Suppression of inositol pyrophosphate toxicosis and hyper-repression of the fission yeast PHO regulon by loss-of-function mutations in chromatin remodelers Snf22 and Sol1.

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA control of pho1 mRNA synthesis is influenced by inositol pyrophosphate (IPP) kinase Asp1, deletion of which results in pho1 hyper-repression. A forward genetic screen for ADS (Asp1 Deletion Suppressor) mutations identified the 14–3–3 protein Rad24 as a governor of phosphate homeostasis. Production of full-length interfering prt lncRNA was squelched in rad24Δ cells, concomitant with increased production of pho1 mRNA and increased Pho1 activity, while shorter precociously terminated non-interfering prt transcripts persisted. Epistasis analysis showed that pho1 de-repression by rad24Δ depends on: (i) 3′-processing and transcription termination factors CPF, Pin1, and Rhn1; and (ii) Threonine-4 of the Pol2 CTD. Combining rad24Δ with the IPP pyrophosphatase-dead asp1-H397A allele caused a severe synthetic growth defect that was ameliorated by loss-of-function mutations in CPF, Pin1, and Rhn1, and by CTD phospho-site mutations T4A and Y1F. Rad24 function in repressing pho1 was effaced by mutation of its phosphate-binding pocket. Our findings instate a new role for a 14–3–3 protein as an antagonist of precocious RNA 3′-processing/termination.

The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Actin and actin‐related proteins (Arps) are critical components of chromatin remodelling and modifying complexes, where they are commonly found in pairs. Yet, little is known about their role in these complexes. In this issue of The EMBO Journal , Fenn et al (2011) report the first structural investigation of two nuclear Arps, the INO80 complex subunits Arp4 and Arp8. Importantly, their results also shed light on the critical role that the nucleotide state of actin may have on its interactions with the Arps.

Inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8 are eukaryal signaling molecules that influence cell physiology, especially phosphate homeostasis. In fission yeast, 1,5-IP8 and 1-IP7 impact gene expression by acting as agonists of RNA 3'-processing and transcription termination. 1,5-IP8 is synthesized by position-specific kinases Kcs1 and Asp1 that convert IP6 to 5-IP7 and 5-IP7 to 1,5-IP8, respectively. Inositol pyrophosphatase enzymes Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase) are agents of inositol pyrophosphate catabolism in fission yeast. Whereas Asp1, Siw14, and Aps1 are individually inessential, double pyrophosphatase mutants asp1-H397A aps1∆ and siw14∆ aps1∆ display severe growth defects caused by overzealous 3'-processing/termination. By applying CE-ESI-MS to profile the inositol pyrophosphate content of fission yeast mutants in which inositol pyrophosphate toxicity is genetically suppressed, we elucidated the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Asp1, which exclusively cleaves the 1-β-phosphate, and Aps1, which prefers to cleave the 1-β-phosphate, play essential overlapping roles in guarding against the accumulation of toxic levels of 1-IP7. Aps1 and Siw14 together catabolize the inositol-5-pyrophosphates, and their simultaneous inactivation results in overaccumulation of 5-IP7. Cells lacking all three pyrophosphatases amass high levels of 1,5-IP8 and 1-IP7, with concomitant depletion of IP6. A genetic screen identified three missense mutations in the catalytic domain of Kcs1 kinase that suppressed inositol-1-pyrophosphate toxicosis. The screen also implicated the 3'-processing factor Swd22, the inositol pyrophosphate sensor Spx1, and the nuclear poly(A)-binding protein Nab2 as mediators of inositol-1-pyrophosphate toxicity.IMPORTANCEInositol pyrophosphates are key effectors of eukaryal cellular phosphate homeostasis. They are synthesized by kinases that add a β-phosphate to the 5- or 1-phosphate groups of IP6 and catabolized by three classes of pyrophosphatases that hydrolyze the β-phosphates of 5-IP7, 1-IP7, or 1,5-IP8. Whereas the fission yeast inositol pyrophosphatases-Asp1 (histidine acid phosphatase), Siw14 (cysteinyl phosphatase), and Aps1 (Nudix hydrolase)-are inessential for growth, Asp1/Aps1 and Aps1/Siw14 double mutations and Asp1/Siw14/Aps1 triple mutations elicit severe or lethal growth defects. By profiling the inositol pyrophosphate content of pyrophosphatase mutants in which this toxicity is genetically suppressed, we reveal the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Their synergies are manifested as excess accumulation of 1-IP7 upon dual inactivation of Asp1 and Aps1 or an excess of 5-IP7 in aps1∆ siw14∆ cells. In the absence of all three pyrophosphatases, cells accrue high levels of 1,5-IP8 and 1-IP7 while IP6 declines.

Fission yeast phosphate homeostasis genes are repressed in phosphate-rich medium by transcription of upstream lncRNAs that interferes with activation of the flanking mRNA promoters. lncRNA control of PHO gene expression is influenced by the Thr4 phospho-site in the RNA polymerase II CTD and the 3′ processing/termination factors CPF and Rhn1, mutations of which result in hyper-repression of the PHO regulon. Here, we performed a forward genetic screen for mutations that de-repress Pho1 acid phosphatase expression in CTD-T4A cells. Sequencing of 18 independent STF (Suppressor of Threonine Four) isolates revealed, in every case, a mutation in the C-terminal pyrophosphatase domain of Asp1, a bifunctional inositol pyrophosphate (IPP) kinase/pyrophosphatase that interconverts 5-IP7 and 1,5-IP8. Focused characterization of two STF strains identified 51 coding genes coordinately upregulated vis-à-vis the parental T4A strain, including all three PHO regulon genes (pho1, pho84, tgp1). Whereas these STF alleles—asp1-386(Stop) and asp1-493(Stop)—were lethal in a wild-type CTD background, they were viable in combination with mutations in CPF and Rhn1, in which context Pho1 was also de-repressed. Our findings implicate Asp1 pyrophosphatase in constraining 1,5-IP8 or 1-IP7 synthesis by Asp1 kinase, without which 1-IPPs can accumulate to toxic levels that elicit precocious termination by CPF/Rhn1.

