MOLECULAR AND CELLULAR BIOLOGY, July 2009, p. 3803–3815
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 13
The Yeast GATA Factor Gat1 Occupies a Central Position in
Nitrogen Catabolite Repression-Sensitive Gene Activation?
Isabelle Georis, Andre ´ Feller, Fabienne Vierendeels, and Evelyne Dubois*
Institut de Recherches Microbiologiques J.-M. Wiame, Laboratoire de Microbiologie, Universite ´ Libre de Bruxelles,
Av. E. Gryson 1, B-1070 Bruxelles, Belgium
Received 27 March 2009/Accepted 6 April 2009
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.
All living cells monitor their environment to ensure that
sufficient nutrients are available to complete a full cell cycle.
Adaptation of growth and metabolism to the nitrogen supply is
a major issue for all organisms, is altered in cancer cells (17),
and has been particularly well documented in the eukaryotic
model organism Saccharomyces cerevisiae.
Yeast cell division is inhibited by nitrogen starvation, and in
contrast, sporulation and hyphal growth are hampered by rich
nitrogen sources. Metabolic reprogramming in the transition
from nitrogen-starved to nitrogen-rich yeast cultures occurs at
two main levels: (i) posttranslationally, affecting the activity of
amino acid permeases through control of their internalization
and degradation in the vacuole (reviewed in references 21 and
27); and (ii) transcriptionally, to restrain the cells’ capacity to
synthesize enzymes and permeases required for using nonpre-
ferred nitrogen sources (proline, allantoin, and GABA) when
readily usable nitrogen sources (glutamine and asparagine) are
available. The latter repressive effect is called nitrogen catab-
olite repression (NCR). Expression of NCR-sensitive genes is
coordinated by the prion-like Ure2 protein and four DNA-
binding proteins possessing homologous GATA-type zinc fin-
gers: two activators (Gln3 and Gat1/Nil1) and two repressors
(Dal80/Uga43 and Gzf3/Deh1/Nil2) (see references 9, 23, 26,
and 27 and references therein). In the presence of good nitro-
gen sources, the GATA activators are sequestered to the cy-
toplasm by Ure2, whereas upon depletion of the repressive
nitrogen sources, NCR is relieved and transcription of NCR-
sensitive genes is activated by Gln3, Gat1, or both (10, 11, 29,
Besides the action of the two GATA activators, modulation
of NCR gene expression is performed by the GATA repressors
Dal80 and Gzf3. Whereas the two activators possess a homol-
ogous asparagine-rich region located approximately 200 amino
acid residues from the N terminus (36), the two repressors
share a C-terminal leucine zipper domain responsible for the
ability of these proteins to homo- and heterodimerize (37).
Both Dal80 and Gzf3 bind in vitro to GATAA-containing
promoter fragments (8). Whereas binding of Dal80 requires
two GATAAG sequences separated by 15 to 30 bp (14), Gzf3
can antagonize the transcription of a lacZ reporter gene con-
trolled by a single GATAAG site (30). Since the zinc finger
regions of Dal80 and Gzf3 are highly homologous to those of
the GATA activators, it was assumed that they repress by
competing with Gat1 or Gln3 for the GATA sites located in
the promoters of NCR genes (2, 8, 15, 30, 34). Nevertheless,
the two GATA repressors play very different roles in the mod-
ulation of NCR-sensitive gene expression. Dal80, whose ex-
pression is NCR regulated (13), limits the activation of NCR-
sensitive gene expression in cells grown on a nonpreferred
source of nitrogen (16). In contrast, Gzf3 seems to inhibit gene
expression specifically under conditions of nitrogen repression
Recent advances in the understanding of the mechanisms
regulating Gln3 and Gat1 function came from studies of the
immunosuppressant drug rapamycin and its target kinases
Tor1 and Tor2, since Gln3 and Gat1 respond similarly to
rapamycin inhibition of Tor1 and Tor2, to nitrogen depriva-
tion, and to growth on a nonpreferred nitrogen source (4, 6,
22). According to the model, a nitrogen shortage, similarly to
rapamycin treatment, would inactivate Tor1 and Tor2, leading
to dephosphorylation of Gln3 and Gat1, causing them to dis-
sociate from Ure2 and enter the nucleus to activate NCR-
sensitive transcription. It was proposed that the Sit4 phos-
* Corresponding author. Mailing address: IRMW-CERIA, Av. E.
Gryson 1, B-1070 Bruxelles, Belgium. Phone: 32 2 526 7277. Fax: 32 2
526 7273. E-mail: Evelyne.firstname.lastname@example.org.
?Published ahead of print on 20 April 2009.
phatase would play a central role in the control of Gln3
phosphorylation and consequently of its subcellular localiza-
tion (3, 5). However, more recently, we demonstrated that the
extent of the Sit4 requirement for Gln3 nuclear localization
was both nitrogen source and strain dependent (39). More-
over, our previous study allowed us to conclude that Tor path-
way regulation of Gat1 differs markedly from that of Gln3: a
yet-unexplored Sit4- and Ure2-independent branch is involved
in Gat1-dependent Tor control of NCR (20). Moreover, no
evidence demonstrating nitrogen-regulated Gat1 phosphoryla-
tion has been published in the literature so far (3, 24).
In order to more thoroughly investigate the involvement of
Gat1 in NCR, we undertook a detailed analysis of the require-
ments for in vivo Gat1 binding to its DNA targets. Our work
also aimed at determining the mechanisms involved in the
antagonism between Gat1 and Gzf3 or Dal80. Our study pro-
vides a wealth of data confirming that GAT1 expression is
NCR sensitive and regulated by the four GATA factors, in-
cluding Gat1 itself. For the first time, DNA binding of Gzf3
and Dal80 could be demonstrated in vivo. Chromatin immu-
noprecipitation (ChIP) analyses confirmed the competition be-
tween Gat1 and Dal80 at the UGA4 promoter. Our ChIP
analyses also confirmed the competition between Gat1 and
Gzf3 under conditions of nitrogen repression, but we could
only demonstrate it at the GAT1 promoter. Since Gzf3 binding
to the GAP1 promoter was Gat1 dependent and increased in
proline-grown cells, it is unlikely that Gzf3 acts by competing
with Gat1 for binding to the GAP1 promoter. The interaction
between Gzf3 and Gat1 led us to propose that Gzf3 prevents
Gat1 from DNA binding by nuclear sequestration.
MATERIALS AND METHODS
Strains and culture conditions. Saccharomyces cerevisiae strains used in this
work are listed in Table 1. Growth conditions were identical to those described
by Scherens et al. (31). Yeast cells (4-ml cultures for quantitative real-time PCR
[qRT-PCR]; 100-ml cultures for ChIP experiments) were grown to mid-log phase
(A660? 0.6) in yeast nitrogen base minimal medium containing the indicated
nitrogen source at a final concentration of 0.1%. Appropriate supplements (50
?g/ml leucine, 25 ?g/ml uracil, histidine, and tryptophan) were added to the
medium as necessary to cover auxotrophic requirements.
Strain construction. All strains constructed in this study are isogenic to the
FY1679 wild-type strain (listed in Table 1). Deletion strains, involving insertion
of KanMX and NatMX cassettes, were constructed using the long flanking
homology strategy of Wach (41) as described in reference 39 using primers listed
in Table 2. Chromosomal DAL80, GAT1, GLN3, and GZF3 gene products were
tagged at their C termini with 13 copies of the Myc epitope (Myc13) as described
by Longtine et al. (25), using primers described in Table 2 or in reference 20.
FV222 was constructed by replacing the wild-type GAT1 allele by GAT1-TAP,
TABLE 1. Strains used in this work
ParentComplete genotype Primer
WTMAT? ura3 his3 trp1
MATa ura3 his3 trp1
MAT? ura3 his3 trp1 gln3::kanMX
MAT? ura3 his3 trp1 gat1::natMX
MAT? ura3 his3 trp1 gln3::kanMX gat1::natMX
MAT? ura3 his3 trp1 GAT1-MYC13?HIS3?
MAT? ura3 his3 trp1 GLN3-MYC13?HIS3?
MAT? ura3 his3 trp1 gat1::natMX GLN3-MYC13?HIS3?
MAT? ura3 his3 trp1 DAL80-MYC13?HIS3?
MAT? ura3 his3 trp1 GZF3-MYC13?HIS3?
MATa ura3 his3 trp1 dal80::kanMX
MAT? ura3 his3 trp1 dal80::kanMX GAT1-MYC13?HIS3?
