Temporal gene activation by TFEB and acetylation mutants result in distinct subsets of newly synthesized lysosomal proteins

The target gene activation of TFEB and acetylation mutants differ from the newly synthesized lysosomal enzymes Lysosomes are central to the degradation and recycling of cellular components. They harbor around 70 hydrolytic enzymes and over 250 membrane proteins, and play a key role in cellular homeostasis. One of the main proteins that regulates lysosomal activity is the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase. Additionally, the microphthalmia-associated transcription factor (MiTF) family members, including TFEB, influence lysosomal and autophagy gene expression by binding to specific DNA sequences. Their functionality is modulated by phosphorylation events. In this study, we discovered acetylation of TFEB at specific lysine residues (K237, K256, and K274) which impacted its DNA binding and localization. Mutations mimicking this acetylation led to cytoplasmic retention of TFEB, independent of mTORC1. Furthermore, while TFEB mutation at these sites didn't hinder its nuclear import, it did alter its gene activation profile. Interestingly, despite these alterations in gene expression, the synthesis of lysosomal proteins within 24 hours remained inconsistent with the transcriptional activity. This suggests that TFEB acetylation acts as a dominant retention signal in the cytoplasm and introduces an additional regulatory layer to phosphorylation-dependent transcriptional activity. The study further indicates a nuanced role for TFEB in modulating lysosomal components in response to cellular conditions. Overall design: TFEB expression was induced with 1 µg/ml doxycycline for 3 hours. Post-induction, cells were either harvested immediately (chase 0 h) or were left without doxycycline for various time periods to track (chase) the expression. HeLa cells induced to express TFEB wt for 3 hours followed by a 21-hour chase (wt 3+21h). HeLa cells induced for a continuous 24-hour period (wt 24h). HeLa cells induced to express K3R for 3 hours followed by a 21-hour chase (K3R 3+21h). HeLa cells induced to express K3R for a continuous 24-hour period (K3R 24h). Control cells not induced by doxycycline. All samples were taken in triplicates.