Major advances have recently occurred in our understanding of GATA factor-mediated, nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Under nitrogen-rich conditions, the GATA family transcriptional activators, Gln3 and Gat1, form complexes with Ure2, and are localized to the cytoplasm, which decreases NCR-sensitive expression. Under nitrogen-limiting conditions, Gln3 and Gat1 are dephosphorylated, move from the cytoplasm to the nucleus, in wild-type but not rna1 and srp1 mutants, and increase expression of NCR-sensitive genes. 'Induction' of NCR-sensitive gene expression and dephosphorylation of Gln3 (and Ure2 in some laboratories) when cells are treated with rapamycin implicates the Tor1/2 signal transduction pathway in this regulation. Mks1 is posited to be a negative regulator of Ure2, positive regulator of retrograde gene expression and to be itself negatively regulated by Tap42. In addition to Tap42, phosphatases Sit4 and Pph3 are also argued by some to participate in the regulatory pathway. Although a treasure trove of information has recently become available, much remains unknown (and sometimes controversial) with respect to the precise biochemical functions and regulatory pathway connections of Tap42, Sit4, Pph3, Mks1 and Ure2, and how precisely Gln3 and Gat1 are prevented from entering the nucleus. The purpose of this review is to provide background information needed by students and investigators outside of the field to follow and evaluate the rapidly evolving literature in this exciting field.

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3⌬ ure2⌬ dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.

The Tor1,2 protein kinases globally influence many cellular processes including nitrogen-responsive gene expression that correlates with intracellular localization of GATA transcription activators Gln3 and Gat1/Nil1. Gln3-Myc 13 and Gat1-Myc 13 are restricted to the cytoplasm of cells provided with good nitrogen sources, e.g. glutamine. Following the addition of the Tor1,2 inhibitor, rapamycin, both transcription factors relocate to the nucleus. Gln3-Myc 13 localization is highly dependent upon Ure2 and type 2A-related phosphatase, Sit4. Ure2 is required for Gln3 to be restricted to the cytoplasm of cells provided with good nitrogen sources, and Sit4 is required for its location to the nucleus following rapamycin treatment. The paucity of analogous information concerning Gat1 regulation prompted us to investigate the effects of deleting SIT4 and URE2 on Gat1-Myc 13 localization, DNA binding, and NCR-sensitive transcription. Our data demonstrate that Tor pathway control of NCR-responsive transcription bifurcates at the regulation of Gln3 and Gat1. Gat1-Myc 13 localization is not strongly influenced by deleting URE2, nor is its nuclear targeting following rapamycin treatment strongly dependent on Sit4. ChIP experiments demonstrated that Gat1-Myc 13 can bind to the DAL5 promoter in the absence of Gln3. Gln3-Myc 13 , on the other hand, cannot bind to DAL5 in the absence of Gat1. We conclude that: (i) Tor pathway regulation of Gat1 differs markedly from that of Gln3, (ii) nuclear targeting of Gln3-Myc 13 is alone insufficient for its recruitment to the DAL5 promoter, and (iii) the Tor pathway continues to play an important regulatory role in NCR-sensitive transcription even after Gln3-Myc 13 is localized to the nucleus.

Induction of the arginase (CAR1) gene expression in Saccharomyces cerevisiae has previously been shown to require participation of a cis-dominantly regulated upstream repression sequence (URS). Deletion of this element results in high-level expression of the CAR1 gene without inducer. To determine the structure of the CAR1 URS element, we performed a saturation mutagenesis. Results of the mutagenic analysis indicated that the CAR1 URS was a 9-base-pair palindromic sequence, 5'-AGCCGCCGA-3'. A DNA fragment containing this sequence was shown to bind one or more proteins by a gel shift assay. DNA fragments containing point mutations that completely eliminated URS function were not effective competitors in this assay, whereas those which supported URS function were effective competitors. Sequences in the 5'-flanking regions of 14 other genes were found to be homologous to the CAR1 URS. These sequences were shown to support varying degrees of URS function in the expression vector assay, to bind protein as demonstrated by the gel shift assay, and to compete with a DNA fragment containing the CAR1 URS for protein binding. These results indicate that the CAR1 URS element possesses the characteristics of a repressor binding site. Further, they are consistent with the suggestion that sites homologous to the CAR1 URS may be situated in the 5'-flanking regions of multiple unrelated yeast genes. The widespread occurrence of this element raises the possibility that it is the target site for one or more negatively acting general transcription factors.

