John M. Lopes

Autoregulated expression of the yeast INO2 and INO4 helix-loop-helix activator genes effects cooperative regulation on their target genes.

In the yeast Saccharomyces cerevisiae, the phospholipid biosynthetic genes are highly regulated at the transcriptional level in response to the phospholipid precursors inositol and choline. In the absence of inositol and choline (derepressing), the products of the INO2 and INO4 genes form a heteromeric complex which binds to a 10-bp element, upstream activation sequence INO (UASINO), in the promoters of the phospholipid biosynthetic genes to activate their transcription. In the presence of inositol and choline (repressing), the product of the OPI1 gene represses transcription dictated by the UASINO element. Curiously, we identified a UASINO-like element in the promoters of both the INO2 and INO4 genes. The presence of the UASINO element in these two promoters suggested that the mechanism for the inositol-choline response would involved regulating expression of the two activator genes. Using a cat reporter gene, we find that INO2-cat expression was regulated 12-fold in response to inositol and choline but that INO4-cat was constitutively expressed. We further observed that INO2-cat was not expressed in either an ino2 or an ino4 mutant strain and was constitutively overexpressed in an opi1 mutant strain. Expression of the INO4-cat gene was affected only by mutation in the INO4 gene itself. Therefore, INO2-cat transcription is regulated by the products of both the INO2 and INO4 genes whereas INO4 must interact with another protein to activate its own transcription. Our data show that derepression of phospholipid biosynthetic gene expression involves two mechanisms: increasing the levels of the INO2 and INO4 gene products and inactivating the OPI1-mediated repression mechanism. We propose a model suggesting that this dual mechanism of regulation accounts for the observed cooperative stimulation of IN01 and CH01 gene expression (phospholipids biosynthetic genes).

Functional characterization of the repeated UASINO element in the promoters of the INO1 and CHO2 genes of yeast

In yeast, INO1 and CHO2 gene expression is subject to repression in response to inositol and choline supplementation. The response by both genes to inositol is controlled by a single set of regulatory factors and the highly conserved and repeated UASINO element (consensus: 5′ CATGTGAAAT 3′) that is found in multiple copies in both promoters. However, none of the native elements found in the INO1 and CHO2 promoters constitutes an exact match to the consensus element and the functionality of individual elements from these two promoters has not been tested. In this study, the function of individual putative UASINO elements from both promoters was tested by placing promoter fragments into a reporter construct which lacked a UAS element but contained the TATA element and start of transcription from the yeast CYC1 gene fused to the Escherichia coli lacZ gene. In addition, a set of oligonucleotides containing the consensus UASINO element with the first position systematically modified was also tested for UASINO function. These studies indicated that elements that contain a C or an A as the first base at the 5′ end are functional to varying degrees. The majority of potential UASINO elements from the INO1 promoter were found to be inactive, whereas all of the elements from the CHO2 promoter tested were active. These results are discussed in light of the differential regulation of the two promoters.

Genomic Analysis of the Opi− Phenotype

Most of the phospholipid biosynthetic genes of Saccharomyces cerevisiae are coordinately regulated in response to inositol and choline. Inositol affects the intracellular levels of phosphatidic acid (PA). Opi1p is a repressor of the phospholipid biosynthetic genes and specifically binds PA in the endoplasmic reticulum. In the presence of inositol, PA levels decrease, releasing Opi1p into the nucleus where it represses transcription. The opi1 mutant overproduces and excretes inositol into the growth medium in the absence of inositol and choline (Opi− phenotype). To better understand the mechanism of Opi1p repression, the viable yeast deletion set was screened to identify Opi− mutants. In total, 89 Opi− mutants were identified, of which 7 were previously known to have the Opi− phenotype. The Opi− mutant collection included genes with roles in phospholipid biosynthesis, transcription, protein processing/synthesis, and protein trafficking. Included in this set were all nonessential components of the NuA4 HAT complex and six proteins in the Rpd3p–Sin3p HDAC complex. It has previously been shown that defects in phosphatidylcholine synthesis (cho2 and opi3) yield the Opi− phenotype because of a buildup of PA. However, in this case the Opi− phenotype is conditional because PA can be shuttled through a salvage pathway (Kennedy pathway) by adding choline to the growth medium. Seven new mutants present in the Opi− collection (fun26, kex1, nup84, tps1, mrpl38, mrpl49, and opi10/yol032w) were also suppressed by choline, suggesting that these affect PC synthesis. Regulation in response to inositol is also coordinated with the unfolded protein response (UPR). Consistent with this, several Opi− mutants were found to affect the UPR (yhi9, ede1, and vps74).

