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. 2005 Jun 1;388(Pt 2):697-703.
doi: 10.1042/BJ20050160.

Rgt1, a glucose sensing transcription factor, is required for transcriptional repression of the HXK2 gene in Saccharomyces cerevisiae

Aaron Palomino  1 , Pilar Herrero, Fernando Moreno
Affiliations

Rgt1, a glucose sensing transcription factor, is required for transcriptional repression of the HXK2 gene in Saccharomyces cerevisiae

Aaron Palomino et al. Biochem J. .

Abstract

Expression of HXK2, a gene encoding a Saccharomyces cerevisiae bifunctional protein with catalytic and regulatory functions, is controlled by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are low. In the present study, we identified Rgt1 as a transcription factor that, together with the Med8 protein, is essential for repression of the HXK2 gene in the absence of glucose. Rgt1 represses HXK2 expression by binding specifically to the motif (CGGAAAA) located at -395 bp relative to the ATG translation start codon in the HXK2 promoter. Disruption of the RGT1 gene causes an 18-fold increase in the level of HXK2 transcript in the absence of glucose. Rgt1 binds to the RGT1 element of HXK2 promoter in a glucose-dependent manner, and the repression of target gene depends on binding of Rgt1 to DNA. The physiological significance of the connection between two glucose-signalling pathways, the Snf3/Rgt2 that causes glucose induction and the Mig1/Hxk2 that causes glucose repression, was also analysed.

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Figures

Figure 1
Figure 1. Rgt1 and Med8 proteins repress HXK2 expression in the absence of glucose
(A) Hxk2 expression was measured by using the lacZ expression as reporter gene. One copy of the HXK2+404::lacZ, containing the Med8 binding downstream regulatory sequence (+DRS), or the HXK2+39::lacZ, lacking the Med8 binding downstream regulatory sequence (−DRS), constructs were integrated in the chromosome at URA3 locus of the wild-type strain DBY1315 or the mutant strain rgt1. β-Galactosidase activities are averages of the results obtained for four to five independent experiments. Average values have standard errors of 10% or less. Yeasts were grown on YEPD medium (open bars) or YEPE medium (solid bars) until the A600 reached 1.0 [3.0 mg (wet weight)/ml]. β-Galactosidase activity was assayed in crude extracts. (B) Effect of RGT1 gene disruption on the expression of HXK2. A wild-type yeast strain (DBY1315) and the isogenic rgt1 mutant strain were grown using glucose (YEPD) or ethanol (YEPE) as carbon source as described above each lane. Total RNA was isolated, size-separated using a horizontal agarose gel and transferred on to a nylon membrane that was then hybridized to probes derived from HXK2 as described in the Materials and methods section. The probe used is indicated at the side of each panel.
Figure 2
Figure 2. Rgt1 protein binds to the RGT1 element of the HXK2 promoter in vitro and in vivo
(A) Gel mobility-shift analysis of Rgt1 binding to the RGT1 regulatory element of the HXK2 promoter. The specific competitor for binding was the unlabelled RGT1-annealed oligonucleotides. The non-specific competitor for binding was 100 ng of calf-thymus DNA (CT). Nucleoprotein complexes were resolved from free DNA by non-denaturing PAGE. For the control (lane 1), the radiolabelled DNA fragment was added alone. F, unbound fragment; CI and CII, positions of shifted bands observed with purified Rgt1 protein. (B) ChIP analysis of Rgt1 binding to the RGT1 regulatory element of the HXK2 promoter. Chromatin was prepared from wild-type yeast cells containing HA-tagged Rgt1 or HA-tagged Med8 proteins. The cells were grown on YEPG (G) or YEPE (E) media as indicated above each lane, and their chromatin was immunoprecipitated using anti-HA monoclonal antibodies. The HXK2 regulatory regions in the immunoprecipitated DNA (IP) was detected by ethidium bromide staining after amplifying it in a PCR by using the primer pair OL3+OL4 (R) or OL5+OL6 (M) and resolving it by electrophoresis on a 2% agarose gel. Lanes show DNA amplified from genomic DNA, whole-cell extract before immunoprecipitation or extracts from cells without tagged protein (Control).
Figure 3
Figure 3. The Rgt1 protein binds to the RGT1 element of the HXK2 promoter in a glucose-dependent manner
(A) Yeast cells were grown in YEPD-2% glucose medium or in YEPE-3% ethanol to an A600 1.0. Nuclear extracts were prepared as indicated in the Materials and methods section and used for EMSA analysis. (B) Yeast cells were grown in YEPD-4% glucose medium to an A600 of 1.0 and shifted to YEPD-0.02% glucose medium for 5 or 30 min; then 2% glucose was added to the medium and cells were collected after 15 min of incubation. Nuclear extracts were prepared as described in the Materials and methods section and used for EMSA analysis. Unlabelled RGT1HXK2 probe (25 ng) was used as a specific competitor in lane 2.
Figure 4
Figure 4. Effect of RGT1 gene disruption on the expression of SUC2
(A) The wild-type yeast strain (DBY1315) and its isogenic rgt1 null mutant were grown using 2% glucose (open bars) or 3% ethanol plus 0.05% glucose (solid bars) as carbon sources, until the A600 reached 1.0 [3.0 mg (wet weight)/ml]. Invertase activity was assayed in whole cells. The average values of the results obtained for four independent experiments have standard errors ≤10%. (B) Yeast rgt1 mutant strain expressing Hxk2–GFP (a and b) from the plasmid YEp352/HXK2::gfp and Mig1–GFP (c and d) from the plasmid YEp352/MIG1::gfp were grown on SD-Ura− medium supplemented with glucose or ethanol as carbon source and fluorescence was imaged. The cells were stained with DAPI for DNA and then imaged for GFP fluorescence and for DAPI fluorescence by phase-contrast optics.
Figure 5
Figure 5. Model for the integration of the glucose signal through distinct signalling pathways
Glucose signals could take several possible routes, namely Snf3/Rgt2 and Mig1/Hxk2 pathways. Binding of glucose to the transmembrane proteins Snf3/Rgt2 inactivates the Rgt1 repressor function, leading to expression of several genes (HXTs, MIG2 and HXK2). Rgt1 is the main downstream repressor of the Snf3/Rgt2 pathway controlling expression in the absence of glucose of several targets genes. Two of the targets, Hxk2 and Mig2, are directly involved, together with Mig1, in glucose signalling through the Mig1/Hxk2 pathway. Genes are depicted by rectangles, proteins by ovals and shaded proteins are discussed in the present work.
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