Nitrogen Catabolite Repression-Sensitive Transcription as a ......The Gat1 require-Figure 1.—Current models describing Tor pathway regula-tion of Gln3 dephosphorylation, localization,

Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.099051

Nitrogen Catabolite Repression-Sensitive Transcription as a Readout ofTor Pathway Regulation: The Genetic Background, Reporter Gene

and GATA Factor Assayed Determine the Outcomes

Isabelle Georis,* André Feller,* Jennifer J. Tate,† Terrance G. Cooper†,1 and Evelyne Dubois*

*Institut de Recherches Microbiologiques J.-M. Wiame, Laboratoire de Microbiologie, Université Libre de Bruxelles, B1070 Brussels, Belgiumand †Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163

Manuscript received December 2, 2008Accepted for publication December 18, 2008

ABSTRACT

Nitrogen catabolite repression (NCR)-sensitive genes, whose expression is highly repressed whenprovided with excess nitrogen and derepressed when nitrogen is limited or cells are treated withrapamycin, are routinely used as reporters in mechanistic studies of the Tor signal transduction pathway inSaccharomyces 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 atthe 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 Sit4plays a role in NCR-sensitive transcription depends upon whether or not (i) Gzf3, a GATA repressorhomologous to Dal80, is active in the genetic background assayed; (ii) Gat1 is able to activate transcriptionof the assayed gene in the absence of Gln3 in that genetic background; and (iii) the gene chosen as areporter is able to be transcribed by Gln3 or Gat1 in the absence of the other GATA factor. Together, thedata indicate that in the absence of these three pieces of information, overall NCR-sensitive genetranscription data are unreliable as Tor pathway readouts.

THE central position of second and third generationrapamycin derivatives in clinical settings hasgreatly stimulated investigation of its target, Tor, andthe mechanisms through which Tor participates in theregulation of cellular processes. One of the mostformative discoveries of these investigations was thefinding that rapamycin induced nuclear localization ofGln3 and activation of nitrogen catabolite repression(NCR)-sensitive transcription under repressive growthconditions where this activator would normally berestricted to the cytoplasm and the transcription ofNCR-sensitive genes (e.g., DAL5, MEP2, GAP1, etc.)would be quiescent (Figure 1A) (Beck and Hall 1999;Cardenas et al. 1999; Hardwick et al. 1999; Bertramet al. 2000; Cox et al. 2000; Shamji et al. 2000). Byinference, Gln3-mediated activation itself has become aprominent reporter of Tor pathway function. In thewild, Saccharomyces cerevisiae uses NCR to selectivelyutilize repressive nitrogen sources (e.g., glutamine orammonia as nitrogen source) in preference to dere-pressive sources that support less robust growth(reviewed in Hofman-Bang 1999; Cooper 2002,2004; Magasanik and Kaiser 2002). In nitrogenexcess, transcription of genes required to transport

and degrade poor nitrogen sources is repressed. Whenpreferred sources are limited or absent, transcription ofthese NCR-sensitive genes is derepressed so that the cellcan scavenge alternative nitrogen sources that might beavailable in its environment.

A highly abridged version of the regulatory modelreported for the Tor pathway and regulation of GATA-factor-activated, NCR-sensitive gene expression is shownin Figure 1A. Although central components of thismodel have not stood up to detailed investigation(Cox et al. 2002, 2004; Wang et al. 2003; Tate et al.2005, 2006,a,b, 2009; Feller et al. 2006; Kulkarni et al.2006; Yan et al. 2006; Georis et al. 2008), it willadequately frame the experiments developed in thiswork. When nitrogen is in excess, Sit4 protein phospha-tase is in a complex with Tor-associated protein (Tap42)and Tor complex 1 (TorC1) (Figure 1A) (Di Como andArndt 1996; Beck and Hall 1999; Jiang and Broach1999; Bertram et al. 2000; Loewith et al. 2002; Düvelet al. 2003; Wang et al. 2003; Reinke et al. 2004;Giannattasio et al. 2005; Di Como and Jiang 2006;Yanet al. 2006; Adami et al. 2007; Aronova et al. 2007). Inthis form, it is inactive and hence incapable of dephos-phorylating Gln3. Under these growth conditions, Gln3remains cytoplasmic and NCR-sensitive gene expressionis repressed. If nitrogen becomes limiting or glutamine-grown cells are treated with the Tor-specific inhibitorrapamycin, the Sit4-Tap42 complex is released from

1Corresponding author: Department of Molecular Sciences, 858 MadisonAve., University of Tennessee, Memphis, TN 38163.E-mail: [email protected]

Genetics 181: 861–874 (March 2009)

TorC1 and thus becomes active. Gln3 can now bedephosphorylated, enter the nucleus, and activate NCR-sensitive catabolic gene expression. Gat1, the secondNCR-sensitive transcription activator, responds to nitro-gen levels and rapamycin similarly to Gln3 (Coffman et al.1995, 1996a,b; Stanbrough et al. 1995). However, it hasbeen recently shown that Tor pathway regulation of Gln3and Gat1 bifurcates at the level of their localization andDNA binding (Figure 1A): (i) Ure2 plays a much moreimportant role in restricting Gln3 to the cytoplasm duringnitrogen excess than it does for Gat1, and (ii) Sit4 isrequired for nuclear localization of Gln3 but not of Gat1following rapamycin treatment (Georis et al. 2008). Oncein the nucleus, Gln3 and Gat1 bind to their target GATAsequences in the promoters of NCR-sensitive genes andactivate their transcription. For DAL5, Gat1 binding isGln3 independent, whereas Gln3 binding is Gat1 de-pendent (Figure 1A) (Georis et al. 2008).

NCR-sensitive gene expression is further regulated bythe finely tuned and integrated action of the twotranscription activators discussed above and two tran-scription repressors (Dal80/Uga43 and Gzf3/Deh1/Nil2) (Figure 1B) (Coffman et al. 1995, 1996a,b, 1997;Stanbrough et al. 1995; Rowen et al. 1997; Soussi-Boudekou et al. 1997; Scherens et al. 2006). While Gln3(Cunningham et al. 1996), and presumably Gat1, bindsto single GATA sequences, the GATA repressors containleucine-zipper motifs near their C termini and formhomo and heterodimers in vivo (Svetlov and Cooper1998). Consistent with the formation of dimers, Dal80binding to DNA requires two GATA sequences thatare properly spaced and oriented (Cunningham andCooper 1993; Cunningham et al. 1994). Gzf3 alsobinds, in a GATA sequence-dependent manner, toDNA fragments (UGA4, GAP1) containing two or moreGATAs (Coffman et al. 1997). With these DNA frag-ments, multiple protein-DNA species were observedwith the one possessing the highest electrophoreticmobility being the most prominent. Gzf3 has also beensuggested as regulating transcription from a singleGATA element (Rowen et al. 1997). Integration of theactions of GATA activators and repressors is achieved bytheir cross and autogenous regulation (Figure 1B)(Daugherty et al. 1993; Coffman et al. 1996a,b, 1997;Rowen et al. 1997; Soussi-Boudekou et al. 1997;Cunningham et al. 2000a,b). Three of the four GATAfactors (Gat1, Dal80, Gzf3/Deh1) are encoded by genescontaining multiple GATA sequences in their pro-moters and their expression is NCR sensitive (Coffmanet al. 1997; Soussi-Boudekou et al. 1997). DAL80 andGAT1 expression is strongly Gln3 dependent as well asDal80 and Gat1 regulated. On the other hand, regu-lation by Gzf3 is very weak (Figure 1B). GZF3 expressionis NCR sensitive and Dal80 regulated and requires atleast one of the two GATA activators (Figure 1B ). Themost striking characteristic of Gzf3 is that its regulationof NCR-sensitive genes occurs in a repressive medium

(Coffman et al. 1996a,b, 1997; Rowen et al. 1997;Soussi-Boudekou et al. 1997).

During investigation of the bifurcated regulation ofGln3 and Gat1 by the Tor pathway discussed above, asurprising observation was made. The Gat1 require-

Figure 1.—Current models describing Tor pathway regula-tion of Gln3 dephosphorylation, localization, and activation ofNCR-sensitive transcription (A) and interactions of the positivelyand negatively-acting GATA factors that fine-tune the levels ofNCR-sensitive transcription (B). The model incorporates con-clusions drawn from many laboratories (see text for references).Green lines and arrows indicate positive regulation, whereas redlines, arrows, and bars indicate negative regulation. In A, redand green letters indicate inactive and active forms of the pro-teins, respectively. Dashed lines indicate weak regulatory rela-tionships. The model in A is not exhaustive—for example,the Tip41 protein ( Jacinto et al. 2001) is not described—anddoes not present all contending views of some steps, for exam-ple, those reported by the Beck and Hall (1999) vs. Carvalhoet al. (2001) or Jacinto et al. (2001) vs. Jiang and Broach(1999). A is modified from Tate et al. (2009) and B is modifiedfrom Coffman et al. (1997).

862 I. Georis et al.

ment for rapamycin-induced DAL5 gene expression wasmuch greater than that for Gln3 (Georis et al. 2008). Infact, DAL5 transcription decreased only about threefoldin a gln3D and was not affected in a sit4D, in which Gln3is cytoplasmic. This contradicted a large body of earlierwork demonstrating just the opposite, i.e., that the Gln3requirement of NCR-sensitive DAL5 expression wasmuch greater than that for Gat1 (Daugherty et al.1993; Coffman et al. 1994, 1996a,b, 1997; Tate et al.2002; Tate and Cooper 2003). Therefore, experimentsdescribed in this work were initiated to resolve thecontradiction.

We were able to effectively explain the apparentparadox surrounding the GATA factor requirements ofDAL5 expression. In doing so, however, we found thatcomparing conclusions about Tor pathway function de-rived from analyses employing NCR-sensitive reportergene or transcriptome assays may be complicated by thegenetic background of the strains analyzed and thespecific NCR-sensitive gene assayed. For example,whether or not Sit4 is required for NCR-sensitive tran-scription of a reporter gene depends on whether GATArepressor Gzf3 effectively functions in the strain assayedand whether or not Gat1 or Gln3, in the absence of theother GATA factor, is able to activate transcription of theparticular NCR-sensitive gene assayed. We also discovered(i) a new regulatory function for GATA repressor Gzf3,(ii) evidence pointing toward the existence of one ormore Tor pathway components acting downstream ofrapamycin-induced nuclear GATA factor localization,and (iii) a potential explanation for variations in the setsof genes identified as NCR-sensitive by transcriptomeanalyses performed in different laboratories.

MATERIALS AND METHODS

Strains and culture conditions: S. cerevisiae strains used inthis work are listed in Table 1 along with the DNA primers usedin their construction. Growth conditions were identical tothose described in Tate et al. (2006a,b), Scherens et al.(2006), and Georis et al. (2008). Rapamycin (Sigma and LCLaboratories) was used at the concentrations and timesindicated in the figure legends.

Strain construction: Deletion strains, involving insertion ofkanMX or natMX cassettes, were constructed as describedearlier (Tate et al. 2006a,b; Georis et al. 2008). ChromosomalGLN3 or GAT1 were tagged at their C termini with 13 copies ofthe Myc epitope (Myc13) as described in Georis et al. (2008).

Steady-state mRNA and DNA-binding analyses: Quantita-tive RT–PCR (qRT–PCR) and chromatin immunoprecipita-tion (ChIP) analyses were performed as described by Georiset al. (2008). Data were analyzed with LightCycler software,version 5.32. The immunoprecipitated DNA/input DNA (IP/IN) ratio corresponds to the concentration of target DNA inthe IP sample relative to that in the corresponding IN sample,multiplied by 10. IP/IN values obtained for the unboundcontrol (DAL5U) were substracted from IP/IN values ob-tained for the DAL5 promoter (DAL5P). To counterbalancevariation generated by the immunoprecipitation step, wetreated all of our data as follows. The wild-type-induced value

was set as 1 and the IP/IN value of every simultaneouslyimmunoprecipitated sample was normalized accordingly. Forevery independent culture, the mean of the IP/IN ratios for oneto three replicate immunoprecipitations was calculated. Valuesin Figures 4, 5, 9, and 11 correspond to the mean IP/IN value ofat least two independent cultures. DAL5U, DAL5O, DAL5P, andTBP1O primers have been described in Georis et al. (2008).ChIP primers amplified a 126-bp region in the promoter ofDAL7 (DAL7P1: 59-AAATCTCCGCTGAAAGTTGC-39; DAL5P2:59-TTTCACGATGTACCTTATCCAAGA-39) or a 141-bp regionin the promoter of GAP1 (GAP1P1: 59-GACCTCATGCAGCAAAGTCA-39; GAP1P2: 59-CCGGTTGCTCCAGAAGATAA-39). qRT–PCR primers amplified a 122-bp region in DAL7(DAL7O1: 59-CACGGAAACAGCTTTTAGCC-39; DAL7O2:59-AGCACCTTGCCATGTAGGAT-59), a 147-bp region in GAP1(GAP1O1: 59-AAATGGCTCCGCTGTTTCTA-39; GAP1O2: 59-GGAGTTTGGGCAGTGATGAT-39), and a 167-bp region inGZF3 (GZF3O1: 59-TTATGGCATCGCAGGCTACA-39; GZF3O2:59-TTGCTCGGCAGATACTGCTT-39).

Indirect immunofluorescence microscopy: Indirect immu-nofluorescence microscopy was performed as described inGeoris et al. (2008). Cells were imaged at room temperatureusing a Zeiss Axioplan 2 imaging microscope with a 3100 Plan-Apochromat 1.40 oil objective at room temperature. Imageswere acquired using a Zeiss Axio camera and AxioVision(Zeiss) software, processed with Adobe Photoshop and Illus-trator programs. Gamma settings were altered where necessaryto avoid any change or loss in cellular detail during processing;changes were applied uniformly to the image presented.

Determination of intracellular Gln3-Myc13 and Gat1-Myc13

distribution: To provide more representative and completedescriptions of Gln3-Myc13 and Gat1-Myc13 localization thancan be obtained from an isolated image of a few cells, wemanually scored Gln3-Myc13 or Gat1-Myc13 localization in$200 cells in multiple, randomly chosen microscopic fieldsfrom which each image presented was taken. Cells wereclassified into one of three categories: cytoplasmic (cyto-plasmic fluorescence only), nuclear cytoplasmic (fluores-cence appeared in the cytoplasm as well as colocalizing withDAPI-positive material), and nuclear (colocalizing only withDAPI-positive material). Limitations placed on interpreta-tions of these descriptions have been described in detail inearlier publications (Tate et al. 2006a,b, 2009; Georis et al.2008).

RESULTS

The requirements of Gln3 and Sit4 for rapamycin-induced DAL5 transcription depends on the geneticbackground: Recent studies, analyzing the effects ofdeleting the SIT4 gene on GATA factor localization,DNA-binding and transcriptional activation revealeda quite surprising and unsettling result. Rapamycin-induced DAL5 transcription was independent of Sit4and even partially independent of Gln3; i.e., in a gln3Dmutant, expression was still one-third of the wild-typelevel (Figure 2A). The latter was a much weaker re-quirement than observed for the gat1D in which DAL5expression was �25-fold lower than wild type (Figure2A). These results differed from multiple previousstudies demonstrating that DAL5 transcription pos-sessed a much greater requirement for Gln3 thanGat1; i.e., transcription had been abolished in a gln3Dwhile being only moderately decreased in a gat1D (e.g.,

Gzf3 Can Compromise NCR Transcription as a Tor Readout 863

TABLE 1

Strains used in this work

Strain Background Parent Complete genotype Primer/reference

25T0bP

MATa, ura3, his3, trp1 —25T0b(a)

P25T0b MATa, ura3, his3, trp1 —

FV022P

25T0b MATa, ura3, his3, trp1, gln3TkanMX gln3: 59, �438 to �421and �15 to �1; 39, 2194–2211and 2597–2614

FV022aP

25T0b(a) MATa, ura3, his3, trp1, gln3TkanMX gln3: 59, �438 to �421and �15 to �1; 39, 2194–2211and 2597–2614

FV023P

25T0b MATa, ura3, his3, trp1, gat1TnatMX gat1: 59, �422 to �405and �15 to �1; 39, 1534–1555and 1879–1896

FV024P

FV023 MATa, ura3, his3, trp1, gln3TkanMX,gat1TnatMX

gln3: 59, �438 to �421and �15 to �1; 39, 2194–2211and 2597–2614

FV025P

25T0b MATa, ura3, his3, trp1, ure2TkanMX ure2: 59, �300 to �279and �21 to �1; 39 1066–1084and 1325–1345

