An Equation to Describe the Competition Between Genes

Caltech researchers develop and verify predictive mathematical model

In biology, scientists typically conduct experiments first, and then develop mathematical or computer models afterward to show how the collected data fit with theory. In his work, Rob Phillips flips that practice on its head. The Caltech biophysicist tackles questions in cellular biology as a physicist would—by first formulating a model that can make predictions and then testing those predictions. Using this strategy, Phillips and his group have recently developed a mathematical model that accounts for the way genes compete with each other for the proteins that regulate their expression.

A paper describing the work appears in the current issue of the journal Cell. The lead authors on the paper are Robert Brewster and Franz Weinert, postdoctoral scholars in Phillips's lab.

"The thing that makes this study really interesting is that we did our calculations before we ever did any experiments," says Phillips, the Fred and Nancy Morris Professor of Biophysics and Biology at Caltech and principal investigator on the study. "Just as it is amazing that we have equations for the orbits of planets around stars, I think it's amazing that we are beginning to be able to write equations that predict the complex behaviors of a living cell."

A number of research teams are interested in modeling gene expression—accurately describing all the processes involved in going from a gene to the protein or other product encoded by that DNA. For simplicity's sake, though, most such models do not take competition into consideration. Instead, they assume that each gene has plenty of whatever it needs in order to be expressed—including the regulatory proteins called transcription factors. However, Phillips points out, there often is not enough transcription factor around to regulate all of the genes in a cell.  For one thing, multiple copies of a gene can exist within the same cell. For example, in the case of genes expressed on circular pieces of DNA known as plasmids, it is common to find hundreds of copies in a single cell. In addition, many transcription factors are capable of binding to a variety of different genes. So, as in a game of musical chairs, the genes must compete for a scarce resource—the transcription factors.

Phillips and his colleagues wanted to create a more realistic model by adding in this competition. To do so, they looked at how the level of gene expression varies depending on the amount of transcription factor present in the cell. To limit complexity, they worked with a relatively simple case—a gene in the bacterium E. coli that has just one binding site where a transcription factor can attach. In this case, when the transcription factor binds to the gene, it actually prevents the gene from making its product—it represses expression.

To build their mathematical model, the researchers first considered all the various ways in which the available transcription factor can interact with the copies of this particular gene that are present in the cell, and then developed a statistical theory to represent the situation.

"Imagine that you go into an auditorium, and you know there are a certain number of seats and a certain number of people. There are many different seating arrangements that could accommodate all of those people," Phillips says. "If you wanted to, you could systematically enumerate all of those arrangements and figure out things about the statistics—how often two people will be sitting next to each other if it's purely random, and so on. That's basically what we did with these genes and transcription factors."

Using the resulting model, the researchers were able to make predictions about what would happen if the level of transcription factor and the number of gene copies were independently varied so that the proteins were either in high demand or there were plenty to go around, for example.

With predictions in hand, the researchers next conducted experiments while looking at E. coli cells under a microscope. To begin, they introduced the genes on plasmids into the cells. They needed to track exactly how much transcription factor was present and the rate of gene expression in the presence of that level of transcription factor. Using fluorescent proteins, they were able to follow these changes in the cell over time: the transcription factor lit up red, while the protein expressed by the gene without the transcription factor attached glowed green. Using video fluorescence microscopy and a method, developed in the lab of Caltech biologist Michael Elowitz, for determining the brightness of a single molecule, the researchers were able to count the level of transcription factor present and the rate at which the green protein was produced as the cells grew and divided.

The team found that the experimental data matched the predictions they had made extremely well. "As expected, we find that there are two interesting regimes," says Brewster. "One is that there's just not enough protein to fill the demand. Therefore, all copies of the gene cannot be repressed simultaneously, and some portion will glow green all the time. In that case, there are correlations between the various copies of the genes. They know, in some sense, that the others exist. The second case is that there is a ton of this transcription factor around; in that case, the genes act almost exactly as if the other genes aren't there—there is enough protein to shut off all of the genes simultaneously."

The data fit so well with their model, in fact, that Phillips and his colleagues were able to use plots of the data to predict how many copies of the plasmid would be found in a cell as it grew and multiplied at various points throughout the cell cycle.

