Béatrice Felenbok

Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon cataboiite repression in Aspergillus nidulans

The CREA repressor responsible for carbon catabolite repression in Aspergillus nidulans represses the transcription of the ethanol regulon. The N‐terminal part of the CREA protein encompassing the two zinc fingers (C2H2 class family) and an alanine‐rich region was expressed in Escherichia colias a fusion protein with giutathione‐S‐transferase. Our results show that CREA is a DNA‐binding protein able to bind to the promoters of both the specific trans‐acting gene, alcR, and of the structural gene, alcA, encoding the alcohol dehydrogenase I. DNase I protection foot‐printing experiments revealed several specific binding sites in the alcR and in the alcA promoters having the consensus sequence 5′‐G/CPyGGGG‐3′. The disruption of one of these CREA‐binding sites in the alcR promoter overlapping the induction target for the trans‐activator ALCR results in a partially derepressed alc phenotype and derepressed alcR transcription, showing that this binding site is functional in vivo. Our data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans‐acting gene, alcR, and the structural gene, alc A.

Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation.

This article reviews our knowledge of the ethanol utilization pathway (alc system) in the hyphal fungus Aspergillus nidulans. We discuss the progress made over the past decade in elucidating the two regulatory circuits controlling ethanol catabolism at the level of transcription, specific induction, and carbon catabolite repression, and show how their interplay modulates the utilization of nutrient carbon sources. The mechanisms featuring in this regulation are presented and their modes of action are discussed: First, AlcR, the transcriptional activator, which demonstrates quite remarkable structural features and an original mode of action; second, the physiological inducer acetaldehyde, whose intracellular accumulation induces the alc genes and thereby a catabolic flux while avoiding intoxification; third, CreA, the transcriptional repressor mediating carbon catabolite repression in A. nidulans, which acts in different ways on the various alc genes; Fourth, the promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA) and the regulatory alcR gene, which exhibit exceptional strength compared to other genes of the respective classes. alc gene expression depends on the number and localization of regulatory cis-acting elements and on the particular interaction between the two regulator proteins, AlcR and CreA, binding to them. All these characteristics make the ethanol regulon a suitable system for induced expression of heterologous protein in filamentous fungi.

The CreA Repressor Is the Sole DNA-binding Protein Responsible for Carbon Catabolite Repression of the alcA Gene inAspergillus nidulans via Its Binding to a Couple of Specific Sites

Carbon catabolite repression is mediated inAspergillus nidulans by the negative acting protein CreA. The CreA repressor plays a major role in the control of the expression of the alc regulon, encoding proteins required for the ethanol utilization pathway. It represses directly, at the transcriptional level, the specific transacting gene alcR, the two structural genes alcA and aldA, and other alc genes in all physiological growth conditions. Among the seven putative CreA sites identified in the alcApromoter region, we have determined the CreA functional targets in AlcR constitutive and derepressed genetic backgrounds. Two different divergent CreA sites, of which one overlaps a functional AlcR inverted repeat site, are largely responsible for alcA repression. Totally derepressed alcA expression is achieved when these two CreA sites are disrupted in addition to another single site, which overlaps the functional palindromic induction target. The fact that derepression is always associated with alcA overexpression is consistent with a competition model between AlcR and CreA for their cognate targets in the same region of the alcApromoter. Our results also indicate that the CreA repressor is necessary and sufficient for the total repression of the alcA gene.