FV027P

25T0b MATa, ura3, his3, trp1, sit4TkanMX sit4: 59, �450 to �429and �23 to �1; 39, 937–955and 1380–1400

FV027aP

25T0b(a) MATa, ura3, his3, trp1, sit4TkanMX sit4: 59, �450 to �429and �23 to �1; 39, 937–955and 1380–1400

FV034P

25T0b MATa, ura3, his3, trp1, GAT1-MYC13

[HIS3]GAT1-F2 and GAT1-R1

(Georis et al. 2008)FV036

P25T0b MATa, ura3, his3, trp1, GLN3-MYC13

[HIS3]GLN3-F2 and GLN3-R1


PFV023 MATa, ura3, his3, trp1, gat1TnatMX,

GLN3-MYC13[HIS3]GLN3-F2 and GLN3-R1


PFV036 MATa, ura3, his3, trp1, sit4TkanMX,

GLN3-MYC13[HIS3]sit4: 59, �450 to �429

and �23 to �1; 39, 937–955and 1380–1400

FV076P

FV069 MATa, ura3, his3, trp1, sit4TkanMX,ure2TnatMX, GLN3-MYC13[HIS3]

ure2: 59, �300 to �279and �21 to �1; 39, 1066–1084and 1325–1345

FV080P

25T0b(a) MATa, ura3, his3, trp1, dal80TkanMX dal80: 59, �533 to �516and �38 to �1; 39, 811–848and 1324–1341

FV083P

25T0b(a) MATa, ura3, his3, trp1, gzf3TkanMX gzf3: 59, �484 to �467and �38 to �1; 39, 1654–1690and 2092–2109

FV086P

FV034 MATa, ura3, his3, trp1, ure2TnatMX,GAT1-MYC13[HIS3]

ure2: 59, �300 to �279and �21 to �1; 39 1066–1084and 1325–1345

FV087P

03803c ura3, his3, trp1, sit4TkanMX,ure2TnatMX, GAT1-MYC13[HIS3]

ure2: 59, �300 to �279and �21 to �1; 39 1066–1084and 1325–1345

FV090P

FV036 MATa, ura3, his3, trp1, ure2TnatMX,GLN3-MYC13[HIS3]

ure2: 59, �300 to �279and �21 to �1; 39 1066–1084and 1325–1345

FV110P

FV080 MATa, ura3, his3, trp1, dal80TkanMX,gln3TnatMX

gln3: 59, �438 to �421and �15 to �1; 39, 2194–2211and 2597–2614

FV113P

FV083 MATa, ura3, his3, trp1, gln3TnatMX,gzf3TkanMX

gln3: 59, �438 to �421and �15 to �1; 39, 2194–2211and 2597–2614

FV114P

FV083 MATa, ura3, his3, trp1, gat1TnatMX,gzf3TkanMX

gat1: 59, �422 to �405and �15 to �1; 39, 1534–1555and 1879–1896

(continued )


Daugherty et al. 1993; Coffman et al. 1994, 1996a,b,1997; Tate et al. 2002; Tate and Cooper 2003).

The extensive use of DAL5 as a reporter in mechanis-tic studies of NCR-sensitive transcription and Torpathway signaling necessitated that we rectify theseapparently conflicting results. Since significant differ-ences could not be detected in growth or assay con-ditions, we shifted our attention to the S. cerevisiaestrains themselves. We noted that S1278b (Sigma)-related strains had been used in most earlier studies ofNCR (Coffman et al. 1995, 1996a,b, 1997; Daughertyet al. 1993; Tate et al. 2002; Tate and Cooper 2003),while those used in more recent Tor studies derivedfrom TB123 (TB)- and occasionally BY4709 (BY)-related

strains (Beck and Hall 1999; Tate et al. 2006a,b, 2009;Georis et al. 2008). This correlation suggested that theconflicting results might derive from the strain back-grounds assayed, a possibility strengthened by reportsdemonstrating a surprisingly high impact of geneticbackground on NCR-sensitive and retrograde transcrip-tional regulation (Tate et al. 2002; Dilova and Powers2006).

Therefore, we compared the requirements of rapa-mycin-induced DAL5 expression in the three geneticbackgrounds. In contrast with data from TB-relatedstrains (Figure 2A), deleting GLN3 (FV022), GAT1(FV023), or SIT4 (FV027) in the Sigma backgrounddecreased rapamycin-induced DAL5 mRNA to almost

TABLE 1

(Continued)

Strain Background Parent Complete genotype Primer/reference

FV126P

FV083 MATa, ura3, his3, trp1, ure2TnatMX,gzf3TkanMX

ure2: 59, �300 to �279and �21 to �1; 39 1066–1084and 1325–1345

03740cP

FV22a 3 FV34 ura3, his3, trp1, gln3TkanMX,GAT1-MYC13[HIS3]

—

03803cP

FV34 3 FV027a ura3, his3, trp1, sit4TkanMX,GAT1-MYC13[HIS3]

—

4709DGLN3 BY BY4709 MATa, ura3, gln3TkanMX Scherens et al. (2006)4709DGAT1 BY BY4709 MATa, ura3, gat1TkanMX Scherens et al. (2006)BY4709 BY BY4709 MATa, ura3 —FV017 BY BY4709 MATa, ura3, GLN3-MYC13[KanMX] GLN3-F2 and GLN3-R1

(Georis et al. 2008)FV001 BY FV017 MATa, ura3, sit4TnatMX,

GLN3-MYC13[KanMX]sit4: 59, �450 to �429

and �23 to �1; 39, 937–955and 1380–1400

TB50 TB MATa, leu2-3,112, ura3-52,trp1, his3, rme1, HMLa

—

FV005 TB TB50 MATa, leu2-3,112, ura3-52, trp1,his3, rme1, HMLa, gln3TkanMX

gln3: 59, �438 to �421and �15 to �1; 39, 2194–2211and 2597–2614

FV006 TB TB50 MATa, leu2-3,112, ura3-52, trp1,his3, rme1, HMLa, gat1TkanMX

gat1: 59, �422 to �405and �15 to �1; 39, 1534–1555and 1879–1896

FV007 TB FV006 MATa, leu2-3,112, ura3-52, trp1,his3, rme1, HMLa, gat1TkanMX,gln3TkanMX

gln3: 59, �438 to �421and �15 to �1; 39, 2194–2211and 2597–2614

FV029 TB TB50 MATa, leu2-3,112, ura3-52, trp1,his3, rme1, HMLa, sit4TnatMX

sit4: 59, �450 to �429and �23 to �1; 39, 937–955and 1380–1400

FV063 TB TB050 MATa, leu2-3,112, ura3-52, trp1,his3, rme1, HMLa, GAT1-MYC13[HIS3]

GAT1-F2 and GAT1-R1(Georis et al. 2008)

FV064 TB FV005 MATa, leu2-3,112, ura3-52, trp1, his3,rme1, HMLa, gln3TkanMXGAT1-MYC13[HIS3]

GAT1-F2 and GAT1-R1(Georis et al. 2008)

FV066 TB FV063 MATa, leu2-3,112, ura3-52, trp1,his3, rme1, HMLa, sit4TkanMX,GAT1-MYC13[HIS3]

sit4: 59, �450 to �429and �23 to �1; 39, 937–955and 1380–1400

FV211 TB TB50 MATa, leu2-3,112, ura3-52, trp1,his3, rme1, HMLa, gzf3TkanMX

gzf3: 59, �484 to �467and �38 to �1; 39, 1654–1690and 2092–2109

Primer coordinates are taken from the DNA sequence of strain S288C.


background levels (Figure 2B). In other words, DAL5expression exhibited strong requirements for all threeproteins rather than for just Gat1. The parallel absenceof DAL5 expression in a BY sit4D (FV001) strainsuggested that this genetic background perhaps pos-sessed requirements that were more similar in thisrespect to those of Sigma than to those of TB strains(Figure 2C). Together, these data indicate that it wasgenetic background differences that accounted for theinconsistent GATA factor and Sit4 requirements ofDAL5 transcription.

