Abstract
Giardia lamblia differentiates into resistant walled cysts for survival outside the host and transmission. During encystation, synthesis of cyst wall proteins is coordinately induced. The E2F family of transcription factors in higher eukaryotes is involved in cell cycle progression and cell differentiation. We asked whether Giardia has E2F-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome database identified one gene (e2f1) encoding a putative E2F protein with two putative DNA-binding domains. We found that the e2f1 gene expression levels increased significantly during encystation. Epitope-tagged E2F1 was found to localize to nuclei. Recombinant E2F1 specifically bound to the thymidine kinase and cwp1-3 gene promoters. E2F1 contains several key residues for DNA binding, and mutation analysis revealed that its binding sequence is similar to those of the known E2F family proteins. The E2F1-binding sequences were positive cis-acting elements of the thymidine kinase and cwp1 promoters. We also found that E2F1 transactivated the thymidine kinase and cwp1 promoters through its binding sequences in vivo. Interestingly, E2F1 overexpression resulted in a significant increase of the levels of CWP1 protein, cwp1-3 gene mRNA, and cyst formation. We also found E2F1 can interact with Myb2, a transcription factor that coordinate up-regulates the cwp1-3 genes during encystation. Our results suggest that E2F family has been conserved during evolution and that E2F1 is an important transcription factor in regulation of the Giardia cwp genes, which are key to Giardia differentiation into cysts.
Highlights
Model for basic studies as its life cycle can be reproduced in vitro by mimicking the intestinal environment [5, 6]
We found that E2F1 can function as a transcriptional activator of the cwp genes and interact with an important encystation-specific Myb2 transcription factor
Our results suggest that E2F1 may be an important transcription factor regulating differentiation in the primitive eukaryote G. lamblia
Summary
G. lamblia Culture—Trophozoites of G. lamblia WB (ATCC 30957), clone C6, were cultured in modified TYI-S33 medium [56]. Cyst count was performed on these stationary phase cultures. Isolation and Analysis of the e2f1 Gene—The G. lamblia genome database [11, 58] was searched with the amino acid sequences of the human E2F1 (GenBankTM accession numbers U47675 and U47646) using the BLAST program [59]. To isolate the cDNA of the e2f1 gene, we performed RT-PCR with e2f1-specific primers using total RNA from G. lamblia. For RT-PCR, 5 g of DNase-treated total RNA from vegetative and 24-h encysting cells was mixed with oligo(dT) and random hexamers and Superscript II RNase H-reverse transcriptase (Invitrogen). For RT-PCR, 5 g of DNase-treated total RNA was mixed with oligo(dT) and random hexamers and Superscript II RNase HϪ reverse transcriptase (Invitrogen). Semi-quantitative RT-PCR analysis of e2f1 (XM_001705535, open reading frame 23756), e2f1-ha, cwp (U09330, open reading frame 5638), cwp (U28965, open reading frame 5435), cwp
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