Replication stress inhibits synthesis of histone mRNAs in yeast by removing Spt10p and Spt21p from the histone promoters.

Proliferating cells coordinate histone and DNA synthesis to maintain correct stoichiometry for chromatin assembly. Histone mRNA levels must be repressed when DNA replication is inhibited to prevent toxicity and genome instability due to free non-chromatinized histone proteins. In mammalian cells, replication stress triggers degradation of histone mRNAs, but it is unclear if this mechanism is conserved from other species. The aim of this study was to identify the histone mRNA decay pathway in the yeast Saccharomyces cerevisiae and determine the mechanism by which DNA replication stress represses histone mRNAs. Using reverse transcription-quantitative PCR and chromatin immunoprecipitation-quantitative PCR, we show here that histone mRNAs can be degraded by both 5' → 3' and 3' → 5' pathways; however, replication stress does not trigger decay of histone mRNA in yeast. Rather, replication stress inhibits transcription of histone genes by removing the histone gene-specific transcription factors Spt10p and Spt21p from histone promoters, leading to disassembly of the preinitiation complexes and eviction of RNA Pol II from histone genes by a mechanism facilitated by checkpoint kinase Rad53p and histone chaperone Asf1p. In contrast, replication stress does not remove SCB-binding factor transcription complex, another activator of histone genes, from the histone promoters, suggesting that Spt10p and Spt21p have unique roles in the transcriptional downregulation of histone genes during replication stress. Together, our data show that, unlike in mammalian cells, replication stress in yeast does not trigger decay of histone mRNAs but inhibits histone transcription.

DNA damage response activates respiration and thereby enlarges dNTP pools to promote cell survival in budding yeast.

The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. Previously, we found that decreased histone expression induces mitochondrial respiration, raising the question whether the DDR also stimulates respiration. Here, using oxygen consumption and ATP assays, RT-qPCR and ChIP-qPCR methods, and dNTP analyses, we show that DDR activation in the budding yeast Saccharomyces cerevisiae, either by genetic manipulation or by growth in the presence of genotoxic chemicals, induces respiration. We observed that this induction is conferred by reduced transcription of histone genes and globally decreased DNA nucleosome occupancy. This globally altered chromatin structure increased the expression of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain, oxidative phosphorylation, elevated oxygen consumption, and ATP synthesis. The elevated ATP levels resulting from DDR-stimulated respiration drove enlargement of dNTP pools; cells with a defect in respiration failed to increase dNTP synthesis and exhibited reduced fitness in the presence of DNA damage. Together, our results reveal an unexpected connection between respiration and the DDR and indicate that the benefit of increased dNTP synthesis in the face of DNA damage outweighs possible cellular damage due to increased oxygen metabolism.

The proto-oncogene Bcl3 induces immune checkpoint PD-L1 expression, mediating proliferation of ovarian cancer cells.

The proto-oncogene Bcl3 induces survival and proliferation in cancer cells; however, its function and regulation in ovarian cancer (OC) remain unknown. Here, we show that Bcl3 expression is increased in human OC tissues. Surprisingly, however, we found that in addition to promoting survival, proliferation, and migration of OC cells, Bcl3 promotes both constitutive and interferon-γ (IFN)-induced expression of the immune checkpoint molecule PD-L1. The Bcl3 expression in OC cells is further increased by IFN, resulting in increased PD-L1 transcription. The mechanism consists of an IFN-induced, Bcl3- and p300-dependent PD-L1 promoter occupancy by Lys-314/315 acetylated p65 NF-κB. Blocking PD-L1 by neutralizing antibody reduces proliferation of OC cells overexpressing Bcl3, suggesting that the pro-proliferative effect of Bcl3 in OC cells is partly mediated by PD-L1. Together, this work identifies PD-L1 as a novel target of Bcl3, and links Bcl3 to IFNγ signaling and PD-L1-mediated immune escape.

Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression.

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4, the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1, which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level.

Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells.