Genetic background specificities rely on mechanismsdownstream of GATA factor binding: Our next objec-tive was to identify the genetic background-specificdifference(s) in the strains. The stronger deleteriouseffect of a sit4D on DAL5 expression in Sigma vs. TBstrains (compare A and B in Figure 2) enabled us to testthe epistatic relationship between sit4 and ure2 muta-tions (Figure 2D). Rapamycin induced DAL5 transcrip-tion to the same high levels in wild type, ure2D andure2Dsit4D cells, whereas DAL5 transcription was mini-mal in the sit4D. This demonstrated that ure2 mutationswere epistatic to those at SIT4. There was also a modest(twofold) positive synergistic effect of additionally de-leting SIT4 in an untreated, glutamine-grown ure2Dmutant. These data were similar to those obtained in theTB-related strains (Georis et al. 2008) and argued thatthe regulatory relationships between Ure2 and Sit4functions were similar in the two backgrounds and thusdid not account for the differences in transcription.

We next turned to the sequential component eventsleading up to transcription, first assaying nuclear GATAfactor localization in Sigma-derived wild-type and mu-tant strains. Rapamycin induced Gln3-Myc13 localizationin the wild type, but not in a sit4D mutant. Deleting

URE2 resulted in constitutive nuclear Gln3-Myc13 local-ization in the absence of rapamycin whether or not Sit4was present; i.e., a ure2D mutation was clearly epistatic toa SIT4 deletion. The behavior of Gat1-Myc13 was distinctfrom that of Gln3-Myc13 in two respects: there was amuch more modest negative effect of Ure2 on nuclearGat1-Myc13 localization in untreated cells than for Gln3-Myc13, and the Sit4 requirement for nuclear Gat1-Myc13

localization was minimal. These data indicated thatSigma and TB strains behaved similarly (compareFigure 3 in this work with Figure 6 in Georis et al.2008). Therefore, requirements for nuclear GATAfactor localization also did not account for the differ-ences in DAL5 transcription.

The next known downstream event in the pathwaywas GATA factor binding to its target sequences withinNCR-sensitive promoters. Here we found that, with onemajor exception, GATA factor binding to the DAL5promoters of Sigma and TB strains was similar (compareFigure 4, A and B, in this work with Figure 7, A and B, inGeoris et al. 2008). The exception occurred in thesit4Dure2D double mutant. Rapamycin induced muchgreater Gln3-Myc13 binding and much weaker Gat1-Myc13 binding to the Sigma DAL5 promoter than it didto the TB promoter where there was no demonstrableeffect. Additionally, as in the TB background, rapamycin-induced Gat1-Myc13 binding to the Sigma DAL5 pro-moter was independent of Gln3 (compare Figure 5A inthis work with Figure 4A in Georis et al. 2008), whereasGln3-Myc13 binding was Gat1 dependent (compareFigure 5B in this work with Figure 4B in Georis et al.2008).

The similarity of rapamycin-induced nuclear Gln3-Myc13 and Gat1-Myc13 localization and binding to theDAL5 promoters of TB and Sigma strains led us to

Figure 2.—Genetic background influen-ces the relative contributions of Gat1 andGln3 to rapamycin-induced DAL5 expres-sion. Wild-type and mutant cells were grownin yeast nitrogen base (YNB)–glutaminemedium. Split cultures were left untreated(Gln) or treated with rapamycin (0.2 mg/ml)for 30 min (1Rap) and processed for qRT–PCR analysis as described in materialsand methods. (A) Total RNA was isolatedfrom wild-type (wt; TB50), gln3D (FV005),gat1D (FV006), and sit4D (FV029) cells ofthe TB genetic background. Data in A ap-peared in Georis et al. (2008). (B) TotalRNA was isolated from wild-type (25T0b),gln3D (FV022), gat1D (FV023), and sit4D(FV027) cells of the Sigma genetic back-ground. (C) Total RNA was isolated fromwild-type (BY4709), gln3D (4709DGLN3),gat1D (4709DGAT1), and sit4D (FV001)

cells of the BY genetic background. (D) Total RNA was isolated from wild-type (25T0b), ure2D (FV025), sit4D (FV027), and ure2-Dsit4D (FV076) cells of the Sigma genetic background. (A–D) DAL5 mRNA levels were quantified by qRT–PCR, as described inmaterials and methods. DAL5 values were normalized with TBP1. Values represent the average of at least three experimentsfrom independent cultures, and error bars indicate standard errors.


conclude, by exclusion, that the genetic background-dependent difference occurred after GATA factorbinding to DAL5. In the TB genetic background, Gln3-independent Gat1-Myc13 binding alone was sufficient toactivate rapamycin-induced DAL5 expression, albeit at a

somewhat diminished level (Georis et al. 2008). Incontrast, the above data showed that in the Sigmabackground Gat1-Myc13 could bind to the DAL5 pro-moter independently of Gln3, but was unable to activatetranscription unless Gln3 was also present. Thus genetic

Figure 3.—Genetic background doesnotaffect rapamycin-induced intracellular local-ization of Gln3-Myc13 and Gat1-Myc13 in wild-type, ure2D, sit4D, and ure2Dsit4D mutantstrains. Wild-type and mutant cells weregrown to mid-log phase (A600nm ¼ 0.50–0.52) in YNB–glutaminemedium. At this celldensity, they were left untreated (Gln) ortreated with rapamycin (0.2 mg/ml) for20 min (1Rap). Samples were then taken,fixed, prepared for indirect immunofluores-cence microscopy, and analyzed as describedin materials and methods. (A and C) Theimages are presented in pairs with Gln3-Myc13 (red) visualized in the top image andDAPI-positive material (blue) in the bottomone. Histograms providing a fuller descrip-tion of the images depicted are shown in Band D. Gln3-Myc13 localization was catego-rized as cytoplasmic (red), nuclear and cyto-plasmic (yellow), and nuclear (green).Strain numbers and pertinent genotypesappearabovethe imagesandbelowthehisto-grams.


background-specific differences in DAL5 transcriptionlikely derived from differences in the requirements forGat1-dependent transcriptional activation.

Differing DAL5 expression requirements in the TBand Sigma genetic backgrounds are not monogenic:The genetic background-specific differences in Gat1’spotential to activate rapamycin-induced DAL5 transcrip-tion prompted us to determine whether one or moregenes were involved. To this end, we crossed SigmaFV027 (MATa, sit4D) with TB FV029 (MATa, sit4D) andassayed rapamycin-induced DAL5 expression in twoindependently isolated diploids and segregants of fourtetrads (03847, 03848, 03849, and 03850) derived fromthem. Low DAL5 expression in heterozygous diploid

cells, equivalent to levels observed in the Sigma-derivedstrains, indicated that Sigma strains possessed thedominant trait, i.e., the inability of Gat1 to mediateGln3-independent transcription (Figure 6). However,consistent 2:2 segregation of the parental phenotypeswas not observed, suggesting that, overall, two or moregenes were involved (Figure 6).

Genetic background-specific requirements forrapamycin-induced DAL5 transcription derive from anegatively-acting GATA factor: The finding that adominant trait was responsible for the inability of Gat1alone to support rapamycin-induced DAL5 transcrip-tion in Sigma strains suggested that a negatively-actinggene product might be responsible for the background-specific difference. Since two negatively-acting proteinsare well known to regulate NCR-sensitive transcription,the GATA repressors Dal80 and Gzf3/Deh1/Nil2 wereour first candidates. Deleting DAL80 has no effect onDAL5 transcription in glutamine-grown Sigma cells,whereas deleting DEH1/GZF3 weakly increases DAL5

Figure 4.—Genetic background does not affect rapamycin-induced in vivo binding of Gln3-Myc13 and Gat1-Myc13 to theDAL5 promoter in wild type, ure2D, sit4D, and ure2Dsit4D mu-tant strains. Wild-type and mutant cells were grown in YNB–glutamine medium. Split cultures were left untreated (Gln)or treated with rapamycin (0.2 mg/ml) for 30 min (1Rap),sampled, and processed for ChIP analyses as described inmaterials and methods. Wild-type untagged cells (25T0b)were used as a negative control. (A) In vivo binding ofGln3-Myc13 to the DAL5 promoter in wild type (FV036), ure2D(FV090), sit4D (FV069), and ure2Dsit4D (FV076) strains of theSigma genetic background. (B) In vivo binding of Gat1-Myc13

to the DAL5 promoter in wild-type (FV034), ure2D (FV086),sit4D (03803c), and ure2Dsit4D (FV087) strains of the Sigmagenetic background. (A and B) ChIP was performed usingantibodies against c-myc as described in materials andmethods. qPCR of IP/IN fractions was performed with pri-mers specific for the DAL5 promoter [DAL5P] and for a re-gion 2.5 kb upstream of the DAL5 ORF as a control[DAL5U]. For each immunoprecipitation, IP/IN values werecalculated as follows: ([DAL5P]IP/[DAL5P]IN � [DAL5U]IP/[DAL5U]IN). Histograms represent the average of two IPs per-formed on at least two experiments from independent cul-tures. Error bars indicate standard errors.

