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Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Long noncoding RNAs (lncRNAs) transcribed across gene promoters have been detected. These regulate transcription by mechanisms that have not been fully elucidated. We herein show that the chromatin configuration is altered into an accessible state within 290 bp downstream from the initiation site of metabolic-stress-induced lncRNAs (mlonRNAs) in the promoter of the fission yeast fbp1 gene, whose transcription is massively induced upon glucose starvation. Chromatin upstream from fbp1 is progressively altered into an open configuration, as a cascade of transcription of three overlapping mlonRNA species (-a, -b and -c in order) occurs with transcriptional initiation sites progressing 5′ to 3′ upstream of the fbp1 promoter. Initiation of the shortest mlonRNA (mlonRNA-c) induces chromatin remodeling around a transcription factor-binding site and subsequent massive induction of fbp1. We identify the cis-element required for mlonRNA-c initiation, and by changing the distance between mlonRNA-initiation site and the transcription factor-binding site, we show that mlonRNA-initiation effectively induces chromatin remodeling in a limited distance within 290 bp. These results indicate that mlonRNAs are transcribed across the fbp1 promoter as a short-range inducer for local chromatin alterations, and suggest that strict chromatin modulation is archived via stepwise mlonRNA-initiations.

Transcription factors (TFs) determine the transcription activity of target genes and play a central role in controlling the transcription in response to various environmental stresses. Three dimensional genome structures such as local loops play a fundamental role in the regulation of transcription, although the link between such structures and the regulation of TF binding to cis-regulatory elements remains to be elucidated. Here, we show that during transcriptional activation of the fission yeast fbp1 gene, binding of Rst2 (a critical C2H2 zinc-finger TF) is mediated by a local loop structure. During fbp1 activation, Rst2 is first recruited to upstream-activating sequence 1 (UAS1), then it subsequently binds to UAS2 (a critical cis-regulatory site located approximately 600 base pairs downstream of UAS1) through a loop structure that brings UAS1 and UAS2 into spatially close proximity. Tup11/12 (the Tup-family corepressors) suppress direct binding of Rst2 to UAS2, but this suppression is counteracted by the recruitment of Rst2 at UAS1 and following delivery to UAS2 through a loop structure. These data demonstrate a previously unappreciated mechanism for the recruitment and expansion of TF-DNA interactions within a promoter mediated by local three-dimensional genome structures and for timely TF-binding via counteractive regulation by the Tup-family corepressors.

The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucose-sensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gα(s) gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC®(1280) library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.

Schizosaccharomyces pombe detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 Gα binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2 K270E -activated Gα relative to Gpa2 + Gα. Deletion of sck1 + or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2 + or Gpa2 K270E , suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2 K270E GDP-GTP exchange rate mutant, GTPase-defective Gpa2 R176H weakly binds Sck1 in the two-hybrid screen and a deletion of sck1 + in a Gpa2 R176H strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deleting sck1 + in a gpa2 Δ strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allow S. pombe to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.

Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High Throughput Screens (HTSs) for PDE inhibitors that possess ‘drug-like’ characteristics, displaying activity in a growth stimulation assay over a 48-hr period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme’s potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium in order to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify if different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.

The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Gα subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gβγ dimer to repress transcription of the glucose-regulated fbp1 + gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2 + chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Gα function, thus extending our understanding of Gα protein structure-function relationships.

The fission yeast Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway. Elevated cAMP levels activate protein kinase A (PKA) to inhibit transcription of genes involved in sexual development and gluconeogenesis, including the fbp1+ gene, which encodes fructose-1,6-bisphosphatase. Glucose-mediated activation of PKA requires the function of nine glucose-insensitive transcription (git) genes, encoding adenylate cyclase, the PKA catalytic subunit, and seven “upstream” proteins required for glucose-triggered adenylate cyclase activation. We describe the cloning and characterization of the git10+ gene, which is identical to swo1+ and encodes the S. pombe Hsp90 chaperone protein. Glucose repression of fbp1+ transcription is impaired by both git10− and swo1− mutant alleles of the hsp90+ gene, as well as by chemical inhibition of Hsp90 activity and temperature stress to wild-type cells. Unlike the swo1− mutant alleles, the git10-201 allele supports cell growth at 37°, while severely reducing glucose repression of an fbp1-lacZ reporter, suggesting a separation-of-function defect. Sequence analyses of three swo1− alleles and the one git10− allele indicate that swo1− mutations alter core functional domains of Hsp90, while the git10− mutation affects the Hsp90 central domain involved in client protein binding. These results suggest that Hsp90 plays a specific role in the S. pombe glucose/cAMP pathway.

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3',5'-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens.

Schizosaccharomyces pombe utilizes two opposing signaling pathways to sense and respond to its nutritional environment. Glucose detection triggers a cyclic AMP signal to activate protein kinase A (PKA), while glucose or nitrogen starvation activates the Spc1/Sty1 stress-activated protein kinase (SAPK). One process controlled by these pathways is fbp1 + transcription, which is glucose repressed. In this study, we isolated strains carrying mutations that reduce high-level fbp1 + transcription conferred by the loss of adenylate cyclase ( git2 Δ), including both wis1 − (SAPK kinase) and spc1 − (SAPK) mutants. While characterizing the git2 Δ suppressor strains, we found that the git2 Δ parental strains are KCl sensitive, though not osmotically sensitive. Of 102 git2 Δ suppressor strains, 17 strains display KCl-resistant growth and comprise a single linkage group, carrying mutations in the cgs1 + PKA regulatory subunit gene. Surprisingly, some of these mutants are mostly wild type for mating and stationary-phase viability, unlike the previously characterized cgs1 - 1 mutant, while showing a significant defect in fbp1-lacZ expression. Thus, certain cgs1 − mutant alleles dramatically affect some PKA-regulated processes while having little effect on others. We demonstrate that the PKA and SAPK pathways regulate both cgs1 + and pka1 + transcription, providing a mechanism for cross talk between these two antagonistically acting pathways and feedback regulation of the PKA pathway. Finally, strains defective in both the PKA and SAPK pathways display transcriptional regulation of cgs1 + and pka1 + , suggesting the presence of a third glucose-responsive signaling pathway.

