梨形鞭毛蟲的E2F同源蛋白質對於Cyst Wall Protein 1和Tymidine kinase基因的轉錄調控之影響

Abstract

The E2F family transcription factors regulate the expression of genes involved in the progression of G1/S transition and DNA replication in higher eukaryotic cells. To understand whether E2F transcription factor is present in the primitive protozoan Giardia lamblia, we searched the Giardia lamblia genome database and found a gene harboring E2F DNA-binding domain (ge2f1). The gE2F1 contains two E2F DNA binding domains and both domains have the RRXYD motif which is conserved in all E2F family proteins. Recently the E2F proteins with two DNA binding-domains were found in human, mouse and plants. gE2F1 may belong to this new E2F subfamily. Epitope-tagged gE2F1 was found to localize to nuclei. During encystation, the levels of the ge2f1 mRNA and protein decreased significantly. Recombinant gE2F1 specifically bound to DNA replication-related thymidine kinase gene promoter in vitro and in vivo. Mutation analysis revealed that TTTCCGCG sequence was required for the binding of gE2F1 and an A-rich sequence can enhance the binding of gE2F1. gE2F1 contains the important RRXYD motif for DNA binding and its binding sequences are similar to those of the known E2F family factors. Like Two DNA binding domain E2Fs in other eukaryotic systems, gE2F1 binds to DNA without the requirement of DP proteins. To further analyze the role of both DNA binding domains in gE2F1, we mutated each of the RRXYD motif in DNA binding domain. Each mutation resulted in a decrease of gE2F1 DNA binding ability, suggesting that both DNA binding domains in gE2F1 are important for its DNA binding. Using cotransfection assay, we found that overexpession of gE2F1 can increase thymidine kinase promoter activity about 30 fold in the luciferase assay and mutation of the TTTCCGCG sequence in the thymidine kinase promoter abolishs the activation, suggesting that gE2F1 acts as an activator in thymidine kinase gene regulation by binding to the positive cis-acting element TTTCCGCG. Using ChIP assay, we also found that gE2F1 was assciated with cwp1 promoter in vivo. In the gE2F1 constitutively expressed cell line, the levels of the cwp1 mRNA and protein gene decreased significantly. In cotransfection assay, overexpressed gE2F1 decreased cwp1 promoter activity. These results suggest that gE2F1 functions as a repressor in cwp1 gene transcriptional regulation. We also compared the cyst number between the gE2F1 overexpressing cell line and the vector only control. We found that overexpression of gE2F1 decreased the cyst formation in both vegetative growth and encysting culture, suggesting that gE2F1 may decrease cwp1 transcription and influence the cyst formation process. Accordingly, our data suggest that DNA binding ability of E2F protein family has been conserved during evolution and gE2F1 is an important transcriptional activator in regulation of Giardia thymidine kinase gene that is involved in DNA replication. In addition, gE2F1 may also play a role in repressing cwp1 gene that is not relative to DNA replication or cell cycle. It is possible that E2F family transcription factor is also involved in Giardia differentiation.