"Many times in science you start out trying to understand something, and then you get so good at understanding it that you are able to use it as a tool to measure something else," says Phillips. "Our model has become a tool for measuring the dynamics of how plasmids multiply. And the dynamics of how they multiply isn't what we would have naively expected. That's a little hint that we're pursuing right now."

Overall, he says, "this shows that the assertion that biology is too complicated to be predictive might be overly pessimistic, at least in the context of bacteria."

The work described in the paper, "The Transcription Factor Titration Effect Dictates Level of Gene Expression," was supported by the National Institutes of Health and by the Fondation Pierre-Gilles de Gennes. Additional coauthors are Mattias Rydenfelt, a graduate student in physics at Caltech; Hernan Garcia, a former member of Phillips's lab who is now at Princeton University; and Dan Song, a graduate student at Harvard Medical School.

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Research Update: Battling Infection With Microbes

A relationship between gut bacteria and blood cell development helps the immune system fight infection, Caltech researchers say.

The human relationship with microbial life is complicated. At almost any supermarket, you can pick up both antibacterial soap and probiotic yogurt during the same shopping trip. Although there are types of bacteria that can make us sick, Caltech professor of biology and biological engineering Sarkis Mazmanian and his team are most interested in the thousands of other bacteria—many already living inside our bodies—that actually keep us healthy. His past work in mice has shown that restoring populations of beneficial bacteria can help alleviate the symptoms of inflammatory bowel disease, multiple sclerosis, and even autism. Now, he and his team have found that these good bugs might also prepare the immune cells in our blood to fight infections from harmful bacteria.

In the recent study, published on March 12 in the journal Cell Host & Microbe, the researchers found that beneficial gut bacteria were necessary for the development of innate immune cells—specialized types of white blood cells that serve as the body's first line of defense against invading pathogens.

In addition to circulating in the blood, reserve stores of immune cells are also kept in the spleen and in the bone marrow. When the researchers looked at the immune cell populations in these areas in so-called germ-free mice, born without gut bacteria, and in healthy mice with a normal population of microbes in the gut, they found that germ-free mice had fewer immune cells—specifically macrophages, monocytes, and neutrophils—than healthy mice.

Germ-free mice also had fewer granulocyte and monocyte progenitor cells, stemlike cells that can eventually differentiate into a few types of mature immune cells. And the innate immune cells that were in the spleen were defective—never fully reaching the proportions found in healthy mice with a diverse population of gut microbes.

"It's interesting to see that these microbes are having an immune effect beyond where they live in the gut," says Arya Khosravi, a graduate student in Mazmanian's lab, and first author on the recent study. "They're affecting places like your blood, spleen, and bone marrow—places where there shouldn't be any bacteria."

Khosravi and his colleagues next wanted to see if the reduction in immune cells in the blood would make the germ-free mice less able to fight off an infection by the harmful bacterium Listeria monocytogenes—a well-studied human pathogen often used to study immune responses in mice. While the healthy mice were able to bounce back after being injected with Listeria, the infection was fatal to germ-free mice. When gut microbes that would normally be present were introduced into germ-free mice, the immune cell population increased and the mice were able to survive the Listeria infection.

The researchers also gave injections of Listeria to healthy mice after those mice were dosed with broad-spectrum antibiotics that killed off both harmful and beneficial bacteria. Interestingly, these mice also had trouble fighting the Listeria infection. "We didn't look at clinical data in this study, but we hypothesize that this might also happen in the clinic," says Mazmanian. "For example, when patients are put on antibiotics for something like hip surgery, are you damaging their gut microbe population and making them more susceptible to an infection that had nothing to do with their hip surgery?"

More importantly, the research also suggests that a healthy population of gut microbes can actually provide a preventative alternative to antibiotics, Khosravi says. "Today there are more and more antibiotic resistant superbugs out there, and we're running out of ways to treat them. Limiting our susceptibility to infection could be a good protective strategy."

These results appear in a paper titled "Gut Microbiota Promote Hematopoiesis to Control Bacterial Infection."

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Battling Infection With Microbes
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Monday, March 31, 2014
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A Changing View of Bone Marrow Cells

Caltech researchers show that the cells are actively involved in sensing infection

In the battle against infection, immune cells are the body's offense and defense—some cells go on the attack while others block invading pathogens. It has long been known that a population of blood stem cells that resides in the bone marrow generates all of these immune cells. But most scientists have believed that blood stem cells participate in battles against infection in a delayed way, replenishing immune cells on the front line only after they become depleted.

