AtrR, an essential regulator of azole resistance in Aspergillus fumigatus, acts via the TR34 element in the cyp51A promoter

Objective:  Previous work identified AtrR as an important regulator of azole resistance in A. fumigatus via its transcriptional control of an ABC transporter gene and the cyp51A locus encoding the azole target enzyme.  
Methods:  Here we used chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) to identify all direct target genes of AtrR.  This analysis was also coupled with higher resolution assays including electrophoretic mobility shift assays (EMSAs) and DNase I protection analysis to precisely locate the AtrR Response Element (ATRE) in target genes.  We constructed two different hyperactive alleles of the atrR gene that support the view that this factor is normally restrained by some type of negative regulation.  RNA-seq experiments revealed the range of AtrR target genes that responded to these hyperactive alleles.  Epistasis analyses were carried out to determine the genetic relationship between the important regulator of ergosterol biosynthetic gene transcription SrbA and AtrR.  Reporter gene experiments were used to functionally map critical ATREs in the abcG1 ATP-binding cassette transporter-encoding gene.
Results:  ChIP-seq identified more than 1000 genes as being potential regulatory targets of AtrR.  Either fusing an epitope tag to the extreme C-terminus or overproducing the wild-type AtrR protein gave rise to an allele that conferred elevated azole resistance.  RNA-seq experiments were consistent with the overproduced wild-type protein behaving as a stronger activating allele than the epitope-tagged atrR allele as 95 genes were expressed more than 2-fold above wild-type while only 59 were similarly induced by the presence of the epitope tag.  Epistasis experiments demonstrated that even hyperactive alleles of atrR could not suppress the azole sensitive phenotype caused by deletion of the srbA gene.  Expression of ergosterol biosynthetic enzyme-encoding genes was routinely found to be responsive to both AtrR and SrbA while only AtrR controlled expression of abcG1.  Proper transcription of abcG1 required tandem ATREs located more than 1000 bp from the transcription start site of this gene.
Conclusion:  AtrR is a crucial regulator of azole resistance and the ATRE is duplicated along with the SrbA-binding site in the drug resistant TR34 allele of cyp51A.  The large number of genes responsive to AtrR is consistent with this transcription factor acting to integrate metabolism with drug tolerance across the genome.  AtrR and SrbA are both necessary for normal azole resistance.

2 / AtrR, an essential regulator of azole resistance in Aspergillus fumigatus, acts via the TR34 element in the cyp51A promoter