Archive for the ‘biology’ category

Gene Enhancers Are Important Despite Apparent Redundancy

January 31, 2018

Cell activity patterns of two gene enhancers (red and green cells). Cells in which both enhancers are active appear yellow. (Credit: Marco Osterwalder).

Every cell in the body has the same DNA and genes, so a cell’s properties and functions are determined by which genes are turned on. That’s why it is critical to understand enhancers, short sections of non-coding DNA that regulate the expression of specific genes.

An enhancer doesn’t have a one-to-one relationship with the gene it controls. Instead, there are many more enhancers than genes and their relationship is unclear. Do many enhancers regulate a given gene’s expression in a given tissue, providing redundancy? Researchers at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) investigated this question, and the overall importance of enhancers to development, in two recent studies.

The researchers answered a long-standing question about the role of enhancers. And by better linking the genomic complement of an organism with its expressed characteristics, their work offers new insights that further the growing field of systems biology, which seeks to gain a predictive understanding of living systems.

Perfect Conservation

In their new study published in Cell, the team investigated enhancers containing “ultraconserved elements,” which are at least 200 base pairs in length and are 100 percent identical in the genomes of humans, mice and rats. Ultraconserved elements have been perfectly conserved for over 80 million years since these mammals shared a common ancestor.

Previously, the group individually deleted four ultraconserved brain enhancers in the mouse genome. All four mouse lines were viable and fertile, which shocked the genomics and evolutionary biology research communities who thought these enhancers were critical for life since they have been so perfectly conserved.

“In our follow-up study, we wanted to dig deeper to test two possible explanations,” said Diane Dickel, a research scientist at Berkeley Lab’s Environmental Genomics and Systems Biology Division. “First, maybe there is some redundancy between these enhancer sequences, and losing two of them will cause the mice to be nonviable or infertile. Second, maybe the mice do have something wrong with them, but it’s more subtle.”

In the new study, the team used the gene-editing tool CRISPR-CAS9 to further investigate enhancers near the Arx gene that, when defective, cause neurological and sexual-development disorders in mice and humans. “We focused on Arx because it has an unusually large number of very long ultraconserved sites nearby,” said Dickel who co-led the studies with Len Pennacchio and Axel Visel, both senior scientists in the same division.

Specifically, they knocked out four of Arx’s brain enhancers, which are active in pairs in either the top or bottom of the forebrain. When these enhancers were individually deleted, all the mice were viable and fertile — confirming that ultraconserved enhancers aren’t essential to life within a given generation. When they were deleted in pairs with similar activity, the mice were still viable and fertile — ruling out redundancy as the primary explanation for the absence of major defects upon knockout of individual ultraconserved enhancers.

But were there more subtle brain defects? To answer this question, the researchers teamed up with John Rubenstein, a neurobiologist at UC San Francisco, who provided in-depth neurological phenotyping.

In three of the four cases with only one enhancer deletion, they found abnormalities —either in overall growth or brain development. In one case, the mice had a severe structural defect in the hippocampus. In another case, the mice had fewer cholinergic neurons.

“The changes to the brain in these mice are reminiscent of those seen in humans with seizure disorders or dementia,” Dickel said. “While we don’t know yet if these mice are affected by such problems, it’s likely that the physiological changes we found are selected against in the wild, and that’s why you maintain a high level of conservation at these sites.”

Pictures of a normal part of the mouse forebrain (left) compared with a mouse missing one ultraconserved enhancer (right). (Credit: Athena Ypsilanti /UCSF)

Protective redundancy

While there are only a few hundred ultraconserved sites in the human and mouse genomes, there are also approximately 100,000 other, less well-conserved enhancers. The defects observed upon deletion of individual ultraconserved enhancers raise the question if deletion of less well-conserved enhancers generally causes similar problems. Are defects the exception or the rule? This question was investigated in a second study, led by postdoctoral researcher Marco Osterwalder, which focused on enhancers for limbs. Limb enhancers were targeted because limbs are easy to assess, unlike neurological phenotyping.

As reported today in Nature, the team deleted ten limb enhancers near genes essential for limb development. They expected to see some anomalies but all ten mouse lines had perfectly normal-looking limbs.

