Regulation Of The TFEB/TFE3 Transcription Factors For Lysosomal Genes

Lysosomes are important organelles in the cell,which function as the major cellular degradation center.Lysosomes play important roles in signal transduction,secretion and maintaining metabolic homeostasis.Dysfunction of lysosomes causes a wide variety of human disorders such as lysosomal storage diseases and neurodegenerative diseases.Transcription factor EB and E3(TFEB/TFE3)are two regulating factors of lysosome biogenesis,which are responsible for the transcription of lysosomal genes.When the cell responds to environmental cues,TFEB/TFE3 are dephosphorylated and translocate from the cytosol to the nucleus and directly bind to the promoter of lysosomal genes.Although a few regulators of TFEB/TFE3 have been identified,more studies are needed to further explore the mechanisms underlying TFEB/TFE3-mediated lysosome biogenesis.TFEB/TFE3 have a homologue,HLH-30,in Caenorhabditis elegans.Surprisingly,RNA interference(RNAi)of let-363(mTOR),gsk-3(GSK3?),and xpo-1(CRM1)in hq247(HLH-30::GFP)did not mimic the behavior of TFEB/TFE3 in mammalian cells.We then heterogeneously expressed human TFEB/TFE3 in the intestine of C.elegans.In the transgenic animals we generated,TFE3::GFP localized in the cytosol of intestinal cells in yqEx1470(Pvha-6 TFE3::GFP),and TFE3(S321A)::GFP localized in the nucleus,consistent with the localization of TFE3 in mammalian cells.We then integrated the extrachromosomal Pvha-6TFE3::gfp into chromosomes using the TMP/UV method,and obtained the integrated strain yqIs213(Pvha-6TFE3::GFP).We therefore performed a candidate-based RNAi screen on yqIs213 to inactivate genes required for nuclear membrane structure and nucleocytoplasmic transport.We found that RNAi of imb-1/KPNB1 induced the nuclear localization of TFE3.KPNB1 and the Ran GTPase function in a complex,which regulates the nuclear import of cargoes.Our results revealed that KPNB1 and the Ran GTPase participate in nuclear import of TFEB/TFE3.Chemical inhibition of KPNB1 by Importazole,a specific inhibitor of KPNB1,similarly induced nuclear retention of TFEB/TFE3 in mammalian cells and promote the biogenesis lysosomes.The research in this thesis established a screening system to identify new regulators of TFEB/TFE3.Our research also provides a potential treatment target for lysosomerelated diseases.