Gln3p is a GATA-type transcription factor responsive to different nitrogen nutrients and starvation in yeast Saccharomyces cerevisiae. Recent evidence has linked TOR signaling to Gln3p. Rapamycin causes dephosphorylation and nuclear translocation of Gln3p, thereby activating nitrogen catabolite repressible-sensitive genes. However, a detailed mechanistic understanding of this process is lacking. In this study, we show that Tor1p physically interacts with Gln3p. An intact TOR kinase domain is essential for the phosphorylation of Gln3p, inhibition of Gln3p nuclear entry and repression of Gln3p-dependent transcription. In contrast, at least two distinct protein phosphatases, Pph3p and the Tap42p-dependent phosphatases, are involved in the activation of Gln3p. The yeast pro-prion protein Ure2p binds to both hyper-and hypo-phosphorylated Gln3p. In contrast to the free Gln3p, the Ure2p-bound Gln3p is signifcantly resistant to dephosphorylation. Taken together, these results reveal a tripartite regulatory mechanism by which the phosphorylation of Gln3p is regulated.

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we ®nd that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to signi®cant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3±Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-speci®c deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3D and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability. Keywords: histone deacetylase/nucleolus/rDNA/RNA polymerase I/target of rapamycin IntroductionControl of cell growth and proliferation requires that cells rapidly change their protein biosynthetic capacity in response to nutrients and mitogens as well as stressful conditions. Modulation of protein synthesis involves coordinated changes in both the rate of translational initiation and the abundance of ribosomes. Ribosome concentration¯uctuates in response to growth conditions (Venema and Tollervey, 1999;Warner, 1999;Leary and Huang, 2001;Fatica and Tollervey, 2002;Moss and Stefanovsky, 2002;Peculis, 2002;Grummt, 2003). When cells are growing under favorable conditions, a high ribosome concentration is needed to meet the demand for protein synthesis. However, ribosome biogenesis is a high energy-and nutrient-consuming process. In yeast, for instance, several hundred genes participate in the production of ribosomes, involving all three RNA polymerases and accounting for a major portion of the total cellular biosynthetic output (Venema and Tollervey, 1999;Warner, 1999;Leary and Huang, 2001;Fatica and Tollervey, 2002;Moss and Stefanovsky, 2002;Peculis, 2002;Grummt, 2003). Ribosomal DNA (rDNA) transcription is an important initial step for ribosome biogenesis, producing rRNA products that represent 60% of total yeast transcripts during normal growth. To conserve resources, cells must limit the production of new ribosomes during nutrient starvation. On the other hand, deregulation of ribosome biogenesis has been implicated in uncontrolled growth and tumorigenesis in mammalian cells (Ruggero and Pandol®, 2003).The nucleolus is the site of rDNA transcription by RNA polymerase I (Pol I), rRNA maturation and assembly of ribosomes (Scheer and Hock, 1999). There are~150 tandem copies of rDNA genes in yeast. In addition to the components of Pol I and rDNAs, the nucleolus contains many other nucleolar proteins and small nucleolar RNAs...

Sir proteins play a critical role in silent chromatin domains. While mutations can cause derepression of heterochromatin, it remains unclear whether silencing is actively involved in transcriptional control under changing environmental conditions. We find that TOR inhibits Sir3 phosphorylation. Rapamycin or stress induced by chlorpromazine leads to activation of MAP kinase Mpk1/Slt2, which phosphorylates Sir3. Sir3 hyperphosphorylation is correlated with reduced subtelomeric silencing, increased subtelomeric cell wall gene expression, and stress resistance to chlorpromazine, but does not affect the silent HML and rDNA loci. Based on these observations, we propose that regulation of silencing may be used to control gene expression at specific silent chromatin domains in response to stress and possibly other environmental changes.

Carbon and nitrogen are two basic nutrient sources for cellular organisms. They supply precursors for energy metabolism and metabolic biosynthesis. In the yeast Saccharomyces cerevisiae, distinct sensing and signaling pathways have been described that regulate gene expression in response to the quality of carbon and nitrogen sources, respectively. Gln3 is a GATA-type transcription factor of nitrogen catabolite-repressible (NCR) genes. Previous observations indicate that the quality of nitrogen sources controls the phosphorylation and cytoplasmic retention of Gln3 via the target of rapamycin (TOR) protein. In this study, we show that glucose also regulates Gln3 phosphorylation and subcellular localization, which is mediated by Snf1, the yeast homolog of AMP-dependent protein kinase and a cytoplasmic glucose sensor. Our data show that glucose and nitrogen signaling pathways converge onto Gln3, which may be critical for both nutrient sensing and starvation responses.Carbon and nitrogen are the two most basic nutrient sources for cellular organisms. They are used to produce energy and synthesize a wide range of biomolecules. Energy metabolism and metabolic biosynthesis are carried out by some 500 individual chemical reactions, which are well organized along the centrally placed glycolysis and tricarboxylic acid (TCA) cycle (1). Individual reactions are well calibrated by a feedback regulation, which fine-tunes the flux of metabolites through a particular pathway by temporarily increasing or decreasing the activity of crucial enzymes. In response to the quality of carbon and nitrogen, cells can also regulate the expression of genes involved in different metabolic pathways, particularly those involved in utilization and transport of the available nutrients.