Expression of the INO2 regulatory gene of Saccharomyces cerevisiae is controlled by positive and negative promoter elements and an upstream open reading frame

The INO2 gene encodes a transcriptional activator of the phospholipid biosynthetic genes of Saccharomyces cerevisiae. Complete derepression of phospholipid biosynthetic gene expression in response to inositol/choline deprivation requires both INO2 and INO4. Ino2p dimerizes with Ino4p to bind the upstream activating sequence (UAS)INO element found in the promoters of the target genes. We have demonstrated previously that transcription from the INO2 promoter is autoregulated 12‐fold in a manner identical to that of the target genes. Here, we show that this regulation occurs at the levels of transcription and translation. Transcription accounts for fourfold regulation, whereas translation accounts for an additional threefold regulation. Regulation of transcription requires a UASINO element. Additional promoter elements include an upstream essential sequence (UES) located upstream of the UASINO element and a negative regulatory element in the vicinity of the UASINO element. Regulation of translation is dependent on an upstream open reading frame (uORF) in the INO2 leader. These data support the model that regulatory gene promoters may display unusual organizations and may be subject to multiple levels of regulation. We have shown previously that the UME6 gene positively regulates INO2 expression. Here, we limit the UME6‐responsive region of the INO2 promoter to nucleotides −217 to −56.

A pleiotropic drug resistance transporter is involved in reduced sensitivity to multiple fungicide classes in Sclerotinia homoeocarpa (F.T. Bennett)

Dollar spot, caused by Sclerotinia homoeocarpa, is a prevalent turfgrass disease, and the fungus exhibits widespread fungicide resistance in North America. In a previous study, an ABC-G transporter, ShatrD, was associated with practical field resistance to demethylation inhibitor (DMI) fungicides. Mining of ABC-G transporters, also known as pleiotropic drug resistance (PDR) transporters, from RNA-Seq data gave an assortment of transcripts, several with high sequence similarity to functionally characterized transporters from Botrytis cinerea, and others with closest blastx hits from Aspergillus and Monilinia. In addition to ShatrD, another PDR transporter showed significant over-expression in replicated RNA-Seq data, and in a collection of field-resistant isolates, as measured by quantitative polymerase chain reaction. These isolates also showed reduced sensitivity to unrelated fungicide classes. Using a yeast complementation system, we sought to test the hypothesis that this PDR transporter effluxes DMI as well as chemically unrelated fungicides. The transporter (ShPDR1) was cloned into the Gal1 expression vector and transformed into a yeast PDR transporter deletion mutant, AD12345678. Complementation assays indicated that ShPDR1 complemented the mutant in the presence of propiconazole (DMI), iprodione (dicarboximide) and boscalid (SDHI, succinate dehydrogenase inhibitor). Our results indicate that the over-expression of ShPDR1 is correlated with practical field resistance to DMI fungicides and reduced sensitivity to dicarboximide and SDHI fungicides. These findings highlight the potential for the eventual development of a multidrug resistance phenotype in this pathogen. In addition, this study presents a pipeline for the discovery and validation of fungicide resistance genes using de novo next-generation sequencing and molecular biology techniques in an unsequenced plant pathogenic fungus.

Expression of the Yeast PIS1 Gene Requires Multiple Regulatory Elements Including a Rox1p Binding Site

The PIS1 gene is required for de novo synthesis of phosphatidylinositol (PI), an essential phospholipid in Saccharomyces cerevisiae. PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline. Relatively little is known about regulation of transcription of the PIS1 gene. We reported previously that PIS1 transcription is sensitive to carbon source. To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1-3). Deletion of either UAS1 or UAS2 resulted in an ∼45% reduction in expression, whereas removal of UAS3 yielded an 84% decrease in expression. A comparison of promoters among several Saccharomyces species shows that these sequences are highly conserved. Curiously, the UAS3 element region (-149 to -138) includes a Rox1p binding site. Rox1p is a repressor of hypoxic genes under aerobic growth conditions. Consistent with this, we have found that expression of a PIS1-cat reporter was repressed under aerobic conditions, and this repression was dependent on both Rox1p and its binding site. Furthermore, PI levels were elevated under anaerobic conditions. This is the first evidence that PI levels are affected by regulation of PIS1 transcription.