However, the team also observed some genes with two enhancers that appeared to be active at the same time during limb development. When the team knocked out such pairs of limb enhancers with similar activity, they saw characteristics like extra digits or differences in bone length — indicating that these enhancers functioned redundantly. “It’s like the pilot and copilot having redundant control sticks in the cockpit,” explained Visel. “Either one can control the plane, but you’re in trouble if you get rid of both control sticks.”

To determine whether or not it’s common to have such a regulatory back-up system, the team computationally analyzed genomic datasets from many different tissues. They determined that genes that control central processes in embryonic development are commonly equipped with sets of enhancers that are likely redundant. In more than 1000 extreme cases, they found sets of five or more enhancers with similar activity patterns controlling the same gene.

Despite the redundancy, these enhancers are evolutionarily conserved, which leads the scientists to surmise that disrupting these enhancers may cause some sort of decrease in fitness in the wild, even if it is so small it can’t be readily detected in the lab.

“We’re not saying these enhancers are perfectly redundant in that one or the other isn’t important, but rather there is a mechanism of protecting against deleterious effects on the order of a given generation,” Pennacchio summarized. “Selection happens over many generations.”

Taken together, these studies demonstrate a varied importance of enhancer redundancy. “The genome is a big place,” Dickel said. “It’s hard to fit every single locus in the genome into one specific model of gene regulation. Some loci have more redundancy than others.”

Broader implications

Complex questions such as these are addressed by biological systems science, where these results can be used to understand the effects of genetic perturbations on inherited and expressed characteristics in a larger context.

Ultimately, the team is interested in whether enhancer mutations contribute to human disease. “With whole human genome sequencing now a reality, we are focusing on studying how human mutations impact health and development in vivo,” Pennacchio said.

The research, funded by the National Institutes of Health, was performed at Berkeley Lab and is a natural evolution of the work that was begun by the DOE and became the Human Genome Project.

The following Berkeley Lab researchers also contributed to the studies: Iros Barozzi, Yoko Fukuda-Yuzawa, Brandon Mannion, Sarah Afzal, Elizabeth Lee, Yiwen Zhu, Ingrid Plajzer-Frick, Catherine Pickle, Momoe Kato, Tyler Garvin, Quan Pham, Anne Harrington, Jennifer Akiyama and Veena Afzal. Also participating in the research were scientists from the University of California San Francisco, University of Basel, and the Centro Andaluz de Biología del Desarrollo.

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Lawrence Berkeley National Laboratory addresses the world’s most urgent scientific challenges by advancing sustainable energy, protecting human health, creating new materials, and revealing the origin and fate of the universe. Founded in 1931, Berkeley Lab’s scientific expertise has been recognized with 13 Nobel Prizes. The University of California manages Berkeley Lab for the U.S. Department of Energy’s Office of Science. For more, visit www.lbl.gov.

DOE’s Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit science.energy.gov.

This is a reposting of my news release, courtesy of Berkeley Lab.

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Drug blocks Zika and other deadly viruses in cells cultures, Stanford study finds

December 12, 2017

Photo of Jan Carette by Paul Sakuma

A team of Stanford researchers is developing approaches to thwart a family of deadly viruses, called flaviviruses, by targeting the human cells that host these invading pathogens.

Flaviviruses include the dengue-fever, yellow-fever, West Nile and Zika viruses transmitted to humans by mosquitoes, as well as encephalitis transmitted by ticks. Unfortunately, approved antiviral drugs for these diseases aren’t currently available.

So, instead of the traditional approach of attacking an individual virus directly, the researchers focused on the cellular factors of their human hosts that are essential to many viral infections.

“Generally, when you develop a drug against a specific protein in dengue virus, for instance, it won’t work for yellow fever or Zika, and you have to develop new antivirals for each,” said Stanford virologist Jan Carette, PhD, in a recent Stanford news release. “Here, by targeting the host rather than a specific virus, we’ve been able to take out multiple viruses at once.”

Earlier, the team genetically profiled human cells to identity the host factors necessary for the viruses to replicate inside the cells — revealing new candidate targets for antiviral drug development. Specifically, they demonstrated the importance of the oligosaccharyltransferase (OST) complex that attaches sugar molecules to proteins. They found flaviviruses did not infect their genetically engineered cells without OST.

In the new study, recently published in Cell Reports, the Stanford researchers collaborated with scientists at Yale University to test the effectiveness of a drug called NGI-1, which inhibits the activity of the OST complex.