MAT? ura3 his3 trp1 dal80::kanMX GLN3-MYC13?HIS3? DAL80-L1-L4
MATa ura3 his3 trp1 gzf3::kanMX
MAT? ura3 his3 trp1 gzf3::kanMX GAT1-MYC13?HIS3?
MAT? ura3 his3 trp1 gat1::natMX dal80::kanMX GLN3-
MAT? ura3 his3 trp1 dal80::kanMX GZF3-MYC13?HIS3?
MAT? ura3 his3 trp1 gat1::kanMX GZF3-MYC13?HIS3?
MAT? ura3 his3 trp1 gln3::kanMX DAL80-MYC13?HIS3?
MAT? ura3 his3 trp1 gat1::kanMX DAL80-MYC13?HIS3?
MATa ura3 his3 trp1 gat1::natMX dal80::kanMX
MATa ura3 his3 trp1 gln3::natMX gzf3::kanMX
MATa ura3 his3 trp1 gat1::natMX gzf3::kanMX
MAT? ura3 his3 trp1 GLN3-?LZ-MYC13?HIS3?
gat1? dal80? GLN3-MYC13
MAT? ura3 his3 trp1 ?TRP1?PGAL1-GAT1-MYC13?HIS3?
MAT? ura3 his3 trp1 gzf3::kanMX ?TRP1?PGAL1-GAT1-
MAT? ura3 his3 trp1 dal80::kanMX ?TRP1?PGAL1-GAT1-
MAT? ura3 his3 trp1 GAT1-TAP?HIS3?
MAT? ura3 his3 trp1 GAT1-TAP?HIS3? GZF3-
MAT? ura3 his3 trp1 GAT1-TAP?HIS3? GLN3-?LZ-
FV022a ? FV034 ura3 his3 trp1 gln3::kanMX GAT1-MYC13?HIS3?
aWT, wild type.
3804 GEORIS ET AL.MOL. CELL. BIOL.
which was obtained by PCR amplification of a GAT1-TAP-tagged strain using
GAT1L1 and GAT1L4 primers (20). Functionality of the tagged constructs was
validated using qRT-PCR to compare NCR regulation in tagged versus untagged
strains (data not shown).
Western blots. Total protein extracts were obtained from 10-ml cultures using
trichloroacetic acid precipitation as described in reference 40. One-twentieth of
the total extracts was loaded on a 4 to 12% NuPage gel (Invitrogen), run for 75
min according to the manufacturer’s instructions, and transferred to a Hybond-
ECL nitrocellulose membrane (GE Healthcare) for 1 h in an XCell II blot
module (Invitrogen) according to the manufacturer’s protocol. Membranes were
incubated with mouse monoclonal anti-Myc antibodies (9E10; Santa Cruz) (1:
800) using an ECL Advance Western blotting detection kit, and Myc-tagged
proteins were visualized using a chemiluminescence camera (Chemi-Smart from
Vilbert-Lourmat). Band intensities were quantified using the Bio-1D algorithm
and normalized with the intensity of two different bands in the corresponding
Coimmunoprecipitation. Cultures (100 ml) were harvested, washed once in 50
mM Tris, pH 8, and resuspended in 2 ml of buffer (50 mM Tris, pH 8, 150 mM
NaCl, 5 mM EDTA, 0.05% NP-40, 1 mM phenylmetyhlsulfonyl fluoride, and
complete protease inhibitor cocktail tablets [Roche]). Lysis was performed by
shaking with 425- to 600-?m acid-washed glass beads (Sigma) on an IKA Vibrax
VXR orbital shaker at maximum speed for 30 min at 4°C. Cell debris and glass
beads were removed by centrifugation. Immunoprecipitation of TAP-tagged
proteins was performed by incubating 500 ?l of total cell extracts with 50 ?l of
prewashed (three times in 0.1% phosphate-buffered saline–bovine serum albu-
min) Dynabeads PAN mouse immunoglobulin G (Invitrogen) and 50 ?l of 1%
phosphate-buffered saline–bovine serum albumin during 2 h of orbital shaking
(800 rpm) at 30°C. Immune complexes were washed three times in lysis buffer,
eluted by boiling in sodium dodecyl sulfate (SDS) sample buffer, and loaded on
SDS-polyacrylamide gel for anti-Myc Western blotting.
Indirect immunofluorescence (IF) microscopy. Gat1-Myc13, Gln3-Myc13, and
Gzf3-Myc13subcellular localization was performed similarly to the Gln3-Myc13
localization described in reference 18.
qRT-PCR. Total RNA was extracted from 4-ml cultures as described previ-
ously (32). cDNA was generated from 100 to 500 ng of total RNA using a
RevertAid H Minus first-strand cDNA synthesis kit with oligo(dT)18primers
from Fermentas using the manufacturer’s recommended protocol. cDNAs were
subsequently quantified by RT-PCR using the Maxima SYBR green qPCR
master mix from Fermentas as described in reference 20. Sequences of the
primers for qPCR are provided in Table 2.
ChIP. Cell extracts, immunoprecipitations, and DNA quantifications were
conducted as described in reference 20, using the Maxima SYBR green qPCR
master mix from Fermentas. Sequences of the primers for qPCR are provided in
Table 2. Immunoprecipitation (IP) and input (IN) values obtained for the un-
bound control (DAL5U) were subtracted from the corresponding IP/IN values
obtained for the GAP1, GAT1, and UGA4 promoters. The mean represents at
least two replicate immunoprecipitations performed on a minimum of two in-
Gat1 binding to the promoters of NCR genes reflects its
abundance. We previously used ChIP to demonstrate induced
binding of Gat1-Myc13to the promoters of well-characterized
NCR-sensitive, permease-encoding genes (DAL5 and GAP1)
upon rapamycin treatment (19, 20). Using the same technique,
we analyzed the in vivo binding of Gat1-Myc13to the GAP1
promoter (PGAP1) upon growth on a nonpreferred nitrogen
source like proline in a wild-type strain of the FY genetic
background (FV034). In vivo binding of Gat1-Myc13to PGAP1
also occurred when cells were grown on proline (Fig. 1A). We
performed similar ChIP experiments in Gat1-Myc13strains
lacking the other NCR transcriptional activator Gln3 or the
Dal80 repressor (in strains 03740c and FV081, respectively).
Gat1-Myc13binding to PGAP1was significantly reduced in pro-
line-grown gln3? cells and increased in proline-grown dal80?
cells (Fig. 1A). Given that GAT1 expression is regulated by the
nitrogen source (7), that Gln3 and Dal80 can bind GATA
sequences upstream of GAT1 in vitro (7), and that a GAT1-
lacZ fusion responds to deletions of GLN3 and DAL80 (8, 16,
30), we hypothesized that the influence of Gln3 and Dal80 on
Gat1 binding to DNA might result from their impact on GAT1
expression. Therefore, we analyzed the expression of the native
TABLE 2. Primers used in this work
Sequence (5? to 3?)
Oligos for deletion strain
Oligos for tagged strain
Oligos for qChIP
Oligos for qRT-PCR
VOL. 29, 2009REVISITING THE GATA REGULATORY NETWORK 3805
GAT1 gene at its own locus using qRT-PCR (Fig. 1B). As
expected, GAT1 expression was derepressed on proline in a
wild-type strain (25T0b). This derepression was reduced when
GLN3 was deleted (in strain FV022) and increased upon
DAL80 deletion (in strain FV080). Since Gat1 localization, like
that of Gln3, was also regulated by the nitrogen supply (3, 7,
24), we verified the subcellular localization of a Gat1-Myc13
protein expressed from its native chromosomal locus by indi-
rect IF in wild-type strains and in strains lacking either Gln3 or
Dal80 (Fig. 1C). On glutamine, Gat1 was mostly cytoplasmic
but, unlike Gln3, it was not excluded from the nucleus. More-
over, its localization was less nuclear in proline-grown cells
than in rapamycin-treated, glutamine-grown cells. Deleting
Gln3 and Dal80 only affected the expression levels of Gat1-
Myc13, as expected from our qRT-PCR data, and consequently
it respectively decreased and increased the amount of nuclear
Gat1-Myc13. Together, these data indicate that Gat1 in vivo
binding to its DNA target in proline-grown cells parallels its
expression level. To confirm this observation, we performed
similar ChIP experiments using a strain in which Gat1-Myc13
was expressed from the GAL1 promoter (strain FV170). Add-
ing glucose to the proline/galactose-containing medium re-
duced the Gat1-Myc13expression levels (Fig. 1D) and concom-
itantly lowered its binding to PGAP1(Fig. 1E).