The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six “upstream” genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Gα) while git5 encodes a Gβ subunit. Multicopy suppression studies with gpa2+ previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Gα followed by βγ. We show here that an activated allele of gpa2 (gpa2R176H, carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2R176H allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S. pombe.

The Schizosaccharomyces pombe pyp1+ gene, encoding a protein tyrosine phosphatase (pyp1), was isolated as a high copy number suppressor of a mutation that results in reduced cAMP-dependent protein kinase (PKA) activity. Overexpression of pyp1+ inhibits both transcription of the fbp1 gene, which is negatively regulated by a glucose-induced activation of PKA, and sexual development, which is negatively regulated by PKA through a nitrogen- and glucose-monitoring mechanism. Overexpression of a catalytically inactive form of pyp1 has little effect on either process. Previous studies suggest that overexpression of pyp1+ results in a mitotic delay by positively regulating wee1 activity. We show that pyp1 repression of fbp1 transcription is independent of wee1. The direct role of the pyp1 protein is to dephosphorylate and inactivate the sty1/spc1 mitogen-activated protein kinase (MAPK) that is activated by the wis1 MAPK kinase. As overexpression of pyp1+ has no further effect upon the mitotic delay observed in a wis1 deletion strain, the role of pyp1 appears to be restricted to negative regulation of the sty1/spc1 MAPK. This study indicates that pyp1 negatively regulates fbp1 transcription, sexual development and mitosis by inactivation of the sty1/spc1 MAPK, but that bifurcations downstream of the MAPK separate these processes as seen by the differential role for the wee1 gene.

We have cloned the his7+ gene of the fission yeast Schizosaccharomyces pombe by complementation of the recessive mutant allele his7-366. The his7+ gene is able to complement a mutation of the Escherichia coli hisI gene, suggesting that his7+ encodes a phosphoribosyl-AMP cyclohydrase. Subcloning experiments localize the gene to a 1.9-kb XbaI-BglII fragment. We describe the construction of plasmids to facilitate the use of his7+ as a selectable marker in S. pombe studies. Plasmid pEA2 carries his7+ cloned into the pUC18 polylinker. From either pEA2 or the original his7+ clone, pMN1, fragments carrying his7+ can be isolated using a variety of restriction enzymes for the construction of gene disruptions. Plasmid pEA500 is a cloning vector that carries his7+ and ars1, yet retains the ability to use the blue/white color screen to identify recombinants.

An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these ‘upstream’ git genes were proposed to act to produce a glucose-induced cAMP signal. We report here that assays of cAMP levels in wild-type and various mutant S. pombe cells, before and after exposure to glucose, show that this is the case. The data suggest that the cAMP signal results from the activation of adenylate cyclase. Therefore these ‘upstream’ git genes appear to encode a glucose-induced adenylate cyclase activation pathway. Assays of cAMP on a strain carrying a mutation in the git6 gene, which acts downstream of adenylate cyclase, indicate that git6 may function to feedback regulate adenylate cyclase activity. Thus git6 may encode a cAMP-dependent protein kinase.

Summary The biological roles of cyclic nucleotide phosphodiesterase 11 (PDE11) enzymes are poorly understood, in part due to the lack of selective inhibitors. To address the need for such compounds, we completed an ∼200,000 compound high-throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay, and identified 4 potent and selective PDE11 inhibitors. One compound, along with two structural analogs, elevates cAMP and cortisol levels in human adrenocortical cells, consistent with gene association studies that link PDE11 activity to adrenal function. As such, these compounds can immediately serve as chemical tools to study PDE11 function in cell culture, and as leads to develop therapeutics for the treatment of adrenal insufficiencies. Our results further validate this yeast-based HTS platform for the discovery of potent, selective, and biologically active PDE inhibitors.

We have constructed a series of plasmids containing a modified form of the phoA gene of Escherichia coli K-12 that have general utility for studies of protein secretion. In these plasmids, the promoter and signal sequence-encoding region of the phoA gene have been deleted; thus, expression of the gene, giving rise to active alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1], is absolutely dependent upon fusion in the correct reading frame to DNA containing a promoter, a translational start site, and a complete signal sequence-encoding region. Alkaline phosphatase, which is normally located in the periplasm of E. coli, is efficiently secreted to the periplasm when fused either to a signal sequence from another periplasmic protein, beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6), or to signal sequences from the outer membrane proteins LamB and OmpF. These heterologous signal sequences are processed during secretion. In the absence of a complete signal sequence, phosphatase becomes localized in the cytoplasm and is inactive. Phosphatase fusion proteins lacking up to 13 amino-terminal amino acids beyond the signal sequence show the same specific activity as that of the wild-type enzyme. However, a significant decrease in activity is seen when 39 or more amino-terminal amino acids are deleted. Addition of approximately 150 amino acids from the enzyme beta-lactamase to the amino terminus of alkaline phosphatase has little effect on the specific activity of the enzyme. The ability to change the amino terminus of phosphatase without altering its activity makes the enzyme particularly useful for construction of protein fusions. The fact that phosphatase is designed for transport across the cytoplasmic membrane makes it an ideal tool for study of protein secretion.