(Received for publication, July 10, 1995; and in revised form, August 11, 1995)
From the
A repeated element, the inositol-sensitive upstream activation
sequence (UAS), having the consensus sequence,
5`-CATGTGAAAT-3`, is present in the promoters of genes encoding enzymes
of phospholipid biosynthesis that are regulated in response to the
phospholipid precursors, inositol and choline. None of the naturally
occurring variants of the UAS
element exactly
recapitulates the consensus (for review, see Carman, G. M., and Henry,
S. A.(1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf,
[Medline]
F., Kolwhein, S., and Henry, S. A.(1992) in Molecular Biology of
the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., and
Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY). The first six bases of the UAS
element are
homologous with canonical binding motif for proteins of the basic
helix-loop-helix (bHLH) family. Two bHLH regulatory proteins, Ino2p and
Ino4p from yeast, were previously shown to bind to promoter fragments
containing this element.
In the present study, an extensive analysis
of UAS function has been conducted. We report that any
base substitution within the putative bHLH binding site resulted either
in a dramatic reduction or in a complete obliteration of UAS
function as tested in an expression assay in vivo. Base
substitutions in the 5` region that flanks the 10-base pair repeat, as
well as sequences within the repeat itself at its 3` end outside the
bHLH core, were also assessed. The two bases immediately flanking the
5` end of the element proved to be very important to its function as a
UAS element as did the two bases immediately 3` of the bHLH core motif.
Substitutions of the final two bases of the original ten base pair
consensus (i.e. 5`-CATGTGAAAT-3`) had less dramatic effects.
We also tested a subset of the altered elements for their ability to
serve as competitors in an assay of Ino2pIno4p binding. The
strength of any given sequence as a UAS
element, as
assayed in vivo, was strongly correlated with its strength as
a competitor for Ino2p
Ino4p binding. We also tested a subset of
the modified UAS
elements for their effects on expression in vivo in a strain carrying an opi1 mutation. The opi1 mutation renders the coregulated enzymes of phospholipid
synthesis constitutive in the presence of phospholipid precursors. All
elements that retained some residual UAS
activity when
tested in the wild-type strain were constitutively expressed at a level
comparable with the wild-type derepressed level when tested in the opi1 mutant. Thus, UAS
appears to be responsible
for OPI1 mediated repression, as well as Ino2p
Ino4p
binding. Furthermore, each of the identified functions of the
UAS
element appears to have the same sequence
specificity, and all require the presence of the intact bHLH motif,
suggesting that transcriptional activation, repression, and
Ino2p
Ino4p binding are all components of a single regulatory
mechanism.
In Saccharomyces cerevisiae, a large number of phospholipid biosynthetic enzyme activities show a common pattern of regulation(1) . These enzymes are fully derepressed in the absence of the soluble phospholipid precursors, inositol and choline. They are partly repressed in the presence of inositol alone and fully repressed in the presence of inositol plus choline. Choline alone appears to have little effect on the expression of these coregulated enzymes, which include the cytoplasmic enzyme, inositol-1-phosphate synthase, product of the INO1 gene(3) . The membrane-associated activities that catalyze the sequence of reactions leading to synthesis of phosphatidylcholine via methylation of phosphatidylethanolamine are also regulated in response to inositol and choline. The structural genes encoding a number of these enzymes have been cloned and coordinate regulation in response to inositol and choline has been shown to occur at the transcriptional level (for review, see Refs. 1 and 2).
The structural genes encoding the
enzymes discussed above also respond to a common set of regulatory
genes, including INO2, INO4, and OPI1(1, 2) . Cells bearing an ino2 or ino4 mutation exhibit inositol auxotrophy due to
inability to derepress the INO1 gene(4) . The ino2 and ino4 mutants also exhibit reduced phosphatidylcholine
synthesis due to the inability to derepress enzymes of
phosphatidylcholine biosynthesis(5) . In contrast, cells
bearing an opi1 mutation constitutively overexpress the
products of the coregulated genes(6, 7) . The INO2 and INO4 gene products, Ino2p and Ino4p, both possess the
basic helix-loop-helix (bHLH) ()motif similar to that seen
in members of myc family of oncogene and protooncogene
proteins (8, 9, 10) . Ino2p and Ino4p have
been shown to form a heterodimer(10, 11, 12) that binds to a repeated element with the consensus
sequence, 5`-CATGTGAAAT-3`, which is found in the promoters of all of
the coregulated genes. The OPI1 gene product contains a
leucine zipper motif and polyglutamine stretches(13) , motifs
that are common to many DNA binding proteins. However, the mechanism of
interaction of the OPI1 gene product with the promoters of the
coregulated genes of phospholipid biosynthesis has not yet been
elucidated.
