Discovery of epigenetic elasticity in neurons may lead to potential chromosomal therapies
Research from the lab of Jeanne B. Lawrence, PhD, shows that differentiated human cells retain the ability to undergo chromosome silencing in response to XIST, a naturally occurring “off switch” for the female X-chromosome. These findings, published in Developmental Cell, address a considerable obstacle in the potential development of chromosomal therapies and provide a platform to directly study the effects of trisomy 21 on neurodevelopment and eventually other cell types.
“Until now, it was believed that XIST expression could only enact chromosome silencing in pluripotent stem cells,” said Dr. Lawrence, professor of neurology and pediatrics. “While XIST is a powerful experimental model, being limited to pluripotent stem cells is an obstacle to developing any kind of chromosomal therapy.
“Our work shows that neural stem cells retain epigenetic plasticity past pluripotent development, allowing silencing to happen farther into cell development than anybody had previously thought,” she said.
In 2013, Lawrence and colleagues were the first to show that the underlying genetic defect responsible for Down syndrome can be suppressed in pluripotent stem cells in culture (in vitro) using XIST. Her current research takes this work further by showing that differentiated neural cells will respond to chromosome silencing using XIST.
“This new finding is paramount for any prospect of developing a potential chromosomal therapy,” said Lawrence. “It also provides a more powerful experimental system to bolster research into the developmental pathology of Down syndrome, as well as Alzheimer’s disease, which is exceptionally common in people with Down syndrome.”
Humans are born with 23 pairs of chromosomes, including two sex chromosomes, but people with Down syndrome are born with three copies of chromosome 21, which is commonly known as trisomy 21. A key challenge for scientists who study trisomy 21, is that hundreds of genes are over-represented in the syndrome, resulting in a vast range of health impacts, including cognitive disability, early-onset Alzheimer’s and dementia, as well as a greater risk of childhood leukemia, heart defects, and immune and endocrine system dysfunction. It is not known which of the more than 200 genes on chromosome 21 are responsible for the various aspects of the syndrome or associated disorders.
Determining the underlying cell pathologies and gene pathways responsible for the Down syndrome is compounded by the normal variation between people and cells. An experimental system created from patient-derived Down syndrome cells that can be selectively turned off could be used to begin answering some of these questions, which is important for developing any type of therapeutics.
In typical females, a long, noncoding RNA from the X-linked XIST gene becomes active very early in development before cell differentiation begins, and this turns off one of the two X chromosomes. XIST RNA coats one of the X chromosomes and triggers irreversible inactivation of nearly all the genes on that chromosome, providing more equivalent gene expression levels between males and females of a species.
The Lawrence lab previously showed that an XIST transgene inserted into one chromosome 21 could silence that chromosome, but this was shown in pluripotent cells.
It was believed that XIST could only work in pluripotent stem cells—before cells started differentiating. Because differentiation happens so early in the development process, any possibility of delivering an XIST-powered chromosome therapy would be nearly impossible, even if all the technical hurdles around delivery could be overcome. Lawrence and colleagues, however, had reason to believe that XIST might be able to conduct silencing even outside of its normal developmental stage.
“Development timing is very tightly controlled,” said Lawrence. “The thinking is that things have to happen during a certain window, or they just won’t happen at all. We wanted to see if we could use XIST to silence a chromosome during neurogenesis in Down syndrome patient-derived cells.
“As an experimental system, this would allow us to start the work of understanding the timing of specific cellular changes and the most important genes that contribute to specific health impacts in Down syndrome, such as dementia and Alzheimer’s disease,” said Jan Czerminski, MD/PhD candidate in the Lawrence lab and first author of the study.
Czerminski used pluripotent stem cells, generated from somatic skin cells from individuals with Down syndrome, which carried an inducible XIST gene on one chromosome 21. He then triggered differentiation for 28 days in a manner that mimics in vivo neurodevelopment. All cells had differentiated to neural stem cells by day 14, and many formed post-mitotic neurons by day 28. To assess the impact XIST RNA has on chromosome silencing, Czerminski turned on the XIST transgene in the cells at different stages of development—days 0, 14, or 21,
Surprisingly, cells treated on day 14 and day 21 showed clear evidence that XIST was working to repress expression of genes across the extra chromosome 21, including in the post-mitotic neurons formed by day 28. Another surprise was the discovery that inducing XIST RNA in neural stem cells enhanced their terminal differentiation to neurons.
The finding that XIST does not require pluripotent cells to initiate the silencing process heightens the power of XIST as an experimental approach, and surmounts a perceived obstacle to potential development of XIST-based therapeutics.
“What we discovered was the neural cells needed to express XIST RNA longer,” said Lawrence. “The key was that after the cells differentiate, it takes XIST longer to silence most genes. In pluripotent stem cells, XIST silences the chromosome in about three days. In differentiated cells it took one to two weeks before most silencing takes effect.”