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

The type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is expresse d abundantly in the CNS, such as in cerebellar Purkinje cells and the hippocampus. We established a tissue-specific cell-free transcription system and studied regulatory properties of the 5' upstream region of the IP(3)R1 gene by use of this system. Deletion analyses of the promo ter revealed several cis elements that function significantly in brain nuclear extracts. Among those elements, sequences from -398 to -295 s howed the most predominant cerebellum-specific positive function. Foot print analyses demonstrated a factor-binding region from -334 to -318, termed box-I, that contained an E-box consensus sequence. Electrophor etic mobility shift assay revealed CNS-related basic helix-loop-helix proteins for the box-1. Mutational studies using the function assay an d competitive electrophoretic mobility shift assays demonstrated a goo d correlation between the box-I-binding factors and the activated tran scription. Box-I-binding factors were present abundantly in adult mous e CNS, whereas their existence was restricted in embryonic and nonneur al tissues. Transient chloramphenicol acetyltransferase assay for the IP(3)R1 promoter revealed the requirement of box-I in Neuro2a neurobla stoma cells. In the postnatal CNS, multiple basic helix-loop-helix fac tors are expressed abundantly, some of which are suggested to activate IP(3)R1 gene expression in the mammalian CNS.