Regulation of the Aldehyde Dehydrogenase Gene (aldA) and Its Role in the Control of the Coinducer Level Necessary for Induction of the Ethanol Utilization Pathway in Aspergillus nidulans

Expression of the structural genes for alcohol and aldehyde dehydrogenase, alcA and aldA, respectively, enables the fungus Aspergillus nidulans to grow on ethanol. The pathway-specific transcriptional activator AlcR mediates the induction of ethanol catabolism in the presence of a coinducing compound. Ethanol catabolism is further subject to negative control mediated by the general carbon catabolite repressor CreA. Here we show that, in contrast to alcA and alcR, thealdA gene is not directly subject to CreA repression. A single cis-acting element mediates AlcR activation ofaldA. Furthermore, we show that the induction of thealc gene system is linked to in situ aldehyde dehydrogenase activity. In aldA loss-of-function mutants, the alc genes are induced under normally noninducing conditions. This pseudo-constitutive expression correlates with the nature of the mutations, suggesting that this feature is caused by an intracellular accumulation of a coinducing compound. Conversely, constitutive overexpression of aldA results in suppression of induction in the presence of ethanol. This shows unambiguously that acetaldehyde is the sole physiological inducer of ethanol catabolism. We hypothesize that the intracellular acetaldehyde concentration is the critical factor governing the induction of the alc gene system.

A newly identified gene cluster in Aspergillus nidulans comprises five novel genes localized in the alc region that are controlled both by the specific transactivator AlcR and the general carbon‐catabolite repressor CreA

Ethanol‐utilization in Aspergillus nidulans is mediated by alcohol dehydrogenase I and aldehyde dehydrogenase encoded by alcA and aldA, respectively. Both genes are under the transcriptional control of the specific activator AlcR and the general carbon catabolite repressor CreA. The alcR and alcA genes are closely linked in chromosome VII; aldA is located in chromosome VIII. We have identified five other transcripts that are expressed from the same genomic region as alcA and alcR. They are inducible by the gratuitous inducer ethyl methyl ketone (EMK), and are carbon catabolite repressed. The corresponding genes, designated alcMalcSalcOalcP, and alcU, are differentially regulated by the specific transcriptional activator AlcR, and they are not all under the direct control by the CreA repressor. Some of the inducible transcripts are very abundant in the cell, whereas others are poorly expressed. Two sets of genes, alcM/alcS and alcR/alcO, are divergently transcribed and probably share a common cis‐acting region, whereas alcP and alcU are individually transcribed from the same strand as alcA and alcR, and have their own promoters. The significance of the alc gene clustering is discussed. At least four of the five novel alc genes in the cluster are not essential for ethanol metabolism.

The Zinc Binuclear Cluster Activator AlcR Is Able to Bind to Single Sites but Requires Multiple Repeated Sites for Synergistic Activation of the alcA Gene in Aspergillus nidulans

The alcA gene which is part of the recently identified ethanol regulon, is one of the most strongly inducible genes in Aspergillus nidulans. Its transcriptional activation is mediated by the AlcR transactivator which contains a DNA-binding domain belonging to the C6 zinc binuclear cluster family. AlcR differs from the other members of this family by several features, the most striking characteristic being its binding to both symmetric and asymmetric DNA sites with the same apparent affinity. However, AlcR is also able to bind to a single site with high affinity, suggesting that unlike the other C6 proteins, AlcR binds as a monomer. In this report, we show that AlcR targets, to be functional in vivo, have to be organized as inverted or direct repeats. In addition, we show a strong synergistic activation of alcAtranscription in which the number and the position of the AlcR-binding sites are crucial. The fact that the AlcR unit for in vitrobinding is a single site whereas the in vivo functional unit is a repeat opens the question of the mechanism of the strongalcA transactivation. These results show that AlcR displays both in vitro and in vivo a new range of binding specificity and provides a novel example in the C6 zinc cluster protein family.