Gat1 conditions Gln3 binding to NCR promoters. The pres-
ence of Gat1 at NCR promoters obviously generates transcrip-
tional activation of NCR genes, since Gat1 is one of the tran-
scriptional activators of NCR genes, but it may also favor the
recruitment of other GATA factors or stabilize activating com-
plexes. Indeed, in our previous study, we provided evidence in
favor of Gat1 participating in the promotion of Gln3 binding
upon rapamycin treatment (20). In order to study the influence
FIG. 1. Gat1 binding to the GAP1 promoter reflects its abundance. (A) Effects of gln3? and dal80? on Gat1-Myc13binding to PGAP1. Untagged
wild-type (wt), GAT1-MYC13wild-type, gln3?, and dal80? cells were grown in glutamine or proline. ChIP was performed using antibodies against
c-myc. qPCR of IP and IN fractions was performed with primers specific for the GAP1 promoter (GAP1P; see Table 2 for primer list) and for a
region 2.5 kb upstream of the DAL5 open reading frame as a control (DAL5U). For each immunoprecipitation, IP/IN values were calculated as
follows: ([GAP1P]IP/[GAP1P]IN? [DAL5U]IP/[DAL5U]IN). Histograms represent the averages of two immunoprecipitations performed on at
least two experiments from independent cultures. Error bars indicate standard errors. (B) Effects of gln3? and dal80? on GAT1 expression. Total
RNA was isolated from glutamine- or proline-grown wild-type, gln3?, and dal80? cells. GAT1 mRNA levels were quantified by qRT-PCR using
primers specific for the open reading frame of GAT1 (GAT1O). GAT1 values were normalized with TBP1. Values represent the averages of at
least three experiments from independent cultures. Error bars indicate standard errors. (C) Effects of gln3? and dal80? on Gat1-Myc13subcellular
localization. GAT1-MYC13wild-type cells, grown in glutamine with or without rapamycin (0.2 ?g/ml) or in proline, and proline-grown GAT1-
MYC13gln3? and dal80? cells were processed for indirect IF. Gat1-Myc13and DNA were detected with anti-myc and DAPI (4?,6?-diamidino-
2-phenylindole), respectively. (D and E) Artificially changing the Gat1 concentration proportionally changes its binding levels. A PGAL1-GAT1-
MYC13strain was grown in 1% galactose and proline with or without 0.1% or 0.2% glucose. (D) Effects of increasing amounts of glucose on
PGAL1-GAT1-MYC13expression. Gat1-Myc13quantification values of anti-myc Western blotting are indicated above the figure. (E) Effects of
decreasing amounts of Gat1-Myc13on its in vivo binding to PGAP1. ChIP was conducted as described for panel A.
3806GEORIS ET AL.MOL. CELL. BIOL.
of Gat1 on Gln3 binding to NCR-sensitive promoters when
cells are grown on proline, we performed ChIP analysis of
Gln3-Myc13binding to PGAP1in wild-type (FV036), gat1?
(FV041), dal80? (FV082), and gat1? dal80? (FV098) strains
grown on glutamine- and proline-containing media. As was the
case upon rapamycin treatment (19, 20), in vivo binding of
Gln3-Myc13to PGAP1was significantly enhanced in wild-type
cells grown on proline versus that with glutamine (Fig. 2A).
Deleting GAT1 drastically decreased the amount of Gln3-
Myc13bound to PGAP1on proline (Fig. 2A), although the
absence of Gat1 does not influence GLN3 expression (28; also
data not shown) and had no effect on Gln3-Myc13subcellular
localization on proline (Fig. 2B). Deleting DAL80 moderately
increased the binding of Gln3-Myc13to PGAP1on proline (Fig.
2A). Given the low Gln3-Myc13binding observed in a proline-
grown strain lacking both DAL80 and GAT1 (Fig. 2A), we
assume that the increase of Gln3-Myc13binding to PGAP1in
proline-grown dal80? cells is caused by the increase of Gat1
binding in these cells. Parallelism between the binding effi-
ciency of Gat1-Myc13(Fig. 1A) and that of Gln3-Myc13(Fig.
2A) strongly suggests interdependence between the two
GATA activators for DNA binding.
Gat1 is the target of Dal80-mediated repression on proline.
Our data and previous observations that Gat1 expression is
tightly regulated and that this regulation influences its in vivo
DNA binding, affecting gene expression directly (as a tran-
scriptional activator) and indirectly (as a modulator of Gln3
binding), led us to hypothesize that Gat1 could occupy a cen-
tral position in NCR-sensitive gene activation. In this context,
it was of high interest to define how Dal80 could interfere with
Gat1-mediated transactivation. We therefore analyzed, by
qRT-PCR, the expression of a gene that is particularly sensi-
tive to Dal80-mediated repression: UGA4 (38). UGA4 expres-
sion was analyzed in a wild-type strain and in cells lacking
GAT1, DAL80, or both (FV111). In proline-grown, wild-type
cells, UGA4 activation was very low (Fig. 3A). Deleting DAL80
increased UGA4 expression 40-fold (Fig. 3A), and gat1? was
epistatic to dal80?, since the expression level in a gat1? dal80?
strain was similar to that in a gat1? strain. To determine
whether Dal80 interfered with Gat1 DNA binding, we per-
formed ChIP experiments in wild-type and dal80? Gat1-Myc13
cells (Fig. 3B). Gat1-Myc13binding to PUGA4was induced in
proline-grown, wild-type cells, compared to levels in glu-
tamine-grown cells, even if UGA4 was only weakly expressed in
the wild-type strain (Fig. 3A and B). Moreover, deleting
DAL80 led to an increase in Gat1-Myc13binding to PUGA4on
proline compared to the level in the wild type. The rise in
Gat1-Myc13DNA binding could be at least partially associated
with an increase in GAT1 expression levels, since the GAT1
FIG. 2. Gat1 conditions Gln3 binding to PGAP1. (A) Effects of
gat1? and dal80? on Gln3-Myc13binding to PGAP1. Glutamine- or
proline-grown, untagged wild-type (wt), GLN3-MYC13wild-type,
gat1?, dal80?, and gat1? dal80? cells were analyzed by ChIP as de-
scribed for Fig. 1A. (B) Effects of gat1? on Gln3-Myc13subcellular
localization. GLN3-MYC13glutamine- or proline-grown wild-type and
proline-grown gat1? cells were processed for indirect IF. Gln3-Myc13
and DNA were detected with anti-myc and DAPI (4?,6?-diamidino-2-
FIG. 3. Dal80 regulates UGA4 expression through the control of
GAT1 transcription. (A) gat1? is epistatic to dal80? for UGA4 expres-
sion. mRNA levels of UGA4 were determined in glutamine- and pro-
line-grown wild-type (wt), gat1?, dal80?, and gat1? dal80? strains as
described for Fig. 1B using qRT-PCR with UGAO primers. (B) Dal80
affects Gat1-Myc13binding to PUGA4. Glutamine- and proline-grown
untagged wild-type, GAT1-MYC13wild-type, and dal80? strains were
subjected to ChIP analysis as described for Fig. 1A using UGA4P
primers. (C) Dal80 binds to PGAT1. Glutamine- and proline-grown
untagged and DAL80-MYC13wild-type cells were subjected to ChIP
analysis as described for Fig. 1A using GAT1P primers.
VOL. 29, 2009REVISITING THE GATA REGULATORY NETWORK3807
transcript level is higher in proline-grown dal80? cells than in
wild-type cells (Fig. 1B). Consistently, Dal80-Myc13binding to
PGAT1was induced in proline-grown, wild-type cells (Fig. 3C).
In order to evaluate the direct action of Dal80 at NCR-sensi-
tive promoters without interference caused by the NCR-sensi-
tive, Dal80-regulated expression of GAT1, we placed GAT1
under the control of the GAL1 promoter, thereby rendering
GAT1 expression no longer NCR sensitive but glucose repress-
ible. We quantified the expression of UGA4 in the PGAL1-
GAT1-MYC13strain carrying either the wild-type DAL80 or a
dal80? (FV190) allele and cultivated it in the presence of
galactose and proline with increasing amounts of glucose.
UGA4 transcript levels remained low in wild-type PGAL1-
GAT1-MYC13cells grown in the presence of proline and ga-
lactose, even when no glucose was added to the culture me-
dium (Fig. 4A). Deletion of DAL80 generated higher UGA4
transcript levels for every concentration of glucose tested (Fig.
