Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription factor.

The ability of the retinoblastoma (Rb) tumor suppressor protein to regulate cell growth is due, at least in part, to its ability to interact with and regulate the E2F family of transcription factors (8, 34). The E2F proteins have been shown to control the expression of a large number of genes involved in DNA replication, cell cycle progression, and cell fate determination. The E2F family is composed of six distinct gene products that form heterodimeric complexes with partners of the DP family. Sequence analysis reveals three distinct subfamilies of E2F genes: the E2F1, E2F2, and E2F3 genes, the E2F4 and E2F5 genes, and the E2F6 gene. This division also coincides with functional distinctions. The E2F1, E2F2, and E2F3 genes are tightly regulated by cell growth and during the cell cycle, whereas E2F4, E2F5 and E2F6 are constitutively expressed. This cell cycle regulation of E2F1, E2F2, and E2F3 transcription is complemented by mechanisms that tightly regulate the accumulation of the proteins. An N-terminal domain unique to E2F1 to E2F3 is responsible for both ubiquitin-mediated degradation of the proteins (29) and targeting by the cyclin A/cdk2 kinase, the latter leading to inhibition of DNA binding capacity (7, 22, 23, 53). The E2F proteins also vary in their role as transcriptional regulatory activities. While E2F1 to E2F3 act as positive regulators of transcription, E2F4 and E2F5 appear to function primarily as transcriptional repressors in concert with Rb family members. E2F6 also appears to function as a transcriptional repressor but in a manner independent of Rb (4, 10, 47, 48). Various experiments have suggested distinct functional roles for the activating E2F proteins E2F1, E2F2, and E2F3. The E2F3 protein appears to be particularly important for cell proliferation, as seen from the inhibition of E2F3 activity by antibody microinjection (25) as well as the results of deletion of the E2F3 gene (18). Moreover, the expression of a number of E2F target genes that encode key cell cycle regulatory proteins, including B-Myb, cyclin A, cdc2, cdc6, and dihydrofolate reductase, are reduced in E2F3 null fibroblasts but not in E2F1 null cells (18). In contrast, E2F1 appears to play a role in triggering an apoptotic response, either when overexpressed in the absence of survival signals (6, 21, 39, 43, 51) or in response to DNA damage (27). In addition, the ability of Myc to induce apoptosis is impaired in the absence of E2F1 function but unaltered by the absence of either E2F2 or E2F3 (26). Given the role of the E2F1, E2F2, and E2F3 proteins as transcriptional activators, the specificity of function might best be explained by an ability of these E2F proteins to activate distinct target genes that then carry out these functions of apoptosis or proliferation. As an example, the p19ARF gene has been shown to be an E2F1 target gene (2, 6), linking the action of the Rb/E2F pathway with the p53 response leading to apoptosis. Similarly, the Apaf1 gene appears to be activated specifically by E2F1 (30). Although one potential mechanism for such specificity could be an ability of these proteins to recognize subtle differences in cis-acting promoter sequences, there is little evidence for distinct DNA sequence recognition among the E2F isoforms. More importantly, analysis of the structure of an E2F-DNA complex did not show a capacity for distinct DNA sequence recognition when the amino acid variation within the E2F family is considered (56). An alternative mechanism for promoter specificity could involve distinct protein-protein interactions. Possibly, sequences within E2F3 allow interaction with a subset of cellular proteins that provide a basis for promoter specificity and that are distinct from the proteins that can interact with E2F1. For example, the interaction of the herpesvirus VP16 transcriptional activator protein with the cellular factor HCF-1 and the cellular Oct-1 transcription factor redirects Oct-1 to herpesvirus immediate-early promoters by virtue of expanded DNA sequence recognition (1). A further example of combinatorial specificity as a mechanism for the specificity of transcription factor function is the pancreatic islet factor STF-1, which interacts with Pbx in a cooperative fashion and targets Pbx to a subset of promoters containing STF-1 binding sites (38). Recently, we identified the YY1-binding protein RYBP as a factor which binds to E2F2 and E2F3 but not E2F1 and recruits these E2Fs to a subset of E2F target promoters containing YY1 binding sites (41). To further explore the mechanistic basis for the specificity of E2F transcription activation, we used yeast two-hybrid screens to identify proteins that specifically interact with E2F3. In so doing, we identified the TFE3 transcription factor as a specific partner for E2F3. Recent experiments identified TFE3 as an activity that can rescue Rb-mediated growth arrest, providing a functional link between TFE3 and the Rb/E2F pathway (M. Nijman, S. Hijmans, and R. Bernards, personal communication). Moreover, previous work identified TFE3 as a fusion partner in chromosomal rearrangements in renal cell carcinomas (14, 45, 50). We further show that TFE3 and E2F3 can act synergistically to activate the p68 subunit gene of DNA polymerase α, dependent on the ability of the two proteins to physically interact, and that the two activities associate with the p68 promoter within intact cells in a mutually dependent manner.