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 (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 -398 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 and 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.
Konishi
Y
Y
Department of Biology, Faculty of Science, Chiba University, Inage-ku, Japan.
Kobayashi
Y
Y
Kishimoto
T
T
Makino
Y
Y
Miyawaki
A
A
Furuichi
T
T
Okano
H
H
Mikoshiba
K
K
Tamura
T
T
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D015220:Calcium Channels / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D005786:Gene Expression Regulation; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic
D015220:Calcium Channels; D004268:DNA-Binding Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D004247:DNA