The heterotrimeric CCAAT-binding complex (CBC) is evolutionary conserved in eukaryotic organisms including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the CBC consists of the subunits HapB, HapC and HapE. All HapC orthologs from eukaryotes contain three conserved cysteine residues in their core histone fold motif. Mutational and in vitro interaction analysis revealed that two of these cysteine residues are indispensable for stable HapC/HapE subcomplex formation and high affinity CBC DNA-binding. Furthermore, oxidised HapC is unable to participate in CBC assembly and located in the cytoplasm as a GFP-fusion, but can be reduced by the thioredoxin A system in vitro and in vivo. The latter was demonstrated by bimolecular fluorescence complementation (BiFC) analysis. Expression of trxA was upregulated in response to oxidative stress. Moreover, in a trxA deletion strain, the GFP-fusion of HapC is also mainly located in the cytoplasm. Therefore, redox regulation via thioredoxin very likely represents a general feature of the CBC in eukaryotes. Recently, a putative fourth CBC subunit with an unknown function was identified in A. nidulans and designated HapX. We found that hapX expression is repressed by iron via the GATA-factor SreA and that various iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron starvation by the interaction of HapX with the CBC. These data suggest a model, in which HapX/CBC interaction is regulated at both transcriptional and post-translational levels. Iron starvation causes expression of hapX. Subsequent binding of HapX to the CBC results in transcriptional repression of iron-dependent pathways. During iron-replete conditions, hapX is repressed and, therefore, iron-dependent pathways are derepressed. Moreover, HapX/CBC interaction is inhibited by increased iron concentrations. This post-translational mechanism allows rapid adjustment to iron availability by disruption of the HapX/CBC complex. Mutual transcriptional control of hapX and sreA coordinates iron acquisition and iron-dependent pathways, thereby serving for both iron supply and prevention of iron toxicity. These data indicate that the CBC has a general role and that HapX function is confined to iron depleted conditions.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)