Multiple Basic Helix-Loop-Helix Proteins Regulate Expression of the ENO1 Gene of Saccharomyces cerevisiae

ABSTRACT The basic helix-loop-helix (bHLH) eukaryotic transcription factors have the ability to form multiple dimer combinations. This property, together with limited DNA-binding specificity for the E box (CANNTG), makes them ideally suited for combinatorial control of gene expression. We tested the ability of all nine Saccharomyces cerevisiae bHLH proteins to regulate the enolase-encoding gene ENO1. ENO1 was known to be activated by the bHLH protein Sgc1p. Here we show that expression of an ENO1-lacZ reporter was also regulated by the other eight bHLH proteins, namely, Ino2p, Ino4p, Cbf1p, Rtg1p, Rtg3p, Pho4p, Hms1p, and Ygr290wp. ENO1-lacZ expression was also repressed by growth in inositol-choline-containing medium. Epistatic analysis and chromatin immunoprecipitation experiments showed that regulation by Sgc1p, Ino2p, Ino4p, and Cbf1p and repression by inositol-choline required three distal E boxes, E1, E2, and E3. The pattern of bHLH binding to the three E boxes and experiments with two dominant-negative mutant alleles of INO4 and INO2 support the model that bHLH dimer selection affects ENO1-lacZ expression. These results support the general model that bHLH proteins can coordinate different biological pathways via multiple mechanisms.

Analysis of Opi1p repressor mutants

Opi1p is the only known repressor protein specific to the phospholipid biosynthetic pathway. Opi1p is required for repression in response to inositol and choline supplementation. However, the mechanism of Opi1p repression is not completely understood. In part, this is because previously identified opi1 mutants contained nonsense mutations and thus provided little insight into the mechanism of Opi1p function. We have recently reported isolating novel opi1 mutants (rum and dim mutants) that contain missense mutations. Here, we show that these opi1 mutants produce Opi1p product at levels comparable to a wild-type strain. However, these mutants mis-regulate expression of two target genes, INO2-HIS3 and INO1-lacZ, and are also defective in autoregulation. An opi1-S339F mutant is particularly interesting because it completely eliminated autoregulation, but only abated regulation of an INO1-lacZ reporter. Two of the mutations in OPI1 (V343Q and S339F) provide genetic evidence for an interaction between Opi1p and the Ino2p activator since they reside in a region of Opi1p recently shown to interact with Ino2p in vitro. A third mutation (L252F) resides in a region of Opi1p with no known function.

Identification of novel dominant INO2 c mutants with an Opi - phenotype

The INO2 gene of Saccharomyces cerevisiae is required for derepression of the phospholipid biosynthetic genes in response to inositol depletion. Conversely, the OPI1 gene is required for repression in response to inositol supplementation. Results of an in vitro assay have led to a model in which Opi1p interacts with Ino2p. However, there is no in vivo evidence to support this model. Additionally, most of the previously isolated ino2 mutants offer little insight into this model. Here, we report the isolation of a new class of dominant mutations in the INO2 gene, which yield constitutive expression of a target gene (i.e. an Opi– mutant phenotype). Two mutations reside in a region of the Ino2p required for interaction with Opi1p in vitro. Three other mutations are at the amino‐terminus in a transcriptional activation domain.

The Promoter of the Yeast INO4 Regulatory Gene: a Model of the Simplest Yeast Promoter

In Saccharomyces cerevisiae, the phospholipid biosynthetic genes are transcriptionally regulated in response to inositol and choline. This regulation requires the transcriptional activator proteins Ino4p and Ino2p, which form a heterodimer that binds to the UAS(INO) element. We have previously shown that the promoters of the INO4 and INO2 genes are among the weakest promoters characterized in yeast. Because little is known about the promoters of weakly expressed yeast genes, we report here the analysis of the constitutive INO4 promoter. Promoter deletion constructs scanning 1,000 bp upstream of the INO4 gene identified a small region (-58 to -46) that is absolutely required for expression. S1 nuclease mapping shows that this region contains the transcription start sites for the INO4 gene. An additional element (-114 to -86) modestly enhances INO4 promoter activity (fivefold). Thus, the region required for INO4 transcription is limited to 68 bp. These studies also found that INO4 gene expression is not autoregulated by Ino2p and Ino4p, despite the presence of a putative UAS(INO) element in the INO4 promoter. We further report that the INO4 steady-state transcript levels and Ino4p levels are regulated twofold in response to inositol and choline, suggesting a posttranscriptional mechanism of regulation.