Abstract
Chlamydomonas reinhardtii fox1 gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify fox1 promoter regulatory elements, an insertional library was generated in a transgenic Chlamydomonas strain (2A38) harboring an arylsulfatase (ARS) reporter gene driven by the fox1 promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted femu2 gene was identified. Activation of the fox1 promoter by the femu2 gene product was confirmed by silencing the femu2 gene using RNA interference. In three femu2 RNAi transgenic lines (IR3, IR6, and IR7), ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore, RT-PCR analysis of both the femu2 mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous fox1 gene under Fe deficient conditions. Amino acid sequence analysis of the femu2 gene product identified three potential C2H2 zinc finger (ZF) motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/T)TTGG(G/T)(G/T)T) was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the fox1 gene in Fe deficient cells.
Highlights
C2H2 zinc finger proteins (ZFPs) comprise an abundant family of nucleic acidbinding proteins in eukaryotic genomes
To identify the C. reinhardtii genes involved in cellular responses to Fe deficiency, C. reinhardtii 2A38 was subjected to random insertion mutagenesis, in which an expression cassette with 2103 to +65 fox1 promoter sequence and an ARS reporter gene was integrated into the genome [25]
The results showed that TTGGGT (WT), Mu1, and Mu3 tetramers were bound to the Femu2-3C2H2 ZF (Fig. 7A), but Mu2 and Mu4 tetramers were not
Summary
C2H2 zinc finger proteins (ZFPs) comprise an abundant family of nucleic acidbinding proteins in eukaryotic genomes. In the photosynthetic eukaryote model Chlamydomonas reinhardtii, iron (Fe)deficiency causes a series of cellular reactions Among these reactions, a highaffinity system that consists of iron reductase (FRE1), a protein involved in iron assimilation (FEA1), and a transport complex comprising a multi-copper oxidase (FOX1) and an Fe transporter (FTR1), are induced [14, 15]. Fox FeRE1 (Fe reaction element) at 287/282 (CACACG) and FeRE2 at 265/261 (CACGCG) were identified These elements are essential in inducing fox expression under Fe-deficient conditions. A plasmid pARG7.8 used in the selection of transformants was cotransformed with the plasmid pJF103, which contained the fox promoter sequence (2103 to +65) and arylsulfatase (ARS) reporter gene, to C. reinhardtii CC425(cw arg). Zeocin-resistant pSP124S plasmid was used to transform C. reinhardtii 2A38, which containing fox promoter::ars chimeric gene, in our study. The Fe-deficiency response-defective mutant mu has been further studied in this work
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