Screening for transposon regulators in mouse development

Abstract

Transposable elements (TEs) are mobile genetic elements that contribute to genome evolution but pose potential threats to genomic stability. Their activity is tightly regulated by host defense mechanisms, including DNA methylation, histone modifications, and transcription factor (TF)-mediated control. This thesis investigates the regulation of TEs in two model systems: mouse embryonic stem cells (mESCs) and the mouse male germline. In the first part of this thesis, we aimed to establish a CRISPR/Cas9-based screening using an innovative endogenous readout to identify TE regulators in mESCs. By generating KAP1-degron and Suv39h-dKO cell lines we aimed to monitor transcriptional and translational activation of endogenous ERV and LINE-1 elements. Despite significant efforts, technical challenges prevented the successful implementation of the screening approach. However, this work highlights the complexities of studying endogenous TE regulation and provides a foundation for future optimization. The second part of this thesis investigates TE reactivation during DNA methylation loss in mouse spermatogenesis. Using Dnmt3CKO/KO mice, we found that young ERVs remain active throughout postnatal germ cell stages in the absence of DNA methylation, and in contrast to other studies, we observed LINE-1 reactivation before meiosis, which further increased during meiosis. DNA pulldown assays identified NRF1, a DNA methylation-sensitive TF, as a potential regulator of unmethylated TEs in the male germline and chromatin profiling analysis confirmed NRF1 binding to unmethylated TEs. Conditional knockout of Nrf1 in Dnmt3CKO/KO mice resulted in significant downregulation of IAP protein expression prior to meiosis, demonstrating NRF1 as a trans-activator of these elements. Additionally, H3K27me3 was identified as a potential barrier to NRF1 binding, suggesting a complex interplay between histone modifications and TF-mediated regulation. Together, these studies enhance our understanding of the multilayered regulatory networks controlling TE activity in the mouse genome. While challenges remain in developing high-throughput approaches for studying TE regulation, our findings emphasize the importance of both epigenetic modifications and TFs in balancing TE repression and activation during development.