Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription

Human annexin A5 is a ubiquitous protein implicated in diverse signal trans duction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function, Promoter transc riptional activity was determined for a wide 5 ' portion of the human annex in A5 gene, from bp -1275 to +79 relative to the most 5 ' of several discre te transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp -202 to +79 as the minimal promoter c onferring optimal transcriptional activity. Two canonical Spl sites in the immediate 5 ' flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region be tween bp -717 to -1153 was attributed to the presence of an endogenous retr oviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid c onstructs abolished activity in transfection assays. Electrophoretic mobili ty-shift assays, Southwestern-blot analysis and affinity chromatography wer e used to identify a protein doublet of relative molecular mass 35 kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-elemen t resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The di scovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor pr comple x, may provide a clue to understanding the initiation of transcription by T ATA-less, multiple start site promoters.