Demonstration of an E-box and its CNS-related binding factors for transcriptional regulation of the mouse type inositol 1,4,5-trisphosphate receptor gene

The type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1) is expressed 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 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 -396 to -295 showed the most predominant cerebellum-specific positive function. Footprint analyses demonstrated a factor-binding region from -334 to -318, termed box-I, that contained an E- box consensus sequence. Electrophoretic mobility shift assay revealed CNS- related basic helix-loop-helix proteins for the box-I. Mutational studies using the function assay end competitive electrophoretic mobility shift assays demonstrated a good correlation between the box-I-binding factors and the activated transcription. Box-I-binding factors were present abundantly in adult mouse CNS, whereas their existence was restricted in embryonic and nonneural tissues. Transient chloramphenicol acetyltransferase assay for the IP₃R1 promoter revealed the requirement of box-I 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.