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Oncogenic rearrangements of theTFE3transcription factor gene are found in two distinct human cancers. These includeASPSCR1–TFE3in all cases of alveolar soft part sarcoma (ASPS) andASPSCR1–TFE3,PRCC-TFE3,SFPQ-TFE3and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1–TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1–TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1–TFE3, identified 2193 genes bound by ASPSCR1–TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1–TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1–TFE3, includingMET(a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1–TFE3. As validation of this approach to identify genuine ASPSCR1–TFE3 target genes, two up-regulated genes bound by ASPSCR1–TFE3,CYP17A1andUPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1–TFE3. As the results indicated that ASPSCR1–TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition toMET, we provide evidence that 11 other ASPSCR1–TFE3 target genes contribute to the growth of ASPSCR1–TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven byTFE3fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors.