Download The amdR product and a CCAAT-binding factor

k.) 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 10 2655
The amdR product and a CCAAT-binding factor bind to
adjacent, possibly overlapping DNA sequences in the
promoter region of the Aspergfflus nidulans amdS gene
Robyn van Heeswijck+ and Michael J.Hynes*
Department of Genetics, University of Melbourne, Parkville, Victoria 3052, Australia
Received February 21, 1991; Revised and Accepted April 22, 1991
ABSTRACT
The amdS gene of Aspergillus nidulans is regulated by
a number of positively acting regulatory genes which
act additively and independently. Using gel mobility
shift assays with crude nuclear extracts we show here
that the product of one of these regulatory genes, the
amdR gene, binds to DNA fragments containing part
of the promoter region of the amdS gene. This confirms
the earlier prediction from DNA sequence data that
amdR encodes a DNA-binding protein containing a
cysteine-rich 'zinc finger' motif. In addition we detected
the binding of another previously unidentified protein
to an adjacent, possibly overlapping region of the amdS
5' sequence at the site of a consensus 'CCAAT-box'
sequence. Replacement of the CCAAT sequence with
CCTTT abolished the binding of this protein which we
have designated as an A. nidulans 'CCAAT-box'
binding factor (AnCF). The 'CCAAT-box' sequence
appears to be involved in determining the basal level
of transcription of amdS (T.G.Littlejohn and M.J.H.,
unpublished data). This suggests that AnCF is a
transcription factor, and that the 'CCAAT-box'
sequences found in the promoters of some filamentous
fungal genes function as binding sites for these factors,
as in other eucaryotes.
containing multiple copies of sub-clones of the 5' region of the
amdS gene has provided support for this (3, 4).
The amdR gene is involved in the induction of amdS and other
A. nidulans genes including gatA, lamA and lamB by omegaamino acids such as I3-alanine (2, 5, 6, 7). The amdI93 mutation,
a 31 bp deletion in the promoter region of amdS (see Fig. 1)
eliminates amdR-mediated regulation of amdS (3, 8). The amdR
gene has been cloned and shown to encode a protein of 765 amino
acids containing a six cysteine 'zinc finger' DNA-binding motif
(9). In this study, we have used a gel mobility shift assay to
demonstrate that the amdR protein binds to sequences that lie
within the amdS 5' region in the vicinity of the region deleted
in the amdI93 mutation.
In the course of determining the binding specificity of the amdR
gene product we unexpectedly detected another DNA-binding
protein. The binding site of this protein does not correspond with
that proposed for any of the regulatory gene products
characterised genetically to date (3), but it does contain a
consensus CCAAT-box motif. We propose that this protein is
a previously unidentified A. nidulans transcription factor and
designate it as AnCF (Aspergillus nidulans CCAAT-box binding
factor) accordingly. The role of AnCF in the regulation of amdS
transcription is not yet known but it appears to set the basal level
of amdS gene expression (T.G.Littlejohn and M.J.H.,
unpublished data).
INTRODUCTION
The amdS gene of Aspergillus nidulans encodes an acetamidase
(1) and is under the control of multiple independent regulatory
circuits (2). The regulation of amdS gene expression has been
extensively studied genetically and both cis-acting and trans-acting
regulatory mutations have been defined and shown to affect
individual circuits. The trans-acting mutations have identified
genes such as amdR and facB which presumably encode specific
regulatory proteins involved in controlling amdS expression. The
cis-acting mutations are located within the 5'-untranslated region
of the amdS gene and identify the binding sites for these specific
regulatory proteins (3). In vivo titration studies of transformants
MATERIALS AND METHODS
Preparation of nuclear extracts
Nuclear extracts were prepared by modification of a protocol
developed for Neurospora crassa (10). Liquid cultures of the
wildtype strain of Aspergillus nidulans (biAl; niiA4) and strain
T142B7 which contains multiple copies of an amdR-lacZ
translational gene fusion (M.J.H., unpublished data) were grown
in minimal medium (11) containing 1% (wt/vol) glucose and
20 mM ammonium tartrate for 16 hours at 37°C. 7 to 10 gm
wet weight mycelia was washed thoroughly with cold water than
homogenized with glass beads in a Bead-Beater (Biospec
To whom correspondence should be addressed
+ Present address: Department of Agriculture, Institute of Plant Sciences, Swan Street,
*
Burnley, Victoria 3121, Australia
2656 Nucleic Acids Research, Vol. 19, No. 10
Products, Bartlesville, OK, USA) in 40 ml 50 mM Tris-HCl
(pH 7.5), 1 M sorbitol, 7% wt/vol ficoll, 20% vol/vol glycerol,
5 mM Mg acetate, 5 mM EGTA, 3 mM CaCl2, 3 mM
dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride
(PMSF) and 2 ytM pepstatin A (Buffer A). Two volumes of
25 mM Tris-HCl (pH 7.5), 10% vol/vol glycerol, 5 mM Mg
Acetate, 5 mM EGTA, 1 mM DTT, 0.25 mM PMSF (Buffer
B) were added gradually with gentle stirring, and then the beads
were allowed to settle. The crude homogenate was layered over
0.6 vol of a 1:1.7 mix of Buffers A and B and centrifuged 4,000
rpm, 7 min in an HB4 rotor. The resultant supernatant was
layered over 5 ml pads of 25 mM Tris-HCl (pH 7.5), 1 M
sucrose, 10% vol/vol glycerol, 5 mM Mg Acetate, 1 mM DTT
(Buffer C) and centrifuged 7,000 rpm, 15 min in an HB-4 rotor.
The crude nuclear pellet was either resuspended in nuclei storage
buffer (25 mM Tris-HCl (pH 7.5), 25 % vol/vol glycerol, 5 mM
Mg Acetate, 3 mM DTT, 0.1 mM EDTA) and stored at -70°C,
or extracted immediately using a modification of the procedure
by Dignam et al. (12). Nuclei were resuspended in 15 mM Hepes
(pH 7.5) containing 0.4 to 0.55 M NaCl, 5 mM MgCl2,
0.1 mM EGTA, 5% (vol/vol) glycerol, 1 mM DTT, 1 mM
PMSF, 2 ,lM pepstatin A and rocked gently on ice for 30 min.
After centrifugation at 100,000 g for 35 min the extracts were
dialysed for 4-16 hours against Binding buffer (25 mM HepesKOH (pH 7.6), 40 mM KCI, 1 mM EDTA, 10% (vol/vol)
glycerol, 1 mM DTT) containing 1 mM PMSF. Concentration
of the extracts was performed either prior to dialysis by addition
of ammonium sulphate to 90% saturation, followed by
resuspension of the precipitate in Binding buffer, or, after dialysis
by centrifugation in a centricon 10 (Amicon). In either case, the
final extract was cleared by centrifugation at 12,000 g for 15 min,
then frozen as aliquots in liquid nitrogen and stored at - 80°C
for up to six months. The protein concentrations were determined
according to Bradford (13) using a Bio-Rad protein assay kit with
-y-globulin as standard. The yield ranged from 0.2-0.8 mg
protein per gram wet weight mycelia.
Gel mobility shift assays
The double stranded DNA fragments used as probes in gel
mobility shift assays are illustrated in Fig. 1. These were
synthesised as complementary pairs of oligonucleotides which,
when annealed, contained 5' single stranded extensions of four
nucleotides (GATC) to facilitate insertion into plasmids
(T.G.Littlejohn and M.J.H., unpublished data). All probes were
end-labelled with a!-32P dATP and the Klenow fragment of
DNA polymerase by standard procedures (14), electrophoresed
on 5-15% polyacrylamide gels containing 15% (vol/vol)
glycerol, then electroeluted from gel slices into 10 mM Tris-HCl
(pH 8.0), 1 mM EDTA. Three to 30 fmol of each probe (104
Cerenkov cpm) was mixed with 5 x Binding buffer, 0.5 to 2 ttg
poly (dI-dC), 0.1 M MgCl2 and nuclear extract (20 ytg protein,
added last) giving final concentrations of 0-5 mM MgCl2 and
1 xBinding buffer in a volume of 20 1l. After incubation at 25°C
for 30 min the binding mixes were loaded onto a 4%
polyacrylamide gel in 6.7 mM Tris-HCl (pH 7.5), 3.3 mM
sodium acetate, 1 mM EDTA which had been preelectrophoresed for 2 hours at 20 mA constant current. After
fiurther electrophoresis for 2-3 hrs at 4°C with continuous buffer
recirculation the gel was transferred to Whatman 3 MM paper
and dried prior to autoradiography. For competition studies the
unlabelled DNA was mixed with the probe and poly (dI-dC) prior
to addition of the nuclear extract.
RESULTS
Oligonucleotide probes spanning the amdI93 mutation of
amdS detect two DNA binding proteins one of which is the
gene product of amdR
The amdI93 deletion (Fig. 1) eliminates amdR-mediated
regulation of amdS (3) thus defining, at least partially, the site
of action of amdR. In order to detect DNA-protein interactions
in this region a set of overlapping oligonucleotides were
synthesised which spanned, and partially dissected the amdI93
mutation (Fig. 1). When these oligonucleotides were used in gel
mobility shift assays with crude nuclear extracts of wild-type A.
nidulms two retarded bands representing protein-DNA complexes
could be seen (Fig. 2). The lower band (b) is detected when using
oligonucleotides 18-19 and 20-21 as probes (Fig. 2, lanes 3
and 4) but not when using 5-6 or 10-11 (Fig. 2, lanes 1 and
A
Pst
-178
-141
-110
Sma
-70
193 deletion
-102 -160
-157 -132
-114
-152
-144
-109
PROBE
5-6
20-2 1
18-19
10-11
B
-.
-157 -
-109
AAAATTC66C6AAGCC914EECACCA6CTA6GCACCAGCTAAACCC
193deletion
20-2 1
18-19
27-28
10-11
rr
Fig. 1. Oligonucleotide probes used in gel mobility shift assays: Location within
the amdS promoter region (A) and DNA sequence (B). Numbers refer to the
number of base pairs from the start point of transcription (bold arrow). The
consensus CCAAT sequence (boxed nucleotides) and the amdl93 deletion atched)
are also indicated. The 21 bp region common to probes 20-21 and 18-19 is
indicated by a dashed line above the DNA sequence, the 8 bp of this region
exclusive to these two probes is indicated by the double dashed line. The 13 bp
common to 20-21, 18-19 and 10-11 is indicated by a dotted line underneath
the sequence.
5610 I118-1920-21
6re.
*44
Fig. 2. Gel mobility shift assay using probes 5 -6 (3 3 fmol), 10- 1-1(26 fmol),
18-19 (12.5 fmnol) or 20 -21 (15 fmol) incubated with a crude nuclear extract
of wild-type A. nidulans in the presence of 0.5 ,sg poly(dl-dC) prior to
electrophoresis as described in Materials and Methods. Arrows indicate the
positions of the two bands (a and b) referred to in the text.
Nucleic Acids Research, Vol. 19, No. 10 2657
2). The sequence specificity of this DNA-protein interaction is
confirmed in competition experiments (Fig. 3, lanes 1-5) using
a large excess of an unlabelled fragment. A reduction in intensity
of band (b) is seen upon addition of an approximately 100-fold
molar excess of unlabelled 18-19 or 20-2 1 to binding reactions
containing labelled 20-21 (Fig. 3, lanes 4 and 5) whilst it
remains unaffected by the presence of similar excesses of
unlabelled 5-6 or 10-11 (Fig. 3, lanes 2 and 3). This localizes
the binding site of the protein in band (b) to the 21 bp sequence
common to both 18-19 and 20-2 1 (single dashed line, Fig.
1) and demonstrates that some, if not all of the first 8 bp (double
dashed line, Fig. 1) is an essential part of the DNA-protein
interaction. The 21 bp sequence common to 18-19 and 20-21
has been shown to be required for the amdR-mediated induction
of an anmdS-lacZ gene fusion and for in vivo titration of the amdR
gene product (3, 15, T.G.Littlejohn and M.J.H., unpublished
data). This suggests that the protein in band (b) is the amdR gene
product.
Further evidence that the amdR gene product is involved in
band (b) comes from gel mobility shift assays performed using
18-19 as labelled probe and an excess of pBR322 containing
the complete amdS gene (p3SR2), or the analogous plasmid with
the amdI93 deletion (p3SR2-DE093) as unlabelled competitors.
A 13-fold molar excess of p3SR2 reduces the intensity of both
bands (a) and (b) (Fig. 4, lane 2) while the same amount of
p3SR2-DE093 results in significantly less competition (Fig. 4,
lane 3). The different abilities of p3SR2 and p3SR2-DE093 to
outcompete both bands (a) and (b) indicates that the amdI93
mutation affects the binding of the proteins involved in both of
these complexes.
Elimination of binding of the amdR gene product (band b) is
consistent with the known effects of the amdI93 mutation on
T142B7
wild-type
extract:
If
probe,
competitor:
5-6
-fold:
loo
20- 21
10-1118-1920-21
100
andR-mediated regulation of amdS (3, 8). Elimination of binding
of the protein involved in band (a) is discussed below. The ability
to detect some competition by p3SR2-DE093 can be attributed
to non-specific binding of protein to the large amounts of vector
pBR322 DNA present in the binding reaction. The detection of
non-specific competition due to large amounts of plasmid DNA
is also seen with pUC18 (Fig. 4, lane 6).
In addition to amdS, amdR also regulates expression of the
lamA, and lamB genes involved in the utilization of lactams, and
the gabA and gatA genes necessary for omega-amino acid
metabolism (5, 6, 7, 16, 17). Competition experiments were
performed using labelled 18-19 as probe and an excess of
unlabelled pUC18 plasmid DNA containing either no insert,
280 bp of the intergenic promoter region of the divergently
transcribed lamA and lamB genes (plam28) or 1 kb of the 5'
region of the gatA gene (pIR12). Both DNA inserts contain
presumed binding sites for the amdR gene product based on in
vivo titration studies (17, I.B. Richardson, M.E. Katz and
M.J.H., unpublished data). Although non-specific protein binding
to pUC18 results in significant competition (Fig. 4, lane 6) it
is clear that plam28 reduces the intensity of band (b) more
efficiently than pUC18 alone (Fig. 4, lane 5) and that pIR12 does
so even more efficiently (Fig. 4, lane 4). The specific competition
by these sequences is consistent with the protein involved in band
(b) being the amdR gene product.
The amdR-mediated regulation of amdS requires omega-amino
acids such as ,B-alanine as co-inducers (5). The presence or
absence of 1-alanine at concentrations up to 200 jtM in the gel
mobility shift assays did not alter the intensity of band (b) (data
not shown). This suggests that (3-alanine does not affect the
affinity of the amdR gene product for DNA. Since expression
of the amdR is constitutive (9), the probable role of inducer is
to cause a conformational change in the amdR protein thus
increasing its capacity to activate transcription.
90
133
5-6
10-11
100
100
20-21
I11 133
n1001
at
0
w
bz>
competitor:-fold:
I,¢
n
QL
13
13
Q
18
23
25
.*_
;
|f&: ..
'
I
-4
it
b>
1
2
3
4
5
6
7
8
9
10
11
1_
12
1
Fig. 3. Binding of the amdR gene product (band b) and the amdR-IacZ fusion
gene product (band bo) to 20-21 and its competition with unlabelled
oligonucleotides added at concentrations resulting in the given fold molar excess
of unlabelled oligonucleotide to labelled probe. All incubations contained 0.5 Ug
poly(dI-dC) and the crude nuclear extract from wild-type A. nidulans or strain
T142B7 (a multi-copy amndR-lacZ transformant). No MgCl2 was added in these
experiments resulting in a weak band (a) since this DNA protein interaction was
strongest in the presence of MgCl2.
2
3
4
5
6
Fig. 4. Competition of the binding of the amdR gene product (band b) to 18-19
by plasmid sub-clones. 12.5 fmoles of labelled 18-19 were incubated with a
crude nuclear extract of wild-type A. nidulans in the presence of the plasmids
indicated at concentrations resulting in the given fold of molar excess of unlabelled
probe over labelled oligonucleotide. No poly(dI-dC) was included in the incubations
(except for lane 1 which contains 0.25 Ag poly(dI-dC)) since the vectors themselves
resulted in considerable non-specific competition.
2658 Nucleic Acids Research, Vol. 19, No. 10
Oligonucleotide probes representing amdS promoter
sequences also bind to the product of an amdR-lacZ gene
fusion
A. nidulans strain T142B7 contains multiple copies of an amdRlacZ gene fusion construct encoding a protein in which 578
N-terminal amino acids of the amdR protein are fused to E. coli
3-galactosidase (9, M.J.H., unpublished data). These copies are
integrated at sites other than the amdR locus, leaving the resident
amdR gene intact. Gel mobility shift assays were performed on
nuclear extracts from this strain and band (b) was detected as
in the wild-type strain. In addition, however, a novel band (b,)
was also detected (Fig. 3, lane 6). Band (b,) was not seen in
identical assays performed on nuclear extracts from the wild-type
strain and presumably contains the amdR-lacZ gene fusion
protein. Competition experiments demonstrate that the protein
in band (br) has the same binding site sequence specificity as
that of the protein in band (b) since the intensity of both bands
is considerably reduced in the presence of an excess of unlabelled
oligonucleotides 18-19 or 20-21, but not 5-6 or 10-11
(Fig. 3, lanes 7-12). Even very high concentrations of
oligonucleotides 5-6 and 10-11 (1000-fold excess) failed to
outcompete band (b) and band (br) (data not shown). The lower
relative mobility of band (b,) compared with band (b) is in
accordance with the much greater molecular weight predicted
for the amdR-lacZ gene fusion product compared with the amdR
gene product itself. The greater intensity of band (br) compared
with band (b) suggests a greater abundance of the amdR-lacZ
fusion product compared with amdR, and is consistent with the
presence of multiple copies of the amdR-lacZ construct in this
transformant. The requirement for higher levels of unlabelled
DNA fragments to outcompete (ba) compared with band (b)
(Fig. 3, lanes 9-12) presumably reflects the difference in
abundance of the two proteins.
Detection of a previously unidentified 'CCAAT-box' binding
factor: AnCF
In addition to the amdR gene product, another protein binds to
oligonucleotides 18 - 19 and 20-21 and is detected as band (a)
in Fig. 2 (lanes 3 and 4). This protein does not bind to 5-6
(Fig. 2, lane 1) but, unlike the amdR gene product, it does bind
to 10-11 (Fig. 2, lane 2). Confirmation of the sequence
specificity observed in Fig. 2 was obtained in competition
experiments using 18-19 as labelled probe (Fig. 5). The intensity
of band (a) is seen to be reduced in the presence of an excess
of unlabelled 10 -11, 18 -19 or 20 -21 (Fig. 5, lanes 3, 4 and
5) but not with an equivalent excess of 5-6 (Fig. 5, lane 2).
Thus, band (a) detected with either 10-11, 18-19 or 20-21
contains a protein component(s) whose binding site lies within
their common 13 bp region (dotted line, Fig. 1). This region does
not correspond with the proposed binding sites of any of the
regulatory gene products characterised to date (3) however it does
contain the consensus 'CCAAT-box' sequence AGCCAAT.
'CCAAT-box' sequences have been shown to function as binding
sites for transcription factors in a number of other cell types (18,
19, 20) and we therefore propose that the protein detected in band
(a) is a previously unidentified transcription factor which we
designate AnCF (Aspergillus nidulans CCAAT box binding
factor).
As described earlier, competition experiments using 18-19
as labelled probe (Fig. 4) showed that plasmid p3SR2 reduced
the intensity of band (a) significantly more than plasmid
p3SR2-DE093 (Fig. 4, lanes 2 and 3) indicating that the amdI93
deletion affects the binding of AnCF.
