Martine Mathieu

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.

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.

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.

Correct intron splicing generates a new type of a putative zinc-binding domain in a transcriptional activator of Aspergillus nidulans

alcR is the pathway‐specific transcriptional activator of the ethanol regulon in the filamentous fungus, Aspergillus nidulans. The deduced amino acid sequence of a cDNA clone, including the 5′ part of the alcR‐mRNA, shows that a putative Zn‐binding of the all‐cysteine class, exemplified by GAL4 is present. This structure presents some striking features. At variance with other structures of this class, the binding domain is strongly asymmetrical. Model building indicates that the zinc‐binding motif of alcR could adopt an helix‐turn‐helix structure. We propose that the DNA binding motif of alcR could participate in two types of DNA‐binding structures: the zinc‐cluster and the helix‐turn‐helix.

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.

Roles of the Aspergillus nidulans homologues of Tup1 and Ssn6 in chromatin structure and cell viability

For three different carbon catabolite repressible promoters, alcA, alcR and the bidirectional promoter prnD-prnB, a deletion of rcoA, the Aspergillus nidulans homologue of TUP1, does not result in carbon catabolite derepression. Surprisingly, it results in disruption of the chromatin default structure of alcR and prnD-prnB promoters. In these promoters, and at variance with the wild type, repression occurs in the absence of nucleosome positioning. For alcR, repression occurs together with a nucleosome pattern identical to that found under conditions of full expression, and for prnD-prnB it occurs with a novel pattern that does not correspond to the pattern seen under conditions of repression in a wild-type strain. Deletion of the putative RcoA partner, SsnF, is lethal in A. nidulans.

Patterns of nucleosomal organization in the alc regulon of Aspergillus nidulans: roles of the AlcR transcriptional activator and the CreA global repressor

We have studied the chromatin organization of three promoters of the alc regulon of Aspergillus nidulans. No positioned nucleosomes are seen in the aldA (aldehyde dehydrogenase) promoter under any physiological condition tested by us. In the alcA (alcohol dehydrogenase I) and alcR (coding for the pathway‐specific transcription factor) promoters, a pattern of positioned nucleosomes is seen under non‐induced and non‐induced repressed conditions. While each of these promoters shows a specific pattern of chromatin restructuring, in both cases induction results in loss of nucleosome positioning. Glucose repression in the presence of inducer results in a specific pattern of partial positioning in the alcA and alcR promoters. Loss of nucleosome positioning depends absolutely on the AlcR protein and it is very unlikely to be a passive result of the induction of transcription. In an alcR loss‐of‐function background and in strains carrying mutations of the respective AlcR binding sites of the alcA and alcR promoters, nucleosomes are fully positioned under all growth conditions. Analysis of mutant AlcR proteins establishes that all domains needed for transcriptional activation and chromatin restructuring are included within the first 241 residues. The results suggest a two‐step process, one step resulting in chromatin restructuring, a second one in transcriptional activation. Partial positioning upon glucose repression shows a specific pattern that depends on the CreA global repressor. An alcR loss‐of‐function mutation is epistatic to a creA loss‐of‐function mutation, showing that AlcR does not act by negating a nucleosome positioning activity of CreA.

DdrO is an essential protein that regulates the radiation desiccation response and the apoptotic‐like cell death in the radioresistant Deinococcus radiodurans bacterium

Deinococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation‐desiccation response (RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif (RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis‐regulation of the RDR gene ddrB in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in Deinococcus deserti, two genes encoding α‐keto‐glutarate dehydrogenase subunits are involved in ddrB regulation. In wild‐type cells, the DdrO cell concentration decreased transiently in an IrrE‐dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO‐depleted cells loose viability and showed morphological changes evocative of an apoptotic‐like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic‐like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE‐dependent DdrO cleavage.

Analysis of the expression of two genes of Dictyostelium discoideum which code for developmentally regulated cysteine proteinases

SummaryWe analysed the expression of two genes encoding two different cysteine proteinases which show a high degree of homology and are regulated differentially during Dictyostelium discoideum development. While absent from growing vegetative cells, the two specific messengers RNAs appear at an early stage of development but accumulate in the polysomes at different later stages of development. The message coding for cysteine proteinase I reaches its maximum level after the aggregative period while cysteine proteinase II mRNA reaches its maximum during the culmination stage. At these times they represent 1% and 0.7%, respectively, of cellular mRNA and belong to the most abundant class of mRNAs.No accumulation of the two messages elsewhere than in the polyribosomes was observed in the cytoplasm of the cells, whatever the stage of development analysed. Results of analysis of the nuclear levels of RNA complementary to the two genes are in agreement with the relative transcription rates determined by in vitro transcription of nuclei isolated at different stages. The combined results of these measurements demonstrate that the expression of the cysteine proteinase I and II genes is mainly under transcriptional control. However, the difference between the RNA synthetic rate in the nucleus and the accumulation of the mRNA in the cytoplasm, indicates an additional post-transcriptional regulation for the cysteine proteinase II gene.

Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha

A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.