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Originally published In Press as doi:10.1074/jbc.M211271200 on December 3, 2002

J. Biol. Chem., Vol. 278, Issue 7, 4646-4653, February 14, 2003
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Calcium Modulates Promoter Occupancy by the Entamoeba histolytica Ca2+-binding Transcription Factor URE3-BP*

Carol A. GilchristDagger , Megan LeoDagger , C. Genghis LineDagger , Barbara J. MannDagger §, and William A. Petri Jr.Dagger §||

From the Departments of Dagger  Internal Medicine, § Microbiology, and  Pathology, University of Virginia, Charlottesville, Virginia 22908

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.

* This work was supported by National Institutes of Health Grant AI 37941. Sequence data pertaining to the URE3-BP genomic context was obtained from the Entamoeba histolytica Genome Data base. The sequences were made freely available by the Pathogen Sequencing Unit at the Sanger Institute (ftp.sanger.ac.uk/pub/pathogens/E_histolytica) and the TIGR Entamoeba histolytica Genome Project (www.tigr.org/tdb/e2k1/eha1/). Preliminary sequence data for E. histolytica are deposited regularly into the GSS division of GenBankTM. The sequencing effort is part of the International Entamoeba Genome Sequencing Project and is supported by an award from NIAID, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Burroughs Wellcome Fund Scholar in Molecular Parasitology. To whom correspondence should be addressed: University of Virginia Health System, MR4 Bldg., Rm. 2115, P. O. Box 801340, Charlottesville, VA 22908-1340. Tel.: 434-824-5621; Fax: 434-924-0075; E-mail: wap3g@virginia.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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