Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Demonstration of an E-box and Its CNS-Related Binding Factors for Transcriptional Regulation of the Mouse Type 1 Inositol 1 ‚4,5-Trisphosphate Receptor Gene Yoshiyuki Konishi, Yasushi Kobayashi, Toshihiko Kishimoto, Yasutaka Makino, *Atsushi Miyawaki, *Tejjchj Furuichi, lHideyuki Okano, *Katsuhjko Mikoshiba, and Taka-aki Tamura Laboratory of Molecular Biology, Department of Biology, Faculty of Science, Chiba University, Chiba; * Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Tokyo; and t Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School, Osaka, Japan Abstract: The type 1 inositol 1 ‚4,5-trisphosphate recep- tor (IP 3R1) is expressed abundantly in the CNS, such as in cerebellar Purkinje cells and the hippocampus. We established a tissue-specific cell-free transcription sys- tem and studied regulatory properties of the 5‘ upstream region of the IP3R1 gene by use of this system. Deletion analyses of the promoter revealed several cis elements that function significantly in brain nuclear extracts. Among those elements, sequences from —398 to —295 showed the most predominant cerebellum-specific posi- tive function. Footprint analyses demonstrated a factor- binding region from —334 to —318, termed box-l, that contained an E-box consensus sequence. Electropho- retic mobility shift assay revealed CNS-related basic he- lix—loop—helix proteins for the box-l. Mutational studies using the function assay and competitive electrophoretic mobility shift assays demonstrated a good correlation be- tween the box-I-binding factors and the activated tran- scription. Box-I-binding factors were present abundantly in adult mouse CNS, whereas their existence was re- stricted in embryonic and nonneural tissues. Transient chloramphenicol acetyltransferase assay for the IP3R1 promoter revealed the requirement of box-l in Neuro2a neuroblastoma cells. In the postnatal CNS, multiple basic helix—loop—helix factors are expressed abundantly, some of which are suggested to activate IP3R1 gene expression in the mammalian CNS. Key Words: Type 1 inositol 1,4,5- trisphosphate receptor—Promoter—Neuronal gene ex- pression— E-box— Basic helix— loop—helix factor— In vitro transcription. J. Neurochem. 69, 476—484 (1997). Inositol 1 ‚4,5-trisphosphate is a second messenger for growth factors and neurotransmitters that cause Ca 2~ release from intracellular Ca2~ stores upon bind- ing to its receptor, the inositol 1,4,5-trisphosphate re- ceptor (IP 3R) (Berridge, 1993). Molecular cloning studies have revealed a gene family of the IP5Rs, and three types of IP3R genes have been identified in verte- brates so far (Ross et al., 1992). Mouse type 1 IP3R (IP3R1) is highly expressed in the CNS, although its expression is observed ubiquitously (Furuichi et al., 1989, 1990; Nakanishi et al., 1991). High IP3R1 ex- pression is observed in many neuronal cell types. In the cerebellum, inositol 1 ‚4,5-trisphosphate is involved in various neural activities (Kasono and Hirano, 1994; Matsumoto et al., 1996). Previous studies demon- strated that, in the CNS, IP3R1 is expressed predomi- nantly in the cerebellum (Furuichi et al., 1993). Cere- bellar Purkinje cells especially have an exceptionally high concentration of the IP3R 1. In contrast, types 2 and 3 IP3Rs have been reported to be poorly expressed in the neural cells (Ross et al., 1992). Hence, the type 1 IP3R is regarded as a nervous system-enriched member of the IP3R family. As expression of IP3R1 is synchronized with devel- opment of the postnatal mouse cerebellum (Furuichi et al., 1990, 1993), we suspect that there are some relationships between cerebellar development and IP3R1 gene expression in Purkinje cells. Moreover, as IP3R1 is significantly concentrated in the hippocampal CAl region (Furuichi et al., 1993), a linkage between IP3R1 gene expression and neuronal plasticity is also Received December 18, 1996; revised manuscript received April 2, 1997; accepted April 2, 1997. Address correspondence and reprint requests to Dr. T. Tamura at Laboratory of Molecular Biology, Department of Biology, Faculty of Science, Chiba University, l-33 Yayoi-cho, Inage-ku, Chiba-263, Japan. Abbreviations used: bHLH, basic helix—loop—helix; CAT, chlor- amphenicol acetyltransferase; DTF, dithiothreitol; EMSA, electro- phoretic mobility shift assay; IP5R, inositol 1 ‚4,5-trisphosphate re- ceptor; IP3RI, type I inositol I ‚4,5-trisphosphate receptor; PCR, polymerase chain reaction. 476