Differing SAGA module requirements for NCR-sensitive gene transcription in yeast.

Nitrogen catabolite repression (NCR) is a means for yeast to adapt its transcriptome to changing nitrogen sources in its environment. In conditions of derepression (under poor nitrogen conditions, upon rapamycin treatment, or when glutamine production is inhibited), two transcriptional activators of the GATA family are recruited to NCR-sensitive promoters and activate transcription of NCR-sensitive genes. Earlier observations have involved the Spt-Ada-Gcn5 acetyltransferase (SAGA) chromatin remodeling complex in these transcriptional regulations. In this report, we provide an illustration of the varying NCR-sensitive responses and question whether differing SAGA recruitment could explain this diversity of responses.

Glutamine transport as a possible regulator of nitrogen catabolite repression in Saccharomyces cerevisiae.

Nitrogen catabolite repression (NCR) is a major transcriptional control pathway governing nitrogen use in yeast, with several hundred of target genes identified to date. Early and extensive studies on NCR led to the identification of the 4 GATA zinc finger transcription factors, but the primary mechanism initiating NCR is still unclear up till now. To identify novel players of NCR, we have undertaken a genetic screen in an NCR-relieved gdh1Δ mutant, which led to the identification of four genes directly linked to protein ubiquitylation. Ubiquitylation is an important way of regulating amino acid transporters and our observations being specifically observed in glutamine-containing media, we hypothesized that glutamine transport could be involved in establishing NCR. Stabilization of Gap1 at the plasma membrane restored NCR in gdh1Δ cells and AGP1 (but not GAP1) deletion could relieve repression in the ubiquitylation mutants isolated during the screen. Altogether, our results suggest that deregulated glutamine transporter function in all three weak nitrogen derepressed (wnd) mutants restores the repression of NCR-sensitive genes consecutive to GDH1 deletion.

Long-term outcomes of patients with ulcerative proctitis: Analysis from a large referral centre cohort.

INTRODUCTION: Long-term outcomes of patients with ulcerative proctitis (UP) have been poorly investigated, since these patients are excluded from participation in randomized controlled clinical trials. OBJECTIVE: The aim of this study was to investigate the prognostic and therapeutic long-term outcomes of patients with UP. METHODS: A retrospective study of patients with UP followed at our referral centre between 1 January 1998 and 1 January 2019 was performed. Treatment success was defined as clinical response (significant improvement in UP-related symptoms) and endoscopic response (mayo endoscopic sub-score of 0 or 1) if available at last follow-up. RESULTS: From a total of 1561 patients with ulcerative colitis, 118 patients with UP were identified. A total of 36 (31%) patients were refractory to rectal and oral therapy with 5-ASA and corticosteroids, necessitating azathioprine as monotherapy in 19 (16%) patients and/or biological therapies in 33 (28%) patients. After a median follow-up of 71 months (interquartile range 29-149 months), treatment success was observed in 103/118 (87%) UP patients and in 25/36 (69%) patients with refractory UP. Clinical response rates were significantly higher for refractory UP patients treated with biologicals (23/33; 70%) compared to ones treated with azathioprine (2/19; 11%; p = 0.001). CONCLUSION: Good clinical outcomes were recorded in UP, with treatment success in 87% of patients. Nevertheless, 28% needed escalation to biologicals. Long-term outcome in patients on biologicals was superior to azathioprine.

Transcription-dependent spreading of the Dal80 yeast GATA factor across the body of highly expressed genes.

GATA transcription factors are highly conserved among eukaryotes and play roles in transcription of genes implicated in cancer progression and hematopoiesis. However, although their consensus binding sites have been well defined in vitro, the in vivo selectivity for recognition by GATA factors remains poorly characterized. Using ChIP-Seq, we identified the Dal80 GATA factor targets in yeast. Our data reveal Dal80 binding to a large set of promoters, sometimes independently of GATA sites, correlating with nitrogen- and/or Dal80-sensitive gene expression. Strikingly, Dal80 was also detected across the body of promoter-bound genes, correlating with high expression. Mechanistic single-gene experiments showed that Dal80 spreading across gene bodies requires active transcription. Consistently, Dal80 co-immunoprecipitated with the initiating and post-initiation forms of RNA Polymerase II. Our work suggests that GATA factors could play dual, synergistic roles during transcription initiation and post-initiation steps, promoting efficient remodeling of the gene expression program in response to environmental changes.

Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production.

The first step in executing the genetic program of a cell is production of mRNA. In yeast, almost every gene is transcribed as multiple distinct isoforms, differing at their 5' and/or 3' termini. However, the implications and functional significance of the transcriptome-wide diversity of mRNA termini remains largely unexplored. In this paper, we show that the GAT1 gene, encoding a transcriptional activator of nitrogen-responsive catabolic genes, produces a variety of mRNAs differing in their 5' and 3' termini. Alternative transcription initiation leads to the constitutive, low level production of 2 full length proteins differing in their N-termini, whereas premature transcriptional termination generates a short, highly nitrogen catabolite repression- (NCR-) sensitive transcript that, as far as we can determine, is not translated under the growth conditions we used, but rather likely protects the cell from excess Gat1.

GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation.

The TorC1 protein kinase complex is a central component in a eukaryotic cell's response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g., proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor-dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3-Myc(13) localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells' nitrogen environment.

Components of Golgi-to-vacuole trafficking are required for nitrogen- and TORC1-responsive regulation of the yeast GATA factors.

Nitrogen catabolite repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae responds to the available nitrogen status and selectively utilizes rich nitrogen sources in preference to poor ones. Expression of NCR-sensitive genes is mediated by two transcription activators, Gln3 and Gat1, in response to provision of a poorly used nitrogen source or following treatment with the TORC1 inhibitor, rapamycin. During nitrogen excess, the transcription activators are sequestered in the cytoplasm in a Ure2-dependent fashion. Here, we show that Vps components are required for Gln3 localization and function in response to rapamycin treatment when cells are grown in defined yeast nitrogen base but not in complex yeast peptone dextrose medium. On the other hand, Gat1 function was altered in vps mutants in all conditions tested. A significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. Further, our results are consistent with the possibility that Ure2 might function downstream of the Vps components during the control of GATA factor-mediated gene expression. These observations demonstrate distinct media-dependent requirements of vesicular trafficking components for wild-type responses of GATA factor localization and function. As a result, the current model describing participation of Vps system components in events associated with translocation of Gln3 into the nucleus following rapamycin treatment or growth in nitrogen-poor medium requires modification.

Constitutive and nitrogen catabolite repression-sensitive production of Gat1 isoforms.

Nitrogen catabolite repression (NCR)-sensitive transcription is activated by Gln3 and Gat1. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and transcription is minimal. In poor nitrogen, Gln3 and Gat1 become nuclear and activate transcription. A long standing paradox has surrounded Gat1 production. Gat1 was first reported as an NCR-regulated activity mediating NCR-sensitive transcription in gln3 deletion strains. Upon cloning, GAT1 transcription was, as predicted, NCR-sensitive and Gln3- and Gat1-activated. In contrast, Western blots of Gat1-Myc(13) exhibited two constitutively produced species. Investigating this paradox, we demonstrate that wild type Gat1 isoforms (IsoA and IsoB) are initiated at Gat1 methionines 40, 95, and/or 102, but not at methionine 1. Their low level production is the same in rich and poor nitrogen conditions. When the Myc(13) tag is placed after Gat1 Ser-233, four N-terminal Gat1 isoforms (IsoC-F) are also initiated at methionines 40, 95, and/or 102. However, their production is highly NCR-sensitive, being greater in proline than glutamine medium. Surprisingly, all Gat1 isoforms produced in sufficient quantities to be confidently analyzed (IsoA, IsoC, and IsoD) require Gln3 and UASGATA promoter elements, both requirements typical of NCR-sensitive transcription. These data demonstrate that regulated Gat1 production is more complex than previously recognized, with wild type versus truncated Gat1 proteins failing to be regulated in parallel. This is the first reported instance of Gln3 UASGATA-dependent protein production failing to derepress in nitrogen poor conditions. A Gat1-lacZ ORF swap experiment indicated sequence(s) responsible for the nonparallel production are downstream of Gat1 leucine 61.

