New Components of a System for Phosphate Accumulation and Polyphosphate Metabolism in Saccharomyces cerevisiae Revealed by Genomic Expression Analysis

Phm1 through 4 proteins clearly play a role in polyP synthesis. The apparently paradoxical increase in the cell’s ability to convert Pi into polyP in response to Pi starvation might represent a strategy for accumulating and holding precious Pi. We therefore measured Pi uptake in the phm1-phm4 mutants (Figure8).

– presumably, under non-starved conditions, cells don’t worry about Pi and therefore only take how much they need (reflected in their metabolism as quickly saturated). When starved, it seems that the strategy is to scavenge and store. This behavior of converting cytoplasmic Pi to polyPi and store them in the vacuole sounds counterintuitive at first, but makes sense if the cells are expecting the bad conditions to get worse (less and less Pi to scavenge from the environment). How interesting!

via New Components of a System for Phosphate Accumulation and Polyphosphate Metabolism in Saccharomyces cerevisiae Revealed by Genomic Expression Analysis.

Mutations in the Pho2 Bas2 Transcription Factor That Differentially Affect Activation with Its Partner Proteins Bas1, Pho4, and Swi5

Introduction –

“The yeast PHO2 gene also known as BAS2 orGRF10 encodes a homeodomain transcription factor that activates transcription in a combinatorial manner. It appears that Pho2 does not act alone, but with several partner proteins. By itself, Pho2 binds DNA with low affinity 4 and comparison of in vitrobinding sites reveals a relatively nonspecific consensus, ATTA or TAAT 4-6, similar to the consensus for Drosophila homeodomain proteins 2.

Pho2 binds DNA cooperatively with the Pho4 helix-loop-helix factor to activate expression of the PHO5 acid phosphatase 7. Similarly, Pho2 and the Swi5 zinc finger protein bind cooperatively to the HO promoter and contribute to its transcriptional activation 4, 8, 9. Bas1, an myb-like transcription factor, works with Pho2 in the activation of a set of biosynthetic genes, includingHIS4, ADE1, and ADE5,7 5, 10-12.In vitro studies have not shown cooperative DNA binding between Bas1 and Bas2 13, however, these experiments were conducted with in vitro expressed proteins that may not reflect their native state in vivo 14. Additionally, two-hybrid analyses detect an interaction between Pho2 and each of its partners, Pho4, Bas1, and Swi5 9, 15-17, and Pho2 Bas1 interaction has been demonstrated by co-immunoprecipitation 18.”

PHO2 with Bas1–

“Almost twenty genes require the combination of Bas1 and Pho2 for expression, including genes required for metabolism of histidine, purine, and pyrimidine nucleotides and one-carbon units (5, 10-12). The HIS4 gene is activated by one of two pathways, either by Pho2 in combination with Bas1 or by Gcn4 acting alone (5). The ADE5,7 gene is activated by Bas1 and Pho2 but does not require Gcn4 (10,28). TheHIS4 and ADE5,7 genes also differ in their dependence on Pho2 in an assay based on activation by Bas1-VP16 (17);HIS4 expression is strongly dependent on Pho2, whereasADE5,7 exhibits a moderate requirement. Several otherADE genes, typified by ADE1, have only a weak dependence on Pho2 (17).”

Discussion–

“Although SwiS and Ace2 show many parallels, they regulate different genes in vivo (8). Swi5 is an activator of HO, whereas Ace2 cannot activate HO unless ACE2 is overexpressed. Similarly, Ace2 can function as an activator of the chitinase gene CTSJ, but Swi5 cannot. The fact that Swi5 and Ace2 can both bind to the HO and CTSI promoters (unpublished observations) leads to the question of what prevents the cross regulation of CTSI by Swi5 and HO by Ace2.

