Upstream regulatory elements controlling expression of the Entamoeba histolytica lectin

Abstract

Entamoeba histolytica genomic organization and putative promoter elements appear to be distinct from both metazoan and better characterized protozoan organisms. The recent development of DNA-mediated transfection for E. histolytica enabled characterization of cis-acting promoter elements required for gene expression. A deletion and replacement analysis was conducted on the promoter of an E. histolytica gene encoding the heavy subunit of the N-acetyl-β-d-galactosamine-specific adhesin (hgl5). Deletion of the DNA from −1000 bases to −272 bases upstream from the start of transcription of hgl5 did not decrease reporter gene expression. Subsequent nested deletions and 10-bp replacement mutagenesis identified four positive upstream regulatory elements between bases −219 to −200, −189 to −160, −69 to −60, and −49 to −40. A negative upstream regulatory element between bases −89 to −80 was conserved upstream of three other E. histolytica genes. Mutation of the previously unidentified ‘GAAC’ element conserved within the putative core promoter decreased reporter gene expression by 75%. Site directed mutagenesis of the putative TATA element decreased reporter gene expression by greater than 50%, while mutation of the putative initiator element resulted in a more modest decrease. This analysis suggests that E. histolytica promoters are unlike other protozoan promoters, with AT-rich upstream regulatory elements, a nonconsensus TATA element, the ‘GAAC’ element, and an unusual initiator element.