The linked niiA and niaD genes of Aspergillus nidulans are transcribed divergently. The expression of these genes is subject to a dual control system. They are induced by nitrate and repressed by ammonium. AreA mediates derepression in the absence of ammonium and NirA supposedly mediates nitrate induction. Out of 10 GATA sites, a central cluster (sites 5-8) is responsible for~80% of the transcriptional activity of the promoter on both genes. We show occupancy in vivo of site 5 by the AreA protein, even under conditions of repression. Sites 5-8 are situated in a pre-set nucleosome-free region. Under conditions of expression, a drastic nucleosomal rearrangement takes place and the positioning of at least five nucleosomes flanking the central region is lost. Remodelling is strictly dependent on the presence of an active areA gene product, and independent from the NirA-specific and essential transcription factor. Thus, nucleosome remodelling is independent from the transcriptional activation of the niiA-niaD promoter. The results presented cast doubts on the role of NirA as the unique transducer of the nitrate induction signal. We demonstrate, for the first time in vivo, that a GATA factor is involved directly in chromatin remodelling.
SummaryThe GATA factor AreA is a wide-domain regulator in Aspergillus nidulans with transcriptional activation and chromatin remodelling functions. AreA interacts with the nitrate-specific Zn 2 -C 6 cluster protein NirA and both proteins cooperate to synergistically activate nitrate-responsive genes. We have previously established that NirA in vivo DNA binding site occupancy is AreA dependent and in this report we provide a mechanistic explanation for our previous findings. We now show that AreA regulates NirA at two levels: (i) through the regulation of nitrate transporters, AreA affects indirectly the subcellular distribution of NirA which rapidly accumulates in the nucleus following induction; (ii) AreA directly stimulates NirA in vivo target DNA occupancy and does not act indirectly by chromatin remodelling. Simultaneous overexpression of NirA and the nitrate transporter CrnA bypasses the AreA requirement for NirA binding, permits utilization of nitrate and nitrite as sole N-sources in an areA null strain and leads to an AreA-independent nucleosome loss of positioning.
Summary In Aspergillus nidulans, the genes coding for nitrate reductase (niaD) and nitrite reductase (niiA), are transcribed divergently from a common promoter region of 1200 basepairs. We have previously characterized the relevant cis‐acting elements for the two synergistically acting transcriptional activators NirA and AreA. We have further shown that AreA is constitutively bound to a central cluster of four GATA sites, and is involved in opening the chromatin structure over the promoter region thus making additional cis‐acting binding sites accessible. Here we show that the asymmetric mode of NirA–DNA interaction determined in vitro is also found in vivo. Binding of the NirA transactivator is not constitutive as in other binuclear C6‐Zn2+‐cluster proteins but depends on nitrate induction and, additionally, on the presence of a wild‐type areA allele. Dissecting the role of AreA further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly exert its effect on NirA via inducer exclusion. We have tested this possibility in a strain accumulating nitrate in the absence of areA. We found that in such a strain the intracellular presence of inducer is not sufficient to promote either chromatin rearrangement or NirA binding, implying that both processes are directly dependent on AreA.
In Aspergillus nidulans, proline can be used as a carbon and nitrogen source, and its metabolism requires the integration of three signals, including proline induction and nitrogen and carbon metabolite derepression. We have previously shown that the bidirectional promoter in the prnD-prnB intergenic region undergoes drastic chromatin rearrangements such that proline induction leads to the loss of positioned nucleosomes, whereas simultaneous carbon and nitrogen metabolite repression results in the partial repositioning of these nucleosomes. In the proline cluster, the inhibition of deacetylases by trichostatin A leads to partial derepression and is associated with a lack of nucleosome positioning. Here, we investigate the effect of histone acetylation in the proline cluster using strains deleted of essential components of putative A. nidulans histone acetyltransferase complexes, namely, gcnE and adaB, the orthologues of the Saccharomyces cerevisiae GCN5 and ADA2 genes, respectively. Surprisingly, GcnE and AdaB are not required for transcriptional activation and chromatin remodeling but are required for the repression of prnB and prnD and for the repositioning of nucleosomes in the divergent promoter region. Chromatin immunoprecipitation directed against histone H3 lysines K9 and K14 revealed that GcnE and AdaB participate in increasing the acetylation level of at least one nucleosome in the prnD-prnB intergenic region during activation, but these activities do not determine nucleosome positioning. Our results are consistent with a function of GcnE and AdaB in gene repression of the proline cluster, probably an indirect effect related to the function of CreA, the DNA-binding protein mediating carbon catabolite repression in A. nidulans.In eukaryotic cells, the acetylation of histones is correlated with both transcriptional activation and the chromatin rearrangements usually associated with it. A turning point in our understanding of the mechanism of histone acetylation was the demonstration of the intrinsic acetylase activity of the Tetrahymena pyriformis p55 protein (7), the homologue of the Saccharomyces cerevisiae Gcn5p protein, which was already known to be involved in the transcriptional activation of a number of genes in this organism (reviewed in reference 33). Homologues, possibly orthologues, of Gcn5p, are universally present in eukaryotes. Gcn5p and its homologues interact with chromatin in large multiprotein complexes, such as ADA and SAGA (17). SAGA is a multiprotein complex whose Gcn5p subunit possesses histone acetyltransferase (HAT) activity. Gcn5p acetylates several lysine residues on the N termini of histones, including K9 and K14 on histone H3 and K8 and K16 on histone H4 (21). The Gcn5-containing complexes share several subunits, such as Ada2p, Ada3p, Spt3p, and Tra1p (24), and it has been estimated that the yeast SAGA complex regulates the expression of ϳ10% of S. cerevisiae genes, with approximately one-third of them being negatively regulated (23). Gcn5p forms a ternary complex with Ada2p an...
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