They showed that low concentrations of NGI-1 could be used to block the viruses from replicating without harming the host cells — successfully reducing the infection by 99 percent when treating cells immediately after they were infected by Zika or dengue virus, and by 80 percent when administered 24 hours after infection.

Their study also indicated that the viruses are unlikely to become resistant to NGI-1. “When you target a host function rather than a viral protein, it’s usually much more difficult for a virus to develop resistance,” Carette said in the release.

The researchers are now busy with follow-up studies to test NGI-1 in small animal models of dengue fever and are also developing similar drugs with improved specificity.

This is a reposing of my Scope blog story, courtesy of Stanford School of Medicine.

Researchers investigate fruit flies as a step towards understanding the human brain

December 7, 2017

Photo by nuzree

Researchers have been trying to map out the brain’s complex neural circuits to understand how diseases like Parkinson’s disrupt healthy brain communication, in hopes of designing better therapies.

That, obviously, is not easy. So a Stanford research team led by biology professor Liqun Luo, PhD, and bioengineer and physicist Stephen Quake, PhD, are instead studying fruit fly brains — yes, those pesky bugs that fly around your bananas.

Specifically, they have mapped out a blueprint for the fruit fly’s olfactory neurons — identifying how specific gene and protein activity correlates with the biological circuitry of different neuronal cell types. The researchers focused on a fruit fly’s sense of smell because the function, physiology and anatomy of its olfactory system are well known, making it a simple and ideal test bed for their research.

They measured the gene expression profiles using a single-cell sequencing technology developed for mice and human cell types, which they modified to work for the smaller and simpler fruit fly cells.

The team determined different types of neurons express genes differently during development, but gene expression between the neuron types becomes indistinguishable as the flies mature, as recently reported in Cell.

“Once the brain is wired up, the fly doesn’t need to express those genes that help them in choosing the connection partners,” said first author and Stanford biology postdoc Hongjie Li, PhD, in a recent news release. “So there is less gene expression in the adult flies.”

The researchers are a long way from using their technique to map the human brain, but they aren’t daunted by the challeng. “By further developing this approach, we hope to one day reverse-engineer and perhaps even repair defective circuitry in the human brain,” Li said in the release.

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.

Stanford researchers grow neural stem cells more efficiently in 3-D gel

November 7, 2017

Photo by luismmolina/Getty Images

Stem cells have the potential to help us understand and treat a range of diseases and injuries — from vision loss to cancers. For instance, a Japanese man in his 60s was recently treated for vision loss due to macular degeneration using stem cells donated by another person. And many other clinical trials involving stem cells are underway.

However, there is still a lot to learn about stem cells and many barriers to overcome before most potential treatments can be realized. One such barrier is how to grow large quantities of stem cells while maintaining their unique properties. Now, Stanford researchers have developed a new gel in which they can grow massive numbers of neural stem cells in less space.

Stem cells are unspecialized cells that can self-renew and develop into many different types of cells in the body. Researchers hope that neural stem cells — that differentiate into neurons and glia cells in the nervous system — can be used to treat spinal cord injuries, Parkinson disease, Huntington disease and other nervous system disorders.

As recently reported in Nature Materials, the Stanford team engineered a new polymer-based gel optimized for neural stem cells, growing them in three dimensions instead of two.

“For a 3-D culture, we need only a 4-inch-by-4-inch plot of lab space, or about 16 square inches. A 2-D culture requires a plot of four feet by four feet, or about 16 square feet,” said the study’s first author Chris Madl, PhD, a postdoctoral research fellow in microbiology and immunology at Stanford, in a recent Stanford news release. In addition to taking 100-times less lab space, the new 3-D process also demands less energy and nutrients to grow the cells, he said.

A key to the development was the realization that neural stem cells need to chemically or physically remodel their surrounding environment to maintain their ability to differentiate into other cells. The researchers discovered this by creating and testing a family of gels with varying stiffness and remodeling susceptibilities. The authors explained in the paper, “Whereas cells cultured in 2-D are unrestricted and free to spread, cells within nanoporous 3-D hydrogels require matrix remodeling to spread, migrate, and proliferate.”

Surprisingly, they also discovered that the neural stem cells weren’t sensitive to the stiffness of the gel, unlike most other stem cells.

These new findings have given the leader of the research group new hope for future stem cell therapies. Sarah Heilshorn, PhD, associate professor of materials science and engineering at Stanford, said in the release, “There’s this convergence of biological knowledge and engineering principles in stem cell research that has me hopeful we might finally actually solve big problems.”