Alteration of the CCAAT sequence to CCTTT abolishes
AnCF binding but does not affect binding of the amdR gene
product
In order to confirm the requirement of the consensus CCAAT
box sequence for AnCF binding the double stranded
oligonucleotide 27-28 (Fig. 1) was synthesised to contain the
-grF
,t
ipmII,f
-- 7. 26
2021
m
Fig. 5. Competition of binding of AnCF to 18-19 (band a) by 5 -6, 10-11,
20-21 and 18- 19 at concentrations giving a 190-, 180-, 170- and 170-fold excess
of unlabelled oligonucleotide over labelled 18-19 respectively. All incubations
contained the crude nuclear extract of wild-type A. nidulans 5 mM MgCl2 and
0.5 jig poly(dI-dC). An arrow indicates the position of band (a). Band (b) was
consistently weak in the presence of MgCl2 and could not be detected in these
experiments.
Fig. 6. Analysis of binding of AnCF (band a) and the amdR gene product (band
b) to 18-19 and 20-21 which contain the CCAAT-box motif and to 27-28
which contains the modified sequence CCTTT. Incubations contained the crude
nuclear extract from wild-type A. nidulans, 0.5 Ag poly(dI-dC), labelled 20-21
(24 fmol) or 27-28 (18 fmol), and unlabelled 27-28 (208-fold molar excess)
or 18-19 (150-fold molar excess) in the presence or absence of 2.5 mM MgCl2.
Nucleic Acids Research, Vol. 19, No. 10 2659
sequence AGCCTTT instead of AGCCAAT. When used as the
labelled probe in gel mobility shift experiments only band (b)
was detected (Fig. 6, lanes 1 and 2). Competition experiments
using labelled 20-21 as probe showed that an excess of
unlabelled 27-28 reduced the intensity of band (b), but had no
specific effect on band (a) (Fig. 6, lanes 5 and 6). An excess
of unlabelled 18-19 which contains the wild-type CCAAT
sequence reduces the intensity of both of these bands (Fig. 6,
lanes 7 and 8). These results indicate that substitution of CCTTT for the sequence CCAAT eliminates the binding of AnCF
(band a), but does not affect the binding of the amdR gene product
(band b).
DISCUSSION
The amdS gene of Aspergillus nidulans is under the control of
multiple regulatory circuits. Gel mobility shift assays provide a
powerful tool for the detection and analysis of DNA-protein
interactions especially when small oligonucleotide probes are
used. Here, they have provided important in vitro confirmation
of the DNA-binding properties of the amdR gene product which
had been inferred from in vivo data and genetic analyses. They
have also enabled detection of the AnCF-CCAAT box
interaction which had not yet been identified by genetic analyses.
The evidence that the DNA-protein complex detected as band
(b) in Fig. 2 does indeed contain the amdR gene product is: (i)
its binding site corresponds to the sequence required for amdRmediated induction of amdS (15, T.G.Littlejohn and M.J.H.,
unpublished data) and lies within the region responsible for in
vivo titration of the amdR gene product (3, 15), (ii) its binding
is reduced or abolished by the amdI93 deletion which is known
to eliminate amdR-mediated regulation of amdS and in vivo
titration of amdR product (3, 8), (iii) it binds to the intergenic
promoter region of lamA and lamB and the promoter region of
gatA all of which contain sequences related to the binding site
proposed here, and all of which mediate regulation by amdR (17,
I.B. Richardson, M.E. Katz and M.J.H., unpublished data), (iv)
its binding site specificity is the same as that of an amdR-lacZ
gene fusion product. The amdR-lacZ gene fusion encodes the Nterminal portion of the amdR protein which contains the 'zincfinger' DNA-binding motif (9). The detection of band (bz) in the
in vitro assays reported here supports the involvement of this
structure in the binding of amdR to specific DNA sequences. No
band of mobility intermediate to band (b) and band (bz) was
detected indicating the absence of mixed dimer formation between
the amdR and amdR-lacZ gene products and suggesting that the
amdR protein may bind to DNA as a monomer.
A 21 bp region of the amdS promoter has been identified which
contains the binding site of the amdR gene product (single dashed
line, Fig. 1). Eight bp at the 5' end of this region contains a
sequence essential for this DNA-protein interaction (double
dashed line, Fig. 1). Nucleotides 3' to this 8 bp region must also
be involved in this interaction since the amdI93 deletion eliminates
DNA binding of the amdR gene product (3, 8). Therefore the
amdR binding site includes nucleotides 3' to this 8 bp region.
