The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition

Abstract

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5′-CSAATN12RWT-3′ and an additional 5′-TKAN-3′ motif positioned 11–23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3′-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.

Structural basis of HapEP88L-linked antifungal triazole resistance in Aspergillus fumigatus

“…A similar, although weaker, tendency was found for cycA and cyp51A. As the promoters of all three of these genes are strong targets of the wt CBC-HapX complex (Furukawa et al, 2020;Gsaller et al, 2014), HapX might compensate the deleterious effects of the mutant HapE subunit and enable their transcription. In agreement, inactivation of HapX in the hapE P88L mutant decreases viability ( Fig 1A).…”

“…Together, these results suggest that HapX plays a dominant role in stabilizing the DNAregulator complex and that the mutation hapE P88L affects transcription of a subset of CBC targets within the genome including cyp51A. Considering that the CBC controls expression of about 30% of all eukaryotic genes (Bucher, 1990;Furukawa et al, 2020), the mutant HapE subunit might cause dysregulation of many biochemical pathways and provoke the observed severe growth retardation of A. fumigatus.…”

“…Despite these structural changes in the HapE P88L mutant, in vivo and SPR experiments suggest that in the presence of HapX, the affinity on CBC-HapX target sequences is partially retained. Although the basic region leucine zipper HapX can act as a transcription factor only when bound to the CBC, it has an own DNA recognition motif downstream of the CCAAT box (Gsaller et al, 2014;Hortschansky et al, 2015;Furukawa et al, 2020). It is, therefore, conceivable that in the hapE P88L mutant strain, HapX guides CBC-HapX complexes to the nucleic acid and by binding to its target sequence may enable correct positioning of the CBC near the CCAAT sequence.…”

“…Recently, patient-acquired azole resistance of the human pathogenic mold A. fumigatus has been linked to the CCAAT-binding complex (CBC) (Arendrup et al, 2010;Camps et al, 2012a;Gsaller et al, 2016), a highly conserved and fundamental eukaryotic transcription factor (Bhattacharya et al, 2003). The core version of the CBC is a heterotrimer of the subunits HapB, HapC, and HapE that binds the CCAAT box, a promoter element present in about 30% of all eukaryotic genes (Bucher, 1990;Marino-Ramirez et al, 2004;Furukawa et al, 2020). The two histone-like subunits HapC and HapE associate with the DNA backbone and bend it in a nucleosome-like manner, whereas HapB with its sensor helix αS and its C-terminal anchor inserts into the minor groove and recognizes the CCAAT box (Huber et al, 2012;Nardini et al, 2013).…”

“…This complex consists of HapB, HapC, and HapE, as well as two copies of HapX. HapX is a basic region leucine zipper (bZIP) that features an additional DNA-binding site 12 bps downstream of the CCAAT box (Hortschansky et al, 2017;Furukawa et al, 2020). CBC-HapX-controlled target genes are involved in iron homeostasis, storage, and consumption as well as ergosterol biosynthesis (Hortschansky et al, 2007;Gsaller et al, 2014Gsaller et al, , 2016.…”