Researchers map 50,000 of DNA's mysterious 'knots' in the human genome

Innovative study of DNA's hidden structures may open up new approaches for treatment and diagnosis of diseases, including cancer.

DNA is well-known for its double helix shape. But the human genome also contains more than 50,000 unusual 'knot'-like DNA structures called i-motifs, researchers at the Garvan Institute of Medical Research have discovered.

Published today in The EMBO Journalis the first comprehensive map of these unique DNA structures, shedding light on their potential roles in gene regulation involved in disease.

In a landmark 2018 study, Garvan scientists were the first to directly visualise i-motifs inside living human cells using a new antibody tool they developed to recognise and attach to i-motifs. The current research builds on those findings by deploying this antibody to identify i-motif locations across the entire genome.

"In this study, we mapped more than 50,000 i-motif sites in the human genome that occur in all three of the cell types we examined," says senior author Professor Daniel Christ, Head of the Antibody Therapeutics Lab and Director of the Centre for Targeted Therapy at Garvan. "That's a remarkably high number for a DNA structure whose existence in cells was once considered controversial. Our findings confirm that i-motifs are not just laboratory curiosities but widespread -- and likely to play key roles in genomic function."

Curious DNA i-motifs could play a dynamic role in gene activity

I-motifs are DNA structures that differ from the iconic double helix shape. They form when stretches of cytosine letters on the same DNA strand pair with each other, creating a four-stranded, twisted structure protruding from the double helix.

The researchers found that i-motifs are not randomly scattered but concentrated in key functional areas of the genome, including regions that control gene activity.

"We discovered that i-motifs are associated with genes that are highly active during specific times in the cell cycle. This suggests they play a dynamic role in regulating gene activity," says Cristian David Peña Martinez, a research officer in the Antibody Therapeutics Lab and first author of the study.

"We also found that i-motifs form in the promoter region of oncogenes, for instance the MYC oncogene, which encodes one of cancer's most notorious 'undruggable' targets. This presents an exciting opportunity to target disease-linked genes through the i-motif structure," he says.

I-motifs hold promise for new type of therapies and diagnostics

"The widespread presence of i-motifs near these 'holy grail' sequences involved in hard-to-treat cancers opens up new possibilities for new diagnostic and therapeutic approaches. It might be possible to design drugs that target i-motifs to influence gene expression, which could expand current treatment options," says Associate Professor Sarah Kummerfeld, Chief Scientific Officer at Garvan and co-author of the study.

Professor Christ adds that mapping i-motifs was only possible thanks to Garvan's world-leading expertise in antibody development and genomics. "This study is an example of how fundamental research and technological innovation can come together to make paradigm-shifting discoveries," he says.

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How evolution tamed a deadly virus and why we should still worry

The story of the rise and fall of western equine encephalitis as a lethal disease offers essential lessons about how a pathogen can gain or lose its ability to jump from animals to humans.

That story is captured in newly published research from Harvard Medical School that identifies the mechanisms the western equine encephalitis virus used to infect humans and matches changes in that ability over time to a decline in illness and deaths caused by the pathogen.

The study results, published July 24 in Nature, offer important lessons for public health experts looking to prepare for future outbreaks, the researchers said.

The work took many unexpected turns, the researchers said. The findings challenge some of the basic assumptions that scientists have relied on in their attempts to understand how viruses interact with human cells and what causes outbreaks to ebb and flow, such as the notion that any given virus targets one host receptor to gain entry and infect cells.

"This was a real scientific detective story," said study senior author Jonathan Abraham, associate professor of microbiology in the Blavatnik Institute at Harvard Medical School. "The virus kept surprising us and taught us some important lessons about how to study viruses."

The researchers identified the specific proteins expressed on host cells that different strains of the virus have used to infect a variety of animals, including horses, humans, and birds over the last century. Their findings tied differences in the virus's ability to sicken humans and horses to changes in the viral genome that left the virus unable to target proteins found in humans and horses, while leaving intact the virus's ability to infect birds and reptiles that serve as reservoirs for the virus.

The surprising diversity and variability in the virus's ability to infect host cells highlights the importance of studying viruses broadly across time, space, and host species to track potential outbreaks and monitor for emerging and re-emerging viruses.

A virus changes

The protagonist in the story is the western equine encephalitis virus (WEEV), a member of a viral family known as alphaviruses.

One key to understanding how a virus interacts with a host is identifying the precise path it takes to enter cells and cause infection.

