Institut für Mikrobiologie, Abt. Genetik und Biochemie, Jahnstr. 15a, D-17487 Greifswald, Germany1
Author for correspondence: Hans-Joachim Schüller. Tel: +49 3834 864154. Fax: +49 3834 864172. e-mail: schuell{at}biologie.uni-greifswald.de
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ABSTRACT |
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Keywords: gluconeogenesis, transcriptional regulation, UAS element
Abbreviations: CSRE, carbon source-responsive element; UAS, upstream activation site
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INTRODUCTION |
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In contrast to UAS1 and its corresponding trans-acting factor, Adr1, UAS2 is much less understood. A DNA-binding factor which is specifically required for UAS2-dependent gene activation and directly interacts with UAS2 has not yet been identified. We have previously analysed the genetic control of structural genes involved in the glyoxylate cycle and gluconeogenesis [ICL1, FBP1 (Schöler & Schüller, 1993 , 1994
), MLS1 (Caspary et al., 1997
) and MDH2 (Roth & Schüller, 2001
)]. This led to the identification of a common cis-acting element, designated CSRE (carbon source-responsive element; consensus CCRTYSRNCCG; reviewed by Entian & Schüller, 1997
; Gancedo, 1998
), which could confer glucose-sensitive gene regulation to a synthetic minimal promoter. CSRE-dependent gene activation requires a functional CAT8 gene (Hedges et al., 1995
; Rahner et al., 1996
), encoding a transcription factor with a binuclear zinc cluster domain at its N terminus and a C-terminal transcription activation domain. Expression of CAT8, as well as transcription activation by Cat8, is affected by carbon source (Rahner et al., 1996
; Randez-Gil et al., 1997
). A functional Cat1/Snf1/Ccr1 protein kinase together with its auxiliary factors Cat3/Snf4 (Celenza & Carlson, 1986
; Schüller & Entian, 1987
, 1988
; Celenza et al., 1989
) and Sip1+Sip2+Gal83 (Yang et al., 1994
; Schmidt & McCartney, 2000
) is absolutely required for Cat8 function. Importantly, Cat8, as well as the related transcription factor Sip4, bind to CSREICL1, CSREFBP1 and CSREMDH2 motifs (Vincent & Carlson, 1998
; Rahner et al., 1999
; Roth & Schüller, 2001
). A CSRE-driven gene has been shown to be no longer regulated by carbon source with constitutively synthesized Cat8 and Sip4 variants containing heterologous activation domains (Vincent & Carlson, 1998
; Rahner et al., 1999
; Hiesinger et al., 2001
).
Previously, we identified a CSRE motif together with an Adr1-binding site in the upstream region of the acetyl-CoA synthetase gene ACS1 (Kratzer & Schüller, 1997 ). While characterizing a cat8 null mutant, we obtained evidence for a Cat8-dependent activation of ADH2 (Rahner et al., 1996
). However, the hypothesis of coregulation of ACS1 and ADH2 by Cat8 and Adr1 was rejected by Donoviel & Young (1996)
who reported a marginal reduction of UAS2-dependent gene expression in a cat8 mutant. In this work we identify a functional and completely Cat8-dependent variant of the CSRE within UAS2. We also demonstrate in vitro binding of Cat8 to UAS2 and therefore suggest a combined derepression of ADH2 by Cat8 and Adr1.
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METHODS |
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Plasmid constructions.