The first six bases at the 5` end of the 10-base pair
(bp) consensus element, 5`-CATGTGAAAT-3`, are homologus to the
canonical bHLH binding site, 5`-CANNTG-3`(2) .
Naturally-occurring variants of this 10-bp element have been shown to
function as an inositol-sensitive upstream activation sequence
(UAS) (for review, see Refs. 2 and 6). However, none of
the naturally occurring copies of the repeated element is identical to
the 10-bp consensus sequence, 5`-CATGTGAAAT-3`, and the various genes
containing this element exhibit widely disparate repression ratios in
response to inositol and choline. In the present study, we have
conducted a systematic analysis of the role of individual bases within
the 10-bp consensus sequence and its 5`-flanking region. We have also
assessed the relationship between UAS
function in
vivo and the binding of the Ino2p
Ino4p complex in cell
extracts. We have also examined the sequence specificity for repression
in response to inositol and choline and compared it with the response
to the opi1 regulatory mutation. In this report, we
demonstrate the functional importance of the bHLH element within the
10-bp UAS
. We have also assessed the function of the
bases within the 10-bp consensus at its 3` end but outside the bHLH
motif, as well as bases flanking the 10-bp repeat on its 5` side. We
demonstrate that the sequence specificity for the OPI1 dependent response, UAS
activity, and
Ino2p
Ino4p binding, are virtually identical, suggesting that they
are all components of a single regulatory mechanism.
X-gal plates
were identical in composition to complete media except that
vitamin-free yeast nitrogen base was replaced with 0.1
MKHPO
, pH 7.0, 15 mM
(NH
)
SO
, 0.8 mM MgSO
, 2 mM FeSO
, 75 mM KOH, and 0.04 g/liter X-gal. These plates were used to score
transformants containing the heterologous CYC1-lacZ reporter
gene fusions.
Escherichia coli strain DH5
transformants were used for production of recombinant plasmid
DNA(14) . The medium used for E. coli cells was Luria
Broth (1% bactotryptone, 0.5% yeast extract, 1% NaCl) with or without
ampicillin (50 mg/ml).
Figure 1: Construction of the expression vector pNB404 used in this study. For full construction protocol of pNB404, see ``Materials and Methods.''
The complementary pairs of single-stranded synthetic oligonucleotides (1-2 µg each) were separately dissolved in 10 mM Tris-HCl, pH 8.0, and phosphorylated by the polynucleotidekinase reaction at 37 °C, followed by ethanol precipitation. DNA was dissolved in 10 mM Tris-HCl buffer, pH 8.0. Two opposite strands of each desired fragment were combined (1 µg each), heated to 85 °C for 10 min and slowly cooled to room temperature (25 °C). Annealed oligonucleotides were then ligated into the expression vector pNB404. Verification of inserts and flanking sequences was carried out by DNA sequencing, as described above.