Alterations in the Ure2 αCap domain elicit different GATA factor responses to rapamycin treatment and nitrogen limitation.

Ure2 is a phosphoprotein and central negative regulator of nitrogen-responsive Gln3/Gat1 localization and their ability to activate transcription. This negative regulation is achieved by the formation of Ure2-Gln3 and -Gat1 complexes that are thought to sequester these GATA factors in the cytoplasm of cells cultured in excess nitrogen. Ure2 itself is a dimer the monomer of which consists of two core domains and a flexible protruding αcap. Here, we show that alterations in this αcap abolish rapamycin-elicited nuclear Gln3 and, to a more limited extent, Gat1 localization. In contrast, these alterations have little demonstrable effect on the Gln3 and Gat1 responses to nitrogen limitation. Using two-dimensional PAGE we resolved eight rather than the two previously reported Ure2 isoforms and demonstrated Ure2 dephosphorylation to be stimulus-specific, occurring after rapamycin treatment but only minimally if at all in nitrogen-limited cells. Alteration of the αcap significantly diminished the response of Ure2 dephosphorylation to the TorC1 inhibitor, rapamycin. Furthermore, in contrast to Gln3, rapamycin-elicited Ure2 dephosphorylation occurred independently of Sit4 and Pph21/22 (PP2A) as well as Siw14, Ptc1, and Ppz1. Together, our data suggest that distinct regions of Ure2 are associated with the receipt and/or implementation of signals calling for cessation of GATA factor sequestration in the cytoplasm. This in turn is more consistent with the existence of distinct pathways for TorC1- and nitrogen limitation-dependent control than it is with these stimuli representing sequential steps in a single regulatory pathway.

Nitrogen-responsive regulation of GATA protein family activators Gln3 and Gat1 occurs by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine.

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

Intranuclear function for protein phosphatase 2A: Pph21 and Pph22 are required for rapamycin-induced GATA factor binding to the DAL5 promoter in yeast.

Protein phosphatase 2A (PP2A), a central Tor pathway phosphatase consisting of a catalytic subunit (Pph21 or Pph22), a scaffold subunit (Tpd3), and one of two regulatory subunits (Cdc55 or Rts1), has been repeatedly shown to play important roles in cytoplasmically localized signal transduction activities. In contrast, its involvement in intranuclear control of mRNA production has heretofore not been reported. Here, we demonstrate for the first time that binding of the nitrogen catabolite repression-responsive GATA transcription activators (Gln3 and Gat1) to the DAL5 promoter and DAL5 expression require Pph21/22-Tpd3-Cdc55/Rts1 in rapamycin-treated glutamine-grown cells. This conclusion is supported by the following observations. (i) Rapamycin-induced DAL5 expression along with Gln3 and Gat1 binding to the DAL5 promoter fails to occur in pph21Δ pph22Δ, tpd3Δ, and cdc55Δ rts1Δ mutants. (ii) The Pph21/22 requirement persists even when Gat1 and Gln3 are rendered constitutively nuclear, thus dissociating the intranuclear requirement of PP2A from its partial requirement for rapamycin-induced nuclear Gat1 localization. (iii) Pph21-Myc(13) (Ppp21 tagged at the C terminus with 13 copies of the Myc epitope) weakly associates with the DAL5 promoter in a Gat1-dependent manner, whereas a similar Pph22-Myc(13) association requires both Gln3 and Gat1. Finally, we demonstrate that a pph21Δ pph22Δ double mutant is epistatic to ure2Δ for nuclear Gat1 localization in untreated glutamine-grown cells, whereas for Gln3, just the opposite occurs: i.e., ure2Δ is epistatic to pph21Δ pph22Δ. This final observation adds additional support to our previous conclusion that the Gln3 and Gat1 GATA factor localizations are predominantly controlled by different regulatory pathways.

Selection systems based on dominant-negative transcription factors for precise genetic engineering.

