Nitrate and the GATA factor AreA are necessary for in vivo binding of NirA, the pathway‐specific transcriptional activator of Aspergillus nidulans

Abstract

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.

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“…Interestingly, the DNA binding of NirA depends on nitrate concentration and on a functional AreA protein. Furthermore, AreA is necessary for intracellular nitrate accumulation and could be exerting its effect indirectly on NirA via inducer exclusion (387).…”

Nuclear Export of the Transcription Factor NirA Is a Regulatory Checkpoint for Nitrate Induction in Aspergillus nidulans

“…nidulans growth conditions and techniques. A. nidulans growth conditions and techniques were as published previously (33,35). Transformation was as described by Tilburn et al (56).…”

“…KapK was in vitro translated using the coupled transcription-translation reticulocyte lysate system provided by Promega (Mannheim, Germany). The reaction was performed with vector pGAD-THKAP as a template for expression and 35 S-labeled methionine (GE Healthcare, Buckinghamshire, United Kingdom) for protein labeling.…”

“…Strains were precultured on 1.25 mM ammonium D(ϩ) tartrate as the sole nitrogen source for 7 h at 37°C as described previously (33,35). After washing and resuspension on fresh minimal medium, 10 mM ammonium D(ϩ) tartrate (noninducing, repressing [NR] conditions) or 5 mM urea (ND conditions) or 10 mM NaNO 3 (inducing, derepressing [ID] conditions) or 10 mM ammonium D(ϩ) tartrate plus 10 mM NaNO 3 (inducing, repressing [IR] conditions) were added.…”

“…In the saprophytic fungus Aspergillus nidulans, nitrate utilization involves transcription of the nitrate transporter and nitrate-and nitrite-reductase genes (42). Transcriptional activation of these genes occurs upon induction by nitrate in the absence of repressing nitrogen sources (ammonium, glutamine), and it is strictly dependent on the synergistic, positive action of the pathway-specific Cys 6 Zn 2 binuclear cluster factor NirA and the GATA factor AreA (3,33,35,50). niaD and niiA, encoding nitrate and nitrite reductases, respectively, are transcribed from a bidirectional promoter containing four NirA and ten AreA-binding sites of varying functional importance (see Fig.…”