Figure 2:
Competition of Ino2pIno4p binding
activity by oligonucleotides containing altered UAS
sequences. The competition assay is described under ``Materials
and Methods.'' Each oligonucleotide used in competition binding
experiments was added to the standard binding assay at a concentration
of 5 µM. The position of the Ino2p
Ino4p-DNA
complexes are indicated with arrows. The strongest level of
competition, and the resulting reduction in Template B binding, is
observed for oligonucleotide NB508 and for JML9/10 (as previously shown
in (9) and (10) ). Oligonucleotide JML13/14, having a
modified sequence that places a G into the first 5` position of the
10-bp consensus sequence, is unable to compete (as previously shown in
Refs. 9 and 10). Addition of poly(dI/dC) also has no effect on
Ino2p
Ino4p binding. For ease of visual comparison, the lanes in
each panel having no competitor are labeled -; lanes having NB508
as a competitor are marked as +; those having poly(dI/dC)
competitor are marked *. A, lane 1, radiolabeled
Template B, no cell extract; lane 2, standard binding assay
(includes radiolabeled Template B), no DNA competitor(-); lane 3, standard binding assay with poly(dI/dC) as competitor
(*); lane 4, standard binding assay with JML9/10 as
competitor; lane 5, standard binding assay with JML13/14 as
competitor; lane 6, standard binding assay with NB503 as
competitor; lane 7, standard binding assay with NB504 as
competitor; lane 8, standard binding assay with NB505 as
competitor; lane 9, standard binding assay with NB506 as
competitor; lane 10, standard binding assay with NB508 as
competitor (+); lane 11, standard binding assay with
NB301 as competitor. B, lane 1, Template B, no cell
extract; lane 2, standard binding assay without DNA
competitor(-); lane 3, standard binding assay with NB508
as competitor (+); lane 4, standard binding assay with
NB607 as competitor; lane 5, standard binding assay with NB617
as competitor; lane 6, standard binding assay with NB610 as
competitor; lane 7, standard binding assay with NB613 as
competitor. C, lane 1, Template B, no cell extract; lane 2, standard binding assay without DNA
competitor(-); lane 3, standard binding assay with NB307
as competitor; lane 4, standard binding assay with NB308 as
competitor; lane 5, standard binding assay with NB505 as
competitor; lane 6, standard binding assay with NB508 as
competitor (+); lane 7, standard binding assay with NB305
as competitor; lane 8, standard binding assay with NB603 as
competitor; lane 9, standard binding assay with NB620 as
competitor; lane 10, standard binding assay with poly(dI/dC)
as competitor (*).
Protein-DNA complexes were resolved on 4% polyacrylamide gels and were visualized by autoradiography.
A systematic modification of the
four bases immediately 5` to the 10-bp element (and, consequently, the
putative HLH binding site, as well) was carried out, and the data are
shown in Table 2. The four bases immediately 5` to the putative
bHLH binding site were initially set as 5`-CGTN-3` followed by the
10-bp consensus element: i.e. 5`-(CGTN)CATGTGAAAT-3`. All four
possible base substitutions for N were constructed and tested (plasmids
pNB501-pNB504; Table 2) for UAS activity by inserting them into
the expression vector pNB404 and transforming them into the wild-type
yeast strain, as described in under ``Materials and
Methods.'' The empty vector, pNB404, gave very low background
expression of -galactosidase under the growth conditions employed
in this study (Table 2).
When the transformants containing
modified UAS elements were grown under derepressing
growth conditions (i.e. absence of the phospholipid precursors
inositol and choline; I
C
condition
on Table 2), constructs pNB502 and pNB503, containing a G or a C
in the 5` position, immediately flanking the consensus element, were
3-fold more active than constructs pNB501 and pNB504, containing an A
or a T. Consequently, G was used in this position in all subsequent
constructs. Modification of the next adjacent base, i.e. 5`-(CGNG)CATGTGAAAT-3`, also had a measurable effect.
Constructs pNB502 and pNB505, containing a T or an A in this position,
were somewhat less active under both repressing (presence of inositol
and choline; I
C
condition on Table 2) and derepressing conditions than constructs pNB506 and
pNB507 containing a G or a C. Again, G was selected as the standard
flanking sequence for subsequent constructs. Substitutions in the next
two bases 5` to the element, i.e. 5`-(NNGG)CATGTGAAAT-3` had much smaller effects (compare
constructs pNB508 and pNB509 with pNB507; Table 2). However,
construct pNB508 containing the sequence 5`-(CCGG)CATGTGAAAT-3` gave
the highest overall level of expression under derepressing growth
conditions. Expression driven by this element was subject to over
10-fold repression in response to inositol and choline. Therefore, in
all of the subsequent experiments described below, in which sequences
within the 10-base pair UAS
element were systematically
modified, we employed the 5`-flanking sequence, 5`-CCGG-3`.