We believe that the GrflO protein may play a critical role in determining the promoter specificity of Swi5. Grf1O may interact only with SwiS at the HO promoter, thus providing the specificity for Swi5 action at HO. This is supported by experimental observations indicating that Grf1O cannot bind cooperatively with Ace2 to the HO promoter and cannot bind at all to the CTSI promoter (P. R. Dohrmann, R.M.B., and D.J.S., unpublished observations). Another factor may also be present in cells that interacts specifically with Ace2 in binding to the CTS1 promoter, and this protein may provide the specificity for Ace2 regulation of CTSJ. Therefore, the cooperative interaction of the zinc-finger DNA-binding domain protein, SwiS, and the homeodomain DNA-binding protein, Grf1O, may provide the specificity required for the proper transcriptional regulation of the HO gene.”

via Mutations in the Pho2 Bas2 Transcription Factor That Differentially Affect Activation with Its Partner Proteins Bas1, Pho4, and Swi5.

## Another paper pointed out that PHO2 also binds to TRP4 promoter. But the potential role of PHO2 there is less clear — it seems to compete with GCN4 for one of the two UAS sites that the latter binds to. The authors tested several conditions X mutant combinations, and only when the cells were both starved for amino acid and phosphate will there be a reduction in the levels of activation of TRP4 gene expression.

Comparative genomics of the environmental stress response in ascomycete fungi – Gasch – 2007 – Yeast – Wiley Online Library

In contrast to the genes induced in the ESR, which participate in a wide variety of functions, most of the genes repressed in these programmes are directly involved in protein synthesis

via Comparative genomics of the environmental stress response in ascomycete fungi – Gasch – 2007 – Yeast – Wiley Online Library.

Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq

…… the majority 69% of strong STARR-seq enhancers were accessible DHS enrichment P ≤ 0.05, binomial test; Fig. 3A and all weak enhancers showed above random DHS enrichment on average fig. S18, suggesting that they are active in their endogenous contexts. Interestingly however, 604 (31%) strong STARR-seq enhancers were not accessible “closed” (Fig. 3A) and occurred next to genes (e.g., the Hox TFs, fig. S19) expressed at significantly lower levels compared to genes next to open STARR-seq enhancers 25-fold difference (P < 2.2×10^-16, Wilcoxon rank-sum test; Fig. 3B). Open and closed enhancers both function in luciferase assays and show the same linear correlation between STARR-seq and luciferase signals fig. S20, suggesting that sequences with enhancer potential can be silenced in their endogenous contexts, presumably at the chromatin level. Indeed, in contrast to open STARR-seq enhancers, closed enhancers are not marked by H3K27ac, a histone modification associated with active enhancers, but lie in broad domains of repressive H3K27me3 Fig. 3A and fig. S19, suggestive of Polycomb-mediated repression 17 or a poised enhancer state 18. Importantly, both open and closed enhancers are marked to similar extents by H3K4me1, which labels enhancers irrespective of their activity 19, 20 Fig. 3A.

via Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq.

Acid Phosphatases of Budding Yeast as a Model of Choice for Transcription Regulation Research

• Analysis of almost 6200 yeast genes revealed 22 genes whose expression is sharply increased for the lack of phosphate. This gene group was designated as PHO-regulon.
• enzymes of phosphate metabolism include the following: isozymes of the nonspecific acid phosphatase (AP), which provide detachment of phosphate group from the phosphate-containing organic compounds in medium; transport proteins, that is, permeases with different phosphate affinity; alkaline phosphatases; polyphosphatases; polyphosphate kinases; also enzymes with phytase activity
• yeast S. cerevisiae synthesize three isozymes of acid phosphatases designated as AP1, AP2, and AP3. AP1 is synthesized constitutively, while others are repressed by high phosphate concentration. AP1 is encoded by gene PHO3 of S. Cerevisiae; it hydrolyses different phosphate-containing substrates in periplasmic space (thiamine pyrophosphates, in particular). Repressible AP2 and AP3, encoded by genes PHO5 and PHO10,11, respectively, are synthesized in conditions of low phosphate only.
• The central role in the regulation of signalling cascades in yeast is executed by nutrients, while in higher eukaryotic cells, the same role is played by hormones and growth factors.
• under the purine starvation conditions, TF Bas1p and Pho4p compete for Pho2p binding that provides the coordinated regulation of nucleotides biosynthesis andP_i uptake in cell.

via Acid Phosphatases of Budding Yeast as a Model of Choice for Transcription Regulation Research.