Overexpression of the pro-angiogenic chemokine IL-8 (CXCL8) is associated with a poor prognosis in several solid tumors, including epithelial ovarian cancer (EOC). Even though histone deacetylase (HDAC) inhibition has shown remarkable antitumor activity in hematological malignancies, it has been less effective in solid tumors, including EOC. Here we report results that may explain the decreased efficiency of HDAC inhibition in EOC, based on our data demonstrating that HDAC inhibition specifically induces expression of IL-8/CXCL8 in SKOV3, CAOV3, and OVCAR3 cells. Suppression or neutralization of vorinostat-induced IL-8/CXCL8 potentiates the vorinostat inhibitory effect on cell viability and proliferation. The IL-8/CXCL8 expression induced by vorinostat in EOC cells is dependent on IκB kinase (IKK) activity and associated with a gene-specific recruitment of IKKß and IKK-dependent recruitment of p65 NFκB to the IL-8/CXCL8 promoter. In addition, HDAC inhibition induces acetylation of p65 and histone H3 and their IL-8/CXCL8 promoter occupancy. In vivo results demonstrate that combining vorinostat and the IKK inhibitor Bay 117085 significantly reduces tumor growth in nude mice compared with control untreated mice or either drug alone. Mice in the combination group had the lowest IL-8/CXCL8 tumor levels and the lowest tumor expression of the murine neutrophil [7/4] antigen, indicating reduced neutrophil infiltration. Together, our results demonstrate that HDAC inhibition specifically induces IL-8/CXCL8 expression in EOC cells and that the mechanism involves IKK, suggesting that using IKK inhibitors may increase the effectiveness of HDAC inhibitors when treating ovarian cancer and other solid tumors characterized by increased IL-8/CXCL8 expression.

Reciprocal Regulation of AMPK/SNF1 and Protein Acetylation.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) serves as an energy sensor and master regulator of metabolism. In general, AMPK inhibits anabolism to minimize energy consumption and activates catabolism to increase ATP production. One of the mechanisms employed by AMPK to regulate metabolism is protein acetylation. AMPK regulates protein acetylation by at least five distinct mechanisms. First, AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) and thus regulates acetyl-CoA homeostasis. Since acetyl-CoA is a substrate for all lysine acetyltransferases (KATs), AMPK affects the activity of KATs by regulating the cellular level of acetyl-CoA. Second, AMPK activates histone deacetylases (HDACs) sirtuins by increasing the cellular concentration of NAD⁺, a cofactor of sirtuins. Third, AMPK inhibits class I and II HDACs by upregulating hepatic synthesis of α-hydroxybutyrate, a natural inhibitor of HDACs. Fourth, AMPK induces translocation of HDACs 4 and 5 from the nucleus to the cytoplasm and thus increases histone acetylation in the nucleus. Fifth, AMPK directly phosphorylates and downregulates p300 KAT. On the other hand, protein acetylation regulates AMPK activity. Sirtuin SIRT1-mediated deacetylation of liver kinase B1 (LKB1), an upstream kinase of AMPK, activates LKB1 and AMPK. AMPK phosphorylates and inactivates ACC, thus increasing acetyl-CoA level and promoting LKB1 acetylation and inhibition. In yeast cells, acetylation of Sip2p, one of the regulatory ß-subunits of the SNF1 complex, results in inhibition of SNF1. This results in activation of ACC and reduced cellular level of acetyl-CoA, which promotes deacetylation of Sip2p and activation of SNF1. Thus, in both yeast and mammalian cells, AMPK/SNF1 regulate protein acetylation and are themselves regulated by protein acetylation.

Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells.

AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects.

Proteasome inhibition increases recruitment of IκB kinase ß (IKKß), S536P-p65, and transcription factor EGR1 to interleukin-8 (IL-8) promoter, resulting in increased IL-8 production in ovarian cancer cells.

Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase ß (IKKß) and an increased recruitment of the nuclear IKKß, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKß and with p65, with preferential binding to S536P-p65. Both IKKß activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKß and EGR-1 as potential new targets in ovarian cancer combination therapies.

Histone hypoacetylation-activated genes are repressed by acetyl-CoA- and chromatin-mediated mechanism.

Transcriptional activation is typically associated with increased acetylation of promoter histones. However, this paradigm does not apply to transcriptional activation of all genes. In this study we have characterized a group of genes that are repressed by histone acetylation. These histone hypoacetylation-activated genes (HHAAG) are normally repressed during exponential growth, when the cellular level of acetyl-CoA is high and global histone acetylation is also high. The HHAAG are induced during diauxic shift, when the levels of acetyl-CoA and global histone acetylation decrease. The histone hypoacetylation-induced activation of HHAAG is independent of Msn2/Msn4. The repression of HSP12, one of the HHAAG, is associated with well-defined nucleosomal structure in the promoter region, while histone hypoacetylation-induced activation correlates with delocalization of positioned nucleosomes or with reduced nucleosome occupancy. Correspondingly, unlike the majority of yeast genes, HHAAG are transcriptionally upregulated when expression of histone genes is reduced. Taken together, these results suggest a model in which histone acetylation is required for proper positioning of promoter nucleosomes and repression of HHAAG.

Protein acetylation and acetyl coenzyme a metabolism in budding yeast.

Cells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation.

Yeast phospholipase C is required for normal acetyl-CoA homeostasis and global histone acetylation.

