Novel plant-GARP-like transcription factors in Giardia lamblia

Abstract

GARP homologues constitute a large family of DNA-binding proteins in plants that may be needed for a variety of key cellular functions including regulation of transcription, phosphotransfer signaling, and differentiation. However, no member of this gene family has been reported to date in yeast, animals, or protozoan parasites. We have identified four genes with putative GARP domains in the Giardia lamblia genome (GARP-like protein or GLP). The glp1 mRNA levels increased slightly during encystation. Epitope-tagged GLP1 localized to both nuclei and the proportion of stained Giardia cells increased by 10-fold during encystation. Recombinant GLP1 specifically bound to both the regulated cwp1 and constitutive ran gene promoters in their double-stranded configurations. The C-terminal region of GLP1 containing the GARP-like domain encoded the DNA binding activity. Mutation analysis revealed that an (A/G)ATCN sequence was required for binding of GLP1 to these promoters. We also found that GLP2 recognized similar binding sites. Using mutated plasmids and transfection assays, we demonstrated that the GLP1/2 binding sites are positive cis-acting elements of the cwp1 and ran gene promoters in both trophozoites and encysting cells. GLP1 is the first GARP family gene found in protozoan parasites. Our results suggest that GLP1 may be an important transcriptional activator and that its binding sites are positive promoter elements for certain Giardia genes.