The Unfolded Protein Response Is Necessary but Not Sufficient to Compensate for Defects in Disulfide Isomerization

The Unfolded Protein Response Is Necessary but Not Sufficient to Compensate for Defects in Disulfide Isomerization.

[Motivation]

• In a previous work, the authors found a puzzling phenomenon: in a mutant strain that carries a truncated PDI protein which is capable of forming disulfide bond but not its isomerization has wild-type growth rate. More than that, this strain, when combined with multiple knock-outs of all potential proteins with isomerase activity can still grow at wildtype rate.
• This work is set to find out how the cells compensate for the compromised isomerase activity

[Results]

• DTT treatment (inducing UPR) results in up-regulation of PDI1 promoter, and the fold-induction is higher in the PDIa’ mutant than in WT strains, presumably to compensate for the partially functional protein.
• Some UPR genes were also up-regulated in PDIa’ mutant strain as compared to wildtype, but not all UPR genes.
• Non-essential UPR genes, including IRE1 and HAC1 became essential in PDIa’ mutant.
• A dSLAM (Diploid synthetic lethality analysis) analysis identified the following number of synthetical lethals with either PDIa’ or tsPDIa’ (the latter produces a 2-fold stronger phenotype when grown in 30C) — from ~6000 genes used in the test, they identified different number of genes in the three sets of comparisons, which may be attributable to both experimental false-positive and -negative as well as potential biological differences due to the difference in phenotype severity between PDIa’ and tsPDIa’

• Focusing on the 34 hits shared by all three experiments and 96 additional shared by the two comparisons involving tsPDIa’, the total number to be looked at further amounts to 130
• “A large proportion (55%) of the clustered genes participate in some aspect of vesicle trafficking, not only into and out of the ER but involving essentially every cellular compartment where ER-synthesized proteins are directed”
• “The induction of the unfolded protein response is required to compensate for defects in disulfide isomerization. However, there is only a small amount of overlap between the 130 genes identified by dSLAM in these experiments and the 381 genes that display expression patters that have been used to define the unfolded protein response based on their HAC1- and IRE1-dependent induction when protein folding in the ER is disturbed by reduction (DTT) or inhibition of glycosylation (tunicamycin) (20). Only 10 of the 130 genes we have identified here are considered part of the canonical UPR.”
• “When the isomerase activity of Pdi1p is compromised in the wtPDIa′ truncation mutant, the initial appearance of FM4-64 in the vacuole may be somewhat slower (Table 4). With the tsPDIa′ mutant, the appearance of FM4-64 in the vacuole is ~3-fold slower than that supported by wild-type Pdi1p (Table 4). Thus, the defect in disulfide isomerization or formation introduced early in the ER by replacing Pdi1p with tsPDIa′ results in a slower trafficking of FM4-64 into the vacuole.”

 

dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae

We have recently developed a technology called dSLAM (heterozygous diploid-based Synthetic Lethality Analysis on Microarrays) that exploits heterozygous diploid YKOs to detect genome-wide synthetic lethality (8). This methodology combines the excellent genetic quality of the heterozygote diploid YKOs, the convenience of handling them in pooled form, and the efficiency of a microarray analysis of abundance of YKOs in the population. The ‘d’ in ‘dSLAM’ also highlights the fact that both the control (single mutant) and experimental (double mutant) pools are derived from the same molecularly manipulated heterozygote diploid pool, which alleviates experimental noises introduced by two separate transformations necessary for a haploid SLAM experiment (9).

Here for the collection: http://www.thermoscientificbio.com/EktronTemplates/ProductLayout.aspx?id=17179927371&terms=yko

via dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae.

