Tox3 Regulates Calcium-dependent Transcription in Neurons We Report the Cloning and Characterization of Tox3, a High Mobility Group Box Protein Involved in Mediating Calcium-dependent Tran- Scription. Tox3 Was Identified as a Calcium-dependent Transactivator Using the Transactivator Trap Screen. We

N euronal activity plays an important role in the development of the nervous system and regulates cell survival, axonal and dendritic growth, and synaptic plasticity. Many of these effects are mediated by calcium-dependent transcription (1). To identify factors that mediate calcium-dependent transcription in neurons, our laboratory developed a screening strategy called Transactivator Trap (2). We have previously described the cloning of CREST (2), NeuroD2 (3), and LMO4 (4) as calcium-dependent transcription using the Transactivator Trap strategy. Here we report the cloning and characterization of TOX3, a cAMP response element (CRE)-binding protein (CREB) and CREB-binding protein (CBP) interacting protein that plays a critical role in regulating calcium-dependent transcription. TOX3 is a high mobility group (HMG) box protein related to TOX, a protein that has been implicated in the regulation of thymocyte selection (5). HMG box proteins bind to the minor groove of DNA and are nonchromosomal nuclear proteins that help to remodel the nucleosome (6). Our investigation of TOX3 was driven by its calcium-activation properties and its association with CREB. CREB has long been known to be the major mediator of stimulus-induced transcription activation in neurons. CREB was first discovered as a protein that binds to the cAMP-responsive element and mediates cAMP-dependent transcription (7, 8). CREB-dependent transcription was subsequently found to be inducible by calcium and growth factors (9, 10). Calcium regulation of CREB-mediated transcription requires phosphorylation of CREB at Ser-133. Phosphorylation of Ser-133 allows CREB to bind to CREB-binding protein (CBP) (11). CBP itself can be phosphorylated by CaMKIV (12–16), and it is a critical mediator of CREB-dependent transcription. We find that TOX3 interacts with both CREB and CBP and plays a critical role in mediating calcium-dependent transcription in neurons. Results Identification of TOX3 as a Calcium-Dependent Transactivator. We used the Transactivator Trap screen (2) to search for novel mediators of calcium-dependent transcription. Brief ly, the screen uses the modular nature of transcription factors that enables the transactivation domain to function without the DNA-binding domain. In addition, the screen takes advantage of the fact that a transcription factor fused to the yeast Gal4DBD (DNA-binding domain) can recognize the upstream activating sequence (UAS) and drive expression of a reporter. In our experiments, a postnatal day 1 (P1) rat cortical cDNA library was fused to Gal4DBD and broken down into 200 pools. Each pool was transfected along with a UAS-CAT (chloramphenicol acetyltransferase) reporter into embryonic day 18 (E18) cortical cultures. Pools containing …

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