![](https://cals.ncsu.edu/international-programs/wp-content/uploads/sites/23/2024/08/sophia-in-lab-wide.jpg)
SUMMER 2024
Sophia Nunez
During my summer in Japan, I studied at Nagoya University with the Laboratory of Genome and Epigenome Dynamics run by Dr. Kenji Ichiyanagi. This research focused on the role of transposable elements (TEs) in the innate immune system, specifically in regards to retrotransposon activation after
cancer treatment.
A compound known as 5-azacytidine is used to treat hematological cancers, but the pathway behind its anti-tumorigenesis activity is largely unknown. This drug acts to inhibit DNA-methylation by incorporating into DNA during replication and binding methyltransferase. At first it was believed that this demethylation of the DNA may directly activate tumor suppressor genes, but recent studies have indicated the important role of the activation of retrotransposons. There are two general functions that retrotransposons are known to have. The first of these is viral mimicry – this is when the demethylation
allows transcription of retrotransposon RNA, which is then recognized as a viral element and triggers an immune response, leading to cell death. However, TEs are also known to have roles as promoters for nearby genes. Thus, it was hypothesized that the regulatory function of TEs may be a key mechanism for
the anti-tumorigenesis activity of 5-azacytidine.
For my project, I studied retrotransposons near various immune genes that were found to have h3k4me3 promoter markers. My goal was to develop the necessary components to use CRISPR/Cas9 for gene knockout on the target sequences, such as the primers and the plasmid containing gRNA. After completing genome analysis and selecting sequences for gRNA and primer sets, I was able to develop 6 gRNA samples for each of my three target retrotransposons. Once primer effectivity was confirmed and the gRNA-containing plasmids were amplified, I prepared 4 knockout cell lines by using CRISPR/Cas9 to remove the target sequence and cloned successful knockout cells. Future studies will use these cell lines to determine the promoter activity of the target retrotransposons based on the impact that the knockout has on the expression of corresponding viral defense genes.
The research completed in my time at Nagoya University will lay the foundation for continued study of the regulatory function of retrotransposons in the innate immune system. This is critical in developing our understanding of cancer treatments, as well as the role of sequences in our DNA that have not been studied in depth. I would like to thank Dr. Kenji Ichiyanagi and Dr. Hitoshi Ohtani for their guidance throughout my time in the lab. Completing my research in Japan helped me learn a lot, and it was fun to explore this country – I really appreciate the role of the lab members in helping me adjust to life abroad.