4A), suggesting that DAL80 can act directly at PUGA4. We then
checked if, in these two PGAL1-GAT1-MYC13strains, Gat1-
Myc13binding to PUGA4was affected by Dal80. Binding of
Gat1-Myc13to PUGA4followed its expression level (Fig. 4B and
C) and was moderately increased in dal80? cells compared to
the level in the wild type (Fig. 4C), indicating that Dal80
interferes with Gat1 binding to DNA, but only very weakly
compared to the expression increase caused by the absence of
Dal80. In other words, at equal binding levels of Gat1-Myc13
(compare 1% Gal for the wild type and 1% Gal plus 0.1% Glu
for the dal80? strain [Fig. 4C]), we noticed a huge difference in
UGA4 transcript levels (compare 1% Gal for the wild type and
1% Gal plus 0.1% Glu for the dal80? strain [Fig. 4A]), sug-
gesting that Dal80 has an additional role in UGA4 repression,
interfering with transcriptional activation. The last step in
demonstrating in vivo competition between Dal80 and Gat1
for binding to NCR-sensitive promoters was to analyze the
profile of Dal80 binding to PUGA4. Therefore, we performed
ChIP analysis of wild-type (FV078), gln3? (FV108), and gat1?
(FV109) strains expressing Dal80-Myc13to determine its bind-
ing at the promoter of UGA4 (Fig. 5A). In proline-grown,
wild-type cells, Dal80-Myc13binding to PUGA4was induced.
Reducing Dal80 expression, by growing cells on glutamine or
in GATA activator mutants (Fig. 5B), resulted in a corre-
sponding decrease of Dal80-Myc13binding (Fig. 5A), suggest-
ing that Dal80-Myc13binding to promoters in vivo is solely
controlled by its expression level.
Gat1 is the target of Gzf3-mediated repression on glu-
tamine. Aiming at the elucidation of the mechanisms regulat-
ing Gat1 activity on glutamine, we quantified the expression of
GAP1—a well-characterized target of Gzf3-mediated repres-
sion (34)—using qRT-PCR in a wild-type strain and in cells
lacking GAT1, GLN3, or GZF3, either in single units (FV023,
FV022, and FV083) or in pairwise combinations (gat1? gln3?,
FV024; gat1? gzf3?, FV114; and gln3? gzf3?, FV113). De-
tailed analysis of Fig. 6 generates the following observations:
(i) expression of GAP1 in proline-grown cells was abolished
only when the two activators were lacking (gat1? gln3?), sug-
gesting that both Gln3 and Gat1 are involved in GAP1 trans-
FIG. 4. Dal80 downregulates UGA4 expression through multiple
mechanisms. Wild-type (wt) and dal80? PGAL1-GAT1-MYC13strains
were grown in 1% galactose and proline with or without 0.1% or 0.2%
glucose. (A) Effects of dal80? on UGA4 expression in a PGAL1-GAT1-
MYC13strain. mRNA levels of UGA4 were determined as described
for Fig. 3A. (B) Effects of increasing amounts of glucose on PGAL1-
GAT1-MYC13expression. Gat1-Myc13quantification values of anti-
myc Western blotting are indicated above the figure. (C) Effects of
dal80? on Gat1-Myc13binding to PUGA4in a PGAL1-GAT1-MYC13
strain. ChIP analysis was conducted as described for Fig. 3B.
FIG. 5. Dal80-Myc13binding to PUGA4parallels its expression level.
(A) Effect of gat1? and gln3? on Dal80-Myc13binding to PUGA4.
Glutamine- or proline-grown untagged wild-type (wt), DAL80-MYC13
wild-type, gln3?, and gat1? cells were subjected to ChIP analysis as
described for Fig. 3B. (B) Effect of gln3? and gat1? on DAL80 expres-
sion. mRNA levels of DAL80 were determined in glutamine- and
proline-grown wild-type, gln3?, and gat1? cells as described for Fig. 1B
using qRT-PCR with DAL80O primers.
3808GEORIS ET AL.MOL. CELL. BIOL.
activation; (ii) maximal expression of GAP1 in proline-grown
cells required the presence of both Gln3 and Gat1; and (iii)
GAP1 expression in glutamine-grown cells lacking GZF3
reached the wild-type derepressed level regardless of the pres-
ence of Gln3 (gln3? gzf3?) but depending on Gat1 (gat1?
gzf3?). These results are consistent with earlier reports sug-
gesting that the negative effect of GZF3 on glutamine occurs
through interference with Gat1-mediated activation (30, 34).
Gat1 binding and GAT1 expression are downregulated by
Gzf3 on glutamine. We next investigated the impact of a GZF3
deletion on Gat1 DNA binding. Glutamine- and proline-grown
GAT1-MYC13wild-type and gzf3? (FV084) cells were sub-
jected to ChIP analysis for the presence of Gat1-Myc13at
PGAP1. Whereas deleting GZF3 had no impact on Gat1-Myc13
binding in proline-grown cells, it moderately increased in glu-
tamine-grown gzf3? cells versus the level in the wild type (Fig.
7A). Consistent with the increased Gat1-Myc13binding to
PGAP1in glutamine-grown gzf3? cells, the observed GAT1 ex-
pression levels were higher than those in wild-type cells grown
in the same conditions (Fig. 7B). As expected, Gat1-Myc13
binding remained proline inducible, given the fact that GAT1
expression (Fig. 7B) and Gat1 localization (data not shown)
were still regulated by the nitrogen source. The elevated GAP1
expression level reached in gzf3? cells (comparable to the
wild-type, derepressed level; Fig. 6), despite rather low Gat1
binding, is explained by Dal80-Myc13binding in glutamine-
grown gzf3? cells that is correspondingly lower than that in
proline-grown, wild-type cells (data not shown). Next, we an-
alyzed the binding of Gat1-Myc13and Gzf3-Myc13(FV079) at
PGAT1. In glutamine-grown, wild-type cells, Gat1-Myc13bound
its own promoter but, as expected, in a rather small amount
compared to the level it reached on proline (Fig. 7C). These
results corroborate previous data indicating that GAT1 is au-
toregulated (30), though the opposite has also been reported
(8). Deleting GZF3 moderately increased Gat1-Myc13binding
at PGAT1. In addition, Gzf3-Myc13significantly bound PGAT1
on glutamine (Fig. 7D), which is in agreement with previous
hypotheses that Gzf3 downregulates GAT1 expression, possi-
bly by preventing its autoactivation (30) through competition
for binding to the same GATA sequences in PGAT1, as it was
presumed to occur at other NCR-sensitive promoters (34).
Gat1 binding to NCR promoters is counteracted by Gzf3. In
order to determine whether Gzf3 controls NCR gene expres-
sion only through GAT1 expression, we quantified GAP1 ex-
pression by qRT-PCR in PGAL1-GAT1-MYC13strains carrying
either a wild-type or a deleted allele of GZF3 (FV172) and
cultivated in the presence of galactose and glutamine with
increasing amounts of glucose (Fig. 8A). In both wild-type and
gzf3? strains, reducing GAT1 expression (Fig. 8B) led to a
decrease in GAP1 expression. Interestingly, at equal Gat1 con-
centrations in wild-type and gzf3? strains (Fig. 8B), GAP1
FIG. 6. Gat1 is the target of Gzf3-mediated repression on glu-
tamine. GAP1 mRNA levels were analyzed in glutamine- and proline-
grown wild-type (wt), gln3?, gat1?, gat1? gln3?, gzf3?, gln3? gzf3?,
and gat1? gzf3? strains as described for Fig. 1B using qRT-PCR with
FIG. 7. Gzf3 affects Gat1 binding to PGAP1through downregulation of GAT1 expression by competing with Gat1 for binding at PGAT1.
(A) Effects of gzf3? on Gat1-Myc13binding to PGAP1. Glutamine- and proline-grown, untagged wild-type (wt), GAT1-MYC13wild-type, and gzf3?
strains were analyzed by ChIP as described for Fig. 1A. (B) Effects of gzf3? on GAT1 expression. GAT1 mRNA levels were analyzed in glutamine-
and proline-grown wild-type and gzf3? strains as described for Fig. 1B. (C) Effects of gzf3? on Gat1-Myc13binding to PGAT1. Glutamine- and
proline-grown, untagged wild-type, GAT1-MYC13wild-type, and gzf3? strains were analyzed by ChIP as described for Fig. 3C. (D) Binding of
Gzf3-Myc13to PGAT1. Glutamine-grown, untagged wild-type, GZF3-MYC13wild-type, gat1?, and GZF3-?LZ-MYC13wild-type strains were
analyzed by ChIP as described for panel C.