Figure 5.—Genetic background does not affect rapamycin-induced in vivo binding of Gln3-Myc13 and Gat1-Myc13 to theDAL5 promoter in gat1D and gln3D mutant strains. The exper-imental format and ChIP analyses were as described in Figure4 using the wild-type and mutant strains listed below. Wild-type untagged cells (25T0b) were used as negative control.(A) In vivo binding of Gat1-Myc13 to the DAL5 promoter inwild-type (FV034) and gln3D (03740c) strains of the Sigma ge-netic background. (B) In vivo binding of Gln3-Myc13 to theDAL5 promoter in wild type (FV036) and gat1D (FV041)strains of the Sigma genetic background.


expression under these culture conditions (Daughertyet al. 1993; Coffman et al. 1995, 1997). The effects ofthese mutations in rapamycin-treated cells, on the otherhand, have never been reported. Therefore, we ob-tained these data and found that loss of either Dal80 orGzf3 modestly increased DAL5 expression (less thantwofold) compared to the wild-type level (Figure 7A).However, the two deletion phenotypes differed greatly ifadditionally accompanied by the deletion of GLN3.Rapamycin-induced DAL5 expression in the dal80Dwas completely Gln3 dependent, whereas in the gzf3Dit was largely Gln3 independent and highly Gat1 de-pendent (Figure 7A). These data were those expected if,in contrast to the situation in Sigma strains, Gzf3 activityin TB strains had little effect on Gat1-mediated tran-scription. They were also in keeping with the observa-tion that Sigma strains possessed the dominant trait.

To test this conclusion further, we compared theeffects of deleting GZF3 in the TB- and Sigma-derivedstrains (Figure 7B). Deleting GZF3 in a TB backgroundhad little, if any, negative effect on rapamycin-inducedDAL5 transcription, whereas in the Sigma backgrounddeleting GZF3 modestly increased transcription. Finally,we determined the effects of rapamycin treatment onexpression of the GZF3 gene itself and found that it hadno effect in either genetic background (Figure 7C).However, there were approximately fourfold greateramounts of GZF3 mRNA in a Sigma wild type comparedto that in the TB background (Figure 7C). In agreementwith this observation it was possible to detect Gzf3protein by Western blot analysis in extracts derived fromSigma but not TB strains (data not shown). From thesedata, we concluded that Gzf3 inhibited the ability ofGat1, in the absence of Gln3, to activate transcriptiononly in the Sigma genetic background.

Genetic background-dependent ability of Gat1 toactivate transcription without Gln3 is gene specific:Since it was conceivable that the difference in Gat1’sability to activate rapamycin-induced DAL5 transcrip-tion independently of Gln3 might derive from somepeculiarity in the DAL5 promoter, we analyzed theexpression of two other NCR-sensitive genes, DAL7and GAP1. As occurred with DAL5, deleting SIT4 didnot adversely affect rapamycin-induced DAL7 expres-sion in the TB genetic background (Figure 8A), but

significantly decreased DAL7 mRNA in the correspondingSigma strain (Figure 8B). Yet, rapamycin-induced Gat1-Myc13 binding to the DAL7 promoter in sit4D cells wascomparable in both genetic backgrounds (Figure 9, A andB). Thus, in the Sigma background, Gat1-Myc13 bindingwas again not sufficient to activate rapamycin-inducedDAL7 expression, showing that genetic background-dependent differences could be demonstrated in morethan one gene and hence were unlikely to be caused bysome pecularity in a single promoter.

On the other hand, the responses and requirementsof DAL5 and DAL7 transcription to rapamycin treat-ment were not general. As shown in Figure 10, A and B,GAP1 transcription occurred in gln3D and gat1D singlemutants, but not in a gln3Dgat1D double mutant. Inother words, a GATA activator was required, but eitherGln3 or Gat1 would do. Consistent with the absence of aGln3 requirement for rapamycin-induced GAP1 expres-sion, Sit4 was also dispensable in both genetic back-grounds (Figure 10, A and B).

These results made a clear prediction that rapamycin-induced Gln3-Myc13 and Gat1-Myc13 binding to theGAP1 promoter should be independent of the otherGATA factor irrespective of the genetic backgroundassayed. As shown in Figure 11, these predictions weresubstantiated experimentally. Gln3-Myc13 binding to theGAP1 promoter occurred at nearly wild-type levels in agat1D of either background (Figure 11, A and B).Similarly, Gat1-Myc13 binding occurred at nearly wild-type levels in both TB gln3D and Sigma gln3D strains(Figure 11, C and 1D).

Finally, we asked how rapamycin-induced GAP1 ex-pression in TB and Sigma strains might be affected bydeletion of GZF3. As shown in Figure 12, there was little toperhaps a slightly negative effect of a gzf3D on rapamycin-induced GAP1 transcription in the TB genetic back-ground, thereby supporting and extending observationsobtained with DAL5. In the Sigma background, there wasa slight increase in rapamycin-induced GAP1 expression,but it was more modest than observed with DAL5. Inmarked contrast, however, GAP1 expression in an un-treated gzf3D was greatly increased relative to wild typeonly in the Sigma genetic background, suggesting thatGzf3 had no repressive effect in glutamine-grown cells ofthe TB genetic background (Figure 12).

Figure 6.—Differing DAL5 expression in TBand Sigma genetic backgrounds are not mono-genic. sit4D cells of the Sigma (FV027) and TB(FV029) genetic backgrounds were crossed.Two diploids (diploids 1 and 2) from this cross(FV027 3 FV029) were assayed along with thesegregants of four tetrads issued from the sporu-lation of diploid 1 (03847, 03848, 03849, and03850). All of the strains were grown in YNB–glutamine medium, treated with rapamycin(0.2 mg/ml) for 30 min, and processed forqRT–PCR analysis as described in Figure 2.


DISCUSSION

Dissecting the detailed molecular events throughwhich the Tor signal transduction pathway influencesNCR-sensitive transcription has been and continues to

be a complex task. However, this work will likelycontribute to rectifying some of the apparently conflict-ing conclusions, such as the differences in Gln3-/Gat1-dependent transcription in Sigma and TB strains, andavoid future inconsistencies between predicted andobserved results. Our experiments have identified andexplained several unexpected variables that potentiallycompromise interpretation of many reporter geneassays and transcriptome analyses employed in the pastto assess Tor pathway function, including (i) the geneticbackground of the strains analyzed, (ii) the regulatorycharacteristics of the reporter genes being assayed, (iii)the nature of their GATA factor requirements, and (iv)the interactions between GATA and other transcriptionfactors that are cumulatively responsible for the tran-scription of NCR-sensitive genes.

The conceptual problem encountered in interpret-ing reporter gene assays is the fact that they measure thegene’s overall expression, which is the cumulative out-come of multiple levels of regulation (Smart et al.1996). While this has long been known, what has notbeen appreciated until recently is that Gln3 and Gat1are regulated quite differently (Kulkarni et al. 2006;Georis et al. 2008). Gln3 is more sensitive than Gat1 tonegative regulation by Ure2. Furthermore, unlike Gln3,nuclear Gat1 accumulation is much less Sit4 dependent(Georis et al. 2008). There are additional variables thatalone or in combination can influence overall NCR-sensitive gene expression and hence conclusions de-rived from their use in investigations of the Tor pathway:(i) the ability of one GATA activator to activate tran-scription of the NCR-sensitive gene assayed in theabsence of the other GATA activator; (ii) the ability ofthe negatively acting GATA factors (Dal80, Gzf3) tocontrol the NCR-sensitive gene assayed; (iii) the abilityof non-GATA transcription factors to function with oneGATA factor, thereby relieving the necessity of the

Figure 7.—Genetic background-specific re-quirements for rapamycin-induced DAL5 transcrip-tionderivefromthenegativeGATAfactorGzf3.Theexperimental formatandqRT–PCRanalyseswereasdescribedinFigure2usingwild-typeandthemutantstrains listed below. (A) Total RNA was extractedfrom wild-type (25T0b), gln3D (FV022), dal80D(FV080), dal80Dgln3D (FV110), gzf3D (FV083),gzf3Dgln3D (FV113),gat1D (FV023),andgzf3Dgat1D(FV114) cells of the Sigma genetic background.DAL5 expression was assayed as described in Figure2. (B) Total RNA was extracted from wild-type TB(TB50) or Sigma (25T0b) and gzf3D TB (FV211)or Sigma (FV083) cells. DAL5 expression was as-sayed as described in Figure 2. (C) Total RNA wasextracted from wild-type TB (TB50) or Sigma(25T0b) cells. GZF3 mRNA levels were quantifiedby qRT–PCR, as described in materials and meth-ods.GZF3valueswerenormalizedwithTBP1.Valuesrepresent the average of at least three experimentsfrom independent cultures, and error bars indicatestandard errors.

