Characterization of mouse Atp6i gene, the gene promoter, and the gene expression

Solubilization of bone mineral by osteoclasts depends on the formation of a n acidic extracellular compartment through the action of a V-type ATPase, W e previously cloned a gene encoding a putative osteoclast-specific proton p ump subunit, termed OC-116 kDa, approved mouse Atp6i (ATPase, Ht transporti ng, [vacuolar proton pump] member I), The function of Atp6i as osteoclast-s pecific proton pump subunit was confirmed in our mouse knockout study.:Howe ver, the transcription regulation of Atp6i remains largely unknown. In this study, the gene encoding mouseiitp6i and the promoter have been isolated a nd completely sequenced. In addition, the temporal and spatial expressions of Atp6i have been characterized. Intrachromosomal mapping studies revealed that the gene contains 20 exons and 19 introns spanning similar to 11 kilo bases (kb) of genomic DNA, Alignment of the mouse Atp6i gene exon sequence and predicted amino acid sequence to that of the human reveals a strong hom ology at both the nucleotide (82%) and the amino acid (80%) levels. Primer extension assay indicates that there is one transcription start site at 48 base pairs (bp) upstream of the initiator Met codon, Analysis of 4 kb of th e putative promoter region indicates that this gene lacks canonical TATA an d CAAT boxes and contains multiple putative transcription regulatory elemen ts. Northern blot analysis of RNAs from a number of mouse tissues reveals t hat Atp6i is expressed predominantly in osteoclasts, and this predominant e xpression was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) assay and immunohistochemical analysis. Whole-mount in situ hybrid ization shows that Atp6i- expression is detected initially in the headfold region and posterior region in the somite stage of mouse embryonic developm ent (E8.5) and becomes progressively restricted to anterior regions and the limb bud by E9.5, The expression level of Atp6i is largely reduced after E 10.5. This is the first report of the characterizations of Atp6i gene,its p romoter, and its gene expression patterns during mouse development. This st udy may provide valuable insights into the function of Atp6i, its osteoclas t-selective expression, regulation, and the molecular mechanisms responsibl e for osteoclast activation.