The second base from the 5` end of
the 10-bp consensus element is an A; (i.e. 5`-CATGTGAAT-3`). Any substitution in this second
position resulted in 10-fold or greater reduction in
-galactosidase expression under derepressing growth conditions as
compared with the activity of construct pNB508, which contains the
10-bp consensus sequence. Constructs pNB605 and pNB607 containing a T
or a C (i.e. CTTGTGAAT or CCTGTGAAAT),
exhibited approximately 38 and 53 units of activity, respectively,
under derepressing growth conditions. This residual expression,
however, was repressed over 3-fold in response to the addition of
inositol and choline to the growth medium. Construct pNB606, containing
a G as the second base, had the lowest level of activity of any of the
constructs containing substitutions in the second base from the 5` end
of the 10-bp element. Under derepressing conditions, construct pNB606
expressed only 17 units of
-galactosidase, and this residual
activity showed little response to inositol and choline. Modification
of the third base from the 5` end (i.e. 5`-CATGTGAAAT-3`) to an A or G (constructs pNB608 and
pNB609) virtually eliminated expression under both derepressing and
repressing growth conditions. The placing of a C in the third position, (i.e. CACGTGAAAT), produced construct pNB610, which
expressed 65 units of
-galactosidase activity under derepressing
conditions. Construct pNB610 showed only 2-fold repression in response
to inositol and choline. Modification of the fourth base (i.e. CATGTGAAAT) to a C or a T (constructs pNB611 and pNB612)
left little
-galactosidase expression above background (13 and 11
units, respectively; Table 2). Placement of an A into the fourth
position, on the other hand, produced construct pNB613, which expressed
67 units of
-galactosidase activity under derepressing conditions,
and this residual expression was repressed nearly 10-fold upon the
addition of inositol and choline to the growth medium. Another
construct (pNB614) that was found, upon sequencing, to have
substitutions in the third and fourth positions (a C and an A,
respectively) gave much lower activity than either of the single
substitutions (compare data in Table 2for pNB614 to pNB613 and
pNB610).
All modifications of the fifth base (i.e. modifications of the consensus; CATGTGAAAT) also resulted
in reduced expression. Construct pNB616 containing a G in this position
had the lowest activity. All three such constructs (i.e. pNB615, pNB616, and pNB617) exhibited reduced expression compared
with the consensus element, but the residual expression was repressed
approximately 2-3-fold in response to inositol and choline.
Likewise, all constructs (pNB618-620) containing substitutions in
the sixth base exhibited very reduced expression compared with pNB508.
Constructs (pNB619 and pNB620) containing an A or a T as the sixth base
appeared to be essentially inactive, while construct pNB618, containing
a C, expressed 28 units of -galactosidase activity under
derepressing conditions. Construct pNB618 was also repressed a little
over 2-fold in response to inositol and choline.
Thus, all six bases
that comprise the putative core binding site for bHLH proteins appeared
to be essential for optimal UAS function. Of the 20
constructs tested that contained base substitutions within this motif,
none retained more than 67 units of
-galactosidase expression
compared with 536 units for the consensus element in pNB508. However,
all of the elements containing modification of the bHLH motif that
retained 20 units or greater expression of
-galactosidase activity
were capable of some repression in response to inositol and choline.
The final two bases at the 3` end of the 10-bp consensus element were tested less rigorously. The sequence that was employed for the two constructs tested (pNB307 and pNB308) had a different flanking sequence at the 5` end than other constructs discussed above. Consequently, their activity levels should not be compared with pNB508 but rather to pNB502 (5`-(CGTG)CATGTGAAAT-3`), which has the same 5`-flanking sequence as pNB307 and pNB308. Construct pNB502 expressed only 280 units of activity under derepressing growth conditions compared with 536 units for pNB508 (Table 2, part A).
In
construct pNB307, a G has been substituted for the A in the second to
last position at the 3` end of the 10-bp consensus element,
5`-(CGTG)CATGTGAAGT-3`. This construct expressed 150 units of
-galactosidase under derepressing growth conditions and was
repressed to 10 units of activity in the presence of inositol and
choline. Thus, construct pNB307 expressed a lower level of
-galactosidase than pNB502, which expresses 280 units. In
construct pNB308, the final T in the consensus sequence has been
replaced by a G, (i.e. 5`-(CGTG)CATGTGAAAG-3`).