To obtain pKW7 variants, the synthetic DNA fragment ADH2-UAS2 (5'-tcgaTGATCTCCTCTGCCGGAACACCGGGCATggtacc-3', together with the complementary oligonucleotide; authentic ADH2 upstream sequence in capital letters) was inserted into promoter test plasmid pJS401 (ICL1-lacZ URA3 2µ; Caspary et al., 1997
), cut at its single XhoI site. Copy number and orientation of inserts were determined by DNA sequencing. The complete ADH2 control region (-926/+3) was amplified as a HindIII/BamHI fragment by PCR and subsequently inserted into lacZ fusion vector YEp356R (Myers et al., 1986
) to give pKW13 (ADH2-lacZ URA3 2µ). Point mutations within UAS2 (alteration of 4 nt essential for a functional CSRE) were generated with the QuikChange site-directed mutagenesis kit (Stratagene), using mutagenic primers ADH2-Mut1 (5'-CTCCTCTGCGTCGACACCGGGCATCT-3'; artificial SalI site underlined) and ADH2-Mut2 (reverse complement). The desired mutation in plasmid pKW14 (ADH2-[CSREMut]-lacZ URA3 2µ) was confirmed by DNA sequencing. Plasmids pAR33, pAR34 and pMH33, encoding deregulated variants of CAT8 and SIP4, have been described previously (carbon source-independent MET25 promoter, constitutive Ino2 or VP16 activation domains together with a FLAG epitope; Rahner et al., 1999
; Hiesinger et al., 2001
).
Gel retardation assay.
For the study of protein/DNA interactions, probe ADH2-UAS2 was radioactively end-labelled by a fill-in reaction using [-32P]dATP and Klenow enzyme. The synthetic DNA fragment OAS12 (containing CSREICL1; Schöler & Schüller, 1994
) was used as competitor. Fusion proteins GST-Gal4 (aa 1300 of Gal4, pMH25-GAL4), GST-Sip4 (aa 1300, pMH25-SIP4; Hiesinger et al., 2001
) and GST-Cat8 (aa 1281, pAR1; Rahner et al., 1996
) were prepared from Escherichia coli lysates by affinity chromatography with glutathione Sepharose as recommended by the manufacturer (Amersham Pharmacia). Yeast protein extract containing Cat8(1281)-Ino2-FLAG was prepared from derepressed transformants of mutant strain SRH24 (carrying plasmid pAR34) as described previously (Schöler & Schüller, 1994
). For supershift experiments, 200 ng monoclonal anti-FLAG antibody M2 (Kodak/IBI) was used.
Miscellaneous procedures.
Strains of S. cerevisiae were transformed according to an established procedure (Soni et al., 1993 ). ß-Galactosidase assays were performed with yeast crude extracts as described previously (Schöler & Schüller, 1994
). Synthetic oligonucleotides were purchased from Gibco-BRL Life Technologies. For the amplification of DNA fragments by PCR, the expand high fidelity system (Roche Molecular Biochemicals) was used.
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RESULTS |
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DISCUSSION |
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Although both almost completely overlapping CSRE motifs within UAS2 deviate from the previously defined consensus sequence, a regulated, 80-fold activation of basal gene expression was found in the presence of at least two CSREADH2 copies (Fig. 1). Recent data derived from the saturation mutagenesis of a strong CSRE suggest that the sequence CCGGTGTTCCG is closer to the modified consensus than the CCGGAACACCG motif on the complementary strand (S. Roth & H.-J. Schüller; unpublished). Gel retardation experiments showed that Cat8 and the related zinc cluster protein Sip4 differ with respect to binding affinity to CSRE sequence variants. Thus, Cat8 must be considered as the dominating regulator acting via UAS2. This conclusion is in agreement with a recently published microarray analysis, comparing mRNA levels from wild-type and cat8 mutant strains on a genome-wide scale (Haurie et al., 2001
). A minor role, even of Adr1, can be derived from the reduction of UAS2-dependent gene expression in an adr1 mutant (Table 1
) as well as from the previous demonstration of low-affinity binding of Adr1 to UAS2 (Donoviel et al., 1995
). Synergistic gene activation is not only observed in the presence of multiple UAS2 copies in a synthetic promoter (Fig. 1
) but also with UAS1 and UAS2 in the natural ADH2 control region (Yu et al., 1989
). Presumably, Cat8 and the UAS1-binding factor Adr1 cooperate in maximal derepression of an ADH2-lacZ reporter gene. This conclusion derived from the analysis of cat8 and adr1 single mutants is in agreement with previous results, comparing UAS2 promoter mutants in the absence or presence of Adr1 (Donoviel et al., 1995
). Synergistic activation of ADH2 by Cat8 and Adr1 contrasts to the additive contribution of these regulators to derepression of ACS1 (Kratzer & Schüller, 1997
). This may simply result from varying distances of Cat8- and Adr1-binding sites upstream of ACS1 (separated by 200 bp) and ADH2 (3 bp apart). Synergistic derepression of ADH2 by Cat8 and Adr1 could be the result of recruiting a common coactivator/mediator of transcriptional activation. Possible candidates may be Ada2 and the histone acetyltransferase Gcn5, as well as basal transcription factors TFIIB and components of TFIID (TBP, Taf90 and Taf130/145) for which direct contacts to activation domains within Adr1 have been demonstrated (Chiang et al., 1996
; Komarnitsky et al., 1998
).
