By Emily Leclerc | Waisman Science Writer
Genetic repeats—a sequence or segment of DNA that is repeated over and over in a row—is a typical part of the genome. However, when there is an atypical number of repeats in the DNA sequence, it can lead to impaired gene function and be the cause of more than 50 expansion conditions including fragile X syndrome (FXS). FXS is an inherited intellectual and developmental disability caused by expansion in the genetic repeats in the FMR1 gene.
New research from the lab of Xinyu Zhao broadens the understanding of the repeats in the FMR1 gene and their function in the hope of applying this knowledge to the development of potential treatments and therapies for fragile X syndrome. Her new paper, “CGG repeats in the human FMR1 gene regulate mRNA localization and cellular stress in developing neurons”, reveals that the typical number of genetic repeats in the FMR1 gene present in healthy humans, may play a significant role in neuronal development and regulating cellular stress responses.
These genetic repeats are officially known as short tandem repeats (STRs). They often occur at the end of genes and are seen in a wide variety of genes across the genome. In the FMR1 gene, the STRs are a series of Cytosine-Guanine-Guanine (CGG) repeats.
A typical person will have 31 CGG repeats in their FMR1 gene. In FXS, that section expands to over 200 which effectively shuts down FMR1. This shows that the CGG repeats likely have a function and purpose in the gene, because when they are disrupted it results in a disorder. But their function at typical numbers was unknown. Previous research conducted by Marsha Mailick, PhD, Waisman investigator and emeritus vice chancellor for research and graduate education, showed that individuals with FXS and individuals with shorter than normal CGG repeat numbers (fewer than 25 repeats) deal poorly with stress in their lives when compared to typical individuals. In order to investigate what CGG repeats do in neurons, Zhao and Carissa Sirois, PhD, a scientist in Zhao’s lab and first author of the paper, turned to stem cells.
Funded by an IDDRC signature project co-led by Zhao and Mailick, PhD scientist Meng Li in the Zhao group created two lines of stem cells. One had the normal complement of CGG repeats while the other had no repeats at all. They snipped the repeats off the end of the gene and named those cells 0CGG cells. The typical cells were called 31CGG cells. Then Zhao and Sirois looked at how the cells were impacted as they differentiated into neurons. “We weren’t exactly sure what to look for but we saw this difference in mRNA localization,” Sirois says.
mRNA, or messenger RNA, is a crucially important molecule used as a template to build proteins. Throughout cell development and everyday cell functioning, mRNA has to be shuttled to the correct place in the cell in order to do its job properly. Localization refers to where the mRNA is being sent in the cell. In the 0CGG cells, the mRNA transcribed from FMR1 had different localization patterns than the 31CGG cells. The mRNA wasn’t being sent where it was supposed to go. The observed differences were most obvious during the early stages of the neurons’ development. “We determined that the CGG repeats are really important for proper mRNA localization,” Zhao says.
Then they started to think about how this different mRNA localization in the 0CGG cells would impact the cell. Zhao and Sirois decided to look at the cells’ stress responses.
Zhao and Sirois recognize that you cannot accurately simulate life stress in a petri dish, but there are ways to mimic stress to stimulate and evaluate cellular responses. They decided to do that by activating the cortisol pathway. “When we’re stressed out, our brain produces cortisol. That binds to a specific receptor which then leads to downstream effects,” Sirois says. “This is a well-known pathway for stress.”
When the 0CGG cells had their cortisol pathway activated, they had distinct differences in their responses when compared to the 31CGG cells. Sirois looked at different protein levels that are implicated in cellular stress and found that not only were the 0CGG cells not responding in a typical way, but that the 0CGG cells seemed to already have changes in cellular stress. “Even at a baseline, before any treatment to simulate stress, there were already differences in the expression of some of these proteins in the 0CGG cells,” Sirois says.
These findings come together to indicate that the CGG repeats do have a purpose in the FMR1 gene and their removal has an impact on developing neurons. The repeats seem to be fairly involved in the mRNA localization process and in how the cells respond to stress. Understanding their function could then lead to insights into FXS as a whole and why neurons behave the way they do in individuals with this condition.
The results also pose important implications for potential therapies down the line. One method of genetic therapy being considered to treat FXS is to remove the CGG repeats from the FMR1 gene in order to turn the gene back on. But you don’t want to entirely remove something before you understand what it does. Zhao and Sirois’ work provides important foundational information for the role of CGG repeats in the cell and the potential risks for removing them with gene therapy. “If you delete the repeats you might solve some FXS symptoms but then create other problems in the process,” Zhao says.
There is still much more work to be done. Zhao and Sirois have plans to continue this thread of research with questions into the impacts on mitochondria and the excitability of neurons. This work also opens doors to encourage other researchers to investigate beyond the CGG repeats in FMR1, and into the multitude of other places in the genome that have these repeats.
“We think this is really exciting. It is also kind of unique thing to study even though there are lots of repeats in the human genome. Most of them we have no idea why they are even there. But we do know that when they’re too long they are pathogenic,” Zhao says. “It’s kind of like the dark side of the moon.”
Other Waisman investigators that contributed to this work include Anita Bhattacharyya, PhD, associate professor of cell and regenerative biology and André Sousa, PhD, assistant professor of neuroscience.
| Your support makes a difference. Donate now to advance knowledge about human development, developmental disabilities, and neurodegenerative diseases through research, services, training, and community outreach. | DONATE NOW |