Construct pNB308 expressed 450 units of
-galactosidase activity
under derepressing growth conditions and 53 units of activity under
repressing conditions, a level of
-galactosidase activity under
both repressing and derepressing conditions that is nearly 2-fold
higher than the consensus construct pNB502, which has the same
5`-flanking sequence as pNB308.
One construct, pNB617, which contained a substitution of a C for the
T as the fifth base of the 10-bp element, was 3 times more active in
the opi1 strain than in the wild-type strain under
derepressing conditions. Other than the case of this one construct, the opi1 mutation rendered constitutive any level of expression
that was associated with a particular construct in the wild-type strain
under derepressing condition. Thus, the sequence specificity for
regulation in response to the OPI1 gene appeared to be
essentially identical to the sequence specificity for UAS function in the wild-type strain, as well as repression in
response to inositol and choline.
Among the oligonucleotides containing base substitutions within the
bHLH core, those tested in the DNA binding competition assay were NB603 (Fig. 2C, lane8); NB607 (Fig. 2B, lane4); NB610 (Fig. 2B, lane6); NB613 (Fig. 2B, lane7); NB617 (Fig. 2B, lane5); and, NB620 (Fig. 2C, lane9). All of these
oligonucleotides had reduced UAS function when tested in
the expression vector (Table 2B) and none were effective
competitors of Ino2p
Ino4p binding as compared to NB508.
Finally, among the oligonucleotides having substitutions in the 3`
region flanking the putative bHLH binding motif, NB301 (Fig. 2A, lane11), NB305 (Fig. 2C, lane7), NB307 (Fig. 2C, lane3), and NB308 (Fig. 2C, lane4), were tested. Among
this group of oligonucleotides, only NB308 was as effective in
competing Ino2pIno4p binding as NB508. Oligonucleotide NB301 (Fig. 2A, lane11) appeared to have
little or no activity as a competitor, which is not surprising since
pNB301 also has very low UAS activity (Table 2, part C). NB307
and NB305 both appeared to have retained a little ability to serve as
competitors to Ino2p
Ino4p binding, but they were clearly much
less active than NB508 of Fig. 2(compare lane6 with lanes3 and 7 in panelC). It is not surprising that NB305 is a weak competitor
since it also has relatively low UAS activity. However, pNB307
expresses an intermediate level of
-galactosidase activity (Table 2) and is considerably more active as a UAS
element than construct pNB305, yet NB307 appears no more active
as a competitor than NB305.
Overall, with the exception noted above,
the binding competition assay revealed a strong correlation between the
ability of a particular oligonucleotide to serve as a competitor of
Ino2pIno4p binding and its ability to serve as a UAS
element. This correlation was particularly strong for
substitutions within the bHLH core motif, where essentially all of the
base changes eliminating the consensus produced a simultaneous
reduction in UAS
function and inability to compete
Ino2p
Ino4p binding. In the 5`- and 3`-flanking regions, however,
some base substitutions resulted in intermediate levels of UAS
function and Ino2p
Ino4p binding.
The element, 5`-CATGTGAAAT-3`, was first identified as a repeated sequence within the promoters of the coregulated genes of phospholipid synthesis during computer-aided homology searches(1, 2) . The sequences within the INO1 promoter that contain this element were shown to be capable of forming specific complexes with the products of the INO2 and INO4 regulatory genes(10, 11) . The INO2 and INO4 gene products are members of the bHLH class of DNA binding proteins (9, 18) and together form a heterodimer (11, 12) that is required for derepression of INO1 and other coregulated genes encoding enzymes of phospholipid biosynthesis(2) .
In the present analysis, we
have shown that the putative core bHLH binding site (CATGTG) contained
within the previously identified UAS, is absolutely
required for its function as a UAS element. Likewise, the putative bHLH
binding site is essential for Ino2p
Ino4p binding as measured by a
competition assay. With respect to UAS
function, as
measured by the ability to support
-galactosidase expression in an
expression vector, all substitutions within the bHLH core, without
exception, were detrimental. None of the elements that had
substitutions in the bHLH motif retained more than 67 units of
-galactosidase activity compared with 536 units for the consensus
element. However, those elements that retained any residual expression
of
-galactosidase above approximately 5% of the activity of NB508 (i.e. above about 20-25 units of
-galactosidase)
were still repressible. All elements having substitutions within the
bHLH core, which were tested in the DNA binding competition assay,
exhibited a substantial reduction in Ino2p
Ino4p binding when
compared with the consensus oligonucleotide NB508. Thus, the putative
bHLH binding site is crucial for both Ino2p
Ino4p binding and
UAS
function.
The data reported here also demonstrated
that sequences flanking the canonical bHLH binding site in UAS are also critical to its function. In the region flanking the 5`
end the 10-bp consensus element, 5`-CATGTGAAAT-3`, the presence of a C
or a G in the first two positions (i.e. 5`-NNCATGTGAAAT-3`)
enhanced the activity of the element. At the 3` end within the 10-bp
consensus, an A was the optimal base in each of the first two positions
immediately 3` to the bHLH core binding site (i.e. 5`-CATGTG(AA)AT-3`). Curiously, however, T, which is the
consensus base for the 3` end of the originally identified 10-bp
UAS
element (i.e. 5`-CATGTGAAAT-3`),
appears to be less active than a G in this position. It is clear,
however, that sequences immediately 5` and 3` to the bHLH consensus
element play a major role in the overall activity of UAS
.
Likewise, there was a general correlation between the level of
UAS
activity and the ability to serve as a competitor for
Ino2p
Ino4p binding. The correlation between binding activity and
UAS
function, however, did not appear to be quite as
strong for substitutions in the 5`- and 3`-flanking regions as it did
within the bHLH core motif itself.
Flanking sequence-dependent
alteration of binding affinity has been shown previously for regulatory
proteins of the bHLH family (8, 19, 20, 21) . Using an in
vitro binding site selection assay, Blackwell et al. (22) demonstrated that c-Myc-Max proteins, members of
a mammalian bHLH protein family, bind not only to canonical motifs
(CACGTG or CATGTG) flanked by variable sequences, but also to
noncanonical sites. However, all of the noncanonical sites to which Myc-Max proteins bind are composed of fixed internal
dinucleotide sequence (CG or TG) in the context of particular
variations in the CANNTG consensus(22) . UAS conforms to the canonical CATGTG bHLH motif. In S.
cerevisiae, other proteins of bHLH family, including the PHO4 activator of yeast phosphatase genes and yeast centromere
promoter-binding factor Cpf1/Cbf1 have been
identified(23, 24, 25) . Both of these
factors bind the core sequence, CACGTG. It is interesting that the
modified UAS
sequence found in oligonucleotide NB610
(CACGTGAAAT), which resembles the sequence to which these two
other yeast factors bind, also retains one of the higher levels of
activity among the elements in this study with substitutions within the
bHLH core. It is possible that the base substitution in pNB610 may have
reduced the specificity of the element, thus allowing it to acquire
some ability to respond to other bHLH regulatory proteins. This could
explain the relatively high residual expression of this element under
derepressing conditions, as well as its relatively low repression ratio
in response to inositol and choline.
Finally, the data presented in
this report demonstrated that the UAS element is involved
in the regulatory response to the OPI1 gene product. Recently,
it has been demonstrated that UAS
is also involved in
responding to the global regulatory element, SIN3(26) . In the case of the OPI1 gene,
previous work had established that fragments of the INO1 promoter containing one or more native UAS
elements
were constitutively expressed in the opi1 genetic
background(16) . However, no DNA binding activity has been
definitively associated with the OPI1 gene product (
)in contrast to the evidence establishing that the
Ino2p
Ino4p complex binds directly to UAS
elements
in the INO1 promoter(10, 11) . In the present
study, we demonstrated that all of the modified UAS
elements that expressed 20 or more units of
-galactosidase
activity under derepressing conditions in wild-type transformants were
also expressed constitutively in the opi1 mutant. These
results demonstrate that repression in response to the OPI1 gene product is mediated by UAS
. Furthermore, the
sequence specificity for the repression response that is mediated by
the OPI1 gene product appears to be concordant with sequence
specificity for UAS
activity and Ino2p
Ino4p DNA binding. These results suggest that Ino2p
Ino4p binding
and transcriptional activation, as well as repression in response to
inositol and choline in the presence of an active OPI1 gene
product, are all components of a single regulatory mechanism that
functions through UAS
.