VOL. 29, 2009REVISITING THE GATA REGULATORY NETWORK 3809
expression was always higher in the absence of Gzf3 (Fig. 8A),
suggesting a negative role for Gzf3 in GAP1 expression in
addition to its control of GAT1 expression. ChIP analysis was
conducted to assess the binding of constitutively expressed
Gat1-Myc13in wild-type and gzf3? strains expressing PGAL1-
Gat1-Myc13grown on galactose and glutamine. Deleting GZF3
increased the in vivo binding of Gat1-Myc13to PGAP1(Fig. 8C),
explaining the raised GAP1 expression levels. All together, our
results suggest that besides affecting GAT1 expression, Gzf3
affects GAP1 expression directly by reducing Gat1 binding at
Gat1 is sequestered by Gzf3 in the nucleus. The next step in
testing the competition between Gat1 and Gzf3 for DNA bind-
ing was to analyze the binding of Gzf3 itself to PGAP1in vivo.
A GZF3-MYC13wild-type strain was grown on glutamine and
shifted to proline for 10, 20, and 60 min or grown on proline
and shifted to glutamine for 10 and 60 min, cross-linked, and
subjected to ChIP analysis (Fig. 9A). Surprisingly, Gzf3-Myc13
was weakly associated with PGAP1in glutamine-grown cells and
was massively recruited only after 20 min of the shift to proline.
Consistently, Gzf3-Myc13bound PGAP1on proline and was
removed by glutamine addition. These unexpected observa-
tions strongly suggested that the current NCR model based on
Gat1-Gzf3 competition for DNA binding to the same GATA
sites required revision. To start, we tried to explain the NCR-
regulated DNA-binding capacity of Gzf3-Myc13. First, we an-
alyzed the expression of GZF3 in a wild-type strain grown on
glutamine or on proline (Fig. 9B), but there was no significant
influence of the nitrogen supply on GZF3 expression, in con-
trast to what was previously observed (30, 34). Second, we
studied the subcellular distribution of Gzf3-Myc13in a wild-
type strain grown in glutamine or in proline (Fig. 9C), but
there was no marked effect of the nitrogen source on Gzf3-
Myc13localization: Gzf3-Myc13was constitutively found in the
nucleus. Third, the unexpected presence of Gzf3-Myc13at
PGAP1on proline could result from it being carried along with
the other GATA factors, whose binding is induced in the same
conditions (Fig. 1A and 2A; also data not shown). Among
these, Dal80 was the most likely candidate, since Gzf3 was
previously shown to form heterodimers with Dal80 through
their leucine zipper domains (37). We therefore tested the
ChIP binding of Gzf3-Myc13in a dal80? mutant strain (FV104;
Fig. 9A). Deleting DAL80 reduced the binding of Gzf3-Myc13
in proline-grown cells, suggesting that proline-induced binding
of Gzf3 could partially result from Dal80-mediated recruit-
ment. However, since Gzf3-Myc13binding to PGAP1remained
inducible on proline, we next assayed its binding in a gat1?
mutant strain (FV106; Fig. 9A). Proline-induced binding of
Gzf3-Myc13to PGAP1was completely abolished when GAT1
was deleted. The weak binding of Gzf3-Myc13observed on
glutamine was also lost in the gat1? strain (Fig. 9A). Given that
the literature about GZF3 expression was contradictory (8, 34),
we assayed GZF3 expression by qRT-PCR and showed that it
was not affected by the deletion of GAT1 (Fig. 9B). Moreover,
Gzf3 subcellular localization was not modified in the absence
of Gat1 (Fig. 9C). It is worth noting that binding of Gzf3-Myc13
to PGAT1occurred in glutamine-grown cells and was not Gat1
dependent (Fig. 7D), suggesting promoter specificities condi-
tioning Gzf3 binding; this could explain the observed Gat1-
Gzf3 competition at this particular promoter.
In sum, the complete lack of Gzf3 binding in gat1? cells
likely results from the joint absence of Gat1 and Dal80, the
latter being poorly induced and not significantly recruited to
NCR-sensitive promoters in proline-grown gat1? cells (Fig. 5B
and A, respectively). On the other hand, the reduction in Gzf3
binding in dal80? cells is moderated due to the increased
GAT1 expression and Gat1 binding in these mutant cells (Fig.
1B and A, respectively).
To test the hypothesis that in vivo DNA binding of Gzf3
occurs as a result of its interaction with Gat1, we assayed the in
vivo interaction between Gat1-TAP and Gzf3-Myc13using co-
immunoprecipitation. Wild-type cells expressing Gat1-TAP
and Gzf3-Myc13from their native loci (FV231) were grown on
glutamine or proline. Gat1-TAP was immunoprecipitated us-
ing magnetic beads coated with monoclonal human anti-mouse
immunoglobulin G, and the presence of Gzf3-Myc13was as-
sayed in the immunopurified fraction by Western blotting. We
demonstrated an interaction between the two GATA factors in
proline- and glutamine-grown cells (Fig. 9D). These results
suggest that, in conditions of nitrogen repression, Gzf3 pre-
FIG. 8. Gzf3 counteracts Gat1 binding to PGAP1. Untagged wild-
type (wt), PGAL1-GAT1-MYC13wild-type, and gzf3? cells were grown
in 1% galactose and glutamine with or without 0.1%, 0.2%, or 0.4%
glucose. (A) Effects of gzf3? on GAP1 expression in a PGAL1-GAT1-
MYC13strain. GAP1 mRNA levels were analyzed as described for Fig.
6. (B) Effects of increasing amounts of glucose on PGAL1-GAT1-
MYC13expression. Gat1-Myc13quantification values of anti-myc
Western blotting are indicated above the figure. (C) Effect of gzf3? on
Gat1-Myc13binding to PGAP1in a PGAL1-GAT1-MYC13strain. Cells
were grown in galactose plus glutamine. ChIP analysis was performed
as described for Fig. 1A.
3810GEORIS ET AL.MOL. CELL. BIOL.
vents nuclear Gat1 from binding to its target GATA sites by
intranuclear sequestration. The observation that the Gzf3-
Gat1 interaction is maintained in proline-grown cells explains
the in vivo binding of Gzf3 to target NCR-sensitive promoters
in conditions of derepression in dal80? cells, probably through
Leucine zipper of Gzf3 is required for its recruitment to
PGAP1on proline and for repression on glutamine. In order to
investigate the domains of Gzf3 that are important for its
recruitment to NCR-sensitive promoters, we compared the
binding of wild-type Gzf3-Myc13to that of Gzf3-Myc13devoid
of its C-terminal leucine zipper domain (GZF3-?LZ-MYC13;
FV135), previously identified to be responsible for its het-
erodimerization with Dal80 (37). In glutamine-grown cells
(Fig. 7D), deleting the leucine zipper of Gzf3 affected its bind-
ing to the GAT1 promoter, which is consistent with its binding
as a homodimer at this particular promoter. In proline-grown
cells (Fig. 9A), Gzf3-?LZ-Myc13binding to PGAP1was severely
affected, indicating that the leucine zipper region of Gzf3 was
involved in the protein-protein interactions responsible for its
recruitment to the GAP1 promoter, probably through its in-
teraction with Dal80. However, Gzf3-?LZ-Myc13binding was
not completely abolished in the way that it was in a gat1?
strain, suggesting that other domains are involved in its recruit-
ment to PGAP1through Gat1, under both repressing and de-
repressing conditions. To test the involvement of the leucine
zipper of Gzf3 in its interaction with Gat1, we performed a
coimmunoprecipitation experiment with wild-type cells ex-
pressing Gat1-TAP and Gzf3-?LZ-Myc13from their native
loci (FV232) and grown on glutamine or proline. In proline-
FIG. 9. Gzf3 is recruited by Dal80 and Gat1 at PGAP1. (A) In vivo binding of Gzf3-Myc13at PGAP1. Untagged wild-type (wt), GZF3-MYC13
wild-type, gat1?, dal80?, and GZF3-?LZ-MYC13wild-type strains were grown in glutamine or proline. The wild-type GZF3-MYC13strain was
grown in glutamine and shifted to proline for 10, 20, and 60 min or in proline and shifted to glutamine for 10 and 60 min. ChIP analysis was
performed as described for Fig. 1A. (B) Effects of gat1? on GZF3 expression. GFZ3 mRNA levels were quantified in glutamine- or proline-grown
wild-type and gat1? strains using qRT-PCR as described for Fig. 1B using GZF3O primers. (C) Effects of gat1? on Gzf3-Myc13subcellular
localization. Glutamine- or proline-grown wild-type and gat1? GZF3-MYC13cells were processed for indirect IF. Gzf3-Myc13and DNA were
detected using anti-myc and DAPI (4?,6?-diamidino-2-phenylindole), respectively. (D) Coimmunoprecipitation of Gzf3-Myc13and Gzf3-?LZ-
Myc13with Gat1-TAP. GZF3-MYC13, GZF3-?LZ-MYC13, GAT1-TAP GZF3-MYC13, and GAT1-TAP GZF3-?LZ-MYC13strains were grown in
glutamine or proline. Total proteins were extracted, immunoprecipitated, and subjected to Western blot analysis. (*, Gzf3-Myc13; °, Gzf3-?LZ-
Myc13). (E) Deleting the leucine zipper of Gzf3 affects its repressing ability. GAP1 mRNA levels were quantified in glutamine- or proline-grown
GZF3-MYC13and GZF3-?LZ-MYC13strains as described for Fig. 6.
