SUC2 Expression Research Articles

Fine-Tuning of Energy Levels Regulates SUC2 via a SNF1-Dependent Feedback Loop

Nutrient sensing pathways are playing an important role in cellular response to different energy levels. In budding yeast, Saccharomyces cerevisiae, the sucrose non-fermenting protein kinase complex SNF1 is a master regulator of energy homeostasis. It is affected by multiple inputs, among which energy levels is the most prominent. Cells which are exposed to a switch in carbon source availability display a change in the gene expression machinery. It has been shown that the magnitude of the change varies from cell to cell. In a glucose rich environment Snf1/Mig1 pathway represses the expression of its downstream target, such as SUC2. However, upon glucose depletion SNF1 is activated which leads to an increase in SUC2 expression. Our single cell experiments indicate that upon starvation, gene expression pattern of SUC2 shows rapid increase followed by a decrease to initial state with high cell-to-cell variability. The mechanism behind this behavior is currently unknown. In this work we study the long-term behavior of the Snf1/Mig1 pathway upon glucose starvation with a microfluidics and non-linear mixed effect modeling approach. We show a negative feedback mechanism, involving Snf1 and Reg1, which reduces SUC2 expression after the initial strong activation. Snf1 kinase activity plays a key role in this feedback mechanism. Our systems biology approach proposes a negative feedback mechanism that works through the SNF1 complex and is controlled by energy levels. We further show that Reg1 likely is involved in the negative feedback mechanism.

Salinity Effects on Sugar Homeostasis and Vascular Anatomy in the Stem of the Arabidopsis Thaliana Inflorescence.

The regulation of sugar metabolism and partitioning plays an essential role for a plant’s acclimation to its environment, with specific responses in autotrophic and heterotrophic organs. In this work, we analyzed the effects of high salinity on sugar partitioning and vascular anatomy within the floral stem. Stem sucrose and fructose content increased, while starch reduced, in contrast to the response observed in rosette leaves of the same plants. In the stem, the effects were associated with changes in the expression of SWEET and TMT2 genes encoding sugar transporters, SUSY1 encoding a sucrose synthase and several FRK encoding fructokinases. By contrast, the expression of SUC2, SWEET11 and SWEET12, encoding sugar transporters for phloem loading, remained unchanged in the stem. Both the anatomy of vascular tissues and the composition of xylem secondary cell walls were altered, suggesting that high salinity triggered major readjustments of sugar partitioning in this heterotrophic organ. There were changes in the composition of xylem cell walls, associated with the collapse and deformation of xylem vessels. The data are discussed regarding sugar partitioning and homeostasis of sugars in the vascular tissues of the stem.

Invertase Suc2-mediated inulin catabolism is regulated at the transcript level in Saccharomyces cerevisiae.

BackgroundInvertase Suc2 was recently identified as a key hydrolase for inulin catabolism in Saccharomyces cerevisiae, whereas the Suc2 activity degrading inulin varies greatly in different S. cerevisiae strains. The molecular mechanism causing such variation remained obscure. The aim of this study is to investigate how Suc2 activity is regulated in S. cerevisiae.ResultsThe effect of SUC2 expression level on inulin hydrolysis was investigated by introducing different SUC2 genes or their corresponding promoters in S. cerevisiae strain BY4741 that can only weakly catabolize inulin. Both inulinase and invertase activities were increased with the rising SUC2 expression level. Variation in the promoter sequence has an obvious effect on the transcript level of the SUC2 gene. It was also found that the high expression level of SUC2 was beneficial to inulin degradation and ethanol yield.ConclusionsSuc2-mediated inulin catabolism is regulated at transcript level in S. cerevisiae. Our work should be valuable for engineering advanced yeast strains in application of inulin for ethanol production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0243-3) contains supplementary material, which is available to authorized users.

The repressor Rgt1 and the cAMP-dependent protein kinases control the expression of the SUC2 gene in Saccharomyces cerevisiae

BackgroundA low level of glucose is required for maximal transcription of the SUC2 gene in Saccharomyces cerevisiae. Although the repressor Rgt1 binds the SUC2 promoter in gel-shift assays, it has been reported that Rgt1 has only minimal effects on SUC2 expression. Rgt1 acts together with Mth1 to repress the HXT genes encoding glucose transporters, and the release of Rgt1 from some HXT promoters requires cAMP-dependent protein kinase (PKA) activity. MethodsThe genes RGT1 and MTH1 have been disrupted and the SUC2 promoter modified in several S. cerevisiae backgrounds. Yeast cells were grown in different carbon sources in the presence or absence of 0.1 or 2% glucose, and invertase was assayed in whole cells. ResultsGalactose, glycerol or ethanol hindered invertase induction by low glucose, but lactate did not. During growth in lactate, deletion of RGT1 or MTH1 caused a marked increase in invertase levels, and elimination of the Rgt1-binding site in the SUC2 promoter caused also invertase induction. PKA activity decreased invertase levels in cells growing in lactate, and increased them during growth in lactate+0.1% glucose. ConclusionsThe low level of expression of SUC2 in the absence of glucose is mainly due to repression by the Rgt1-Mth1 complex. Repression is dependent on PKA activity, but not on any specific Tpk isoenzyme. General significanceThe results show that previously overlooked regulatory elements, such as Rgt1 and Tpks, participate in the control of SUC2 expression in S.cerevisiae.

Effect of exogenous gibberellin on reserve accumulation during the seed filling stage of oilseed rape

Exogenous gibberellins (GAs) are widely applied to increase crop yields, with knowledge about the physiological functioning and biochemistry mechanisms of these phytohormones improving; however, information remains limited about the effect of GAs on seed filling. In this study, the siliques (containing the seeds) of oilseed rape (Brassica napus L.) were treated with GA3 at 3 stages of seed filling. We confirmed that GA3 regulates the deposition of storage reserves in developing seeds. The percentage of crude fat in the seeds increased during the early stage, but remained stable during the middle and late stages. In comparison, the percentage of total protein decreased during the early and middle stages, but significantly increased during the late stage. In addition, Q-PCR was employed to analyze the expression level of related genes in response to GA3. It was found that the expression of WRI and ABI3 transcription factors corresponded to crude fat content and total protein content, respectively. The expression of storage reserve related genes DGAT, MCAT, SUC2, and GPT was consistent with crude fat content, whereas the expression of Napin corresponded to total protein content. The results of this study indicate that exogenous GA3 has a different effect on storage reserve deposition in seed during different stages of seed filling, and the effect might be achieved via changing the expression of related genes.

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter

Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipid-derived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1IN promoter (FBA1 sequence of -826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (-831 to -1). The transcriptional enhancement by the 5'-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC+ phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC+ strains, our engineered stains secreted most of invertase into the medium.

Alterations at Dispersed Sites Cause Phosphorylation and Activation of SNF1 Protein Kinase during Growth on High Glucose

The SNF1/AMP-activated protein kinases are central energy regulators in eukaryotes. SNF1 of Saccharomyces cerevisiae is inhibited during growth on high levels of glucose and is activated in response to glucose depletion and other stresses. Activation entails phosphorylation of Thr(210) on the activation loop of the catalytic subunit Snf1 by Snf1-activating kinases. We have used mutational analysis to identify Snf1 residues that are important for regulation. Alteration of Tyr(106) in the αC helix or Leu(198) adjacent to the Asp-Phe-Gly motif on the activation loop relieved glucose inhibition of phosphorylation, resulting in phosphorylation of Thr(210) during growth on high levels of glucose. Substitution of Arg for Gly(53), at the N terminus of the kinase domain, increased activation on both high and low glucose. Alteration of the ubiquitin-associated domain revealed a modest autoinhibitory effect. Previous studies identified alterations of the Gal83 (β) and Snf4 (γ) subunits that relieve glucose inhibition, and we have here identified a distinct set of Gal83 residues that are required. Together, these results indicate that alterations at dispersed sites within each subunit of SNF1 cause phosphorylation of the kinase during growth on high levels of glucose. These findings suggest that the conformation of the SNF1 complex is crucial to maintenance of the inactive state during growth on high glucose and that the default state for SNF1 is one in which Thr(210) is phosphorylated and the kinase is active.

Increased biomass production of industrial bakers' yeasts by overexpression of Hap4 gene

HAP4 encodes a transcriptional activator of respiration-related genes and so, redirection from fermentation to respiration flux should give rise to an increase in biomass production in Saccharomyces cerevisiae transformants that overexpress HAP4. With this aim, three bakers' yeasts, that is, V1 used for lean doughs, its 2-deoxy-D-glucose resistant derivative DOG21, and V3 employed for sweet doughs, were transformed with integrative cassettes that carried HAP4 gene under the control of constitutive promoter pTEF2; in addition VTH, DTH and 3TH transformants were selected and characterized. Transformants showed increased expression of HAP4 and respiration-related genes such as QCR7 and QCR8 with regard to parental, and similar expression of SUC2 and MAL12; these genes are relevant in bakers' industry. Invertase (Suc2p) and maltase (Mal12p) activities, growth and sugar consumption rates in laboratory (YPD) or industrial media (MAB) were also comparable in bakers' strains and their transformants, but VTH, DTH and 3TH increased their final biomass production by 9.5, 5.0 and 5.0% respectively as compared to their parentals in MAB. Furthermore, V1 and its transformant VTH had comparable capacity to ferment lean doughs (volume increase rate and final volume) while V3 and its transformant 3TH fermented sweet doughs in a similar manner. Therefore transformants possessed increased biomass yield and appropriate characteristics to be employed in bakers' industry because they lacked drug resistant markers and bacterial DNA, and were genetically stable.

An RT-qPCR approach to study the expression of genes responsible for sugar assimilation during rehydration of active dry yeast

A short reactivation period in aqueous media is required for active dry yeast (ADY) to be utilised in winemaking. Rehydration restores the active metabolic conditions necessary for good fermentative and competitive abilities. We used a reverse transcription-quantitative PCR (RT-qPCR) method with relative quantification to investigate the expression of seven hexose transporter genes ( HXT1-7) and one invertase-encoding gene ( SUC2) during ADY rehydration in water with or without sucrose. For this, seven candidate reference genes were evaluated, and the three most stably expressed genes were selected and used for mRNA normalisation. The results show that, during the rehydration in the presence of sucrose, yeast cells are able to immediately hydrolyse this sugar into glucose and fructose as soon as they are introduced in the medium. Subsequently, differential glucose/fructose uptake occurs, which is mediated by hexose transporters. At the transcriptomic level, there is a strong induction of the high-affinity transporters, HXT2 and HXT4, and the low-affinity transporters, HXT3 and HXT1, when ADY is rehydrated with sucrose, while HXT5 and HXT6/7 are expressed at high levels with a moderate tendency to decrease. In water, the HXT2 gene was the only one of the transporter genes studied that showed significant variations. These results suggest that during rehydration, expression is not simply regulated by the affinity to hexose but is also controlled by other mechanisms that allow the cell to bypass glucose control. Moreover, the expression of SUC2 showed little variation in media with sucrose, suggesting that other invertases and/or posttranscriptional controls exist.

Steady-state analysis of glucose repression reveals hierarchical expression of proteins under Mig1p control in Saccharomyces cerevisiae.

Glucose repression is a global transcriptional regulatory mechanism commonly observed in micro-organisms for the repression of enzymes that are not essential for glucose metabolism. In Saccharomyces cerevisiae, Mig1p, a homologue of Wilms' tumour protein, is a global repressor protein dedicated to glucose repression. Mig1p represses genes either by binding directly to the upstream repression sequence of structural genes or by indirectly repressing a transcriptional activator, such as Gal4p. In addition, some genes are repressed by both of the above mechanisms. This raises a fundamental question regarding the physiological relevance of the varied mechanisms of repression that exist involving Mig1p. We address this issue by comparing two well-known glucose-repression systems, that is, SUC2 and GAL gene expression systems, which encompass all the above three mechanisms. We demonstrate using steady-state analysis that these mechanisms lead to a hierarchical glucose repression profile of different family of genes. This switch over from one carbon source to another is well-calibrated as a function of glucose concentration through this hierarchical transcriptional response. The mechanisms prevailing in this repression system can achieve amplification and sensitivity, as observed in the well-characterized MAPK (mitogen-activated protein kinase) cascade system, albeit through a different structure. A critical feature of repression predicted by our steady-state model for the mutant strain of S. cerevisiae lacking Gal80p agrees well with the data reported here as well as that available in the literature.

Gln3p and Nil1p regulation of invertase activity and expression in

In Saccharomyces cerevisiae, sensing and signalling pathways regulate gene expression in response to quality of carbon and nitrogen sources. One such system, the target of rapamycin (Tor) proteins, senses nutrients and uses the GATA activators Gln3p and Nil1p to regulate translation in response to low-quality carbon and nitrogen. The signal transduction, triggered in response to nitrogen nutrition that is sensed by the Tor proteins, operates via a regulatory pathway involving the cytoplasmic factor Ure2p. When carbon and nitrogen are abundant, the phosphorylated Ure2p anchors the also phosphorylated Gln3p and Nil1p in the cytoplasm. Upon a shift from high- to low-quality nitrogen or treatment with rapamycin all three proteins are dephosphorylated, causing Gln3p and Nil1p to enter the nucleus and promote transcription. The genes that code for yeast periplasmic enzymes with nutritional roles would be obvious targets for regulation by the sensing and signalling pathways that respond to quality of carbon and nitrogen sources. Indeed, previous results from our laboratory had shown that the GATA factors Gln3p, Nil1p, Dal80p, Nil2p and also the protein Ure2 regulate the expression of asparaginase II, coded by ASP3. We also had observed that the activity levels of the also periplasmic invertase, coded by SUC2, were 6-fold lower in ure2 mutant cells in comparison to wild-type cells collected at stationary phase. These results suggested similarities between the signalling pathways regulating the expression of ASP3 and SUC2. In the present work we showed that invertase levels displayed by the single nil1 and gln3 and by the double gln3nil1 mutant cells, cultivated in a sucrose-ammonium medium and collected at the exponential phase, were 6-, 10- and 60-fold higher, respectively, in comparison to their wild-type counterparts. RT-PCR data of SUC2 expression in the double-mutant cells indicated a 10-fold increase in the mRNA(SUC2) levels.

Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae

The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.

Gts1p stabilizes oscillations in energy metabolism by activating the transcription of TPS1 encoding trehalose-6-phosphate synthase 1 in the yeast Saccharomyces cerevisiae.

We reported previously that Gts1p regulates oscillations of heat resistance in concert with those of energy metabolism in continuous cultures of the yeast Saccharomyces cerevisiae by inducing fluctuations in the levels of trehalose, but not in those of Hsp104 (heat shock protein 104). Further, the expression of TPS1, encoding trehalose-6-phosphate synthase 1, and HSP104 was activated by Gts1p in combination with Snf1 kinase, a transcriptional activator of glucose-repressible genes, in batch cultures under derepressed conditions. Here we show that, in continuous cultures, the mRNA level of TPS1 increased 6-fold in the early respiro-fermentative phase, while that of HSP104 did not change. The expression of SUC2, a representative glucose-repressible gene encoding invertase, also fluctuated, suggesting the involvement of the Snf1 kinase in the periodic activation of these genes. However, this possibility was proven to be unlikely, since the oscillations in both TPS1 and SUC2 mRNA expression were reduced by approx. 3-fold during the transient oscillation in gts1Delta (GTS1-deleted) cells, in which the energy state determined by extracellular glucose and intracellular adenine nucleotide levels was comparable with that in wild-type cells. Furthermore, neither the mRNA level nor the phosphorylation status of Snf1p changed significantly during the oscillation. Thus we suggest that Gts1p plays a major role in the oscillatory expression of TPS1 and SUC2 in continuous cultures of Saccharomyces cerevisiae, and hypothesized that Gts1p stabilizes oscillations in energy metabolism by activating trehalose synthesis to facilitate glycolysis at the shift from the respiratory to the respiro-fermentative phase.

A Short-range Gradient of Histone H3 Acetylation and Tup1p Redistribution at the Promoter of the Saccharomyces cerevisiae SUC2 Gene

Chromatin immunoprecipitation assays are used to map H3 and H4 acetylation over the promoter nucleosomes and the coding region of the Saccharomyces cerevisiae SUC2 gene, under repressed and derepressed conditions, using wild type and mutant strains. In wild type cells, a high level of H3 acetylation at the distal end of the promoter drops sharply toward the proximal nucleosome that covers the TATA box, a gradient that become even steeper on derepression. In contrast, substantial H4 acetylation shows no such gradient and extends into the coding region. Overall levels of both H3 and H4 acetylation rise on derepression. Mutation of GCN5 or SNF2 lead to substantially reduced SUC2 expression; in gnc5 there is no reduction in basal H3 acetylation, but large reductions occur on derepression. SNF2 mutation has little effect on H3 acetylation, so SAGA and SWI/SNF recruitment seem to be independent events. H4 acetylation is little affected by either GCN5 or SNF2 mutation. In a double snf2/gcn5 mutant (very low SUC2 expression), H3 acetylation is at the minimal level, but H4 acetylation remains largely unaffected. Transcription is thus linked to H3 but not H4 acetylation. Chromatin immunoprecipitation assays show that Tup1p is evenly distributed over the four promoter nucleosomes in repressed wild type cells but redistributes upstream on derepression, a movement probably linked to its conversion from a repressor to an activator.

Non-Histone Proteins Nhp6A and Nhp6B Are Required for the Regulated Expression of SUC2 Gene of Saccharomyces cerevisiae

Transcription of the SUC2 gene that encodes invertase enzyme is controlled by glucose repression and derepression mechanisms in Saccharomyces cerevisiae. Several regulatory factors such as Mig1p complex, Gcr1p, Hxk2p, nucleosomes, and the Snf1p kinase complex have been identified as the regulators of SUC2 transcription. The results presented in this study indicate that the non-histone proteins Nhp6A and Nhp6B were also required for the regulated expression of SUC2 gene. Expression of the SUC2 gene reduced to one-fiftieth–one-tenth in the Δnhp6A Δnhp6B double mutant strain depending on the growth conditions. Moreover, SUC2 expression and invertase synthesis became constitutive after long-term derepression, and decreased to a low level in Δnhp6A Δnhp6B double deletion mutant. A time course analysis of the invertase synthesis revealed that both the repression and derepression rates were very slow in the Δnhp6A Δnhp6B double mutant yeast. These results indicate that the architectural transcription factors Nhp6A and Nhp6B play a very critical role in the regulation of SUC2 gene expression.

Isolation of AtSUC2 promoter-GFP-marked companion cells for patch-clamp studies and expression profiling.

K+ channels control K+ homeostasis and the membrane potential in the sieve element/companion cell complexes. K+ channels from Arabidopsis phloem cells expressing green fluorescent protein (GFP) under the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RT-PCR. Single green fluorescent protoplasts were selected after being isolated enzymatically from vascular strands of rosette leaves. Companion cell protoplasts, which could be recognized by their nucleus, vacuole and chloroplasts, and by their expression of the phloem-specific marker genes SUC2 and AHA3, formed the basis for a cell-specific cDNA library and expressed sequence tag (EST) collection. Although we used primers for all members of the Shaker K+ channel family, we identified only AKT2, KAT1 and KCO6 transcripts. In addition, we also detected transcripts for AtPP2CA, a protein phosphatase, that interacts with AKT2/3. In line with the presence of the K+ channel transcripts, patch-clamp experiments identified distinct K+ channel types. Time-dependent inward rectifying K+ currents were activated upon hyperpolarization and were characterized by a pronounced Ca2+-sensitivity and inhibition by protons. Whole-cell inward currents were carried by single K+-selective channels with a unitary conductance of approximately 4 pS. Outward rectifying K+ channels (approximately 19 pS), with sigmoidal activation kinetics, were elicited upon depolarization. These two dominant phloem K+ channel types provide a versatile mechanism to mediate K+ fluxes required for phloem action and potassium cycling.

Mutations in GCR1 affect SUC2 gene expression in Saccharomyces cerevisiae.

Transcription of SUC2, the gene that encodes the cytoplasmic and secreted forms of the enzyme invertase, is controlled by glucose repression and derepression mechanisms in Saccharomyces cerevisiae. Several regulatory factors such as the Mig1p-Tup1p-Ssn6p repressor complex and the Snf1p kinase complex have been identified previously as regulators of SUC2 expression. We show that, in addition to these factors, expression of SUC2 is affected by mutations in the gene GCR1 that encodes the glycolysis regulatory protein Gcr1p. Expression of Suc2-LacZ was not repressed by glucose in gcr1 mutant yeast cells exposed to glucose. Furthermore, secreted invertase activity was constitutively expressed under glucose-repressed and derepressed conditions in gcr1 mutants. DNA gel mobility shift assays and in-vitro DNase I protection experiments mapped a DNA binding site for Gcr1p in the transcriptional control region of the SUC2 gene, next to a previously mapped Mig1p binding site. However, the mechanism by which gcr1 mutations relieve glucose repression remains obscure.

Interaction of the repressors Nrg1 and Nrg2 with the Snf1 protein kinase in Saccharomyces cerevisiae.

The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C(2)H(2) zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

Functional interaction between GCN5 and polyamines: a new role for core histone acetylation.

Polyamines are organic polycations essential for a wide variety of cellular functions, including nuclear integrity and chromosome condensation. Here we present genetic evidence that depletion of cellular polyamines partially alleviates the defects in HO and SUC2 expression caused by inactivation of the GCN5 histone acetyltransferase. In addition, the combination of polyamine depletion and a sin(-) allele of the histone H4 gene leads to almost complete bypass of the transcriptional requirement for GCN5. In contrast, polyamine depletion does not alter the transcriptional requirements for the SWI/SNF chromatin remodeling complex nor does depletion lead to global defects in transcriptional regulation. In addition to these genetic studies, we show that polyamines facilitate oligomerization of nucleosomal arrays in vitro, and that polyamine-mediated condensation requires intact core histone N-terminal domains and is inhibited by histone hyperacetylation. Our studies suggest that polyamines are repressors of transcription in vivo, and that one role of histone hyperacetylation is to antagonize the ability of polyamines to stabilize highly condensed states of chromosomal fibers.