WEEV and others in the alphavirus family typically attach a spike protein to a compatible protein -- the receptor -- on the surface of a host cell. Once attached to the host receptor, the virus enters the cell. Once inside the cell, the virus hijacks the cells' armamentarium to enable its own replication, spread, and survival.

The researchers made harmless replicas of various viral strains collected from different times and places and tested their ability to infect host cells in lab dishes. They also tested some of the strains in mice.

Several deadly strains of WEEV are known to cause severe brain inflammation in both horses and humans. Some years, thousands of horses were killed and hundreds of humans were sickened. Case fatality rates for people were as high as 15 percent in North America in the early and middle decades of the 20th century.

Abraham's group found that some of these early strains could stick their spike proteins to several different types of receptors to enter animal cells. That was an unexpected finding because the prevailing dogma in virology thus far has been that viruses typically attack by targeting only one type of host cell receptor.

The team observed that the strains circulating during the years of frequent outbreaks could use multiple receptors that are expressed on brain cells of humans and horses, including proteins known as PCDH10 and VLDLR.

Although the virus still circulates between birds, mosquitoes, and other animals, the most recent outbreak in the United States in humans was in 1987, according to the Centers for Disease Control and Prevention. Since then, there have been only five cases identified in the United States.

By contrast, when the researchers tested more recently isolated strains recovered from mosquitos in California in 2005, they found that the viral spike protein failed to recognize the human receptors, but could still interact with similar proteins found in birds.

Based on these findings, the researchers hypothesize that the virus had evolved, perhaps because horses can be vaccinated and are no longer prevalent enough in the agriculture or transportation industries to serve as effective amplifiers for the virus. Alternatively, the researchers note, the virus may have evolved through simple antigenic drifting, a process by which random mutations cause a series of small changes to a viral genome that, over time, may end up changing the way a virus interacts with its host. Whatever the reason, the researchers said, subtle shifts in the shape of the viral spike proteins changed the cellular receptors with which the virus could connect.

This change in targetable host receptors is likely the central reason why the virus "submerged" as a human pathogen in North America, the research team said. This newly gleaned appreciation of the dynamic complexity of viral receptors is an essential tool for understanding how this virus or others like it might one day re-emerge, the scientists said.

"We need to understand what happens to viruses when they submerge, to better prepare for when they re-emerge," said first author Wanyu Li, a Harvard Kenneth C. Griffin Graduate School of Arts and Sciences doctoral student in the virology program in the Harvard Division of Medical Sciences at HMS.

For example, knowing whether dangerous versions of the pathogen persist in isolated populations of insects, or if the virus has gained the ability to infect other animals, could provide important early warning signs for potential resurgences of illnesses that are thought to have disappeared.

A virus's complex behavior

Through their experiments, the researchers discovered that certain old WEEV strains behaved differently than expected.

The team used eastern equine encephalitis virus -- a deadlier cousin of WEEV -- as a control in some experiments. In one test, the team found that an old strain of WEEV could use the same receptor as the eastern virus, which is something that newer WEEV strains could not do. They also found different strains of WEEV that used different receptors. Some strains could stick to avian versions of the receptor protein but not those expressed in human or equine cells.

The findings serve as an important reminder that viruses are part of a dynamic system and that the viruses themselves are dynamic, with subtle but significant differences across time and geography -- a notion that was powerfully underscored by the rapidly shapeshifting SARS-CoV-2 virus that fueled the COVID-19 pandemic, the researchers said.

"It was a wake-up call," Abraham said. "It's telling us that we can't just study one strain of a virus and assume we know the whole story. Viruses seem simple, but they're quite complex, and they're constantly changing."

Applying lessons to pandemic preparedness

In standard virology, researchers often only check a limited number of viral strains. These new findings show that that's not enough to truly understand the virus.

"There's so much more biology to be learned by exploring the diversity of these complex systems," Abraham said. He also noted that it's necessary to explore as much of that viral diversity as possible in order to prepare for possible outbreaks.

Many viruses circulate in insects and animals that live around us, Abraham said. Some, like the tick-borne infection Powassan, which is endemic in New England, occasionally flare up to cause deadly or debilitating disease.

There could be many reasons for the flare-ups, Abraham said. Are there different strains of Powassan that carry different levels of risk? Is it an environmental change or an evolutionary shift in the pathogen itself that causes new outbreaks? Looking at all these aspects and the breadth of viral diversity will help researchers predict and protect against outbreaks.