Bacterial infections are a leading cause of global health loss. The prerequisites for bacteria to colonize and establish systemic infection in the host are environmental adaptability and the expression of virulence factors. Phosphorus constitutes the fifth most important element in terms of its cellular content, which is pivotal for DNA replication, metabolism, signal transmission, and microbial cell composition. The phosphate (Pho) regulon is a well-established unique mechanism in response to inorganic phosphate (Pi) starvation. The Pho regulon is strongly related to bacterial pathogenicity, except for the simple regulatory system for Pho balance. The PhoBR two-component regulatory system of the Pho regulon has documented the effects of affecting virulence in microbes. This review emphasizes the impact of the absence of PhoB and virulence-related gene regulation by PhoB on pathogenicity in common pathogens of Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, Vibrio cholerae, etc. Collectively, Pho regulon is a regulatory network connecting Pho homeostasis with bacterial virulence. This study may offer valuable information for understanding the regulation of bacterial virulence by the Pho regulon, providing novel insights for the development of antimicrobial strategies.

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes – pho1, pho84, and tgp1 – that are repressed under phosphate-replete conditions by 5′ flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor ‘precocious’ lncRNA 3′-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.

Many Proteobacteria possess the paralogous PTS(Ntr), in addition to the sugar transport phosphotransferase system (PTS). In the PTS(Ntr) phosphoryl-groups are transferred from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. The PTS(Ntr) has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS(Ntr) plays a role in potassium homeostasis. In particular, EIIA(Ntr) binds to and stimulates activity of a two-component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high-affinity K(+) transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS(Ntr). The pho regulon, which comprises more than 30 genes, is activated by the two-component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA(Ntr) are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild-type level in mutants deficient for EIIA(Ntr) phosphorylation suggesting that non-phosphorylated EIIA(Ntr) modulates pho. Protein interaction analyses reveal binding of EIIA(Ntr) to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA(Ntr) is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.

ABSTRACTInositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1–385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1’s kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro, by virtue of its antagonism of IP8 turnover.

Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

The fission yeast phosphate acquisition (PHO) regulon is repressed under phosphate-replete conditions by upstream lncRNA-mediated transcriptional interference. Inositol-1-pyrophosphates control phosphate homeostasis via their action as agonists of precocious PHO lncRNA 3'-processing/termination. Inositol pyrophosphatase-inactivating mutations that increase inositol-1-pyrophosphates elicit derepression of the PHO genes and a severe growth defect in YES medium. Previous studies demonstrated suppression of inositol pyrophosphate toxicosis by targeted deletion or loss-of-function mutations in the nonessential Ssu72, Ppn1, Swd22, and Ctf1 subunits of the fission yeast cleavage and polyadenylation factor (CPF) complex. Here we conducted a selection for spontaneous mutations that suppress the precocious PHO lncRNA termination underlying the sickness of asp1-STF pyrophosphatase mutants. We thereby recovered and characterized novel hypomorphic missense mutations in five essential CPF subunits: Ysh1 (the cleavage endonuclease), Pta1 (an armadillo/HEAT-repeat protein), Pfs2 (a WD repeat protein), Cft1 (a WD repeat protein), and Msi2 (a tandem RRM RNA-binding protein). The suppressor screen also yielded an intron branchpoint mutation in the gene encoding essential CPF subunit Iss1. In addition, we found that asp1-STF toxicosis was suppressed by a missense mutation in the active site of Pla1, the essential poly(A) polymerase subunit of CPF. Genetic crosses revealed a hierarchy of mutational synergies between the essential CPF subunits, the inessential CPF subunits, termination factor Rhn1, the Thr4 "letter" of the RNA polymerase II CTD code, and the Asp1 kinase that synthesizes inositol-1-pyrophosphates. The synthetic lethality of msi2-G252E with ctf1Δ, swd22Δ, ppn1Δ, ssu72-C13S, rpb1-CTD-T4A, and asp1Δ establishes Msi2 as a central agent of 3'-processing/termination, functioning in parallel to inositol-1-pyrophosphates.

In Caulobacter crescentus, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration. Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium. To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration. The disrupted genes were identified as homologues of the high-affinity phosphate transport genes pstSCAB of Escherichia coli. In E. coli, pst mutants have a constitutively expressed phosphate (Pho) regulon. To determine if stalk elongation is regulated by the Pho regulon, the Caulobacter phoB gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated. While phoB was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation. Both pstS and phoB mutants were deficient in phosphate transport. When a phoB mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium. Thus, the phenotypes of phoB and pst mutants were opposite. phoB mutants were unable to elongate stalks during phosphate starvation, whereas pst mutants made long stalks in both high- and low-phosphate media. Analysis of double pst phoB mutants indicated that the long-stalk phenotype of pst mutants was dependent on phoB. In addition, analysis of a pstS-lacZ transcriptional fusion showed that pstS transcription is dependent on phoB. These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB.