The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator TFEB revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. Here, we report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag GTPases and exhibited mTORC1-dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. Following starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders.

The activation of transcription factors is critical to ensure an effective defense against pathogens. In this study we identify a critical and complementary role of the transcription factors TFEB and TFE3 in innate immune response. By using a combination of chromatin immunoprecipitation, CRISPR-Cas9-mediated genome-editing technology, and in vivo models, we determined that TFEB and TFE3 collaborate with each other in activated macrophages and microglia to promote efficient autophagy induction, increased lysosomal biogenesis, and transcriptional upregulation of numerous proinflammatory cytokines. Furthermore, secretion of key mediators of the inflammatory response (CSF2, IL1B, IL2, and IL27), macrophage differentiation (CSF1), and macrophage infiltration and migration to sites of inflammation (CCL2) was significantly reduced in TFEB and TFE3 deficient cells. These new insights provide us with a deeper understanding of the transcriptional regulation of the innate immune response.

To reestablish homeostasis and mitigate stress, cells must activate a series of adaptive intracellular signaling pathways. The participation of the transcription factors TFEB and TFE3 in cellular adaptation to starvation is well established. Here, we show that TFEB and TFE3 also play an important role in the cellular response to ER stress. Treatment with ER stressors causes translocation of TFEB and TFE3 to the nucleus in a process that is dependent on PERK and calcineurin but not on mTORC1. Activated TFEB and TFE3 enhance cellular response to stress by inducing direct transcriptional upregulation of ATF4 and other UPR genes. Under conditions of prolonged ER stress, TFEB and TFE3 contribute to cell death, thus revealing an unexpected role for these proteins in controlling cell fate. This work evidences a broader role of TFEB and TFE3 in the cellular response to stress than previously anticipated and reveals an integrated cooperation between different cellular stress pathways.