: We propose to study gene expression in Entamoeba histolytica controlled by the upstream regulatory element 3 binding protein (URE3-BP). We hypothesize that URE3-BP regulates virulence by a coordinated regulation of.qene expression in response to changes in intracellular Ca 2*. This sequence-specific DNA binding protein recognizes the URE3 sequence present in the promoters of the Gal/GalNAc-inhibitible lectin hgl5 and ferredoxin 1 fdx genes. URE3-BP contains two Ca2*-binding EF-hand motifs, and increases in Ca 2v block its ability to bind to URE3 in vitro and to URE3-containing promoters in vivo. Mutation of the second EF hand motif in URE3-BP leads to a loss of Ca 2. inhibition of DNA binding. These results demonstrate that elevated intracellular Ca 2vblocks the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by intracellular Ca 2v may represent an important mechanism of control of gene expression in E. histolytica.
Three specific aims are proposed to test our hypothesis: (1) Study of the URE3-BP - URE3 cisacting DNA sequence interaction, including definition of the DNA motif required for the interaction as determined by experimental and computer-assisted modeling; (2) Study of the structure and function of URE3-BP, including mapping residues important in binding to calcium, DNA and membranes as well as domains of the molecule important for intracellular localization; and (3) Determination of gene expression controlled by URE3-BP, including Entamoeba genomic micro-array analysis of global gene expression and chromatin immunoprecipitation using anti-URE3-BP mAb. Successful completion of these studies will provide insight into transcription regulation of virulence factors by the novel mechanism of direct calcium control of an EF-hand containing transcription factor.
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