Abstract
The Schizosaccharomyces pombe fep1(+) gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations. This transcriptional response is altered only in strains harboring a combined deletion of both tup11(+) and tup12(+) genes. This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa. The tup11(+)- and tup12(+)-encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor. Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other. The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11. Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association. Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1. Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted beta-propeller structure of this motif. Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated. In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain. Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes.
Highlights
Iron is required for a number of biological functions in most life forms, from microbes to mammals [1, 2]
Tup11 Associates with the S. pombe Fep1 Iron-sensing Transcription Factor—We have previously shown by genetic analysis that Fep1 requires the presence of the cofactor Tup11 or Tup12 to repress the expression of the iron transport genes under conditions of iron repletion [16]
The first three constructs in which the last 522, 313, and 258 amino acids of Tup11 were removed showed no -galactosidase activity when coexpressed with VP16-319Fep1564 (Fig. 3B). These results suggest that the predicted Ssn6-binding region and another region that is potentially required for interaction with histones H3 and H4 were not needed to establish contacts with the Fep1 C-terminal region
Summary
Fe protein 1; AD, activation domain; BPS, bathophenanthrolinedisulfonic acid; GST, glutathione S-transferase; MBP, maltose-binding protein; TAP, tandem affinity purification; PCNA, proliferating cell nuclear antigen. Elimination of either Tup or Tup alone was not sufficient to annihilate the iron-mediated repression of fio1ϩ [16] These observations suggest that, tup11ϩ and tup12ϩ, known to encode transcriptional corepressors, are functionally redundant in down-regulating the expression of the iron transport genes. The Ssn6-Tup corepressor complex is incapable of binding to DNA, it is attracted to the regulatory regions of different genes by interacting with transcription factors that function in specific metabolic pathways [28]. Based on our previous findings that the S. pombe Tup protein has the potential to regulate the expression of the fio1ϩ iron transport gene [16], we sought to determine its ability to interact with the iron-sensing DNA-binding repressor Fep. Our findings indicate that Tup and Fep are components of a heteromeric complex that is required for transcriptional down-regulation of genes that are critical for iron acquisition in fission yeast
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