In vivo studies of upstream regulatory cis‐acting elements of the alcR gene encoding the transactivator of the ethanol regulon in Aspergillus nidulans

The alcR gene of Aspergillus nidulans, which encodes the specific transactivator of the ethanol utilization pathway, is positively autoregulated and carbon catabolite repressed. Regulation by these two circuits occurs at the transcriptional level via the binding of the two regulators, AlcR and CreA, to their cognate targets respectively. We demonstrate here that out of two clustered putative AlcR repeated consensus sequences, only the palindromic target is functional in vivo. Hence, it is solely responsible for the alcR positive autogenous activation loop. Transcript mapping of the alcR gene showed that transcription initiation can occur at 553 bp and at or near 86 bp upstream of the start codon. These transcription start sites yield a transcript of 3.0 kb, which appears only under induced growth conditions, and of 2.6 kb, which is present under both induced and non‐induced growth conditions respectively. Nine CreA consensus sites are present in the alcR promoter but only two pairs of two sites are functional in vivo. One of them is located in close proximity to the AlcR functional target. Within this pair, both sites are necessary to mediate a partial repression of alcR transcription. Disruption of either site results in an overexpression of alcR due to the absence of direct competition between AlcR and CreA for the same DNA region. The second functional pair of CreA sites is located between the two transcription initiation sites. Disruption of either of the two sites results in a totally derepressed alcR transcription, showing that they work as a pair constituting the more efficient repression mechanism. Thus, CreA acts by two different mechanisms: by competing with AlcR for the same DNA region and by an efficient direct repression. The latter mechanism presumably interfers with the general transcriptional machinery.

In vitro recognition of specific DNA targets by AlcR, a zinc binuclear cluster activator different from the other proteins of this class.

AlcR is the transactivator mediating transcriptional induction of the alc gene cluster inAspergillus nidulans. The AlcR DNA-binding domain consists of a zinc binuclear cluster different from the other members of the Zn2Cys6 family by several features. In particular, it is able to bind to symmetric and asymmetric sites with the same affinity, with both sites being functional in A. nidulans. Here, we show that unlike the other proteins of the Zn2Cys6 binuclear cluster family, AlcR binds most probably as a monomer to its cognate targets. Two molecules of the AlcR protein can simultaneously bind in a noncooperative manner to inverted repeats. The consensus core has been determined precisely (5′-CCGCN-3′), and the AlcR-binding site in the aldApromoter has been localized. The sequence downstream of the zinc cluster is necessary for high affinity binding. Furthermore, our data show that the use of the carrier protein glutathioneS-transferase in AlcR binding experiments introduces an important bias in the recognition of DNA sites due to its tertiary dimeric structure.

The basal level of transcription of the alc genes in the ethanol regulon in Aspergillus nidulans is controlled both by the specific transactivator AlcR and the general carbon catabolite repressor CreA.

In the A. nidulans ethanol utilization pathway, specific induction is mediated by the transactivator AlcR which is subject to strong positive autogenous regulation and activates the transcription of the two structural genes alcA and aldA. Carbon catabolite repression is mediated by CreA which represses directly the transacting gene alcR and the two structural genes. We show here that the basal expression of the alcR and alcA genes is also controlled by the two regulatory circuits, positively by the transactivator AlcR and negatively by the repressor CreA, the aldA gene being subject only to the control of the CreA repressor.

Unique DNA Binding Specificity of the Binuclear Zinc AlcR Activator of the Ethanol Utilization Pathway in Aspergillus nidulans

AlcR is the transcriptional activator inAspergillus nidulans, necessary for the induction of thealc gene cluster. It belongs to the Zn2Cys6 zinc cluster protein family, but contains some striking differences compared with other proteins of this group. In this report, we show that no dimerization element is present in the entire AlcR protein which occurs in solution as a monomer and binds also to its cognate sites as a monomer. Another important feature of AlcR is its unique specificity for single sites occurring naturally as inverted or direct repeats and sharing a common motif, 5′-(T/A)GCGG-3′. Like most other Zn2Cys6proteins, AlcR contacts directly with the CGG triplet and, in addition, the upstream adjacent guanine is required for high affinity binding. We also establish that the flanking regions outside the core play an essential role in tight binding. From our in vitroanalysis, we propose an optimal AlcR-binding site which is 5′-PuNGCGG-AT rich 3′.