The level of DAL80 expression down-regulates GATA factor-mediated transcription in Saccharomyces cerevisiae

Abstract

Nitrogen-catabolic gene expression in Saccharomyces cerevisiae is regulated by the action of four GATA family transcription factors: Gln3p and Gat1p/Nil1p are transcriptional activators, and Dal80 and Deh1p/Gzf3p are repressors. In addition to the GATA sequences situated upstream of all nitrogen catabolite repression-sensitive genes that encode enzyme and transport proteins, the promoters of the GAT1, DAL80, and DEH1 genes all contain multiple GATA sequences as well. These GATA sequences are the binding sites of the GATA family transcription factors and are hypothesized to mediate their autogenous and cross regulation. Here we show, using DAL80 fused to the carbon-regulated GAL1,10 or copper-regulated CUP1 promoter, that GAT1 expression is inversely regulated by the level of DAL80 expression, i.e., as DAL80 expression increases, GAT1 expression decreases. The amount of DAL80 expression also dictates the level at which DAL3, a gene activated almost exclusively by Gln3p, is transcribed. Gat1p was found to partially substitute for Gln3p in transcription. These data support the contention that regulation of GATA-factor gene expression is tightly and dynamically coupled. Finally, we suggest that the complicated regulatory circuit in which the GATA family transcription factors participate is probably most beneficial as cells make the transition from excess to limited nitrogen availability.

FIG. 1

Proposed regulatory circuit of GATA-factor-dependent transcription in S. cerevisiae. Open arrows and closed bars indicate positive and negative regulation, respectively.

FIG. 2

Constructs for the production and assay of Dal80p and Gat1p from the GAL1,10 promoter. Genomic loci are shown prior to (A) and following (B) integration of a construct in which a GAL1,10 promoter fragment (derived from pTSC645 or pTSC674) replaces the native promoter of the gene; open bars, yeast DNA; grey bars, GAT1 or DAL80 open reading frame (ORF); black bars, yeast selectable marker; stippled bars, GAL1,10 promoter DNA; dashed lines, vector sequences. Strain numbers of the resulting strains are also indicated. lacZ fusion plasmids were derived from GAL1,10-GAT1 (C) and GAL1,10-DAL80 (D), pTSC666 and pTSC679, respectively. Symbols are as indicated for panels A and B above except vertical lines, indicating lacZ DNA. (E) CUP1-DAL80 plasmid.

FIG. 3

GAT1-lacZ expression driven from the GAL1,10 promoter in cultures to which increasing amounts of glucose were added. The detailed protocol for the experiment is described in Materials and Methods. Data in Fig. 3B are those from Fig. 3A plotted as the reciprocal of the glucose concentration to facilitate understanding of the results.

FIG. 4

Characterization of the Dal80p-production system. (A) Northern blot analysis of DAL80 mRNA driven by the GAL1,10 promoter. Total RNA (10 μg/lane) was extracted from TCY65. The experimental protocol was as described in Materials and Methods. The ³²P-dCTP-labeled DAL80 probe was synthesized from the 0.8-kb NdeI-EcoRI fragment of pTSC417. HHT1 and HHT2 standards were probed with a radioactive complementary oligonucleotide. (B) Effect of increasing glucose addition on lacZ expression in strain TCY36 transformed with GAL1,10-DAL80-lacZ pTSC679. (C) Expression of DAL80-lacZ from pTSC572 in WT (TSC46) and dal80 (TCY53) strains growing in YNB–2% glucose–2% galactose–0.1% proline medium. Cells were grown under standard conditions and harvested for β-galactosidase assays at a cell density A₆₀₀ of 0.4 to 0.8.

FIG. 5

Inverse regulation of GAL80 and GAT1 expression. (A) GAT1 expression at different levels of DAL80 expression from pTSC624 in strain TCY65. (B) DAL80 expression at different levels of GAT1 expression from pTSC572 in strains TCY46 (WT) or TCY53 (dal80). Growth and assay conditions were as described in Fig. 3. Steady-state β-galactosidase production derived from DAL80-lacZ and GAT1-lacZ fusion genes in WT, dal80, and gat1 mutant backgrounds have been reported (6). However, the values obtained in those experiments should be only generally compared with the values obtained here because of the differences in the experimental conditions used.

FIG. 6

DAL3 expression at various levels of CUP1-DAL80 expression. Strains TCY5 (WT) and TCY29 (dal80::hisG) were transformed with reporter pTSC560 alone or together with CUP1-DAL80 pTSC565. Cultures were grown overnight in 2% glucose–0.1% proline medium, containing the indicated concentrations of copper sulfate, to a cell density (A₆₀₀) of 0.4 to 1.0, at which time samples were removed for the assay of β-galactosidase, using standard assay methods.

FIG. 7

The effect of GAT1 expression on DAL80 expression in gln3Δ strains. β-Galactosidase production was supported by a pTSC572 transformant of strain TCY55 (gln3Δ GAL1,10-GAT1) or TCY64 (gln3Δ dal80 GAL1,10-GAT1) with proline or ammonium sulfate as the nitrogen source and 2% galactose plus the indicated concentration of glucose as the carbon source as per Fig. 3 and Materials and Methods.

FIG. 8

Expression of DAL5-lacZ and a UGA4 promoter fragment in a heterologous expression vector following a shift of cultures from YNB glucose-asparagine to glucose-proline medium. Strains TCY36 (WT) and TCY38 (dal80) were transformed with DAL5-lacZ pJD52 (A) or UGA4_GATA pTSC491 (B).

FIG. 9

β-Galactosidase production from DAL80-lacZ pTSC572 (A) and GAT1-lacZ pTSC624 (B) transformed into strains TCY36 (WT) and TCY38 (dal80). Conditions were as described in the legend to Fig. 8.