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Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms.Protein isoforms generated by alternative splicing contribute to proteome diversity. Because of the lack of effective techniques, the isoform-specific function, expression, localization, and signaling of endogenous proteins are unknown for most genes. Here, we report a genetic method, isoTarget, for multi-purpose studies of targeted isoforms in select cells. Applying isoTarget to two isoforms of Drosophila Dscam, Dscam[TM1] and [TM2], we found that, in neurons, endogenous Dscam[TM1] is in dendrites, whereas Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization, rather than biochemical properties, leads to the two isoforms' functional differences. Moreover, we show that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade in axons. We further apply isoTarget to study two isoforms of a GABA receptor to demonstrate its general applicability. isoTarget is an effective technique for studying how alternative splicing enhances proteome complexity.Expansion of a CAG repeat in ATXN3 causes the dominant polyglutamine disease spinocerebellar ataxia type 3 (SCA3), yet the physiological role of ATXN3 remains unclear. Here, we focus on unveiling the function of Ataxin-3 (ATXN3) in the retina, a neurological organ amenable to morphological and physiological studies. Depletion of Atxn3 in zebrafish and mice causes morphological and functional retinal alterations and, more precisely, photoreceptor cilium and outer segment elongation, cone opsin mislocalization, and cone hyperexcitation. ATXN3 localizes at the basal body and axoneme of the cilium, supporting its role in regulating ciliary length. HexaDarginine Abrogation of Atxn3 expression causes decreased levels of the regulatory protein KEAP1 in the retina and delayed phagosome maturation in the retinal pigment epithelium. We propose that ATXN3 regulates two relevant biological processes in the retina, namely, ciliogenesis and phagocytosis, by modulating microtubule polymerization and microtubule-dependent retrograde transport, thus positing ATXN3 as a causative or modifier gene in retinal/macular dystrophies.Endometriosis affects 1 in 10 women and is characterized by the presence of abnormal endometrium at ectopic sites. ARID1A mutations are observed in deeply invasive forms of the disease, often correlating with malignancy. To identify epigenetic dependencies driving invasion, we use an unbiased approach to map chromatin state transitions accompanying ARID1A loss in the endometrium. We show that super-enhancers marked by high H3K27 acetylation are strongly associated with ARID1A binding. ARID1A loss leads to H3K27 hyperacetylation and increased chromatin accessibility and enhancer RNA transcription at super-enhancers, but not typical enhancers, indicating that ARID1A normally prevents super-enhancer hyperactivation. ARID1A co-localizes with P300 at super-enhancers, and genetic or pharmacological inhibition of P300 in ARID1A mutant endometrial epithelia suppresses invasion and induces anoikis through the rescue of super-enhancer hyperacetylation. Among hyperactivated super-enhancers, SERPINE1 (PAI-1) is identified as an essential target gene driving ARID1A mutant endometrial invasion. Broadly, our findings provide rationale for therapeutic strategies targeting super-enhancers in ARID1A mutant endometrium.Cohesin, a critical mediator of genome organization including sister chromatid cohesion, is a ring-shaped multi-subunit ATPase that topologically embraces DNA. Its loading and function on chromosomes require the Scc2-Scc4 loader. Using biochemical reconstitution, we show here that the ability of the loader to bind DNA plays a critical role in promoting cohesin loading. Two distinct sites within the Mis4Scc2 subunit are found to cooperatively bind DNA. Mis4Scc2 initially forms a tertiary complex with cohesin on DNA and promotes subsequent topological DNA entrapment by cohesin through its DNA binding activity, a process that requires an additional DNA binding surface provided by Psm3Smc3, the ATPase domain of cohesin. Furthermore, we show that mutations in the two DNA binding sites of Mis4 impair the chromosomal loading of cohesin. These observations demonstrate the physiological importance of DNA binding by the loader and provide mechanistic insights into the process of topological cohesin loading.Tissue injury is one of the most severe environmental perturbations for a living organism. When damage occurs in adult Drosophila, there is a local response of the injured tissue and a coordinated action across different tissues to help the organism overcome the deleterious effect of an injury. We show a change in the transcriptome of hemocytes at the site of tissue injury, with pronounced activation of the Toll signaling pathway. We find that induction of the cytokine upd-3 and Toll receptor activation occur in response to injury alone, in the absence of a pathogen. Intracellular accumulation of hydrogen peroxide in hemocytes is essential for upd-3 induction and is facilitated by the diffusion of hydrogen peroxide through a channel protein Prip. Importantly, hemocyte activation and production of reactive oxygen species (ROS) at the site of a sterile injury provide protection to flies on subsequent infection, demonstrating training of the innate immune system.