Identify interaction partners of acetylated and non-acetylated TFEB and their role in transcriptional activation of lysosomal and autophagy genes. TFEB_RNASeq

TFEB, which is a Transcription Factor, is mostly involved on the regulation of lysosomal homeostasis. Since it is known that the phosphorylation is the main posttranslational modification that regulates TFEB activity, we were wondering on the role of others posttranslational modifications in TFEB regulation. Then, we found several lysine residues that can be acetylated/de-acetylated. So we generated TFEB-acetylated/de-acetylated mutants to better understand the role of the acetylation in TFEB regulation. In particular, we observed that TFEB-de-acetylated-mutant shows the same behaviour of TFEB-WT (goes into the nuclei under certain stimuli), while TFEB-acetylated-mutant can´t translocate into the nuclei. Since the TFEB-de-acetylated-mutant is able to migrate into the nuclei, we wanted to investigate its transcriptional activity compared to the -WT one. For that reason we performed the RNA sequencing analysis. The cell line used is a human (HeLa) inducible (Tet-On) cell line, where TFEB-WT and TFEB-de-acetylated-mutant (TFEB-K3R) are stably expressed. Their expression is regulated by adding a doxycycline to the medium, so for that reason we wanted to investigate TFEB-WT and mutant transcription activity while the proteins are induced for 24 hours continously and while they are induced for 3 hours followed by 21hours of medium chase; and then we wanted to compare not only the differences between the -WT and the mutant, but also how the different amount of TFEB protein (after 24hrs of induction = huge amount of TFEB; 3hours followed by 21hrs medium chase = less amount of protein) change the activity of TFEB-target genes. We used as a negative control HeLa Tet-On cell line expressing only TFEB-endogenous. So we prepared the samples in triplicate for each condition (24hrs and 3+21hrs), extract the RNA using one kit of RNA extraction, quantified the RNA amount by Nanodrop and then give the samples to the people who carries out the RNA sequencing analysis.