Diverse tools are available for performing genetic modifications of microorganisms. However, new methods still need to be developed for performing precise genomic engineering without introducing any undesirable side-alteration. Indeed for functional analyses of genomic elements, as well as for some industrial applications, only the desired mutation should be introduced at the locus considered. This article describes a new approach fulfilling these requirements, based on the use of selection systems consisting in truncated genes encoding dominant-negative transcription factors. We have demonstrated dominant-negative effects mediated by truncated Gal4p and Arg81p proteins in Saccharomyces cerevisiae, interfering with galactose and arginine metabolic pathways, respectively. These genes can be used as positive and negative markers, since they provoke both growth inhibition on substrates and resistance to specific drugs. These selection markers have been successfully used for precisely deleting HO and URA3 in wild yeasts. This genetic engineering approach could be extended to other microorganisms.

Distinct phosphatase requirements and GATA factor responses to nitrogen catabolite repression and rapamycin treatment in Saccharomyces cerevisiae.

In yeast, rapamycin (Rap)-inhibited TorC1, and the phosphatases it regulates (Sit4 and PP2A) are components of a conserved pathway regulating the response of eukaryotic cells to nutrient availability. TorC1 and intracellular nitrogen levels regulate the localization of Gln3 and Gat1, the activators of nitrogen catabolite repression (NCR)-sensitive genes whose products are required to utilize poor nitrogen sources. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and NCR-sensitive transcription is repressed. During nitrogen limitation or Rap treatment, Gln3 and Gat1 are nuclear, and transcription is derepressed. We previously demonstrated that the Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for nuclear Gln3 localization differ. We now show that Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for NCR-sensitive and Rap-induced nuclear Gat1 localization markedly differ from those of Gln3. Our data suggest that Gln3 and Gat1 localizations are controlled by two different regulatory pathways. Gln3 localization predominantly responds to intracellular nitrogen levels, as reflected by its stronger NCR-sensitivity, weaker response to Rap treatment, and strong response to methionine sulfoximine (Msx, a glutamine synthetase inhibitor). In contrast, Gat1 localization predominantly responds to TorC1 regulation as reflected by its weaker NCR sensitivity, stronger response to Rap, and immunity to the effects of Msx. Nuclear Gln3 localization in proline-grown (nitrogen limited) cells exhibits no requirement for Pph21/22-Tpd3/Cdc55, whereas nuclear Gat1 localization under these conditions is absolutely dependent on Pph21/22-Tpd3/Cdc55. Furthermore, the extent to which Pph21/22-Tpd3-Cdc55 is required for the TorC1 pathway (Rap) to induce nuclear Gat1 localization is regulated in parallel with Pph21/22-Tpd3-Cdc55-dependent Gln3 dephosphorylation and NCR-sensitive transcription, being highest in limiting nitrogen and lowest when nitrogen is in excess.

The yeast GATA factor Gat1 occupies a central position in nitrogen catabolite repression-sensitive gene activation.

Saccharomyces cerevisiae cells are able to adapt their metabolism according to the quality of the nitrogen sources available in the environment. Nitrogen catabolite repression (NCR) restrains the yeast's capacity to use poor nitrogen sources when rich ones are available. NCR-sensitive expression is modulated by the synchronized action of four DNA-binding GATA factors. Although the first identified GATA factor, Gln3, was considered the major activator of NCR-sensitive gene expression, our work positions Gat1 as a key factor for the integrated control of NCR in yeast for the following reasons: (i) Gat1 appeared to be the limiting factor for NCR gene expression, (ii) GAT1 expression was regulated by the four GATA factors in response to nitrogen availability, (iii) the two negative GATA factors Dal80 and Gzf3 interfered with Gat1 binding to DNA, and (iv) Gln3 binding to some NCR promoters required Gat1. Our study also provides mechanistic insights into the mode of action of the two negative GATA factors. Gzf3 interfered with Gat1 by nuclear sequestration and by competition at its own promoter. Dal80-dependent repression of NCR-sensitive gene expression occurred at three possible levels: Dal80 represses GAT1 expression, it competes with Gat1 for binding, and it directly represses NCR gene transcription.

Nitrogen catabolite repression-sensitive transcription as a readout of Tor pathway regulation: the genetic background, reporter gene and GATA factor assayed determine the outcomes.

