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

Chih-Hung Wang,Li-Hsin Su,Chin-Hung Sun

doi:10.1074/jbc.m611170200

Chih-Hung Wang, Li-Hsin Su + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m611170200

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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.

Highlights

The ability of trophozoites to encyst in the intestine is key to Giardia pathophysiology
Some of the key contact residues identified by structural studies of gARID1 and the human ARID3 subfamily or Drosophila DRI is limited to their AT-rich interaction domain (ARID) domains
We found that the purified gARID1 mutant (gARID1m) did not bind to cwp1 Ϫ45/ Ϫ1, cwp1 Ϫ90/Ϫ46, ran Ϫ51/Ϫ20, or the other probes listed in Table 1, indicating that the Tyr-82 is important for DNA binding

Summary

The abbreviations used are

Initiator; ARID, AT-rich interaction domain; RT, reverse transcription; RACE, rapid amplification of cDNA ends. The ARID protein family has been found in yeast, Caenorhabditis elegans, plants, Drosophila, and mammals [26, 27]. They have been shown to have AT-rich sequence-specific DNA binding activity and have been reported to function as transcriptional activators or repressors [26, 27]. The binding sites of the ARID3 subfamily Bright and DRI are (A/G)AT(T/A)AA, and (A/G)ATTAA or TATTGAT, respectively, all of which contain the sequence ATT embedded within a larger AT-rich sequence [27, 28, 31]. Because most giardial promoters contain AT-rich sequences spanning the transcription start sites, we asked whether Giardia has ARID family proteins and whether they influence gene expression. We found that gARID1 bound to specific AT-rich Inr sequences and functioned as a transcriptional activator in G. lamblia

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION