Multiple Mechanisms Of Transcriptional Regulation Of The GAL System In Saccharomyces Cerevisiae

Yeast galactose metabolism genes are induced by galactose, and based on different external carbon sources there are three modes in gene expression:when on glucose, the GAL switch is repressed by various inhibition components, such as Migl,on the other hand, the transcription activator Gal4 also binds the UAS sequence with the transcription inhibitor Gal80 in its transcriptional activation (TAD); when on the glycerol/lactate or raffinose as carbon source in non-inducing conditions, the repression of the glucose-caused is relieved, but the GAL switch is remain turned off because the transcriptional activity of Gal4 is inhibited by Gal80;when on galactose as the carbon source, galactose, ATP, and the signal sensing protein Gal3 can relieve the inhibition of Gal80 in transcription activator Gal4,and at this time various transcription co-activator and RNA polymerase can be recruited on the promoter of the gal genes by the expose of Gal4-TAD. This induction is fast and efficient, within a few minutes the GAL mRNAs are up to 1000 times than in the non-inducing condition. Above all, transcription of galactose-inducible genes in yeast is regulated by the prototypical transcription activator Gal4p by interactions with Gal80,and during the GAL genes transcription, it has been also demonstrated that Gal4p becomes phosphorylated upon activation by polâ…¡-associated kinases. Among the identified sites of phosphorylation within Gal4p, the most notable ones are serine 699 (S699) and serine 837 (S837),and S699 and S837 phosphorylations have been shown not to be required for Gal4p activity but as a consequence of activation in its transcriptional activity. Moreover, the stability of transcriptionally active forms of Gal4p reflected by S699 phosphorylation has been demonstrated to be regulated by a F box protein Dsgl and on non-inducing condition the phosphorylation of Gal4 in 837 serine is regulated by another F-box, Grr1. In the absence of Dsg1, ubiquitin-mediated destruction of transcription-coupled Gal4p and productive GAL mRNA synthesis was compromised, thus a Dsgl-meiated proteolysis-dependent mode of regulating Gal4p activity was proposed. On the other hand, non-proteolytic mode of regulation of Gal4p has also been reported to participate in regulating the formation of activator-promoter complexes to limit the number rounds of transcription without continued responsiveness to environmental signals.petite mutant also referred to as a small colony, is a mutant yeast. In the sixties and seventies, researchers found some petites defect in utilization of galactose, maltose and other carbon sources, and speculated that there must be some signals that from mitochondria responsible for this phenotype. Dsgl/Mdm30 targeted for Gal4 degradation in inducing condition has been originally shown to associate with mitochondria and to be required for maintenance of fusion-competent mitochondria. Deletion of Dsgl leads to aggregated mitochondria, loss of mtDNA and a failure to respire. Besides the selective degradation of Fzo1 (GTPase) associated with the mitochondrial outer membrane fusion is also by Dsg1. In this respect, some unidentified signal(s) derived from mitochondria have been reported to be involved in the regulation of GAL gene expression. But the correlation with the galactose metabolism and mitochondrial dysfunction are not clear at the moment. About 95% of the energy needed for cell life activity is produced in mitochondria. While In the galactose metabolism, ATP directly involved in regulation of GAL gene expression that on the other hand implies the regulation role of mitochondrial function in the GAL system.; NADPH is an important coenzyme not only involved in the biological synthesis reaction is responsible for providing reducing power, and also has an important role in maintaining resistance to reactive oxygen species toxic to cell survive. And recently it has been reported that the cofactors NAD (H)/NADP (H) can directly bind the transcription inhibitor Gal80 and as a result regulate the interaction of Gal4 and Gal80:the binding of NAD (H) in Gal80 can stabilize Gal4-Gal80 relations while the binding of NADP (H) in Gal80 can destabilize the relationship between the Gal4-Gal80 facilitating GAL genes expression. As the mitochondrion is the main place to provide the reducing power NADPH, the level of intracellular NAD (H)/NADP (H) in specific regulation of GAL gene expression may have relationship with motochondria.Saccharomyces cerevisiae is the most simple single cell model, it has readily manipulable genetic system. Since many cellular processes such as protein folding and quality control system, membrane trafficking, and cellular stress responses are highly conserved in many fundamental aspects between human and budding yeast, pathophysiological processes of quite a few neurodegenerative disorders including Creutzfeldt-Jacob, Parkinson's disease and cancer have been modeled in yeast and useful insights regarding the fundamental mechanisms have thus been gained in the past decades. At the same time, Saccharomyces cerevisiae is also one of the three major model organisms in molecular biology. This thesis work on the galactose metabolic regulation on the basis of a systematic study on the transcriptional activator Gal4, and interaction of Gal4-Gal80 was carried out to exploring the possible mechanism in the regulation of the GAL. In this paper, The major results of the thesis are as follows:1. By analyzing different gene deletion strains and site-directed mutagenesis, or part deletion mutations of the Gal4, exploring the role of the alterations in the interaction between GAL4 and GAL80 in regulation of the yeast GAL regulon mediated by the F box protein Dsg1To test the role of S699 phosphorylation in Dsgl-mediated GAL gene expression, transcriptional activities of Gal4 mutants bearing changes of S699 or S837 to glutamate were analyzed. Similar to wild-type Gal4p, productive activation of transcription by Gal4 S699E, S837E as well as a double mutant (S699E S837E) was severely impaired with deletion of dsgl, though a relative higher level of reporter gene expression driven by these mutants was observed as compared with wild-type Gal4 under both non-inducing and inducing conditions in the presence of Dsg1, This results suggests that S699 phosphorylation is only correlated with, but not required for, the Dsg1-mediated regulation of GAL gene activation; In contrast to previous results, accumulation of multi-ubiquitylated Gal4p was not affected by the absence of dsgl in response to galactose. Moreover, the induction defect with deletion of dsg1 was partly rescued by deletion of dnml, whose simultaneous absence has been shown to rescue the defect in mitochondria fusion caused by defective dsgl,and our results revealed that the dsg1-deleted strain displayed a slow induction phenotype and effects the early-onset expression of GAL genes in response to galactose. While the requirement for Dsg1 can be suppressed by defective GAL80 as well as by GAL4 mutations effecting Gal4p-Gal80p interaction. Also change of lysine 23 to arginine in Gal4p DBD results in a weakened interaction between Gal4p and Gal80p which may partly bypass the requirement for Dsg1 at the immediate early stage of induction.2. By constructing and analyzingâ–³rip1 andâ–³atp1 gene deletion strains, the mechanism of energy state in regulation of the GAL systemIn order to assess the importance of cellular energetic status on the induction of GAL regulon in response to galactose, aâ–³rip1 strain which lacks the functional respiratory chain, and anâ–³atp1 strain which carries a nonfunctional ATP synthase were constructed. The intracellular ATP concentration in both strains drastically decreased after the glucose-to-galactose switch. Productive activation of transcription by Gal4p as measured by the endogenous GAL1 mRNA as well as the GAL1-LacZ reporter gene expression was almost abolished in both strains in response to galactose. The lack of transcription upon induction was further found to correlate with the drastically reduced distribution of RNA polymeraseâ…¡(polâ…¡) across the GAL1 gene. These results indicate that maintenance of an appropriate level of intracellular energy charge is essential for the yeast cells to rapidly turn on the GAL genes through enabling the efficient Gal3p-Gal80p interaction and adapt to a new carbon substrate.3. By constructing and analyzingâ–³pos5 andâ–³utr1 strains exploring the effect the mechanism of intracellular NAD (H)/NADP (H) levels in regulation GAL gene expressionTo address the role of the NAD(H)/NADP(H) in regulation of the GAL system, we constructed yeasts deleted for the two major NAD kinase genes, pos5 and utr1, whose products play a critical role in determining the levels of NADP in mitochondria and cytoplasm respectively. The coding sequence of Nma2 which participates in a nuclear salvage way of NAD synthesis was also deleted as a control. The intracellular levels of both NADP and NADPH dropped dramatically in pos5 deleted strain and were slightly reduced in utr1 deleted strain. Analysis of the GAL1 transcription as well as the GAL1-LacZ reporter gene expression revealed that, in contrast to deletion of nma2, transcriptional activation in response to galactose did not occur in pos5 mutant and was severely impaired in utr1 deletion strain. Similar to ATP synthesis mutants, distribution of RNA polymeraseâ…¡(polâ…¡) across the GAL1 gene was drastically reduced in both mutants. To ask whether other gene targets might be affected in these mutants includingâ–³rip1 andâ–³atp1, we examined both the transcription of and polymerase density across two other genes, ADH1 and PMA1. This analysis showed that neither the transcription nor the appearance of polâ…¡across the genes was affected in these deletion mutants under both uninducing and inducing conditions. To further test the effect of changes in levels of NADP(H) on the ability of yeasts to use galactose as a carbon source, yeasts cells were spotted onto plates containing various concentrations of hydrogen peroxide. In contrast to deletion of pos5 which virtually eliminated the yeast growth on galactose, yeasts deleted for utr1 are still able to use galactose as a carbon source. Notably, the potential nonspecific, toxic effect of H2O2 is not responsible for the inability of yeast to grow on galactose media because all strains grew well on glucose media. These results suggest that an appropriate level of intracellular NADP(H) is critical for the yeast cells to effciently turn on the GAL genes.4. Overexpression of Gal3p or Gal4p mutants with altered Gal80p-binding characteristics restores GAL gene expression in mutants deficient in ATP or NADPH synthesisAnalysis of RNA species corresponding to the 3'ends of the GAL3 gene or GAL80 gene revealed that, although the modest induction of both genes did not occur inâ–³rip1 andâ–³pos5 cells in response to galactose as did in wild-type, the expression levels of GAL3 and GAL80 in these yeasts were not affected under non-inducing conditions. Together with previous reports, the results indicated that the perturbed GAL switch due to the disabled dual feedback loops is not responsible for the inability of yeast to respond to galactose induction. To probe further into the possibility that the normal dissociation of Gal80p from Gal4p might be affected upon induction in mutant yeasts, we asked whether destabilizing Gal80p-Gal4p interaction rescues the defective GAL gene expression. In contrast to the induction defect displayed by pos5 or rip1 deletion in GAL80+ cells, induction of a GAL1-LacZ reporter in pos5 or rip1-deleted strains simultaneously lacking Gal80p (gal80-) was as efficient as in wild-type yeast. Moreover, activation of a GAL1-LacZ reporter gene was largely recovered after galactose induction in the mutant cells overexpressing Gal3p. Similar results were also observed for mutant cells expressingâ–³683 or K23R, both of which have been shown to have a weakened interaction with Gal80p. This rescued activation of expression was consistent with the increased distribution of the polâ…¡across the GAL1 gene. These results demonstrate that cellular ATP and NADP(H) are involved in regulating the interaction between Gal4p and Ga80p.5. Gal3p synergizes with NADPH to dissociate Gal80p from DNA-bound Gal80p-Gal4p complexThe results above implicate that the defect in GAL gene activation in ATP or NADP(H) synthesis mutant is to a larger extent caused by the failure of Gal80p to dissociate from Gal4p. Either Gal3p or NADP(H) has been shown to destabilize the Gal80p-Gal4p interaction. To address whether NADP(H) synergizes with Gal3p in the destabilization of DNA-bound Gal4p-Gal80p complex, we performed the following experiment. A preformed DNA-Gal4p-Gal80p complex with recombinant miniGal4p and Gal80p bound to oligonucleotides containing three consensus Gal4p binding sites was incubated either with NADP alone or with Gal3p in the presence of ATP and galactose. The amount of Gal80p retained by biotin-conjugated DNA was determined by Western blot. Our results reveales that, in accordance with previous results, NADP alone caused a marked decrease in the retention of Gal80p by the DNA-bound Gal4p. In contrast, such a destabilizing effect did not occur with the addition of NAD. The preformed complex was further incubated with varying concentrations of NADP in the presence of a constant concentration of Gal3p (1 nM) plus galactose and ATP. The dissociation of Gal80p occurred at a lower concentration of NADP. Similarly, a more efficient dissociation of Gal80p from the complex was achieved in the presence of NADP with one half of the amount required for the same dissociation by Gal3p alone. On the basis of these results, we suggest that NADP(H) synergizes with ATP-Gal3p in assuring the efficient dissociation of Gal80p from Gal4p in response to galactose induction. Because the association of Gal80p with Gal4p on the promoter of GAL gene under noninducing conditions and rapid dissociation from Gal4p in response to galactose is critical for the GAL gene transcription regulation, we asked whether the association of Gal80p with Gal4p is changed in vivo with cellular ATP or NADP(H) shortage after the addition of galactose. In vivo chromatin immunoprecipitation (Chip) was used to monitor the extent of the association Gal4p and Gal80p with the UASGAL of the GAL1 promoter. In wild-type cells, the association of Gal80p with the promoter decreased dramatically upon galactose induction whereas there was almost no change for the occupancy by Gal4p before and after galactose addition. In rip1â–³or pos5â–³cells, however, the Gal80p association with the UASGAL was only slightly decreased in response to galactose.The failure of rip1â–³or pos5â–³yeasts to achieve the dissociation of Gal80p from Gal4p demonstrates that ATP and NADP(H) are physiological relevant in playing a role in regulating transcriptional activation of GAL genes in response to the change of carbon sources. Analysis of the GAL1 mRNA revealed that the efficient transcription of Gal4p target genes in the absence of dsgl was severely impaired and was correlated with increased association of Gal80p with the promoter when Adsgl cells were induced with galactose under microanaerobic conditions. These results suggest that the incomplete disscociation of Gal80p as compared with that in wild-type strain is sufficient for the transcriptional activation in the absence of dsgl. Considering that the induction defect in dsg1-null was rescued by Gal4 mutants with decreased interaction with Gal80p, we asked whether the dissociation of Gal80p as well as the distribution of Ser5-phosphorylated polâ…¡are restored inâ–³dsg1 cells by Gal4p K23R. In accordance with previous results, while Ser5-phosphorylated CTD of polâ…¡was dramatically reduced within the GAL1 ORF in the absence of Dsgl, robust Ser5 phosphorylation was restored by Gal4p K23R. Importantly, the dissociation of Gal80p from the promoter occurred as efficiently as in wild-type strain upon galactose addition. These results again provide evidence that the efficient dissociation of Gal80p from Gal4p not only represents a key step in determining the on-off of transcription of GAL genes in response to galactose addition, but also suggest that the appropriate spatial distribution of Gal80p relative to Gal4p may also be involved in the fine tuning of the whole transcription process including the formation of mature mRNAs.