The master regulator MAT1-1-1 of fungal mating binds to its targets via a conserved motif in the human pathogen Aspergillus fumigatus

Abstract

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 α-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 AfMAT1-1-178–235 variant that harbors an extended alpha-box domain. AfMAT1-1-178–235 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 µM were determined for AfMAT1-1-178–235 and fusion protein GST-AfMAT1-1-178–235. 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.

The mating type transcription factor MAT1-1-1 from the fungal human pathogen Aspergillus fumigatus: synthesis, purification, and crystallization of the DNA binding domain

“…This domain is also present in the well-characterized Mat1α (MAT1-1-1) mating-type TF from the yeast Saccharomyces cerevisiae , which was used previously as a model to get a mechanistic understanding of eukaryotic signal transduction pathways (Bardwell, 2005; Engelberg et al ., 2014). Recently, we characterized the DNA binding motif of MAT1-1-1 derived from promoter regions of several developmentally regulated genes, including the one for the pheromone receptor PreA (Ramšak et al ., 2021). Several attempts have been undertaken to crystallize MAT1-1-1-like proteins (Jackson et al ., 2013; Dyer et al ., 2016), however no successful report has been published anywhere.…”

“…Plasmid vector pGEX-AfMAT1-1-1_F5 for inducible expression of recombinant AfMAT1-1-1 protein (residues 78-235) has previously been reported (Ramšak et al ., 2021). The vector contains a GST-affinity tag fused to the region encoding N-terminus of the AfMAT1-1-1 78-235 .…”

“…The sequence encoding the tobacco etch virus (TEV) protease cleavage site separates the GST-tag and the AfMAT1-1-1 78-235 coding sequence, leaving three additional residues (GMA) at the N-terminus. The AfMAT1-1-1 78-235 synthesis and purification was performed as described in detail in Ramšak et al . (2021).…”

“…For crystallization trials, the complementary 29 bp oligonucleotides preA-2_f and preA-2_r (Ramšak et al ., 2021) were purchased from Sigma-Aldrich in HPLC purity grade. The preA-2 DNA duplex consisted of the -CTATTGAG-consensus motif and 5’-AT overhang ( Fig.…”

“…The preA-2 DNA duplex consisted of the -CTATTGAG-consensus motif and 5’-AT overhang ( Fig. 1 ) and was previously shown to be bound by the truncated AfMAT1-1-1 78-235 (Ramšak et al ., 2021). Prior to crystallization the complementary strands were annealed in equimolar ratios in 10 m M Tris pH 7.5, 50 m M NaCl, 1 m M EDTA, to a final concentration of 2.7 m M .…”