Nitrogen catabolic gene expression in Saccharomyces cerevisiae has been reported to be regulated by three GATA family proteins, the positive regulators Gln3p and Gat1p/Nil1p and the negative regulator Dal80p/ Uga43p. We show here that a fourth member of the yeast GATA family, the Dal80p homolog Deh1p, also negatively regulates expression of some, but not all, nitrogen catabolic genes, i.e., GAP1, DAL80, and UGA4 expression increases in a deh1⌬ mutant. Consistent with Deh1p regulation of these genes is the observation that Deh1p forms specific DNA-protein complexes with GATAA-containing UGA4 and GAP1 promoter fragments in electrophoretic mobility shift assays. Deh1p function is demonstrable, however, only when a repressive nitrogen source such as glutamine is present; deh1⌬ mutants exhibit no detectable phenotype with a poor nitrogen source such as proline. Our experiments also demonstrate that GATA factor gene expression is highly regulated by the GATA factors themselves in an interdependent manner. DAL80 expression is Gln3p and Gat1p dependent and Dal80p regulated. Moreover, Gln3p and Dal80p bind to DAL80 promoter fragments. In turn, GAT1 expression is Gln3p dependent and Dal80p regulated but is not autogenously regulated like DAL80. DEH1 expression is largely Gln3p independent, modestly Gat1p dependent, and most highly regulated by Dal80p. Paradoxically, the high-level DEH1 expression observed in a dal80::hisG disruption mutant is highly sensitive to nitrogen catabolite repression.

Expression of the yeast arginase gene (CARl) responds to both induction and nitrogen catabolite repression. Regulation is mediated through sequences that both positively and negatively modulate CAR] transcription. A short sequence, 5'-TAGCCGCCGAGGG-3', possessing characteristics of a repressor binding site, plays a central role in the induction process. A fragment containing this upstream repression sequence (URSI) repressed gene expression when placed either 5' or 3' to the upstream activation sequences of the heterologous gene CYCL. Action of the URS and its cognate repressor was overcome by CARI induction when the URS was situated cis to the CAR] flanking sequences. This was not observed, however, when it was situated downstream of a heterologous CYCI upstream activation sequence indicating that URS function is specifically neutralized by cis-acting elements associated with CARI induction. Searches of sequences in various gene banks revealed that URSI-like sequences occur ubiquitously in genetic regulatory regions including those of bacteriophage A, yeast, mammalian, and viral genes. In a significant number of cases the sequence is contained in a region associated with negative control of yeast gene regulation. These data suggest the URS identified in this work is a generic repressor target site that apparently has been conserved during the evolution of transcriptional regulatory systems.Identification of sequences and proteins that mediate control of eukaryotic gene expression is central to our understanding of this process. Recent work has shown that sequences responsible for gene activation and control, upstream activation sequences (UAS) as they are called, tend to be short (1, 2). For yeast genes responding to regulation by the general amino acid control system, for example, a 6-base-pair (bp) sequence appears to be sufficient for activation and control (3). This conclusion is based on the observation that placement of this short sequence 5' to the TATA box of a heterologous gene lacking its UAS sequence supported gene expression (4). Moreover, expression responded to amino acid starvation just as it does for HIS4 and a variety of other genes encoding amino acid biosynthetic enzymes (4). Recently, Hope and Struhl have shown that the GCN4 gene product binds to this sequence (5), an observation they interpreted to indicate that this protein was responsible for activation of the genes in this regulon (set of similarly regulated genes). A similar motif of regulation is being found for the GAL, PHO, SUC, DAL, CAR, and mating type responsive genes, to name just a few.The number of yeast genes responding to well-characterized negative control systems is considerably smaller. The most notable representatives are those associated with mating type (6), CYC7 (7), enolase (8), the genes encoding arginase (CARl) and ornithine aminotransferase (CAR2) (9), and CTTJ (10). The CAR] and CAR2 genes are the focus of this work. Expression of these genes is induced by arginine and is subject to nitrogen catabolite rep...

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