Nitrogen catabolite repression (NCR)-sensitive genes, whose expression is highly repressed when provided with excess nitrogen and derepressed when nitrogen is limited or cells are treated with rapamycin, are routinely used as reporters in mechanistic studies of the Tor signal transduction pathway in Saccharomyces cerevisiae. Two GATA factors, Gln3 and Gat1, are responsible for NCR-sensitive transcription, but recent evidence demonstrates that Tor pathway regulation of NCR-sensitive transcription bifurcates at the level of GATA factor localization. Gln3 requires Sit4 phosphatase for nuclear localization and NCR-sensitive transcription while Gat1 does not. In this article, we demonstrate that the extent to which Sit4 plays a role in NCR-sensitive transcription depends upon whether or not (i) Gzf3, a GATA repressor homologous to Dal80, is active in the genetic background assayed; (ii) Gat1 is able to activate transcription of the assayed gene in the absence of Gln3 in that genetic background; and (iii) the gene chosen as a reporter is able to be transcribed by Gln3 or Gat1 in the absence of the other GATA factor. Together, the data indicate that in the absence of these three pieces of information, overall NCR-sensitive gene transcription data are unreliable as Tor pathway readouts.

Rapamycin-induced Gln3 dephosphorylation is insufficient for nuclear localization: Sit4 and PP2A phosphatases are regulated and function differently.

Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Delta22Delta, tpd3Delta, or cdc55Delta cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.

Tor pathway control of the nitrogen-responsive DAL5 gene bifurcates at the level of Gln3 and Gat1 regulation in Saccharomyces cerevisiae.

The Tor1,2 protein kinases globally influence many cellular processes including nitrogen-responsive gene expression that correlates with intracellular localization of GATA transcription activators Gln3 and Gat1/Nil1. Gln3-Myc(13) and Gat1-Myc(13) are restricted to the cytoplasm of cells provided with good nitrogen sources, e.g. glutamine. Following the addition of the Tor1,2 inhibitor, rapamycin, both transcription factors relocate to the nucleus. Gln3-Myc(13) localization is highly dependent upon Ure2 and type 2A-related phosphatase, Sit4. Ure2 is required for Gln3 to be restricted to the cytoplasm of cells provided with good nitrogen sources, and Sit4 is required for its location to the nucleus following rapamycin treatment. The paucity of analogous information concerning Gat1 regulation prompted us to investigate the effects of deleting SIT4 and URE2 on Gat1-Myc(13) localization, DNA binding, and NCR-sensitive transcription. Our data demonstrate that Tor pathway control of NCR-responsive transcription bifurcates at the regulation of Gln3 and Gat1. Gat1-Myc(13) localization is not strongly influenced by deleting URE2, nor is its nuclear targeting following rapamycin treatment strongly dependent on Sit4. ChIP experiments demonstrated that Gat1-Myc(13) can bind to the DAL5 promoter in the absence of Gln3. Gln3-Myc(13), on the other hand, cannot bind to DAL5 in the absence of Gat1. We conclude that: (i) Tor pathway regulation of Gat1 differs markedly from that of Gln3, (ii) nuclear targeting of Gln3-Myc(13) is alone insufficient for its recruitment to the DAL5 promoter, and (iii) the Tor pathway continues to play an important regulatory role in NCR-sensitive transcription even after Gln3-Myc(13) is localized to the nucleus.

Saccharomyces cerevisiae Sit4 phosphatase is active irrespective of the nitrogen source provided, and Gln3 phosphorylation levels become nitrogen source-responsive in a sit4-deleted strain.

