A Novel ARID/Bright-like Protein Involved in Transcriptional Activation of Cyst Wall Protein 1 Gene in Giardia lamblia

Abstract

The capability of protozoan parasite Giardia lamblia to encyst is critical for survival outside the host and its transmission. AT-rich interaction domain (ARID) or Bright homologs constitute a large family of transcription factors in higher eukaryotes that regulate cell proliferation, development, and differentiation. We asked whether Giardia has ARID-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome data base identified two genes with putative ARID/Bright domains (gARID1 and 2). Epitope-tagged gARID1 was found to localize to nuclei. Recombinant gARID1 specifically bound to the encystation-induced cyst wall protein (cwp) gene promoters. Mutation analysis revealed that AT-rich initiators were required for binding of gARID1 to the cwp promoters. gARID1 contains several key residues for DNA binding, and its binding sequences are similar to those of the known ARID family proteins. The gARID1 binding sequences were positive cis-acting elements of the cwp1 promoter during both vegetative growth and encystation. We also found that gARID1 transactivated the cwp1 promoter through its binding sequences in vivo. Our results suggest that the ARID family has been conserved during evolution and that gARID1 is an important transactivator in regulation of the Giardia cwp1 gene, which is key to Giardia differentiation into cysts.Giardia lamblia is an important human intestinal parasite that causes outbreaks of waterborne diarrheal disease (1, 2). It has two life cycle stages that are well adapted to survival in different inhospitable environments (3-6). The motile flagellated trophozoites attach to and colonize the human small intestine to cause the symptoms of giardiasis. The dormant infective cysts, which are protectively walled and resistant to external hostile conditions, are responsible for transmission of giardiasis.The ability of trophozoites to encyst in the intestine is key to Giardia pathophysiology. However, little is known of the regulation of encystation. Synthesis and secretion of specific proteins and polysaccharide required for the formation of a protective cyst wall is a major process of encystation (3, 5-7). Expression of genes encodes three cyst wall structural proteins (Cwp1, Cwp2, Cwp3) (8 -10), and glucosamine-6-phosphate isomerase-B, the first enzyme in the cyst wall polysaccharide biosynthetic pathway (11, 12), increases with similar kinetics during encystation (6, 9, 11, 12), suggesting the importance of regulation at the transcriptional level.G. lamblia is of significant evolutionary interest because it has been proposed as one of the most early diverging eukaryotes (13,14). The giardial transcription mechanism may be unusual because relatively short 5Ј-flanking regions (Ͻ65 bp) are sufficient for the expression of many constitutive and encystationinduced giardial genes (11,(15)(16)(17). No classical TATA or CCAAT boxes or other cis-acting elements have been found in the promoters of many giardial protein-coding genes (16, 18). AT-rich...

A Novel WRKY-like Protein Involved in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

“…4, lanes 15-19). We also found that WRKY did not bind to the poly(A) probe (AAAAA-AAAAAAAAAAAAAAAAAA), poly(A) ϩ 1T probe (AAAA-AAAAAAATAAAAAAAAAAA), or poly(A) ϩ TC probe (AAAAAAAAAAATCAAAAAAAAAA) (data not shown), suggesting that the WRKY binding site is different from the ARID1 binding site (25).…”

“…Binding reaction mixtures contained the components described (25). Labeled probe (0.02 pmol) was incubated for 15 min at room temperature with 5 ng of purified WRKY protein in a 20-l volume supplemented with 0.5 g of poly(dI-dC) (Sigma).…”

“…Few transcription factors have been characterized to date in G. lamblia (23)(24)(25). A Myb family transcription factor (Myb2) is encystation-induced and is involved in coordinating up-regulation of the cwp1-3 genes (23,26).…”

“…Two GARP (named from the maize GOLDEN2, Arabidopsis response regulator proteins and the Chlamydomonas Psr1 protein) family transcription factors may be involved in transcriptional regulation of many different genes, including the encystation-induced cwp1 gene and constitutive ran gene (24). An ARID (AT-rich interaction domain) family transcription factor can bind to specific AT-rich Inr sequences and function as an important transactivator in the regulation of the cwp1 gene (25).…”

Encystation commitment inGiardia duodenalis: a long and winding road

“…In spite of lacking 8 of 12 general eukaryotic transcription factors (Best et al, 2004), Giardia possesses at least four other transcription factors that are overexpressed in encystation and bind to and transactivate gene promoters of encystation markers: GARP-like protein 1 (GLP1) and AT-rich interaction domain protein 1 (ARID1) for cwp1 promoter; Myb2 for promoters of cwp 1-3, g6pi-b and myb2 itself and a WRKY protein homolog for promoters of cwp 1-2, myb2 and wrky itself (Sun et al, 2002;Sun et al, 2006;Wang et al, 2007;Pan et al, 2009). The manipulated overexpression of some of these transcription factors provided further insights: for instance, the constitutive over-expression of Myb2 moderately increased cwp1 expression (≈ 3.7-fold) as compared with levels observed during bile-induced encystation (≈ 47-fold; Huang et al, 2008).…”