Phospholipase C (Plc1p) is required for the initial step of inositol polyphosphate (InsP) synthesis, and yeast cells with deletion of the PLC1 gene are completely devoid of any InsPs and display aberrations in transcriptional regulation. Here we show that Plc1p is required for a normal level of histone acetylation; plc1Δ cells that do not synthesize any InsPs display decreased acetylation of bulk histones and global hypoacetylation of chromatin histones. In accordance with the role of Plc1p in supporting histone acetylation, plc1Δ mutation is synthetically lethal with mutations in several subunits of SAGA and NuA4 histone acetyltransferase (HAT) complexes. Conversely, the growth rate, sensitivity to multiple stresses, and the transcriptional defects of plc1Δ cells are partially suppressed by deletion of histone deacetylase HDA1. The histone hypoacetylation in plc1Δ cells is due to the defect in degradation of repressor Mth1p, and consequently lower expression of HXT genes and reduced conversion of glucose to acetyl-CoA, a substrate for HATs. The histone acetylation and transcriptional defects can be partially suppressed and the overall fitness improved in plc1Δ cells by increasing the cellular concentration of acetyl-CoA. Together, our data indicate that Plc1p and InsPs are required for normal acetyl-CoA homeostasis, which, in turn, regulates global histone acetylation.

IFNγ-Induced Bcl3, PD-L1 and IL-8 Signaling in Ovarian Cancer: Mechanisms and Clinical Significance.

IFNγ, a pleiotropic cytokine produced not only by activated lymphocytes but also in response to cancer immunotherapies, has both antitumor and tumor-promoting functions. In ovarian cancer (OC) cells, the tumor-promoting functions of IFNγ are mediated by IFNγ-induced expression of Bcl3, PD-L1 and IL-8/CXCL8, which have long been known to have critical cellular functions as a proto-oncogene, an immune checkpoint ligand and a chemoattractant, respectively. However, overwhelming evidence has demonstrated that these three genes have tumor-promoting roles far beyond their originally identified functions. These tumor-promoting mechanisms include increased cancer cell proliferation, invasion, angiogenesis, metastasis, resistance to chemotherapy and immune escape. Recent studies have shown that IFNγ-induced Bcl3, PD-L1 and IL-8 expression is regulated by the same JAK1/STAT1 signaling pathway: IFNγ induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in OC cells, resulting in their increased proliferation and migration. In this review, we summarize the recent findings on how IFNγ affects the tumor microenvironment and promotes tumor progression, with a special focus on ovarian cancer and on Bcl3, PD-L1 and IL-8/CXCL8 signaling. We also discuss promising novel combinatorial strategies in clinical trials targeting Bcl3, PD-L1 and IL-8 to increase the effectiveness of cancer immunotherapies.

Acetyl-CoA carboxylase regulates global histone acetylation.

Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA in the nucleocytosolic compartment. However, because nucleocytosolic acetyl-CoA is also used for de novo synthesis of fatty acids, histone acetylation and synthesis of fatty acids compete for the same acetyl-CoA pool. The first and rate-limiting reaction in de novo synthesis of fatty acids is carboxylation of acetyl-CoA to form malonyl-CoA, catalyzed by acetyl-CoA carboxylase. In yeast Saccharomyces cerevisiae, acetyl-CoA carboxylase is encoded by the ACC1 gene. In this study, we show that attenuated expression of ACC1 results in increased acetylation of bulk histones, globally increased acetylation of chromatin histones, and altered transcriptional regulation. Together, our data indicate that Acc1p activity regulates the availability of acetyl-CoA for histone acetyltransferases, thus representing a link between intermediary metabolism and epigenetic mechanisms of transcriptional regulation.

Hydroxyurea and inactivation of checkpoint kinase MEC1 inhibit transcription termination and pre-mRNA cleavage at polyadenylation sites in budding yeast.

The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. The transcription changes triggered by DDR depend on the nature of DNA damage, activation of checkpoint kinases, and the stage of cell cycle. The transcription changes can be localized and affect only damaged DNA, but they can be also global and affect genes that are not damaged. While the purpose of localized transcription inhibition is to avoid transcription of damaged genes and make DNA accessible for repair, the purpose and mechanisms of global transcription inhibition of undamaged genes are less well understood. We show here that a brief cell treatment with hydroxyurea (HU) globally inhibits RNA synthesis and transcription by RNA polymerase I, II, and III (RNAPI, RNAPII, and RNAPIII). HU reduces efficiency of transcription termination and inhibits pre-mRNA cleavage at the polyadenylation (pA) sites, destabilizes mRNAs, and shortens poly(A) tails of mRNAs, indicating defects in pre-mRNA 3' end processing. Inactivation of the checkpoint kinase Mec1p downregulates the efficiency of transcription termination and reduces the efficiency of pre-mRNAs clevage at the pA sites, suggesting the involvement of DNA damage checkpoint in transcription termination and pre-mRNA 3' end processing.