VOL. 29, 2009REVISITING THE GATA REGULATORY NETWORK 3811
grown cells, Gzf3-?LZ-Myc13interacted with Gat1-TAP, al-
though to a lesser extent than the wild-type Gzf3-Myc13(Fig.
9D). Surprisingly, deleting the leucine zipper of Gzf3 severely
reduced the interaction between Gat1-TAP and Gzf3-?LZ-
Myc13in glutamine-grown cells. This result indicates that the
leucine zipper region of Gzf3 is mostly required to enable the
Gat1-Gzf3 interaction in repressing conditions, whereas it is
dispensable on proline, suggesting that another domain is re-
sponsible for the interaction. Since a previous two-hybrid anal-
ysis has shown a weak but significant interaction between the
zinc fingers of Gln3 and Gzf3 (37), it is possible that the Gzf3
zinc finger region alone can allow an interaction with Gat1
and/or basal level DNA binding. Finally, deleting the leucine
zipper of Gzf3 impaired its ability to repress GAP1 expression
on glutamine (Fig. 9E) without affecting its basal binding to
PGAP1, demonstrating that low-level binding of Gzf3 to PGAP1
is not responsible for its repressing function on glutamine.
This study generates a thorough understanding and uncov-
ered novel aspects of the network of four GATA zinc finger
proteins that are responsible for the fine tuning of yeast’s
response to the quality of nitrogen supply (Fig. 10). Cross-
regulation of these four GATA factors has been extensively
documented, but some of the results obtained differed from
one lab to another, mainly due to differences in the techniques
used (7, 8, 13, 30, 34). Our qRT-PCR results corroborate
previous observations that GLN3 was constitutively expressed
and that GAT1 and DAL80 expression was NCR sensitive.
However, whereas some reports in the literature describe
GZF3 as being regulated by the nitrogen source (8, 34), we
could not demonstrate any significant difference in GZF3 ex-
pression under various nitrogen conditions and in cells lacking
Gat1, with qRT-PCR and Western blotting (data not shown).
On proline, Dal80 function involves GAT1 expression con-
trol, competition for DNA binding, and direct repression.
Dal80-negative action can take place in vivo at three different
levels (Fig. 10B). First, our results confirmed earlier data show-
ing that Gat1 and Dal80 regulators are linked by reciprocal
transcriptional control (7, 12, 34). Indeed, GAT1 expression
increased in dal80? proline-grown cells compared to that in
the wild type and DAL80 expression was severely affected in
proline-grown gat1? cells. Thus, in proline-grown cells, Dal80
downregulates NCR-sensitive genes by lowering the amount of
FIG. 10. GATA factor involvement in NCR-sensitive gene regulation. (A) Nuclear GATA factors controlling NCR genes in glutamine-grown
cells. GAT1 is expressed at low levels. Gzf3 represses NCR-sensitive transcription at two levels: (a) it represses GAT1 expression by binding to its
promoter (competition with Gat1); and (b) it sequestrates nuclear Gat1, thereby preventing it from binding GATA sites. DAL80 is not expressed,
and Gln3 is sequestered in the cytoplasm. (B) GATA factors controlling NCR genes in proline-grown cells. The Gat1 and Gln3 activators are
nuclear and transactivate NCR genes, including GAT1 and DAL80. Gat1 is required for Gln3 binding to NCR promoters. Dal80-dependent
repression occurs at three levels: (a) it represses GAT1 expression; (b) it competes with Gat1 for binding; and (c) it directly represses NCR gene
transcription. (C) Nitrogen-regulated Gzf3 complexes in wild-type (WT) and mutant cells. In glutamine-grown, wild-type cells, Gzf3 is mostly
present as a leucine zipper-dependent dimer capable of sequestrating Gat1. A Gat1-Gzf3 heterodimer, which forms independently from the leucine
zipper, is found in much smaller amounts and is able to bind NCR promoters (not represented on the model). Growth with proline generates higher
DAL80 and GAT1 expression. Dal80 efficiently competes with Gzf3 for the formation of a Dal80-Gzf3 heterodimer, responsible for most of the
DNA binding of Gzf3. The Gat1-Gzf3 heterodimer also accounts for part of the Gzf3 recruitment to DNA. In the absence of Dal80, the Gat1-Gzf3
heterodimer is solely responsible for the residual recruitment of Gzf3 to NCR promoters. In the absence of Gat1, only the Gzf3 homodimer is
found, and it is unable to bind to DNA. Deleting the leucine zipper region of Gzf3 abolishes the formation of the Gat1-Gzf3 heterotrimer and
also of the Gzf3-Dal80 heterodimer. In these conditions, only the Gat1-Gzf3 heterodimer is formed and binds to DNA.
3812 GEORIS ET AL.MOL. CELL. BIOL.
Gat1 produced. Second, at the UGA4 promoter, which con-
tains a canonical URSGATA(1, 2, 15), Dal80 competes with
Gat1 for binding to similar or overlapping sites: (i) Dal80 binds
in vitro to PUGA4(14); (ii) the in vivo binding of Dal80 and
Gat1 to PUGA4was elevated in nitrogen derepression condi-
tions; and (iii) the binding of constitutively expressed Gat1-
Myc13to PUGA4was raised upon DAL80 deletion. Third, im-
pressive changes in UGA4 expression have been observed
following DAL80 deletion (about 20?), although constitutively
expressed Gat1-Myc13binding was only weakly affected (about
2?). These data suggest that Dal80 could have a third, yet-
unreported effect that does not rely on GAT1 expression nor
on its binding and rather corresponds to Dal80-mediated re-
pression interfering with transcriptional activation.
On glutamine, Gzf3 represses at two levels, by two different
modes of action. Initial observations of Gzf3 function, using
GAP1-lacZ fusions, have demonstrated an antagonistic role for
Gzf3 on activation by Gat1 in cells grown under nitrogen
repression conditions (30, 34). Our qRT-PCR results confirm
that derepression of GAP1 expression in glutamine-grown
gzf3? cells requires Gat1 and not Gln3. The absence of in-
volvement of Gln3 is explained by its cytoplasmic sequestration
by Ure2 in these conditions. Our study provides more details
on how Gzf3 negatively influences NCR-regulated gene ex-
pression (Fig. 10A). First, several lines of evidence validate
previous assumptions that Gzf3 competes with Gat1 at its own
promoter (30), thereby reducing the expression of the activator
in conditions of nitrogen abundance: (i) GAT1 expression was
higher in glutamine-grown gzf3? cells than in wild-type cells,
confirming earlier lacZ fusion data (30); (ii) rendering GAT1
expression constitutive diminished the difference between
wild-type and gzf3? glutamine-grown strains; and (iii) in vivo
binding of Gzf3-Myc13to PGAT1was elevated on glutamine,
whereas binding of Gat1-Myc13to its own promoter was weak.
Second, the fact that GAP1 expression still responded to Gzf3
in cells constitutively expressing GAT1 indicates that Gzf3 also
acts downstream of GAT1 expression.
A revised model for Gzf3-mediated repression on glutamine.
Our data disagree with earlier hypotheses accounting for the
negative effect of Gzf3 by its competition with Gat1 for binding
to similar GATA sites in NCR-sensitive promoters (34). In-
deed, on glutamine, the in vivo binding of Gzf3-Myc13to PGAP1
was, like that of Gat1-Myc13, very low. In glutamine-grown
cells, a physical interaction between Gzf3-Myc13and Gat1-
TAP was detected, which suggests that Gzf3, which we showed
to be expressed constitutively and located exclusively in the
nucleus, negatively acts on nuclear Gat1 by sequestrating it,
thereby preventing it from binding to its target GATA sites in
conditions of nitrogen repression.