Tor1,2 control of type 2A-related phosphatase activities in Saccharomyces cerevisiae has been reported to be responsible for the regulation of Gln3 phosphorylation and intracellular localization in response to the nature of the nitrogen source available. According to the model, excess nitrogen stimulates Tor1,2 to phosphorylate Tip41 and/or Tap42. Tap42 then complexes with and inactivates Sit4 phosphatase, thereby preventing it from dephosphorylating Gln3. Phosphorylated Gln3 complexes with Ure2 and is sequestered in the cytoplasm. When Tor1,2 kinase activities are inhibited by limiting nitrogen, or rapamycin-treatment, Tap42 can no longer complex with Sit4. Active Sit4 dephosphorylates Gln3, which can then localize to the nucleus and activate transcription. The paucity of experimental data directly correlating active Sit4 and Pph3 with Gln3 regulation prompted us to assay Gln3-Myc(13) phosphorylation and intracellular localization in isogenic wild type, sit4, pph3, and sit4pph3 deletion strains. We found that Sit4 actively brought about Gln3-Myc(13) dephosphorylation in both good (glutamine or ammonia) and poor (proline) nitrogen sources. This Sit4 activity masked nitrogen source-dependent changes in Gln3-Myc(13) phosphorylation which were clearly visible when SIT4 was deleted. The extent of Sit4 requirement for Gln3 nuclear localization was both nitrogen source- and strain-dependent. In some strains, Sit4 was not even required for Gln3 nuclear localization in untreated or rapamycin-treated, proline-grown cells or Msx-treated, ammonia-grown cells.

Identification of direct and indirect targets of the Gln3 and Gat1 activators by transcriptional profiling in response to nitrogen availability in the short and long term.

Nitrogen catabolite repression (NCR) consists in the specific inhibition of transcriptional activation of genes encoding the permeases and catabolic enzymes needed to degrade poor nitrogen sources. Under nitrogen limitation or rapamycin treatment, NCR genes are activated by Gln3 or Gat1, or by both factors. To compare the sets of genes responding to rapamycin or to nitrogen limitation, we used DNA microarrays to establishing the expression profiles of a wild type strain, and of a double gln3Delta-gat1Delta strain, grown on glutamine, after addition of rapamycin, on proline, or after a shift from glutamine to proline. Analysis of microarray data revealed 392 genes whose expression was dependent on the nitrogen source quality. 91 genes were activated in a GATA factor-dependent manner in all growth conditions, suggesting a direct role of Gln3 and Gat1 in their expression. Other genes were only transiently up-regulated (stress-responsive genes) or down-regulated (genes encoding ribosomal proteins and translational factors) upon nitrogen limitation, and this regulation was delayed in a gln3Delta-gat1Delta strain. Repression of amino acid and nucleotide biosynthetic genes after a nitrogen shift did not depend on Gcn4. Several transporter genes were repressed as a consequence of enhanced levels of NCR-responsive permeases present at the plasma membrane.

Transduction of the nitrogen signal activating Gln3-mediated transcription is independent of Npr1 kinase and Rsp5-Bul1/2 ubiquitin ligase in Saccharomyces cerevisiae.

Nitrogen Catabolite Repression (NCR) allows the adaptation of yeast cells to the quality of nitrogen supply by inhibiting the transcription of genes encoding proteins involved in transport and degradation of nonpreferred nitrogen sources. In cells using ammonium or glutamine, the GATA transcription factor Gln3 is sequestered in the cytoplasm by Ure2 whereas it enters the nucleus after a shift to a nonpreferred nitrogen source like proline or upon addition of rapamycin, the TOR complex inhibitor. Recently, the Npr1 kinase and the Rsp5, Bul1/2 ubiquitin ligase complex were reported to have antagonistic roles in the nuclear import and Gln3-mediated activation. The Npr1 kinase controls the activity of various permeases including transporters for nitrogen sources that stimulate NCR such as the Mep ammonium transport systems. Combining data from growth tests, Northern blot analysis and Gln3 immunolocalization, we show that the Npr1 kinase is not a direct negative regulator of Gln3-dependent transcription. The derepression of Gln3-activated genes in ammonium-grown npr1 cells results from the reduced uptake of the nitrogen-repressing compound because NCR could be restored in npr1 cells by repairing ammonium-uptake defects through different means. Finally, we show that the impairment of the ubiquitin ligase complex does not prevent induction of NCR genes under nonpreferred nitrogen conditions. The apparent Rsp5-, Bul1/2-dependent Gln3 activation keeps to the cellular status, as it is only observed in cells having left the balanced phase of exponential growth.