In this respect, we note that probe 27-28 which contains the
sequence CCTTT instead of CCAAT still binds the amdR gene
product. Further analysis of the DNA-protein complex detected
as band (b) by DNAseI or hydroxyl radical 'footprinting' is
required to provide more precise information on which
nucleotides are actually involved in protein binding (21).
The CCAAT sequence motif is found in the promoter regions
of many eucaryotic genes and is known to be a crucial component
of several of these promoters (18, 19, 20). A number of different
nuclear proteins, some of which are composed of heterologous
subunits, have been found to interact with the CCAAT sequence
(22, 23, 24 and references therein). It appears that the CCAAT
sequence is not a target for a specific ubiquitous factor, and that
CCAAT sequences of different genes bind distinct transcription
factors. The multiplicity of factors may explain the variety of
functions that have been attributed to the CCAAT sequence in
modulation of transcript levels in eucaryotic cells (20, 24, 25,
26, 27, 28, 29, 30). Despite the heterogeneity in 'CCAAT-box'
binding proteins, the subunit interaction and DNA-binding
properties of at least one of these factors has been evolutionarily
conserved between mammals and the yeast Saccharomyces
cerevisiae (31). 'CCAAT-box' sequences have been found in the
promoters of a number of genes from filamentous fungi but their
relevance to the regulation of transcription has not been shown
(32). The detection of AnCF in the experiments reported here
suggests that they too will be found to function as binding sites
for transcription factors. Elucidation of the structure and function
of AnCF awaits further analyses but studies in vivo suggest that
the 13 bp sequence identified here as the AnCF binding site is
important in setting the basal level of amdS expression
(T.G.Littlejohn and M.J.H., unpublished data). Consistent with
this is the observation that the amdI93 deletion, as well as
abolishing amdR mediated regulation, also results in a general
'down promoter' effect on amdS expression (8). This may in fact
be attributable to the loss of AnCF binding and action. In addition
to having an effect on the constitutive level of amdS expression,
AnCF may also have a regulatory function. Specifically, it has
been suggested to play a role in carbon control since the general
up-promoter effect conferred by the sequence containing the
AnCF binding site is greater under conditions of carbon starvation
(15).
The binding sites for the amdR gene product and AnCF have
been found to consist of adjacent, possibly overlapping sequences
(Fig. 1). Adjacent binding sites for regulatory proteins can result
in synergistic effects on transcription activation. This has been
demonstrated for example with steroid hormone response
elements in combination with a number of different transcription
factor binding sites, including those containing 'CCAAT-box'
motifs (30, 33). There is some evidence that the adjacent location
of amdR and AnCF binding sites occurs in the promoters of other
genes in A. nidulans. The 5'-region of the gatA gene, for
example, contains a sequence identical to the 8 bp of the amdS
promoter containing an essential part of the amdR binding site
(double dashed line, Fig. 1) (17). Adjacent to this gatA sequence
is a 'CCAAT-box' motif and the spacing between these two
elements is precisely the same as that found in amdS. This gatA
'CCAAT-box' motif has also been shown to bind a protein in
gel mobility shift assays (I.B. Richardson and M.J.H.,
unpublished data). Other regions of gatA and lamAIlamB which
contain sequences related to the amdR binding site also contain
more or less degenerate forms of the 'CCAAT-box' motif in this
same position (17, I.B. Richardson, M.E. Katz and M.J.H.,
unpublished data). The function of AnCF in the regulation of
gene expression in A. nidulans remains to be elucidated. Its role,
if any, in the amdR-mediated regulation of amdS awaits analysis
of the in vivo effects of the CCTTT mutation which, unlike the
amdI93 mutation, abolishes only the binding of AnCF and not
that of the amdR gene product.
2660 Nucleic Acids Research, Vol. 19, No. 10
ACKNOWLEDGEMENTS
We thank M.E.Katz, T.G.Littlejohn, A.Andrianopoulos and
I.B.Richardson for their helpful discussions and supply of
materials, and especially M.E.K., T.G.L. and M.A.Davis for
critical reading of the manuscript.
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