Since Gzf3 and Gat1 are apparently associated in both re-
pressive and derepressive growth conditions, despite the fact
that the negative effect of Gzf3 toward Gat1 function is only
obvious in conditions of repression, one has to hypothesize that
binding of Gzf3 to Gat1 can occur in two manners: one being
productive (repressive, on glutamine), and the other being
nonproductive (not repressive, on proline). The deletion of
Gzf3’s leucine zipper, which is known to mediate its ho-
modimerization, affected its repressing capability and impaired
its interaction with Gat1, which suggests that the form of Gzf3
able to sequestrate Gat1 is a Gzf3 homodimer. In contrast,
deleting Gzf3’s leucine zipper did not severely impair its inter-
action with Gat1 in proline-grown cells, which suggests that a
Gzf3 monomer can also interact with Gat1. Our model is
summarized in Fig. 10C. In wild-type, glutamine-grown cells,
Gzf3 is more abundant than Gat1, and the most common form
of Gzf3 is the repressive one. The Gzf3 homodimer can bind to
PGAT1(Fig. 10A) and also interacts with Gat1, sequestrating it
in the nucleus. Some Gzf3-Gat1 heterodimers can be found at
NCR-sensitive promoters. Upon a shift to proline, NCR ex-
pression increases, leading to induced expression of DAL80
and GAT1. In these conditions, the Gzf3-Dal80 heterodimer
dominates over the Gzf3 homodimer and the Gzf3-Gat1 het-
erodimer due to a stronger affinity (37). The two heterodimers
are responsible for the high-level DNA binding of Gzf3. Low-
ered DNA binding of Gzf3 in dal80? cells is explained by the
lack of DAL80, partially compensated for by a higher GAT1
expression. Lack of Gzf3 DNA binding in gat1? cells is due to
the joined absence of the two proteins responsible for its re-
cruitment to PGAP1. Finally, deleting Gzf3’s leucine zipper
resulted in the lack of formation of its repressive form, and of
the Dal80-Gzf3 heterodimer, without impairing low-level,
Gat1-mediated Gzf3 recruitment to PGAP1.
An unanticipated role for Gzf3 on proline. Our results indi-
cate that proline-induced Gzf3 binding to NCR-sensitive pro-
moters occurs via Dal80- and Gat1-mediated recruitment or is
stabilized by them. Attempts to identify a function for Gzf3
during growth on poor nitrogen sources gave contradictory
results: in proline-grown cells, deletion of GZF3 reduced
DAL5 and GAP1 expression (Fig. 6; also data not shown),
suggesting that Gzf3 may carry out an activating function or be
required to stabilize activating complexes when bound on some
NCR-sensitive promoters. On the other hand, we have also
demonstrated that Gzf3 could perform an inhibitory task at the
promoter of DAL5 in glutamine-grown cells treated with rapa-
mycin (19). Hints of a repressive function for Gzf3 have pre-
viously been reported in derepressive growth conditions for
DAL80 (34) and CIS2 (35) regulation.
Binding of all four GATA factors is induced upon growth on
poor nitrogen sources. Although we have observed a concom-
itant increase of binding of the four GATA factors in proline-
grown cells, it does not necessarily imply that all are bound to
the same promoter at the same time. However, hints of pro-
tein-protein interaction have been provided throughout our
work: (i) the fact that Gln3-Myc13binding to DNA required
Gat1 suggests that the latter may help in recruiting the former
or in stabilizing its binding; (ii) Dal80 interference with tran-
scriptional activation, besides its competitive effect with Gat1-
Myc13binding to DNA, suggests that it might bind simulta-
neously and alter the activator potency; and (iii) Dal80- and
Gat1-mediated recruitment of Gzf3-Myc13to NCR-sensitive
promoters has been demonstrated, with no clearly established
function for the latter during transcriptional activation. It is,
however, conceivable that Gzf3 may relay transduction signals
to Gat1, due to its demonstrated physical interactions with
Ure2 (33) and Tor1 (5). However, these interaction data, ob-
tained using the two-hybrid technique, must be taken with care
since Ure2 is currently thought to be cytoplasmic, whereas
Gzf3 is nuclear.
Controlling Gat1 function is a good strategy to fine-tune
NCR. This study reveals a central role for Gat1 in NCR con-
VOL. 29, 2009 REVISITING THE GATA REGULATORY NETWORK3813
trol. Multiple-level control of Gat1 activity enables yeasts to
perform subtle adaptation of their metabolism in response to
the wide variety of nitrogen sources they can utilize. First,
GAT1 transcription is controlled by the nitrogen supply by the
four GATA factors. The existence of an autoactivation loop on
GAT1 expression makes it an efficient target to control NCR.
Attempts to reproduce wild-type variations in GAT1 expres-
sion demonstrated that Gat1 DNA binding and GAP1 expres-
sion levels paralleled GAT1 expression, suggesting that Gat1
concentration is the limiting factor for NCR activation. Thus,
small changes in GAT1 expression have a high impact on
NCR-sensitive gene expression.
Our study demonstrates that cells constitutively expressing
Gat1 are still sensitive to NCR, although to a lesser extent,
suggesting an important role for sequestration, competition,
and presumed posttranslational modifications affecting Gat1
function. Unlike Gln3, Gat1 localization on glutamine is not
exclusively cytoplasmic, is only weakly Ure2 dependent (20),
and is not exclusively nuclear on proline. Preventing Gat1 from
accessing its binding sites occurs not only via cytoplasmic re-
tention but also by nuclear sequestration involving Gzf3. Com-
petition of Gat1 with the two negative GATA factors for DNA
binding has also been demonstrated. Conflicting results about
Gat1 phosphorylation have been presented in the literature,
some suggesting nitrogen-regulated Gat1 phosphorylation (3;
data not shown) and others showing indistinguishable SDS-
polyacrylamide gel electrophoresis patterns (24; also our un-
Once having integrated these multiple signals, Gat1 activity
in stimulating NCR-sensitive gene transcription occurs not
only by direct transcriptional activation but also by controlling
the binding of the other transcriptional activator, Gln3.
In sum, Gat1 function does integrate the information about
the activity of all four GATA factors, not only at the level of its
expression (all four GATA factors are involved in GAT1 ex-
pression control) but also through presumed protein-protein
interactions with Gln3, Dal80, and Gzf3.
We are grateful to T. G. Cooper for critical reading and improve-
ment of the manuscript. We thank D. L. Lafontaine for providing the
Work by E.D., I.G., and F.V. is supported by the Commission
Communautaire Franc ¸aise (COCOF).
1. Andre ´, B., C. Hein, M. Grenson, and J. C. Jauniaux. 1993. Cloning and
expression of the UGA4 gene coding for the inducible GABA-specific trans-
port protein of Saccharomyces cerevisiae. Mol. Gen. Genet. 237:17–25.
2. Andre ´, B., D. Talibi, S. Soussi Boudekou, C. Hein, S. Vissers, and D. Coor-
naert. 1995. Two mutually exclusive regulatory systems inhibit UASGATA, a
cluster of 5?-GAT(A/T)A-3? upstream from the UGA4 gene of Saccharomy-
ces cerevisiae. Nucleic Acids Res. 23:558–564.
3. Beck, T., and M. N. Hall. 1999. The TOR signalling pathway controls nuclear
localization of nutrient-regulated transcription factors. Nature 402:689–692.
4. Beck, T., A. Schmidt, and M. N. Hall. 1999. Starvation induces vacuolar
targeting and degradation of the tryptophan permease in yeast. J. Cell Biol.
5. Bertram, P. G., J. H. Choi, J. Carvalho, W. Ai, C. Zeng, T. F. Chan, and X. F.
Zheng. 2000. Tripartite regulation of Gln3p by TOR, Ure2p, and phosphata-
ses. J. Biol. Chem. 275:35727–35733.
6. Cardenas, M. E., N. S. Cutler, M. C. Lorenz, C. J. Di Como, and J. Heitman.
1999. The TOR signaling cascade regulates gene expression in response to
nutrients. Genes Dev. 13:3271–3279.
7. Coffman, J. A., R. Rai, T. Cunningham, V. Svetlov, and T. G. Cooper. 1996.
Gat1p, a GATA family protein whose production is sensitive to nitrogen
catabolite repression, participates in transcriptional activation of nitrogen-
catabolic genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:847–858.
