Isolation, sequencing, and functional analysis of the TATA-less murine ATPase II promoter and structural analysis of the ATPase II gene

Abstract

The P-type Mg²⁺-ATPase, termed ATPase II (Atp8a1), is a putative aminophospholipid transporting enzyme, which helps to maintain phospholipid asymmetry in cell membranes. In this project we have elucidated the organization of the mouse ATPase II gene and identified its promoter. Located within chromosome 5, this gene spans about 224 kb and consists of 38 exons, three of which are alternatively spliced (exons 7, 8 and 16), giving rise to two transcript variants. Translation of these transcripts results in two ATPase II isoforms (1 and 2) composed of 1164 and 1149 amino acids, respectively. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) we identified multiple transcription start sites (TSS) in messages obtained from heart, lung, liver, and spleen. The mouse ATPase II promoter is TATA-less and lacks a consensus initiator sequence. Luciferase reporter analysis of full and core promoters revealed strong activity and little cell type specificity, possibly because more flanking, regulatory sequences are required to cause such tissue specificity. In the neuronal HN2, N18, SN48 cells and the NIH3T3 fibroblast cells, but not in the B16F10 melanoma cells, the core promoter (− 318/+ 193 with respect to the most common TSS) displayed significantly higher activity than the full promoter (− 1026/+ 193). Serial 5′ deletion of the core promoter revealed significant cell type-specific activity of the fragments, suggesting differential expression and use of transcription factors in the five cell lines tested. Additionally distribution of the TSS was organ specific. Such observations suggest tissue-specific differences in transcription initiation complex assembly and regulation of ATPase II gene expression. Information presented here form the groundwork for further studies on the expression of this gene in apoptotic cells.