Tolerance to replication stress requires Dun1p kinase and activation of the electron transport chain.

One of the key outcomes of activation of DNA replication checkpoint (DRC) or DNA damage checkpoint (DDC) is the increased synthesis of the deoxyribonucleoside triphosphates (dNTPs), which is a prerequisite for normal progression through the S phase and for effective DNA repair. We have recently shown that DDC increases aerobic metabolism and activates the electron transport chain (ETC) to elevate ATP production and dNTP synthesis by repressing transcription of histone genes, leading to globally altered chromatin architecture and increased transcription of genes encoding enzymes of tricarboxylic acid (TCA) cycle and the ETC. The aim of this study was to determine whether DRC activates ETC. We show here that DRC activates ETC by a checkpoint kinase Dun1p-dependent mechanism. DRC induces transcription of RNR1-4 genes and elevates mtDNA copy number. Inactivation of RRM3 or SGS1, two DNA helicases important for DNA replication, activates DRC but does not render cells dependent on ETC. However, fitness of rrm3Δ and sgs1Δ cells requires Dun1p. The slow growth of rrm3Δdun1Δ and sgs1Δdun1Δ cells can be suppressed by introducing sml1Δ mutation, indicating that the slow growth is due to low levels of dNTPs. Interestingly, inactivation of ETC in dun1Δ cells results in a synthetic growth defect that can be suppressed by sml1Δ mutation, suggesting that ETC is important for dNTP synthesis in the absence of Dun1p function. Together, our results reveal an unexpected connection between ETC, replication stress, and Dun1p kinase.

Activated heme synthesis regulates glycolysis and oxidative metabolism in breast and ovarian cancer cells.

Heme is an essential cofactor for enzymes of the electron transport chain (ETC) and ATP synthesis in mitochondrial oxidative phosphorylation (OXPHOS). Heme also binds to and destabilizes Bach1, a transcription regulator that controls expression of several groups of genes important for glycolysis, ETC, and metastasis of cancer cells. Heme synthesis can thus affect pathways through which cells generate energy and precursors for anabolism. In addition, increased heme synthesis may trigger oxidative stress. Since many cancers are characterized by a high glycolytic rate regardless of oxygen availability, targeting glycolysis, ETC, and OXPHOS have emerged as a potential therapeutic strategy. Here, we report that enhancing heme synthesis through exogenous supplementation of heme precursor 5-aminolevulinic acid (ALA) suppresses oxidative metabolism as well as glycolysis and significantly reduces proliferation of both ovarian and breast cancer cells. ALA supplementation also destabilizes Bach1 and inhibits migration of both cell types. Our data indicate that the underlying mechanisms differ in ovarian and breast cancer cells, but involve destabilization of Bach1, AMPK activation, and induction of oxidative stress. In addition, there appears to be an inverse correlation between the activity of oxidative metabolism and ALA sensitivity. Promoting heme synthesis by ALA supplementation may thus represent a promising new anti-cancer strategy, particularly in cancers that are sensitive to altered redox signaling, or in combination with strategies that target the antioxidant systems or metabolic weaknesses of cancer cells.

Plc1p is required for SAGA recruitment and derepression of Sko1p-regulated genes.

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p-controlled osmoinducible genes upon osmotic shock. Although plc1Delta mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock-induced recruitment of the SAGA complex. Like plc1Delta cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p-repressed promoters in plc1Delta cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p-controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.

Probing Metabolic Changes in IFNγ-Treated Ovarian Cancer Cells.

Interferon-γ (IFNγ) is a pleiotropic cytokine that signals to many different cell types. IFNγ has both antitumor functions and pro-tumorigenic effects and regulates different aspects of cell physiology, including metabolism. Cancer cells undergo a complex rearrangement of metabolic pathways that allows them to satisfy the needs of increased proliferation, and many cancer cells redirect glucose metabolism from oxidative phosphorylation to aerobic glycolysis. In this chapter, we describe a protocol that utilizes the Agilent Seahorse XFp Analyzer to assess mitochondrial respiration and glycolysis in ovarian cancer cells treated with IFNγ.

Plc1p is required for proper chromatin structure and activity of the kinetochore in Saccharomyces cerevisiae by facilitating recruitment of the RSC complex.

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p's involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function.

Saccharomyces cerevisiae phospholipase C regulates transcription of Msn2p-dependent stress-responsive genes.

Phosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1 Delta cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1 Delta cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1 Delta cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated G(alpha) protein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.