Evolutionary Dynamics of Gene and Isoform Regulation in Mammalian Tissues

To explore this connection to phosphorylation, we used Scansite (23) to predict phosphorylation sites in peptides encoded by different subsets of exons. Tissue-specific alternatively spliced exons, including both the conserved and nonconserved subsets, contained about 40% more predicted phosphorylation sites than other classes of exons (Fig. 4B and fig. S9). A comparable degree of enrichment for phosphorylation sites was observed in these exons with the curated PhosphoSite database (24) of experimentally determined phosphorylation sites (Fig. 4B). Phosphorylation site density in exons was correlated with phylogenetic breadth of alternative splicing (Fig. 4C and fig. S10). These observations suggest that tissue-specific alternative splicing is often used to alter the potential for protein phosphorylation, which can alter protein stability, enzymatic activity, subcellular localization, and other properties.

via Evolutionary Dynamics of Gene and Isoform Regulation in Mammalian Tissues.

Pre-Disposition and Epigenetics Govern Variation in Bacterial Survival upon Stress

In the case of ampicillin ‘persisters’, a sub-population of bacteria transiently enter a dormant state in a non-stressing environment and can thus survive ampicillin treatment that attacks only growing cells [14]. Such persister cells pay a cost to express a phenotype which is less fit in the current environment but more fit for a particular environmental change. This example was interpreted as a bet-hedging strategy anticipating the arrival of future stress conditions. Yet whether it is beneficial to apply a bet-hedging strategy depends on the phenotypic switching rate, the time scale of environmental change and the fitness cost [15], or on rather stochastic events inherent to cellular physiology rather than resulting from a positive evolutionary fitness gain. In our observations, there is no sign of a pre-disposition factor working to hedge phenotypic bets. Higher stress responses prior to induction do not prime our cells for the stress to come. Instead, cells with relatively higher basal RpoH transcriptional activity are more likely to give rise to more stressed progeny (Figure S11, S12, S13, S14). This suggests that under non-stressed conditions, cells with a higher basal stress level may be paying a cost which will not help them to survive the upcoming stress. It is the weaker cells that simply suffer more, while fitter cells prevail, suggesting a simple performance-based selection.

via Pre-Disposition and Epigenetics Govern Variation in Bacterial Survival upon Stress.

PLOS Genetics: Contrasting Properties of Gene-Specific Regulatory, Coding, and Copy Number Mutations in Saccharomyces cerevisiae: Frequency, Effects, and Dominance

we examine the spectrum of mutations affecting a focal gene in Saccharomyces cerevisiae by characterizing 231 novel haploid genotypes with altered activity of a fluorescent reporter gene. 7% of these genotypes had a nonsynonymous mutation in the coding sequence for the fluorescent protein and were classified as “coding” mutants; 2% had a change in the S. cerevisiae TDH3 promoter sequence controlling expression of the fluorescent protein and were classified as “cis-regulatory” mutants; 10% contained two copies of the reporter gene and were classified as “copy number” mutants; and the remaining 81% showed altered fluorescence without a change in the reporter gene itself and were classified as “trans-acting” mutants. As a group, coding mutants had the strongest effect on reporter gene activity and always decreased it. By contrast, 50%–95% of the mutants in each of the other three classes increased gene activity, with mutants affecting copy number and cis-regulatory sequences having larger median effects on gene activity than trans-acting mutants. When made heterozygous in diploid cells, coding, cis-regulatory, and copy number mutant genotypes all had significant effects on gene activity, whereas 88% of the trans-acting mutants appeared to be recessive.

Figure 3. Effects on YFP fluorescence in haploid cells differ among mutational classes.
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The effects on YFP fluorescence of the 4 cis-regulatory (black), 16 coding (green), 22 CNV (red), and 179 trans-acting (blue) mutants are summarized in histograms. For each mutational class, the height of each bar indicates the number of mutants with the corresponding effect (as measured by Z-score) on YFP fluorescence in haploid cells. Positive Z-scores indicate increases in YFP fluorescence relative to control cells and negative Z-scores indicate decreases in YFP fluorescence relative to control cells. The relative frequency of mutants in each of the four mutational classes is also shown in the inset pie chart.

via PLOS Genetics: Contrasting Properties of Gene-Specific Regulatory, Coding, and Copy Number Mutations in Saccharomyces cerevisiae: Frequency, Effects, and Dominance.