In another twist, as Abraham and his team conducted their experiments, a new outbreak of WEEV occurred in South America, which had also seen steep declines in the disease in recent years. The viral populations in South and North America appear to be genetically distinct, and the South American strain of the virus doesn't remain viable long enough for migrating birds to transfer it from one continent to the other regularly. Still, Abraham noted, the new outbreak in South America emphasizes the importance of vigilance and of improving scientific understanding of these volatile, shapeshifting viruses.

"WEEV's return caught everyone by surprise," Li said. "Now with its cellular host receptors known, we have the tools to understand the molecular aspects of WEEV's re-emergence."

Abraham and collaborators are now investigating the strains associated with recent outbreak in South America.

"One small shift in the viral genome, in the intensity of a rainy season that allows mosquitos to proliferate, or in the place humans live or work, could trigger an outbreak," Abraham said. "The more we know, the better we'll be able to protect ourselves."

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Scientists define new type of memory loss in older adults

Researchers at Mayo Clinic have established new criteria for a memory-loss syndrome in older adults that specifically impacts the brain's limbic system. It can often be mistaken for Alzheimer's disease. The good news: Limbic-predominant Amnestic Neurodegenerative Syndrome, or LANS, progresses more slowly and has a better prognosis, and is now more clearly defined for doctors working to find answers for memory loss patients.

Prior to the researchers developing clinical criteria published in the journal Brain Communications, the hallmarks of the syndrome could be confirmed only by examining brain tissue after a person's death. The proposed criteria provide a framework for neurologists and other experts to classify the condition in patients living with symptoms, offering a more precise diagnosis and potential treatments. They consider factors such as age, severity of memory impairment, brain scans, and biomarkers indicating the deposits of specific proteins in the brain.

The criteria were developed and validated using data from more than 200 participants in databases for the Mayo Clinic Alzheimer's Disease Research Center, the Mayo Clinic Study of Aging and the Alzheimer's Disease Neuroimaging Initiative.

Understanding the condition will lead to better management of symptoms and more tailored therapies for patients suffering from this type of cognitive decline, distinct from Alzheimer's disease, says David T. Jones, M.D., a Mayo Clinic neurologist and senior author of the study.

"In our clinical work, we see patients whose memory symptoms appear to mimic Alzheimer's disease, but when you look at their brain imaging or biomarkers, it's clear they don't have Alzheimer's. Until now, there has not been a specific medical diagnosis to point to, but now we can offer them some answers," Jones says. "This research creates a precise framework that other medical professionals can use to care for their patients. It has major implications for treatment decisions, including amyloid-lowering drugs and new clinical trials, and counseling on their prognosis, genetics and other factors."

Decades of work to understand and classify different types of dementia is ongoing, says Nick Corriveau-Lecavalier, Ph.D., the paper's first author. These findings build upon scientists' continued efforts to untangle neurological conditions that often have similar symptoms or can occur simultaneously, but can have drastically different treatments and prognoses.

"Historically, you might see someone in their 80s with memory problems and think they may have Alzheimer's disease, and that is often how it's being thought of today," Corriveau-Lecavalier says. "With this paper, we are describing a different syndrome that happens much later in life. Often, the symptoms are restricted to memory and will not progress to impact other cognitive domains, so the prognosis is better than with Alzheimer's disease."

Without signs of Alzheimer's disease, the researchers looked at the involvement of one possible culprit -- a buildup of a protein called TDP-43 in the limbic system that scientists have found in the autopsied brain tissue of older adults. Researchers have classified the build-up of these protein deposits as limbic-predominant age-related TDP-43 encephalopathy, or LATE. These protein deposits could be associated with the newly defined memory loss syndrome, but there are also other likely causes and more research is needed, the authors say.

With clinical criteria established by Jones, Corriveau-Lecavalier and co-authors, practitioners could soon diagnose LANS in patients so those living with memory loss might better understand options for treatment and potential progression of the disease, opening doors for research to further illuminate the characteristics of the disease.

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Nerve cells not entirely 'young at heart'

Most human nerve cells last a lifetime without renewal. A trait echoed within the cells' components, some enduring as long as the organism itself. New research by Martin Hetzer, molecular biologist and president of the Institute of Science and Technology Austria (ISTA), and colleagues discovered RNA, a typical transient molecule, in the nerve cells of mice that remain stable for their entire lives. Published in Science, these findings contribute to unraveling the complexities of brain aging and associated diseases.

