotics," can help treat digestive disorders and may even play a beneficial role in mental health. For instance, a separate Caltech-led study found that probiotics can alleviate autism-like behaviors in mice—a finding that could potentially lead to new therapies for the disorder in humans.

The entire collection of bacteria in our gut can include 1,000 different species or more and weigh a total of three pounds. Exactly how these microscopic organisms influence our health, for good and bad, is an area of active research with many unanswered questions. The White House recently announced the National Microbiome Initiative, with federal funding worth $121 million, to investigate the mysteries of microbes not only living in our bodies but all over the planet. In addition, more than 100 nonfederal agencies have pledged money and support toward researching microbial communities.

"Our study gives biologists and scientists studying diseases of the gut something else to think about," says Datta. "Now they can take the structure of the gut mucus, and how it responds to its environment, into account."

This research was funded by the Defense Advanced Research Projects Agency (DARPA) and National Science Foundation.

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Microbes & Dietary Fiber Change the Gut Lining
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The study is the first to look at the structure of the gut's mucus gel lining and how it morphs in the presence of other substances naturally found in the gut.

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