Lina Riego

GDH1 expression is regulated by GLN3, GCN4, and HAP4 under respiratory growth.

In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenase isoenzymes encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and alpha-ketoglutarate. In this work we analyzed GDH1 transcriptional regulation, in order to deepen the studies in regard to its physiological role. Our results indicate that: (i) GDH1 expression is strictly controlled in ethanol-grown cultures, constituting a fine-tuning mechanism that modulates the abundance of Gdh1p monomers under this condition, (ii) GDH1 expression is controlled by transcriptional activators that have been considered as exclusive of either nitrogen (Gln3p and Gcn4p) or carbon metabolism (HAP complex), and (iii) chromatin remodeling complexes play a role in GDH1 expression; ADA2 and ADA3 up-regulated GDH1 expression on ethanol, while that on glucose was ADA3-dependent. SPT3 and SNF2 activated GDH1 expression on either carbon source whereas GCN5 played no role in any condition tested. The above described combinatorial control results in a refined mechanism that coordinates carbon and nitrogen utilization.

Swi/SNF‐GCN5‐dependent chromatin remodelling determines induced expression of GDH3, one of the paralogous genes responsible for ammonium assimilation and glutamate biosynthesis in Saccharomyces cerevisiae

It is accepted that Saccharomyces cerevisiae genome arose from complete duplication of eight ancestral chromosomes; functionally normal ploidy was recovered because of the massive loss of 90% of duplicated genes. There is evidence that indicates that part of this selective conservation of gene pairs is compelling to yeast facultative metabolism. As an example, the duplicated NADP‐glutamate dehydrogenase pathway has been maintained because of the differential expression of the paralogous GDH1 and GDH3 genes, and the biochemical specialization of the enzymes they encode. The present work has been aimed to the understanding of the regulatory mechanisms that modulate GDH3 transcriptional activation. Our results show that GDH3 expression is repressed in glucose‐grown cultures, as opposed to what has been observed for GDH1, and induced under respiratory conditions, or under stationary phase. Although GDH3 pertains to the nitrogen metabolic network, and its expression is Gln3p‐regulated, complete derepression is ultimately determined by the carbon source through the action of the SAGA and SWI/SNF chromatin remodelling complexes. GDH3 carbon‐mediated regulation is over‐imposed to that exerted by the nitrogen source, highlighting the fact that operation of facultative metabolism requires strict control of enzymes, like Gdh3p, involved in biosynthetic pathways that use tricarboxylic acid cycle intermediates.

The UGA3-GLT1 intergenic region constitutes a promoter whose bidirectional nature is determined by chromatin organization in Saccharomyces cerevisiae

Transcription of an important number of divergent genes of Saccharomyces cerevisiae is controlled by intergenic regions, which constitute factual bidirectional promoters. However, few of such promoters have been characterized in detail. The analysis of the UGA3‐GLT1 intergenic region has provided an interesting model to study the joint action of two global transcriptional activators that had been considered to act independently. Our results show that Gln3p and Gcn4p exert their effect upon cis‐acting elements, which are shared in a bidirectional promoter. Accordingly, when yeast is grown on a low‐quality nitrogen source, or under amino acid deprivation, the expression of both UGA3 and GLT1 is induced through the action of both these global transcriptional modulators that bind to a region of the bidirectional promoter. In addition, we demonstrate that chromatin organization plays a major role in the bidirectional properties of the UGA3‐GLT1 promoter, through the action of an upstream Abf1p‐binding consensus sequence and a polydAdTtract. Mutations in these cis‐elements differentially affect transcription of UGA3 and GLT1, and thus alter the overall relative expression. This is the first example of an intergenic region constituting a promoter whose bidirectional character is determined by chromatin organization.

Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae

The transcriptional activation response relies on a repertoire of transcriptional activators, which decipher regulatory information through their specific binding to cognate sequences, and their capacity to selectively recruit the components that constitute a given transcriptional complex. We have addressed the possibility of achieving novel transcriptional responses by the construction of a new transcriptional regulator--the Hap2-3-5-Gln3 hybrid modulator--harbouring the HAP complex polypeptides that constitute the DNA-binding domain (Hap2-3-5) and the Gln3 activation domain, which usually act in an uncombined fashion. The results presented in this paper show that transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions is achieved through the action of the novel Hap2-3-5-Gln3 transcriptional regulator. We propose that the combination of the Hap DNA-binding and Gln3 activation domains results in a hybrid modulator that elicits a novel transcriptional response not evoked when these modulators act independently.

Salt-dependent expression of ammonium assimilation genes in the halotolerant yeast, Debaryomyces hansenii

Debaryomyces hansenii is adapted to grow in saline environments, accumulating high intracellular Na+ concentrations. Determination of the DhGDH1-encoded NADP-glutamate dehydrogenase enzymatic activity showed that it increased in a saline environment. Thus, it was proposed that, in order to overcome Na+ inhibition of enzyme activity, this organism possessed salt-dependent mechanisms which resulted in increased activity of enzymes pertaining to the central metabolic pathways. However, the nature of the mechanisms involved in augmented enzyme activity were not analyzed. To address this matter, we studied the expression of DhGDH1 and DhGLN1 encoding glutamine synthetase, which constitute the central metabolic circuit involved in ammonium assimilation. It was found that: (1) expression of DhGDH1 is increased when D. hansenii is grown in the presence of high NaCl concentrations, while that of DhGLN1 is reduced, (2) DhGDH1 expression in Saccharomyces cerevisiae takes place in a GLN3- and HAP2,3-dependent manner and (3) salt-dependent DhGDH1 and DhGLN1 expression involves mechanisms which are limited to D. hansenii and are not present in S. cerevisiae. Thus, salt-dependent regulation of the genes involved in central metabolic pathways could form part of a strategy leading to the ability to grow under hypersaline conditions.

Gln3-Gcn4 hybrid transcriptional activator determines catabolic and biosynthetic gene expression in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is able to sense the availability and quality of nitrogen sources and the intrinsic variation of amino acid disponibility for protein synthesis. When this yeast is provided with secondary nitrogen sources, transcription of genes encoding enzymes involved in their catabolism is elicited through the action of Gln3, which constitutes the main activator of the Nitrogen Catabolite Repression network (NCR). Activation of genes encoding enzymes involved in the amino acid biosynthetic pathways is achieved through the action of the GCN4-encoded transcriptional modulator whose transcriptional activation is induced at the translational level by limitation for any amino acid. Thus the role of each one of these activators had been secluded to either catabolic or biosynthetic pathways. However, some observations have suggested that under peculiar physiological conditions, Gln3 and Gcn4 could act simultaneously in order to contemporaneously increase expression of both sets of genes. This paper addresses the question of whether Gln3 and Gcn4 cooperatively determine expression of their target genes. Results presented herein show that induced expression of catabolic and biosynthetic genes when cells are grown under nitrogen derepressive conditions and amino acid deprivation is dependent on the concurrent action of Gln3 and Gcn4, which form part of a unique transcriptional complex. We propose that the combination of Gln3 and Gcn4 results in the constitution of a hybrid modulator which elicits a novel transcriptional response, not evoked when these modulators act in a non-combinatorial fashion.