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.

New study shines light on how to better engineer fluorescent proteins

September 22, 2017

Researchers have now captured the ultrafast changes of green fluorescent proteins as they transition between a dark and fluorescent state, using an X-ray laser at the SLAC National Accelerator Laboratory.

Green fluorescent proteins (GFPs), originally found in the jellyfish Aequorea victoria, have helped transform biomedical research. Their green glow has acted like a flashlight on the inner workings of cells, illuminating pathways and processes in lab dishes and living animals since it was discovered in 1961. The protein acts as a molecular switch depending on the conditions, flipping from dark to glowing when excited by light. Scientists attach these fluorescent tags to other proteins to track their activity — studying how cancer cells spread, how HIV infections progress, how genes are expressed and much more.

Although researchers have used these proteins for decades, they were unable to observe how GFPs flipped between their dark and glowing states until now. The transition was too fast for traditional X-ray imaging techniques. So an international collaboration of scientists recently used SLAC’s Linac Coherent Light Source, one of the world’s fastest and brightest X-ray lasers, to excite the proteins and take snapshots of the fluorescent molecules in action.

These images were used to investigate what happened as GFP flipped states — with the hope of engineering GFP to make this happen even faster. They found that the protein became momentarily stuck between a dark and glowing state, as reported in Nature Chemistry.

“After a picosecond, a very short time, this molecular switch is stuck between on and off,” said Martin Weik, PhD, a scientist at the Institute of Structural Biology in Grenoble, France, in a recent news release. “People have predicted this, but to actually confirm it experimentally is extremely exciting. It’s as if there is a door and it’s neither closed nor completely open; it’s half open. And now we are learning what can go through the door, what might be blocking it and how it works in real-time.”

The team discovered that an amino acid partially blocked the doorway, slowing the GFP’s ability to flip states. Using this knowledge, they then engineered a mutated version of the protein with a smaller amino acid that could switch more quickly — creating a brighter and more efficient fluorescent tag that can observe cellular processes more precisely.

“We think that this approach will open a world of possibilities to tailor and create proteins,” Weik said in the release. “We not only have the structure of the molecule, but now we can see what is happening between one static state and the other.”

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.

The skinny on how chickens grow feathers and, perhaps, on how humans grow hair

July 24, 2017

How do skin cells make regularly spaced hairs in mammals and feathers in birds? Scientists had two opposing theories, but new research at the University of California, Berkeley surprisingly links them.

The first theory contends that the timing of specific gene activation dictates a cell’s destiny and predetermines tissue structure — for example, in the skin, gene activation determines whether a skin cell becomes a sweat gland cell or hair cell, or remains a skin cell. The second theory asserts that a cell’s fate is determined instead by interacting with other cells and the material that it grows on.

Now, Berkeley researchers have found that the creation of feather follicles (like hair follicles) is initiated by cells exerting mechanical tension on each other, which then triggers the necessary changes in gene expression to create the follicles. Their results were recently reported in Science.  

“The cells of the skin in the embryo are pulling on each other and eventually pull one another into little piles that each go on to become a follicle,” said first author Amy Shyer, PhD, a post-doctoral fellow in molecular and cell biology at the University of California, Berkeley, in a recent news release. “What is really key is that there isn’t a particular genetic program that sets up this pattern. All of these cells are initially the same and they have the same genetic program, but their mechanical behavior produces a difference in the piled-up cells that flips a switch, forming a pattern of follicles in the skin.”

The research team grew skin taken from week-old chicken eggs on different materials with varying stiffness. They found that the stiffness of the substrate material was critical to forming feather follicles — material that was too stiff or too soft yielded only one follicle, whereas material with intermediate stiffness resulted in an orderly array of follicles.

“The fundamental tension between cells wanting to cluster together and their boundary resisting them is what allows you to create a spaced array of patterns,” said co-author Alan Rodgues, PhD, a biology consultant and former visiting scholar at Berkeley.

The researchers also showed that when the cells cluster together, this activated genes in those cells to generate a follicle and eventually a feather.

Although the study used chicken skin, the researchers suggest that they have discovered a basic mechanism, which may be used in the future to help grow artificial skin grafts that look like normal human skin with hair follicles and sweat pores.

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.


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