After two decades in the United States, Martin Hetzer returned home to Austria in 2023 to become the 2nd President of the Institute of Science and Technology Austria (ISTA). A year into his new role, the molecular biologist remains engaged in the realm of aging research.

Hetzer is fascinated by the biological puzzles surrounding the aging processes in organs like the brain, heart, and pancreas. Most cells comprising these organs are not renewed throughout a human's entire life span. Nerve cells (neurons) in the human brain, for instance, can be as old as the organism, even up to more than a century, and must function for a lifetime. This remarkable age of neurons might be a major risk factor for neurodegenerative disorders such as Alzheimer's disease. Crucial to comprehending these kinds of ailments is a deeper understanding of how nerve cells function over time and maintain control. This potentially opens doors to therapeutically counteract the aging processes of these specific cells.

The latest collaborative publication by Hetzer, Tomohisa Toda from the Friedrich-Alexander University Erlangen-Nürnberg (FAU), who is also associated with the Max Planck Center for Physics and Medicine, Erlangen, and colleagues, gives new insights into this underexplored field of intricate mechanisms. For the first time in mammals, the study shows that RNA -- an essential group of molecules important for various biological processes inside the cell -- can persist throughout life. The scientists identified specific RNAs with genome-protecting functions in the nuclei of nerve cells of mice that remain stable for two years, covering their entire lives. The findings, published in the journal Science, underpin the importance of long-lived key molecules for maintaining a cell's function.

Longevity of key molecules

The inside of cells is a very dynamic place. Some components are constantly renewed and updated; others stay the same their whole lives. It is like a city in which the old buildings blend in with the new ones. DNA found in the nucleus -- the city's heart -- for instance, is as old as the organism. "DNA in our nerve cells is identical to DNA within the developing nerve cells in our mother's womb," explains Hetzer.

Unlike stable DNA, which is constantly being repaired, RNA, especially messenger RNA (mRNA), which forms proteins upon DNA's information, is characterized by its transient nature. The cellular scope, however, extends beyond mRNA to a group of so-called non-coding RNAs. They do not turn into proteins; instead, they have specific duties to contribute to the overall organization and function of the cell. Intriguingly, their lifespan remained a mystery. Until now.

RNAs that last the whole life

Hetzer and Co. set out to decipher that secret. Therefore, RNAs were labeled, i.e. "marked," in the brains of newborn mice. "For this labeling, we used RNA analogs -- structurally similar molecules -- with little chemical hooks that click fluorescent molecules on the actual RNAs," explains Hetzer. This assured efficient tracking of the molecules and powerful microscopic snapshots at any given time point in the mice's lives.

"Surprisingly, our initial images revealed the presence of long-lived RNAs, in various cell types within the brain. We had to further dissect the data to identify the ones in the nerve cells," Hetzer explains. "Fruitful collaboration with Toda's lab enabled us to make sense of that chaos during brain mapping." Collaboratively, the researchers were able to focus solely on long-lived RNAs in neurons. They quantified the molecules' concentration throughout a mouse's life, examined their composition and analyzed their positions.

While humans have an average life expectancy of around 70 years, the typical lifespan of a mouse is 2.5 years. After one year, the concentration of long-lived RNAs was slightly reduced compared to newborns. However, even after two years, they remained detectable indicating a lifelong persistence of these molecules.

RNAs help protect the genome

Additionally, the scientists proved long-lived RNAs' prominent role in cellular longevity. They found out that long-lived RNAs in neurons consist of mRNAs and non-coding RNAs and accumulate near the heterochromatin -- the densely packed region of the genome, typically homing inactive genes. Next they further investigated the function of these long-lived RNAs.

In molecular biology, the most effective approach to achieve this is by reducing the molecule of interest and observing its subsequent effects. "As their name and our previous experiments suggest, these long-lived RNAs are extremely stable," says Hetzer. The scientists, therefore, employed an in vitro (outside a living organism) approach, using neuronal progenitor cells -- stem cells with the capacity to give rise to neural cells, including neurons. The model system allowed them to effectively intervene with these long-lived RNAs. A lower amount of long-lived RNAs caused problems in the heterochromatin architecture and stability of genetic material, eventually affecting the cells' viability. Thus, the important role of long-lived RNAs' in cellular longevity was clarified.

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