Giardia lamblia becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in Giardia. Due to the tetraploid genome in two nuclei of Giardia, it could be hard to disrupt a gene completely in Giardia. We only generated knockdown but not knockout mutants. We found that knockdown of the mlf gene resulted in a significant decrease of cwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used mlf as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in cwp gene expression. Our results provide insights into a positive role of MLF in inducing Giardia differentiation and a useful tool for studies in Giardia.

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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...

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.