Validation of Degron Tag-Mediated Depletion of YY1 and MSL1 in Mouse Embryonic Stem Cells

Abstract

This study explores the efficiency of degron tag-dTAG mediated depletion of YY1 and MSL1 proteins in mouse embryonic stem cells (mESCs) and its impact on histone modifications and LINE-1 transposable elements expression. Using CRISPR-Cas9, mESCs were engineered to express the FKBP12F36V tag, followed by treatment with PROTAC drugs to induce YY1 and MSL1 degradation. Western blot analysis revealed a strong depletion of YY1, whereas MSL1 depletion was not observed but showed a clear reduction in H4K16ac. YY1 depletion did not significantly affect H4K16ac levels but substantially reduced LINE-1 expression, indicating YY1’s role in gene regulation rather than histone modification. Conversely, the reduction in H4K16ac levels without significant alteration in LINE-1 expression underscores MSL1’s critical function in histone acetylation and chromatin structure modulation. These findings highlight the differential roles of YY1 and MSL1 in mESCs, where YY1 primarily influences gene regulation and MSL1 affects histone modifications. The study demonstrates the utility of the dTAG system in providing temporal control over protein degradation, offering valuable insights into the molecular mechanisms governing gene regulation and genomic stability in pluripotent cells. The ability of the dTAG system to target specific proteins and study their immediate and downstream effects in real-time makes it a powerful tool for functional genomics. This research enhances our understanding of protein functions in gene regulation, paving the way for future studies aimed at manipulating these processes in stem cell biology and other fields.