Journal
G3-GENES GENOMES GENETICS
Volume 11, Issue 2, Pages -Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/g3journal/jkaa012
Keywords
mating type; transcription factor; DNA-binding motif; Escherichia coli protein synthesis; human pathogen Aspergillus fumigatus
Categories
Funding
- German Research Foundation (DFG) (Bonn Bad-Godesberg, Germany) [KU 517/15-1, KR 2294/3-2]
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The DNA-binding sequence of the MAT1-1-1 transcription factor from Aspergillus fumigatus was identified and characterized. The study explored MAT1-1-1 DNA-binding targets, revealing insights into gene expression regulation in fungi through the alpha-box domain regulators. The specific DNA-binding specificity and affinity were confirmed through mutational analysis and equilibrium dissociation constant determination.
Mating-type transcription factors are master regulators of sexually related signal transduction pathways in fungi; however, their recognition of specific DNA sequences from target genes is widely undetermined. Here, we identified and characterized the DNA-binding sequence of the MAT1-1-1 alpha-box domain transcription factor from the human pathogen Aspergillus fumigatus. In order to explore MAT1-1-1 DNA-binding targets, we used the previously reported MAT1-1-1 binding motif from Penicillium chrysogenum, in a bioinformatics approach. We identified 18 A. fumigatus genes carrying the MAT1.1 sequence in their upstream region, among them genes for the alpha-pheromone precursor (PpgA), G-protein-coupled pheromone receptor (PreA), and for TomA, an unidentified protein. To validate our prediction further, quantification of transcript levels showed a decrease in expression of ppgA, tomA, and others in a MAT1-1 deletion strain. For a functional analysis of the binding sites, truncated variants of the A. fumigatus MAT1-1-1 gene were introduced into Escherichia coli for heterologous expression. The yield of recombinant protein was further optimized for the AfMAT1-1-1(78-235) variant that harbors an extended alpha-box domain. AfMAT1-1-1(78-235) bound to a subset of the most strongly upregulated genes: ppgA, preA, and tomA. The DNA-binding specificity was confirmed by testing mutated binding sequences, as well as performing competition experiments with specific and non-specific sequences. Finally, equilibrium dissociation constants of 1.83 +/- 0.1 and 1.45 +/- 0.26 mu M were determined for AfMAT1-1-1(78-235) and fusion protein GST-AfMAT1-1-1(78-235). Collectively, these findings provide further insights into AfMAT1-1-1-mediated gene expression and imply that alpha-box domain regulators from other members of Eurotiales control fungal development in a conserved manner.
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