Figure 8.—Genetic background-specific requirements forrapamycin-induced transcription are not restricted to DAL5.The experimental format and qRT–PCR analyses were per-formed as described in Figure 2 using wild-type and sit4D cells.(A) Total RNA was extracted from wild-type (TB50) and sit4D(FV029) cells of the TB genetic background. (B) Total RNAwas extracted from wild-type (25T0b) and sit4D (FV027) cellsof the Sigma genetic background. (A and B) DAL7 mRNA lev-els were quantified by qRT–PCR, as described in materialsand methods. DAL7 values were normalized with TBP1.


second one’s participation in supporting transcriptionwhen both GATA factors are required; and (iv) theinfluence of the genetic background of the strain beinginvestigated.

The above variables and potential problems of in-terpretation that derive from them are exemplified bythe two strains (TB and Sigma) and three genes (DAL5,DAL7, and GAP1) assayed in this work. Gat1, in theabsence of Gln3, can activate DAL5 and DAL7 transcrip-tion in TB- but not Sigma-derived strains. The lattergenetic background requires both Gln3 and Gat1 toactivate DAL5 and DAL7 transcription. Since Gln3 butnot Gat1 requires Sit4 for nuclear localization, DAL5and DAL7 transcription in TB strains exhibits no Sit4requirement, whereas in Sigma strains the Sit4 require-ment is nearly absolute. Therefore, apparent participa-tion of Tor regulation in this scenario depends uponwhich strain is assayed. Consider a second example, i.e.,assaying two genes in the Sigma genetic background.DAL5 and DAL7 transcription requires both Gln3 andGat1 and hence there is a strong Sit4 requirement forDAL5 and DAL7 transcription. On the other hand,

either Gln3 or Gat1, in the absence of the other GATAactivator, is capable of activating GAP1 transcription inboth TB and Sigma strains. Therefore, if DAL5 andDAL7 expression is assayed in Sigma strains, a strongSit4 requirement is observed, but if GAP1 is assayed inthe same genetic background, no such requirement isobserved. For results employing NCR-sensitive genetranscription as a reporter of Tor pathway activity to beinterpretable, measurements of the reporter gene’sexpression in gln3D and gat1D single mutants as wellas in the gln3Dgat1D double mutant is necessary. Datawith the double mutant alone are insufficient.

The fact that one can obtain conflicting results is clearfrom these examples. However, the disparate conclu-sions may be rectified by considering the characteristicsof the strains and genes assayed: (i) Sit4 was required forGln3, but not Gat1 nuclear localization in both the TBand Sigma genetic backgrounds; (ii) Gzf3 preventedGat1 from activating GATA-factor-dependent gene tran-scription in Sigma but not in the TB strain; (iii) withoutGat1, Gln3 was able to bind to the promoters of someNCR-sensitive genes but not others and activate theirtranscription even in the Sigma genetic background;and (iv) Gat1 did not require Gln3 to bind to NCR-sensitive promoters but was not always able to activatetranscription.

The apparently paradoxical nature of the last twocharacteristics may derive from the participation of bothGATA and non-GATA factors in the activation of genes

Figure 9.—Genetic background does not affect rapamycin-induced in vivo binding of Gat1-Myc13 to the DAL7 promoterin a sit4D mutant strain. The experimental format and ChIPanalyses were as described in Figure 4 using the wild-type andmutant strains listed below. (A) In vivo binding of Gat1-Myc13

to the DAL7 promoter in wild-type (FV063) and sit4D (FV066)strains of the TB genetic background. Wild-type untaggedcells (TB50) were used as negative control. (B) In vivo bindingof Gat1-Myc13 to the DAL7 promoter in wild-type (FV034) andsit4D (03803c) strains of the Sigma genetic background. Wild-type untagged cells (25T0b) were used as negative control.qPCR of IP/IN fractions was performed with primers specificfor the DAL7 promoter [DAL7P] and for a region 2.5 kbupstream of the DAL5 ORF as a control [DAL5U]. IP/INvalues were calculated as follows: ([DAL7P]IP/[DAL7P]IN �[DAL5U]IP/[DAL5U]IN).

Figure 10.—Genetic background-specific requirements forrapamycin-induced transcription are not identical for allNCR-sensitive genes. The experimental format and qRT–PCR analyses were as described in Figure 2 using wild-typeand the mutant strains listed below. (A) Total RNA was ex-tracted from wild-type (TB50), gln3D (FV005), gat1D(FV006), gat1Dgln3D (FV007), and sit4D (FV029) cells ofthe TB genetic background. (B) Total RNA was extractedfrom wild type (25T0b), gln3D (FV022), gat1D (FV023),gat1Dgln3D (FV024), and sit4D (FV027) cells of the Sigma ge-netic background.


whose transcription exhibits these characteristics. Sev-eral examples of such functional cooperation in GATA-factor-mediated transcription have been previouslydocumented:

i. The CAR1 promoter consists of at least 14 functionalupstream activation sequences working togetherto produce the overall CAR1 expression (Smartet al. 1996). Two UASNTR GATA elements are re-sponsible for its NCR sensitivity, while three UASIelements are responsible for inducibility by argi-nine. Under some culture conditions, all of theseelements function, while with others only a subset ofthem are operating (Smart et al. 1996; Dubois andMessenguy 1997).

ii. In the case of the PUT1 promoter, a syntheticfragment from the promoter, containing a single

GATA element and a Put3 binding site, supportsheterologous reporter gene transcription. In thiscase, only a single GATA factor can be functioningbecause there is only one binding site present (Raiet al. 1995).

iii. In the case of DAL5, there are required upstreamactivation sequences immediately downstream (twoand one turns, 21 and 11 bp, respectively) of each ofthe two GATA elements that account for themajority (�90%) of DAL5 transcription (Rai et al.2004).

iv. Finally, placing a wild-type Dal82-binding site (cis-acting element responsible for allophanate-induciblegene expression) adjacent to a mutated GATAelement (Gln3/Gat1-binding site) suppressed themutant phenotype (Van Vuuren et al. 1991).

This work makes three additional contributions thatfurther our understanding of nitrogen regulation in S.cerevisiae. The first is identification of a new regulatoryfunction for the negatively acting GATA factor, Gzf3.Although the existence of Gzf3 and the regulation of itsproduction have been known for some time, little wasknown about why two homologous, negatively actingGATA factors (Dal80 and Gzf3/Deh1) existed. Thiswork provides one such function. Why, however, Gzf3 ispresent and functional in the Sigma but not in the TBgenetic background remains to be elucidated. Second isthe remarkably robust ability of rapamycin to increaseDAL5 transcription and GATA factor binding to thepromoters of these genes in ure2D cells as well as thegenetic background-specific effects brought about byadditionally deleting SIT4 in both the presence andabsence of rapamycin. These observations forecast thatone or more yet-to-be-discovered components of theTor1,2 regulatory pathway function downstream of

Figure 11.—In vivo binding profiles of Gat1-Myc13 and Gln3-Myc13 to the GAP1 promoterare similar in TB and Sigma genetic backgroundsand are only weakly affected by the deletion ofone or the other GATA activator. The experimen-tal format and ChIP analyses were as described inFigure 4, using the wild-type and mutant strainslisted below. qPCR of IP/IN fractions was per-formed with primers specific for the GAP1 pro-moter [GAP1P] and for a region 2.5 kb upstreamof the DAL5 ORF as a control [DAL5U]. IP/INvalues were calculated as follows: ([GAP1P]IP/[GAP1P]IN � [DAL5U]IP/[DAL5U]IN). (A) Invivo binding of Gln3-Myc13 to the GAP1 promoterin wild-type (TB123) and gat1D (FV018) strainsof the TB genetic background. Wild-type un-tagged cells (TB50) were used as negative con-trol. (B) In vivo binding of Gln3-Myc13 to theGAP1 promoter in wild-type (FV036) and gat1D(FV041) strains of the Sigma genetic back-

ground. Wild-type untagged cells (25T0b) were used as negative control. (C) In vivo binding of Gat1-Myc13 to the GAP1 promoterin wild-type (FV063) and gln3D (FV064) strains of the TB genetic background. Wild-type untagged cells (TB50) were used asnegative control. (D) In vivo binding of Gat1-Myc13 to the GAP1 promoter in wild-type (FV034) and gln3D (03740c) strains ofthe Sigma genetic background. Wild-type untagged cells (25T0b) were used as negative control.