Now, using a novel microfluidic technique, researchers at Caltech have shown that these stem cells might be more actively involved, sensing danger signals directly and quickly producing new immune cells to join the fight.

"It has been most people's belief that the bone marrow has the function of making these cells but that the response to infection is something that happens locally, at the infection site," says David Baltimore, president emeritus and the Robert Andrews Millikan Professor of Biology at Caltech. "We've shown that these bone marrow cells themselves are sensitive to infection-related molecules and that they respond very rapidly. So the bone marrow is actually set up to respond to infection."

The study, led by Jimmy Zhao, a graduate student in the UCLA-Caltech Medical Scientist Training Program, will appear in the April 3 issue of the journal Cell Stem Cell.

In the work, the researchers show that blood stem cells have all the components needed to detect an invasion and to mount an inflammatory response. They show, as others have previously, that these cells have on their surface a type of receptor called a toll-like receptor. The researchers then identify an entire internal response pathway that can translate activation of those receptors by infection-related molecules, or danger signals, into the production of cytokines, signaling molecules that can crank up immune-cell production. Interestingly, they show for the first time that the transcription factor NF-κB, known to be the central organizer of the immune response to infection, is part of that response pathway.

To examine what happens to a blood stem cell once it is activated by a danger signal, the Baltimore lab teamed up with chemists from the lab of James Heath, the Elizabeth W. Gilloon Professor and professor of chemistry at Caltech. They devised a microfluidic chip—printed in flexible silicon on a glass slide, complete with input and output ports, control valves, and thousands of tiny wells—that would enable single-cell analysis. At the bottom of each well, they attached DNA molecules in strips and introduced a flow of antibodies—pathogen-targeting proteins of the immune system—that had complementary DNA. They then added the stem cells along with infection-related molecules and incubated the whole sample. Since the antibodies were selected based on their ability to bind to certain cytokines, they specifically captured any of those cytokines released by the cells after activation. When the researchers added a secondary antibody and a dye, the cytokines lit up. "They all light up the same color, but you can tell which is which because you've attached the DNA in an orderly fashion," explains Baltimore. "So you've got both visualization and localization that tells you which molecule was secreted." In this way, they were able to measure, for example, that the cytokine IL-6 was secreted most frequently—by 21.9 percent of the cells tested.

"The experimental challenges here were significant—we needed to isolate what are actually quite rare cells, and then measure the levels of a dozen secreted proteins from each of those cells," says Heath. "The end result was sort of like putting on a new pair of glasses—we were able to observe functional properties of these stem cells that were totally unexpected."

The team found that blood stem cells produce a surprising number and variety of cytokines very rapidly. In fact, the stem cells are even more potent generators of cytokines than other previously known cytokine producers of the immune system. Once the cytokines are released, it appears that they are able to bind to their own cytokine receptors or those on other nearby blood stem cells. This stimulates the bound cells to differentiate into the immune cells needed at the site of infection.

"This does now change the view of the potential of bone marrow cells to be involved in inflammatory reactions," says Baltimore.

Heath notes that the collaboration benefited greatly from Caltech's support of interdisciplinary work. "It is a unique and fertile environment," he says, "one that encourages scientists from different disciplines to harness their disparate areas of expertise to solve tough problems like this one."

Additional coauthors on the paper, "Conversion of danger signals into cytokine signals by hematopoietic stem and progenitor cells for regulation of stress-induced hematopoiesis," are Chao Ma, Ryan O'Connell, Arnav Mehta, and Race DiLoreto. The work was supported by grants from the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, a National Research Service Award, the UCLA-Caltech Medical Scientist Training Program, a Rosen Fellowship, a Pathway to Independence Award, and an American Cancer Society Research Grant.

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Friday, March 14, 2014
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Caltech's "Secrets" to Success

Everyone who really knows Caltech understands that it is unique among universities around the world. But just what makes Caltech so special? We've asked that question before, and the numbers don't tell the full story. So, is it our focus? Our culture? Our people?

The UK's Times Higher Education magazine recently tackled the topic, asking more specifically, "How does a tiny institution create such an outsized impact?" Caltech faculty share their perspectives in the cover story of the magazine's latest issue.

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