The influence of Cat8 on UAS2-dependent gene activation is further confirmed by the obvious deregulation of a reporter gene by the MET25-CAT8-INO2TAD variant, encoding a protein with a constitutive activation domain (Ino2TAD) driven by a carbon source-independent promoter (MET25). Compared to a wild-type control, gene expression was clearly increased under repressing, but not under derepressing conditions (Table 2). An increased expression of UAS2-dependent ADH2 was previously shown in the presence of multicopy plasmids containing SPT6 and MEU1 genes (Donoviel & Young, 1996
). While the essential SPT6 gene may be involved in chromatin modification during transcriptional elongation (Hartzog et al., 1998
), the function of MEU1 (multicopy enhancer of UAS2) remained unclear. A high copy number of MEU1 increased ADH2 expression about three- to fivefold under either condition, while its deletion led to a twofold reduction (Donoviel & Young, 1996
). Our data on the contribution of Cat8 (which are in agreement with the findings of Haurie et al., 2001
) completely differ from results reported by these authors. In contrast to the complete loss of UAS2-driven gene expression documented in this work (Table 1
), Donoviel & Young (1996)
merely describe a slight reduction by a factor of two. The reason for this discrepancy remains unclear.
Assuming UAS2 is the sole Cat8-dependent activating element, equivalent and non-additive effects of cis and trans mutations (CSREADH2 and cat8) on ADH2 gene expression should be expected. However, in the absence of Cat8, gene expression was clearly more affected than with a mutant UAS2 (Fig. 2). Possibly, the site-directed mutagenesis did not abolish all UAS2 activity. This appears unlikely since positions critical for a functional CSRE had been altered. Instead, we suggest the existence of (at least) one additional CSRE motif in the ADH2 upstream region. We were able to identify the sequence motif CCGTCTCTCCG (-478/-468) which fits the weakly stringent consensus CCN6CCG typical of a subfamily among zinc cluster proteins. This element may be considered as a weak CSRE, being responsible for Cat8-dependent ADH2 derepression in the absence of UAS2.
We finally asked whether at least one among five aldehyde dehydrogenase genes (ALD2, ALD3, ALD4/ALD7, ALD5 and ALD6) in S. cerevisiae may also require transcriptional activation by Cat8 and Adr1. Mutant phenotype (no growth with ethanol as sole carbon source; Meaden et al., 1997 ), as well as expression pattern (Haurie et al., 2001
), indicates that the ALD6 gene encoding a cytoplasmic, NADP-dependent aldehyde dehydrogenase is a possible candidate. In silico analysis of its upstream region revealed the existence of two CSRE-related motifs (CCTCTTGCCCG, -615/-605 and CCTTATTCCCG, -484/-474) and a partially palindromic sequence similar to an Adr1-binding site (TCTCCN10GGTGA; according to the consensus as defined by Cheng et al., 1994
). Thus, the metabolic pathway responsible for the cytoplasmic synthesis of acetyl-CoA from ethanol involves structural genes ADH2, ALD6 and ACS1, which may be transcriptionally coregulated by Adr1 and Cat8 (summarized in Fig. 5
).
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ACKNOWLEDGEMENTS |
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Received 8 March 2001;
accepted 30 April 2001.