8. Coffman, J. A., R. Rai, D. M. Loprete, T. Cunningham, V. Svetlov, and T. G.
Cooper. 1997. Cross regulation of four GATA factors that control nitrogen
catabolic gene expression in Saccharomyces cerevisiae. J. Bacteriol. 179:3416–
9. Cooper, T. G. 2002. Transmitting the signal of excess nitrogen in Saccharo-
myces cerevisiae from the Tor proteins to the GATA factors: connecting the
dots. FEMS Microbiol. Rev. 26:223–238.
10. Courchesne, W. E., and B. Magasanik. 1988. Regulation of nitrogen assim-
ilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes. J.
11. Cox, K. H., R. Rai, M. Distler, J. R. Daugherty, J. A. Coffman, and T. G.
Cooper. 2000. Saccharomyces cerevisiae GATA sequences function as TATA
elements during nitrogen catabolite repression and when Gln3p is excluded
from the nucleus by overproduction of Ure2p. J. Biol. Chem. 275:17611–
12. Cunningham, T. S., R. Andhare, and T. G. Cooper. 2000. Nitrogen catabolite
repression of DAL80 expression depends on the relative levels of Gat1p and
Ure2p production in Saccharomyces cerevisiae. J. Biol. Chem. 275:14408–
13. Cunningham, T. S., and T. G. Cooper. 1991. Expression of the DAL80 gene,
whose product is homologous to the GATA factors and is a negative regu-
lator of multiple nitrogen catabolic genes in Saccharomyces cerevisiae, is
sensitive to nitrogen catabolite repression. Mol. Cell. Biol. 11:6205–6215.
14. Cunningham, T. S., and T. G. Cooper. 1993. The Saccharomyces cerevisiae
DAL80 repressor protein binds to multiple copies of GATAA-containing
sequences (URSGATA). J. Bacteriol. 175:5851–5861.
15. Cunningham, T. S., R. A. Dorrington, and T. G. Cooper. 1994. The UGA4
UASNTRsite required for GLN3-dependent transcriptional activation also
mediates DAL80-responsive regulation and DAL80 protein binding in Sac-
charomyces cerevisiae. J. Bacteriol. 176:4718–4725.
16. Cunningham, T. S., R. Rai, and T. G. Cooper. 2000. The level of DAL80
expression down-regulates GATA factor-mediated transcription in Saccha-
romyces cerevisiae. J. Bacteriol. 182:6584–6591.
17. Deberardinis, R. J., J. J. Lum, G. Hatzivassiliou, and C. B. Thompson. 2008.
The biology of cancer: metabolic reprogramming fuels cell growth and pro-
liferation. Cell Metab. 7:11–20.
18. 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 and Rsp5-Bul1/2 ubiquitin ligase in Saccharomyces cerevisiae.
J. Biol. Chem. 281:28546–28554.
19. Georis, I., A. Feller, J. J. Tate, T. G. Cooper, and E. Dubois. 2009. NCR-
sensitive transcription as a readout of Tor pathway regulation—the genetic
background, reporter gene, and GATA-factor assayed determine the out-
comes. Genetics 181:861–874.
20. Georis, I., J. J. Tate, T. G. Cooper, and E. Dubois. 2008. Tor pathway control
of the nitrogen-responsive DAL5 gene bifurcates at the level of Gln3 and
Gat1 regulation in Saccharomyces cerevisiae. J. Biol. Chem. 283:8919–8929.
21. Haguenauer-Tsapis, R., and B. Andre ´. 2004. Membrane trafficking of yeast
transporters: mechanisms and physiological control of downregulation. Top-
ics Curr. Genet. 9:273–323.
22. Hardwick, J. S., F. G. Kuruvilla, J. K. Tong, A. F. Shamji, and S. L.
Schreiber. 1999. Rapamycin-modulated transcription defines the subset of
nutrient-sensitive signaling pathways directly controlled by the Tor proteins.
Proc. Natl. Acad. Sci. USA 96:14866–14870.
23. Hofman-Bang, J. 1999. Nitrogen catabolite repression in Saccharomyces
cerevisiae. Mol. Biotechnol. 12:35–73.
24. Kulkarni, A., T. D. Buford, R. Rai, and T. G. Cooper. 2006. Differing
responses of Gat1 and Gln3 phosphorylation and localization to rapamycin
and methionine sulfoximine treatment in Saccharomyces cerevisiae. FEMS
Yeast Res. 6:218–229.
25. Longtine, M. S., A. McKenzie III, D. J. Demarini, N. G. Shah, A. Wach, A.
Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for
versatile and economical PCR-based gene deletion and modification in Sac-
charomyces cerevisiae. Yeast 14:953–961.
26. Magasanik, B. 2005. The transduction of the nitrogen regulation signal in
Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 102:16537–16538.
27. Magasanik, B., and C. A. Kaiser. 2002. Nitrogen regulation in Saccharomy-
ces cerevisiae. Gene 290:1–18.
28. Minehart, P. L., and B. Magasanik. 1991. Sequence and expression of
GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae en-
coding a protein with a putative zinc finger DNA-binding domain. Mol. Cell.
29. Mitchell, A. P., and B. Magasanik. 1984. Regulation of glutamine-repress-
ible gene products by the GLN3 function in Saccharomyces cerevisiae. Mol.
Cell. Biol. 4:2758–2766.
30. Rowen, D. W., N. Esiobu, and B. Magasanik. 1997. Role of GATA factor
Nil2p in nitrogen regulation of gene expression in Saccharomyces cerevisiae.
J. Bacteriol. 179:3761–3766.
31. Scherens, B., A. Feller, F. Vierendeels, F. Messenguy, and E. Dubois. 2006.
Identification of direct and indirect targets of the Gln3 and Gat1 activators
3814GEORIS ET AL.MOL. CELL. BIOL.
by transcriptional profiling in response to nitrogen availability at short and Download full-text
long term. FEMS Yeast Res. 6:777–791.
32. Schmitt, M. E., T. A. Brown, and B. L. Trumpower. 1990. A rapid and simple
method for preparation of RNA from Saccharomyces cerevisiae. Nucleic
Acids Res. 18:3091–3092.
33. Shewmaker, F., L. Mull, T. Nakayashiki, D. C. Masison, and R. B. Wickner.
2007. Ure2p function is enhanced by its prion domain in Saccharomyces
cerevisiae. Genetics 176:1557–1565.
34. Soussi-Boudekou, S., S. Vissers, A. Urrestarazu, J. C. Jauniaux, and B.
Andre ´. 1997. Gzf3p, a fourth GATA factor involved in nitrogen-regulated
transcription in Saccharomyces cerevisiae. Mol. Microbiol. 23:1157–1168.
35. Springael, J. Y., and M. J. Penninckx. 2003. Nitrogen-source regulation of
yeast gamma-glutamyl transpeptidase synthesis involves the regulatory net-
work including the GATA zinc-finger factors Gln3, Nil1/Gat1 and Gzf3.
Biochem. J. 371:589–595.
36. Stanbrough, M., D. W. Rowen, and B. Magasanik. 1995. Role of the GATA
factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of
nitrogen-regulated genes. Proc. Natl. Acad. Sci. USA 92:9450–9454.
37. Svetlov, V. V., and T. G. Cooper. 1998. The Saccharomyces cerevisiae GATA
factors Dal80p and Deh1p can form homo- and heterodimeric complexes. J.
38. Talibi, D., M. Grenson, and B. Andre ´. 1995. Cis- and trans-acting elements
determining induction of the genes of the gamma-aminobutyrate (GABA) uti-
lization pathway in Saccharomyces cerevisiae. Nucleic Acids Res. 23:550–557.
39. Tate, J. J., A. Feller, E. Dubois, and T. G. Cooper. 2006. Saccharomyces
cerevisiae Sit4 phosphatase is active irrespective of the nitrogen source pro-
vided, and Gln3 phosphorylation levels become nitrogen source-responsive
in a sit4-deleted strain. J. Biol. Chem. 281:37980–37992.
40. Volland, C., D. Urban-Grimal, G. Geraud, and R. Haguenauer-Tsapis. 1994.
Endocytosis and degradation of the yeast uracil permease under adverse
conditions. J. Biol. Chem. 269:9833–9841.
41. Wach, A. 1996. PCR-synthesis of marker cassettes with long flanking homol-
ogy regions for gene disruptions in S. cerevisiae. Yeast 12:259–265.
VOL. 29, 2009REVISITING THE GATA REGULATORY NETWORK3815