Lysosome biogenesis and homeostasis

National Heart, Lung, And Blood Institute

Investigators

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Abstract

Animals have the ability to adapt to numerous internal and external perturbations, thus ensuring organismal homeostasis throughout their lifetime. In recent years, the MiT/TFE family of basic helixloophelix leucine-zipper transcription factors has emerged as a critical component of the cellular response to stress. The MiT/TFE family includes four members, MITF, TFEB, TFE3, and TFEC, which are present in most metazoan organisms and can heterodimerize with each other. In contrast, only one member of the family is present in D. melanogaster and C. elegans, termed Mitf and HLH-30, respectively. The transcription factors TFEB and TFE3 control lysosomal biogenesis and autophagy by positively regulating genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. We previously described that the main regulatory mechanism for TFEB and TFE3 is the control of their translocation from the cytosol to the nucleus. Under basal (non-stressed) conditions, TFEB and TFE3 are recruited to the surface of lysosomes through interaction with active Rag GTPases. This brings TFEB and TFE3 in close proximity to the serine/threonine kinase mTORC1, which phosphorylates the transcription factors on multiple residues. mTORC1dependent phosphorylation of TFEB on serine 211 (S211) and TFE3 on serine 321 (S321) creates a binding site for 1433, resulting in sequestration of TFEB and TFE3 in the cytosol. Under stress conditions, dephosphorylation of TFEB and TFE3, either by inactivation of mTORC1 or activation of specific phosphatases, causes a rapid translocation of the transcription factors to the nucleus, where they activate multiple transcriptional networks with the goal of eliminating damaged organelles, preserving cellular functions and ultimately, restoring cellular homeostasis. Our laboratory has identified a growing list of stressors that induce TFEB and TFE3 activation, including nutrient deprivation, pathogens, accumulation of unfolded proteins pathogens, DNA damage, and oxidative stress. Other groups have also reported TFEB and TFE3 activation in response to mitochondrial damage, physical exercise, and increased cytosolic Ca2+. These observations clearly suggest an essential role of these transcription factors in cellular response to stress. We have also investigated the potential mechanisms required to maintain sustained TFEB and TFE3 activation under chronic stress. We recently characterized a novel mechanism of TFEB and TFE3 regulation. We identified a cysteine-based redox switch that controls the shift between TFEB and TFE3 oligomeric states. Detachment of 14-3-3 following stress exposes a single cysteine residue that undergoes ROS-dependent disulfide-bond formation, resulting in the assembly of TFEB and TFE3 oligomers. Oligomer formation is rapid and reversible and occurs in response to a variety of stresses both in vitro and in vivo. Oligomers are further stabilized under prolonged stress conditions and show increased resistance to inactivation. In C. elegans, inhibition of oligomers assembly also affects HLH-30 activity, resulting in deleterious phenotypes like developmental delay, altered dauer function, and increased pathogen susceptibility. These findings reveal a novel and evolutionary conserved mechanism important to maintain MiT/TFE transcription factors activation under prolonged stress conditions. While several of the mechanisms of TFEB/TFE3 nuclear translocation are well characterized, little is known about the nuclear factors that modulate their transcriptional activity. To address this question, we recently performed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) and identified a novel interaction between TFEB/TFE3 and the Facilitating Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone that mediates nucleosome disassembly to facilitate rapid transcriptional elongation of target genes. We found that several stimuli, including nutrient deprivation, Torin1-induced mTORC1 inactivation and oxidative stress, induced nuclear translocation of TFEB and TFE3, which then associated with the FACT complex to regulate stress-induced gene transcription. Depletion or inactivation of FACT did not affect TFEB/TFE3 activation, stability, or ability to bind to the promoter of target genes. In contrast, by using a combination or RNA-seq and q-PCR, we found that the TFEB-mediated induction of lysosomal and antioxidant genes was significantly impaired in FACT-depleted cells. Furthermore, the transcriptional elongation rates of numerous TFEB/TFE3 targets were decreased by FACT depletion or inactivation, thus suggesting that the FACT complex functions as a TFEB/TFE3 transcriptional activator. This work highlights the importance of chromatin remodeling for a sustained and efficient stress response, and sheds new light on the epigenetic regulation of redox homeostasis and lysosomal biogenesis.