Figure 12.—Gzf3 does not control GAP1 expression in theTB genetic background. The experimental format and qRT–PCR analyses were as described in Figure 2 using wild-type andthe mutant strains listed below. Total RNA was extracted fromwild-type (25T0b) and gzf3D (FV083) cells of the Sigma ge-netic background and from wild-type (TB50) and gzf3D(FV211) cells of the TB genetic background.


intracellular transcription factor localization. Third, theabove observations may help to explain why lists of NCR-sensitive genes that appear in the results of varioustranscriptome analyses vary so much and also frequentlyfail to identify multiple genes whose transcription ispreviously known to be NCR sensitive.

We thank Michael Hall for strains, Tim Higgins for preparing theartwork, Fabienne Vierendeels for excellent technical assistance, andthe University of Tennessee Yeast Group for suggestions for improvingthe manuscript. Work by E.D. and I.G. was supported by theCommission Communautaire Francxaise. Work by J.J.T. and T.G.C.was supported by National Institutes of Health (NIH) grant GM-35642and NIH/National Science Foundation grant DMS-0443901.

LITERATURE CITED

Adami, A., B. Garcı́a-Alvarez, E. Arias-Palomo, D. Barford and O.Llorca, 2007 Structure of TOR and its complex with KOG1.Mol. Cell 27: 509–516.

Aronova, S., K. Wedaman, S. Anderson, J. Yates, III and T. Powers,2007 Probing the membrane environment of the TOR kinasesreveals functional interactions between TORC1, actin, and mem-brane trafficking in Saccharomyces cerevisiae. Mol. Biol. Cell 18:2779–2794.

Beck, T., and M. N. Hall, 1999 The TOR signalling pathway con-trols nuclear localization of nutrient-regulated transcription fac-tors. Nature 402: 689–692.

Bertram, P. G., J. H. Choi, J. Carvalho, W. Ai, C. Zeng et al.,2000 Tripartite regulation of Gln3p by TOR, Ure2p, and phos-phatases. J. Biol. Chem. 275: 35727–35733.

Cardenas, M. E., N. S. Cutler, M. C. Lorenz, C. J. Di Como andJ. Heitman, 1999 The TOR signaling cascade regulates geneexpression in response to nutrients. Genes Dev. 13: 3271–3279.

Carvalho, J., P. G. Bertram, S. R. Wente and X. F. Zheng,2001 Phosphorylation regulates the interaction between Gln3pand the nuclear import factor Srp1p. J. Biol. Chem. 276: 25359–25365.

Coffman, J. A., H. M. el Berry and T. G. Cooper, 1994 The URE2protein regulates nitrogen catabolic gene expression throughthe GATAA-containing UASNTR element in Saccharomyces cerevi-siae. J. Bacteriol. 176: 7476–7483.

Coffman, J. A., R. Rai and T. G. Cooper, 1995 Genetic evidence forGln3p-independent, nitrogen catabolite repression-sensitivegene expression in Saccharomyces cerevisiae. J. Bacteriol. 177:6910–6918 (erratum: J. Bacteriol. 178: 2159).

Coffman, J. A., R. Rai, T. Cunningham, V. Svetlov and T. G.Cooper, 1996a Gat1p, a GATA family protein whose produc-tion is sensitive to nitrogen catabolite repression, participatesin transcriptional activation of nitrogen-catabolic genes in Saccha-romyces cerevisiae. Mol. Cell. Biol. 16: 847–858.

Coffman, J., R. Rai, T. Cunningham, V. Svetlov and T. G. Cooper,1996b NCR-sensitive transport gene expression in S. cerevisiaeis controlled by a branched regulatory pathway consisting ofmultiple NCR-responsive activator proteins. Folia Microbiol. 41:85–86.

Coffman, J. A., R. Rai, D. M. Loprete, T. Cunningham, V. Svetlovet al., 1997 Cross regulation of four GATA factors that controlnitrogen catabolic gene expression in Saccharomyces cerevisiae.J. Bacteriol. 179: 3416–3429.

Cooper, T. G., 2002 Transmitting the signal of excess nitrogen inSaccharomyces cerevisiae from the Tor proteins to the GATA factors:connecting the dots. FEMS Microbiol. Rev. 26: 223–238.

Cooper, T. G., 2004 Integrated regulation of the nitrogen-carbon in-terface, pp. 225–257 in Nutrient-Induced Responses in Eukaryotic Cells:Topics in Current Genetics, Vol. 7, edited by J. Winderickx andP. M. Taylor. Springer-Verlag, Berlin/Heidelberg, Germany/New York.

Cox, K. H., R. Rai, M. Distler, J. R. Daugherty, J. A. Coffman et al.,2000 Saccharomyces cerevisiae GATA sequences function as TATA

elements during nitrogen catabolite repression and when Gln3pis excluded from the nucleus by overproduction of Ure2p. J. Biol.Chem. 275: 17611–17618.

Cox, K. H., J. J. Tate and T. G. Cooper, 2002 Cytoplasmic compart-mentation of Gln3 during nitrogen catabolite repression and themechanism of its nuclear localization during carbon starvation inSaccharomyces cerevisiae. J. Biol. Chem. 277: 37559–37566.

Cox, K. H., A. Kulkarni, J. J. Tate and T. G. Cooper, 2004 Gln3phosphorylation and intracellular localization in nutrient limita-tion and starvation differ from those generated by rapamycin in-hibition of Tor1/2 in Saccharomyces cerevisiae. J. Biol. Chem. 279:10270–10278.

Cunningham, T. S., and T. G. Cooper, 1993 The Saccharomyces cer-evisiae DAL80 repressor protein binds to multiple copies ofGATAA-containing sequences (URSGATA). J. Bacteriol. 175:5851–5861.

Cunningham, T. S., R. A. Dorrington and T. G. Cooper, 1994 TheUGA4 UASNTR site required for GLN3-dependent transcriptionalactivation also mediates DAL80-responsive regulation and Dal80protein binding in Saccharomyces cerevisiae. J. Bacteriol. 176: 4718–4725.

Cunningham, T. S., V. V. Svetlov, R. Rai, W. Smart and T. G. Cooper,1996 Gln3p is capable of binding to UASNTR elements and ac-tivating transcription in Saccharomyces cerevisiae. J. Bacteriol.178: 3470–3479.

Cunningham, T. S., R. Andhare and T. G. Cooper, 2000a Nitrogencatabolite repression of DAL80 expression depends on the rela-tive levels of Gat1p and Ure2p production in Saccharomyces cerevi-siae. J. Biol. Chem. 275: 14408–14414.

Cunningham, T. S., R. Rai and T. G. Cooper, 2000b The level ofDAL80 expression down-regulates GATA factor-mediated tran-scription in Saccharomyces cerevisiae. J. Bacteriol. 182: 6584–6591.

Daugherty, J. R., R. Rai, H. M. el Berry and T. G. Cooper,1993 Regulatory circuit for responses of nitrogen catabolic geneexpression to the Gln3 and Dal80 proteins and nitrogen cataboliterepression in Saccharomyces cerevisiae. J. Bacteriol. 175: 64–73.

Di Como, C. J., and K. T. Arndt, 1996 Nutrients, via the Tor pro-teins, stimulate the association of Tap42 with type 2A phospha-tases. Genes Dev. 10: 1904–1916.

Di Como, C. J., and Y. Jiang, 2006 The association of Tap42 phos-phatase complexes with TORC1: another level of regulation inTor signaling. Cell Cycle 5: 2729–2732.

Dilova, I., and T. Powers, 2006 Accounting for strain-specific dif-ferences during RTG target gene regulation in Saccharomyces cer-evisiae. FEMS Yeast Res. 6: 112–119.

