Calcium modulates promoter occupancy by the Entamoeba histolytica Ca2+-binding transcription factor URE3-BP

Abstract

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) binds to the URE3 sequence of the Gal/GalNAc-inhibitable lectin hgl5 and ferredoxin 1 (fdx) gene promoters. This binding can be inhibited in vitro by addition of calcium. Two EF-hand motifs, which are associated with the ability to bind calcium, are present in the amino acid sequence of URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in the loss of calcium inhibition of DNA binding as monitored by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays revealed that URE3-BP was physically bound to the hgl5 and fdx promoters in vivo. Parasite intracellular calcium concentrations were altered by changes in extracellular calcium. Promoter occupancy was lost when intracellular calcium levels were increased by coordinate increases in extracellular calcium. Increased intracellular calcium also resulted in decreased levels of URE3-BP mRNA. Together these results demonstrate that changes in extracellular calcium result in changes in URE3-BP mRNA and in the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by calcium may represent an important mechanism of control of gene expression in E. histolytica.