Gln3p is a nitrogen catabolite repression-sensitive GATA-type transcription factor. Its nuclear accumulation was recently shown to be under the control of TOR signaling. Gln3p normally resides in the cytoplasm. When cells are starved from nitrogen nutrients or treated with rapamycin, however, Gln3p becomes translocated into the nucleus, thereby activating the expression of genes involved in nitrogen utilization and transport. To identify other genes under the control of Gln3p, we searched for the Gln3p-binding GATAA motifs within 500 base pairs of the promoter sequences upstream of the yeast open reading frames in the Saccharomyces Genome Database. APG14, a gene essential for autophagy, was found to have the most GATAA motifs. We show that nitrogen starvation or rapamycin treatment rapidly causes a more than 20-fold induction of APG14. The expression of APG14 is dependent on Gln3p; deletion of Gln3p severely reduced its induction by rapamycin, whereas depletion of Ure2p caused its constitutive expression. However, overexpression of APG14 led to only a slight increase in autophagy in nitrogen-rich medium. Therefore, these results define a signaling cascade leading to the expression of APG14 in response to the availability of nitrogen nutrients and suggest that the regulated expression of APG14 contributes to but is not sufficient for the control of autophagy.In response to nutrient starvation conditions, particularly nitrogen starvation, autophagy acts as an emergency measure to generate an internal supply of nutrients. It may also serve to reduce energy-consuming cellular activities, such as protein synthesis, by nonselectively delivering cytosolic materials to the lysosome (higher eukaryotes) or vacuole (yeast) for degradation (reviewed in Ref. 1). Failure to undergo autophagy severely compromises the viability of cells during starvation. In addition, autophagy is also involved in selectively removing aged organelles such as mitochondria under normal growth conditions in higher eukaryotes. During autophagy, a portion of the cytoplasm is sequestered by a double-or multilayered membrane structure referred to as the autophagosome or autophagy body. Autophagosomes are then transported to and fused with the lysosome or vacuole, resulting in the eventual degradation of cytoplasmic materials by the lysosomal or vacuolar proteases (reviewed in Ref. 1).Genetic approaches have been undertaken to identify the players involved in autophagy. A number of autophagy (APG or AUT) genes have been identified that are required for autophagy (2, 3). In a separate genetic screen for genes involved in cytoplasm to vacuole targeting, it was found that there is an overlap of many common components between the cytoplasm to vacuole targeting and autophagy pathways (4). Emerging biochemical evidence indicates that the autophagy genes are involved in various steps in the formation and delivery of autophagosomes. For example, Apg6p/Vps30p/Vpt30p/Lph7p and Apg14p form a peripheral membrane-associated complex. No autophagosomes were observed ...

Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion. Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements. In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements. Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter. Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements. In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity. Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression. We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements. Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.Histamine is a bioamine that plays an important role in many physiological processes, including allergy, inflammation, neurotransmission, and gastric acid secretion (1-3). Histidine decarboxylase (HDC) 1 is the single enzyme that converts histidine to histamine (4). HDC is expressed in many different cell types, including mast cells, skin cells, platelets, and basophils. However, in the adult mammals, HDC is highly expressed in enterochromaffin-like cells, where the HDC activity is tightly regulated by a gut peptide hormone, gastrin (5). HDC regulation occurs at both the transcriptional and post-translational levels, the latter by proteolytic processing through the ubiquitin-proteasome pathway (6, 7).HDC promoter activity is up-regulated by several different stimuli, including gastrin (8), phorbol ester phorbol 12-myristate 13-acetate (8 -11), oxidative stress (12), thrombopointin (13), and Helicobacter pylori infection (14, 15). Whereas not all of the cis-acting DNA elements or the transcriptional factors involved in regulation of HDC transcription have been identified, three GC-rich gastrin responsive elements located downstream of the transcription initiation site have been characterized in the human HDC promoter region (16,17). Through the use of Southwestern blot and UV cross-linking, the sizes of the three gastrin responsive element-binding factors have previously been assessed at 53, 33, and 110 kDa of apparent molecular mass, respectively (16,17). Recently, a neural peptide pituitary adenylate cyclase-activating polypeptide has been reporte...

Hypergastrinemia in INS-GAS mice leads to accelerated carcinogenesis of the stomach, but the mechanisms have not been well defined. We investigated the possible role of gastrin-induced gastric cell apoptosis in the development of gastric cancer. We examined apoptosis and the expression of Bcl-2 family proteins in INS-GAS mice of different ages, as well as in gastrin-deficient (GAS-KO) mice after gastrin-17 (G-17) infusion. In addition, we studied the effects of the gastrin/cholecystokinin-2 (CCK-2) receptor antagonist YF476 and/or histamine H2 (H-2) receptor antagonist loxtidine on apoptosis and atrophy in INS-GAS mice with or without Helicobacter felis (H. felis) infection. INS-GAS mice had age-associated increases in Bax protein expression and decreases in Bcl-2 protein expression, along with increased glandular and epithelial cell apoptosis. At 8-week gastrin infusions in GAS-KO mice resulted in a similar pattern of altered Bax and Bcl-2 expression, followed by gastric cell apoptosis. H. felis infection of INS-GAS mice led to increased apoptosis and the development of atrophy, whereas treatment with either YF476 and/or loxtidine strongly inhibited both apoptosis and atrophy. In vitro studies with Fas-expressing RGM1 cells showed that gastrin stimulation alone directly induced apoptosis via gastrin/CCK-2 receptor and synergized with FasL stimulation. These results indicate that gastrin can induce apoptosis in gastric epithelial cells and contribute to the development of gastric carcinogenesis.

Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.