Dubois, E., and F. Messenguy, 1997 Integration of the multiplecontrols regulating the expression of the arginase gene CAR1of Saccharomyces cerevisiae in response to different nitrogen sig-nals: role of Gln3p, ArgRp-Mcm1p, and Ume6p. Mol. Gen.Genet. 253: 568–580.

Düvel, K., A. Santhanam, S. Garrett, L. Schneper and J. R. Broach,2003 Multiple roles of Tap42 in mediating rapamycin-inducedtranscriptional changes in yeast. Mol. Cell 11: 1467–1478.

Feller, A., M. Boeckstaens, A. M. Marini and E. Dubois,2006 Transduction of the nitrogen signal activating Gln3-mediated transcription is independent of Npr1 kinase andRsp5-Bul1/2 ubiquitin ligase in Saccharomyces cerevisiae. J. Biol.Chem. 281: 28546–28554.

Georis, I., J. J. Tate, T. G. Cooper and E. Dubois, 2008 Tor pathwaycontrol of the nitrogen-responsive DAL5 gene bifurcates at thelevel of Gln3 and Gat1 regulation in Saccharomyces cerevisiae. J.Biol. Chem. 283: 8919–8929.

Giannattasio, S., Z. Liu, J. Thornton and R. A. Butow,2005 Retrograde response to mitochondrial dysfunction is sep-arable from TOR1/2 regulation of retrograde gene expression.J. Biol. Chem. 280: 42528–42535.

Hardwick, J. S., F. G. Kuruvilla, J. K. Tong, A. F. Shamji andS. L. Schreiber, 1999 Rapamycin-modulated transcriptiondefinesthe subset of nutrient-sensitive signaling pathways directly controlledby the Tor proteins. Proc. Natl. Acad. Sci. USA 96: 14866–14870.

Hofman-Bang, J., 1999 Nitrogen catabolite repression in Saccharo-myces cerevisiae. Mol. Biotechnol. 12: 35–73.


Jacinto, E., B. Guo, K. T. Arndt, T. Schmelzle and M. N. Hall,2001 Tip41 interacts with Tap42 and negatively regulates theTOR signaling pathway. Mol. Cell 8: 1017–1026.

Jiang, Y., and J. R. Broach, 1999 Tor proteins and protein phospha-tase 2A reciprocally regulate Tap42 in controlling cell growth inyeast. EMBO J. 18: 2782–2792.

Kulkarni, A., T. D. Buford, R. Rai and T. G. Cooper,2006 Differing responses of Gat1 and Gln3 phosphorylationand localization to rapamycin and methionine sulfoximine treat-ment in Saccharomyces cerevisiae. FEMS Yeast Res. 6: 218–229.

Loewith, R., E. Jacinto, S. Wullschleger, A. Lorberg, J. L. Crespoet al., 2002 Two TOR complexes, only one of which is rapamycinsensitive, have distinct roles in cell growth control. Mol. Cell 10:457–468.

Magasanik, B., and C. A. Kaiser, 2002 Nitrogen regulation in Sac-charomyces cerevisiae. Gene 290: 1–18.

Rai, R., J. R. Daugherty and T. G. Cooper, 1995 UASNTR func-tioning in combination with other UAS elements underlies ex-ceptional patterns of nitrogen regulation in Saccharomycescerevisiae. Yeast 11: 247–260.

Rai, R., J. R. Daugherty, J. J. Tate, T. D. Buford and T. G. Cooper,2004 Synergistic operation of four cis-acting elements mediatehigh level DAL5 transcription in Saccharomyces cerevisiae. FEMSYeast Res. 5: 29–41.

Reinke, A., S. Anderson, J. M. McCaffery, J. Yates, III, S. Aronovaet al., 2004 TOR complex 1 includes a novel component,Tco89p (YPL180w), and cooperates with Ssd1p to maintain cel-lular integrity in Saccharomyces cerevisiae. J. Biol. Chem. 279:14752–14762.

Rowen, D. W., N. Esiobu and B. Magasanik, 1997 Role of GATAfactor Nil2p in nitrogen regulation of gene expression in Saccha-romyces cerevisiae. J. Bacteriol. 179: 3761–3766.

Scherens, B., A. Feller, F. Vierendeels, F. Messenguy and E.Dubois, 2006 Identification of direct and indirect targets ofthe Gln3 and Gat1 activators by transcriptional profiling in re-sponse to nitrogen availability in the short and long term. FEMSYeast Res. 6: 777–791.

Shamji, A. F., F. G. Kuruvilla and S. L. Schreiber, 2000 Partitioningthe transcriptional program induced by rapamycin among the ef-fectors of the Tor proteins. Curr. Biol. 10: 1574–1581.

Smart, W. C., J. A. Coffman and T. G. Cooper, 1996 Combinatorialregulation of the Saccharomyces cerevisiae CAR1 (arginase) pro-moter in response to multiple environmental signals. Mol. Cell.Biol. 16: 5876–5887.

Soussi-Boudekou, S., S. Vissers, A. Urrestarazu, J.-C. Jauniauxand B. André, 1997 Gzf3p, a fourth GATA factor involved innitrogen-regulated transcription in Saccharomyces cerevisiae. Mol.Microbiol. 23: 1157–1168.

Stanbrough, M., D. W. Rowen and B. Magasanik, 1995 Role ofthe GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in

the expression of nitrogen-regulated genes. Proc. Natl. Acad.Sci. USA 92: 9450–9454.

Svetlov, V., and T. G. Cooper, 1998 The Saccharomyces cerevisiaeGATA factors Dal80p and Deh1p can form homo- and heterodi-meric complexes. J. Bacteriol. 180: 5682–5688.

Tate, J. J., and T. G. Cooper, 2003 Tor1/2 regulation of retrogradegene expression in Saccharomyces cerevisiae derives indirectly as aconsequence of alterations in ammonia metabolism. J. Biol.Chem. 278: 36924–36933.

Tate, J. J., K. H. Cox, R. Rai and T. G. Cooper, 2002 Mks1p is re-quired for negative regulation of retrograde gene expression inSaccharomyces cerevisiae but does not affect nitrogen catabolite re-pression-sensitive gene expression. J. Biol. Chem. 277: 20477–20482.

Tate, J. J., R. Rai and T. G. Cooper, 2005 Methionine sulfoximinetreatment and carbon starvation elicit Snf1-independent phos-phorylation of the transcription activator Gln3 in Saccharomycescerevisiae. J. Biol. Chem. 280: 27195–27204 (erratum: J. Biol.Chem. 282: 13139).

Tate, J. J., A. Feller, E. Dubois and T. G. Cooper,2006a Saccharomyces cerevisiae Sit4 phosphatase is active irrespec-tive of the nitrogen source provided, and Gln3 phosphorylationlevels become nitrogen source-responsive in a sit4-deleted strain.J. Biol. Chem. 281: 37980–37992.

Tate, J. J., R. Rai and T. G. Cooper, 2006b Ammonia-specific regu-lation of Gln3 localization in Saccharomyces cerevisiae by proteinkinase Npr1. J. Biol. Chem. 281: 28460–28469.

Tate, J. J., I. Georis, A. Feller, E. Dubois and T. G. Cooper,2009 Rapamycin-induced Gln3 dephosphorylation is insuffi-cient for nuclear localization: Sit4 and PP2A phosphatases areregulated and function differently. J. Biol. Chem. 284: 2522–2534.

van Vuuren, H. J., J. R. Daugherty, R. Rai and T. G. Cooper,1991 Upstream induction sequence, the cis-acting element re-quired for response to the allantoin pathway inducer and en-hancement of operation of the nitrogen-regulated upstreamactivation sequence in Saccharomyces cerevisiae. J. Bacteriol. 173:7186–7195.

Wang, H., X. Wang and Y. Jiang, 2003 Interaction with Tap42 is re-quired for the essential function of Sit4 and type 2A phospha-tases. Mol. Biol. Cell. 14: 4342–4351.

Yan, G., X. Shen and Y. Jiang, 2006 Rapamycin activates Tap42-associated phosphatase by abrogating their association withTor complex 1. EMBO J. 25: 3546–3555.

Communicating editor: A. P. Mitchell


Documents

Nitrogen Catabolite Repression-Sensitive Transcription as a ......The Gat1 require-Figure 1.—Current models describing Tor pathway regula-tion of Gln3 dephosphorylation, localization,