‘Jumping Genes’ – Roberts’ Lab Makes Ground-breaking Finding in Exercise Genetic Research with LINE-1 Retrotransposon Activity in Skeletal Muscle

August 17, 2018

Pictured from left to right are Chris Vann, doctoral student; Cody Haun, doctoral student; Michael Roberts, Ph.D. & MASL Director; and Petey Mumford, doctoral student.
Pictured from left to right are Chris Vann, doctoral student; Cody Haun, doctoral student; Michael Roberts, Ph.D. & MASL Director; and Petey Mumford, doctoral student.

‘Jumping genes’ (or transposable elements) have been known to exist in organisms since the 1950s when Barbara McClintock, Ph.D. at Cold Spring Harbor Laboratory theorized that they were responsible for the pigmentation of corn kernels. Given that this discovery was around the time Watson, Crick, Franklin and others elucidated the chemical structure of DNA, the idea that genes could randomly jump around the genome was a bit abstract to most scientists. Suffice it to say McClintock’s work went unnoticed for several years until other scientists began observing that transposable elements existed in numerous species. By the 1970s it was generally accepted that McClintock’s discovery was indeed a real phenomenon, and she was eventually awarded the Nobel Prize in Medicine in 1983 for her work in transposable elements.

LINE-1 (or Long Interspersed Nuclear Element-1) is a type of transposable that exists in humans and animals. Traditional transposable elements physically remove themselves from one genomic region and re-insert themselves into a different region. On the other hand, LINE-1 is a retrotransposon, meaning that it can copy itself and then re-insert said copy into a different genomic region while still maintaining the original copy. As a conceptual example, if you were born with 10 genomic DNA LINE-1 copies in a long-living brain cell, by the age of 50 this same cell possesses the 10 original copies along with 10 more new copies randomly inserted into its genome.

The LINE-1 jumping gene has been somewhat of a mystery to scientists. First, it has been estimated that upwards of 500,000 copies of the LINE-1 gene (not 10 copies) can exist in a single cell’s genome. Alternatively stated, about 17 percent of a cell’s DNA is likely comprised of 500,000 copies of the LINE-1 gene. Fortunately, most of these LINE-1 copies do not perform the copy-paste mechanism due to structural defects, otherwise cellular DNA would likely turn into an uncontrollable growing mass and the afflicted cell would either proliferate uncontrollably or die. However, scientists have estimated that 5 to 20 LINE-1 copies within a given cell are “hot,” meaning they are capable of making copies that randomly re-insert themselves into the genome. As you can imagine, based upon the underlying mechanism described above, it is becoming more appreciated that LINE-1 retrotransposition is likely deleterious for cells because it has the potential to generate random genetic mutations. In this regard, a recent review paper in Genetics in Medicine (2016) cites studies where markers of increased LINE-1 activity have been observed in certain cases of breast, liver, and colon cancers. Further, these authors concluded that LINE-1-induced genetic mutations were likely the cause of these cancer cases.

Michael D. Roberts, Ph.D., an Associate Professor in the School of Kinesiology and the Director of the Molecular and Applied Sciences Laboratory, became fascinated with the idea of jumping genes when he was a Ph.D. student at the University of Oklahoma in 2009. According to Roberts, he was an exercise physiology graduate student who was out of his element enrolled in a developmental genetics course taught by David Durica, Ph.D. Roberts remembers the day that Durica brought an article to class discussing jumping genes.

“This blew my mind,” said Roberts. “I immediately began to wonder if exercise was doing anything to affect this process in skeletal muscle.”

Roberts finished his doctoral studies at Oklahoma, but put the idea on hold. He undertook a three-year postdoctoral fellowship at the University of Missouri in order to strengthen his background in genetics, and in 2013 he accepted his first faculty position in the School of Kinesiology at Auburn University. A few years went by where Roberts’ research primarily focused on examining the physiological effects of dietary protein. Then, he came across a publication which immediately re-ignited his interest in jumping genes.

“I immediately emailed my graduate students and told them this is the best scientific article that I’ve ever read,” Roberts recalls.

The article was a 2013 paper in Aging by Marco De Cecco, Ph.D. and Jill Kreiling, Ph.D. These authors examined skeletal muscles of younger, middle-aged, and older mice and noted that the number of LINE-1 copies in the DNA increased in an age-dependent fashion. While there were no firm mechanisms tied to these observations (for instance, the authors were not able to state increases in muscle LINE-1 directly caused a dysregulation in muscle function), the authors did rightfully speculate that this phenomenon likely contributes to maladies observed with muscle aging (e.g., increased insulin resistance, decrements in muscle-building mechanisms, decrements in mitochondrial function).

“This article is paradigm shifting in the field of muscle aging,” says Roberts. “Most of the science indicates that decrements in hormones, an increase in oxidative stress, or the inability of muscle to turn on muscle-building mechanisms in response to feeding causes age-related muscle dysfunction. Instead, this article implies that the LINE-1 jumping gene may be central to muscle aging.”

As a laboratory interested in how exercise promotes muscle health, Roberts and Matthew Romero, one of Roberts’ doctoral students, immediately sought to analyze LINE-1 retrotransposition markers in muscle tissue. At the time, Roberts’ laboratory had just completed two human studies where muscle biopsy tissue was preserved. The first study involved three consecutive days of high-intensity leg resistance exercise training, and biopsies were collected prior to, during, and after the training protocol. The second study involved a 12-week resistance exercise training study, and biopsies were collected prior to and following the training protocol. Romero immediately seized the opportunity to analyze LINE-1 markers in these muscle samples. After months of tedious work, Romero noted that almost all markers of LINE-1 retrotransposition were reduced in muscle samples collected after the exercise interventions. This is to say LINE-1 markers from subjects’ post-training muscle biopsies were 20 to 50 percent lower compared to pre-intervention biopsies.

“In other words, we were batting for a single in hoping that we would observe something interesting and we ended up hitting a grand slam,” says Roberts, “Our laboratory at Auburn University was the first in the world to demonstrate that exercise reduces markers of LINE-1 retrotransposition in human skeletal muscle.”

While Romero’s findings were extraordinarily exciting, Roberts and Romero admit their study has some limitations. First, like De Cecco’s work mentioned above, Roberts’ laboratory did not demonstrate an exercise-induced reduction in skeletal muscle LINE-1 markers led to improvements in other processes related to muscle health (for instance, increased muscle-building mechanisms). Second, increased skeletal muscle LINE-1 retrotransposition is seemingly a problem that occurs with aging, and Romero’s paper examined muscle biopsies from younger, college-aged males (given that this was the only available human tissue at the time). Notwithstanding, Romero’s paper was viewed with great enthusiasm by the editors and reviewers of the American Journal of Physiology – Cell Physiology, and the contribution was awarded the “APSselect” distinction by the American Physiological Society in March 2018.

Roberts’ initial excitement in this area has grown into a multi-university collaboration, and he is currently exploring numerous research avenues related to skeletal muscle LINE-1 retrotransposition. Currently, Roberts’ laboratory is examining skeletal muscle from rats ranging from three months of age (adolescent rats) to 24 months of age (rats in their 70’s in terms of human age equivalency). Petey Mumford, another doctoral student in Roberts’ laboratory who is pursuing this area as a dissertation topic, is working with Romero on analyzing how aging affects skeletal muscle LINE-1 retrotransposition markers in these specimens. While Roberts says that this project is essentially repeating De Cecco’s 2013 work performed in mice mentioned above, a similar observation in rats would implicate this muscle aging pathway is conserved across different species, which strengthens its potential importance. Mumford and Romero are at the beginning of their analyses, although the preliminary data already collected has confirmed two critical markers indicative of skeletal muscle LINE-1 retrotransposition are increasing with aging.

“Both Petey and Matt have taken this idea to a whole new level,” says Roberts. “Nowadays they’re teaching me the literature and assays that are involved in this area of research. I’m very proud of both of them.”

The laboratory’s second project is Romero’s dissertation and involves examining how exercise in rats at an early age in life (through voluntary wheel running) affects skeletal muscle LINE-1 retrotransposition markers. Dr. Frank Booth from the University of Missouri shipped Roberts’ laboratory skeletal muscle from rats that began running at one month of age and ceased running around three to four months of age. Roberts’ laboratory also received muscle from age-matched rats that never had access to a voluntary running wheel. This research design will allow the researchers to examine if initiating exercise at an early age in life has an appreciable effect on LINE-1-related mechanisms.

“Matt’s hypothesis is that exercise at a very early age in life will dramatically reduce skeletal muscle LINE-1 retrotransposition markers and, relating these findings to humans, this phenomenon may be one of the reasons why children that exercise at a younger age in life typically exhibit markers that are indicative of better muscle health,” says Roberts.

Finally, Roberts has established collaborations with John McCarthy, Ph.D., who is a world-renowned muscle biologist at the University of Kentucky Medical School, as well as Jef Boeke, Ph.D., who is a world-renowned geneticist and Director of the Institute for Systems Genetics at NYU. The team will generate a special mouse breed which, according to Roberts, will unequivocally inform the researchers as to how increased skeletal muscle LINE-1 activity affects mechanisms of muscle aging.

“Dr. Boeke is a world-renowned LINE-1 researcher and geneticist, so his involvement is crucial,” says Roberts.

Beyond these collaborators, Roberts recruited the help of Andreas Kavazis, Ph.D. (School of Kinesiology), Mike Miller, Ph.D. (Harrison School of Pharmacy), Kaelin Young, Ph.D. (VCOM-Auburn), and Shawn Levy, Ph.D. (HudsonAlpha Institute for Biotechnology). The collective research team recently worked together to submit a Tier 1 PAIR grant application related to this project, and the team is slated to submit a grant to the National Institutes of Health (NIH) this October. Despite recent criticism regarding the translation potential of mice studies to human physiology, Roberts believes that his published data in humans demonstrating exercise reduces skeletal muscle LINE-1 retrotransposition markers makes this project highly competitive.

“Through a spark of fortune the hard part is essentially done. We’ve reverse-engineered the story from humans to rodents by first showing that this mechanism is exercise-responsive in humans, and now we’re proposing to use the mouse model to see how LINE-1 overexpression may cause muscle deterioration,” says Roberts.

Roberts says the ultimate goal of these efforts are two-fold. First, he and his students believe that they have uncovered a mechanism that is critically responsible for muscle aging. Thus, he believes that confirming this hypothesis through their potential PAIR and NIH work will be paradigm-shifting in the field of muscle aging. Second, and more importantly, Roberts thinks that exercise can reduce LINE-1 retrotransposition in skeletal muscle.

“If we continue to confirm this hypothesis with our upcoming experiments then this will provide another example codifying the current-day adage that ‘exercise is medicine,’” explained Roberts, adding that work in this area can easily extend to physiology beyond skeletal muscle. “What if we also observe that exercise reduces markers of LINE-1 activity in colon or liver tissue?! Such findings will potentially demonstrate that exercise can mechanistically reduce the odds of acquiring certain types of cancers!” says Roberts.

While the laboratory is still in the preliminary phases of this project, it is undeniable that the work done in this area at Auburn University could be potentially ground-breaking and lead to an entirely new field of exercise genetics.