Transcription factor FfmA interacts both physically and genetically with AtrR to properly regulate gene expression in the fungusAspergillus fumigatus

Abstract

Transcriptional regulation of azole resistance in the filamentous fungusAspergillus fumigatusis a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility and expression of an ATP-binding cassette transporter gene calledabcG1. Null alleles offfmAexhibit a strongly compromised growth rate even in the absence of any external stress. Here we employ an acutely repressible doxycycline-off form offfmAto rapidly deplete FfmA protein from the cell. Using this approach, we carried out RNA-seq analyses to probe the transcriptome ofA. fumigatuscells that have been deprived of normal FfmA levels. We found that 2000 genes were differentially expressed upon depletion of FfmA, consistent with the wide-ranging effect of this factor on gene regulation. Chromatin immunoprecipitation coupled with high throughput DNA sequencing analysis (ChIP-seq) identified 530 genes that were bound by FfmA using two different antibodies for immunoprecipitation. More than 300 of these genes were also bound by AtrR demonstrating the striking regulatory overlap with FfmA. However, while AtrR is clearly an upstream activation protein with clear sequence specificity, our data suggest that FfmA is a chromatin-associated factor that may bind to DNA in a manner dependent on other factors. We provide evidence that AtrR and FfmA interact in the cell and can influence one anothers expression. This interaction of AtrR and FfmA is required for normal azole resistance inA. fumigatus.