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J Biol Chem, Vol. 273, Issue 51, 34463-34471, December 18, 1998
From the Abteilung für Mikrobielle Biochemie, Institut
für Biochemische Technologie und Mikrobiologie, Technische
Universität Wien, Getreidemarkt 9/1725,
A-1060 Wien, Austria
The cellulase system of the filamentous fungus
Hypocrea jecorina (Trichoderma reesei) consists
of several cellobiohydrolases, endoglucanases, and Cellulose is the most abundant renewable carbon source on earth.
Its recycling in nature occurs in a variety of habitats by the action
of various pro- and eukaryotic microorganisms, which hydrolyse this
homopolymer with extracellular enzyme systems (1). Among the best
characterized of these is the cellulase system of the saprophytic
Ascomycete Hypocrea jecorina (=Trichoderma reesei, because the teleomorphic form of T. reesei has
now been identified by Kuhls et al. (2), we prefer to name
the organism accordingly), which contains three classes of enzymes (3). 1,4- H. jecorina cellulases are not formed during growth on
easily metabolizable carbon sources but induced in the presence of cellulose (4). The mechanism by which this extracellular, insoluble polysaccharide triggers the biosynthesis of cellulases has been investigated for decades. Some workers claim a role for low
constitutive levels of cellulases in the initial attack on cellulose,
thereby releasing the inducer (5, 6), whereas others presented evidence for an involvement of carbon catabolite derepression (7) or of
conidial-bound cellulases (8) in this process. The nature of the actual
inducer is still unknown, but depending on the conditions, either the
cellulolytic end product cellobiose itself or its transglycosylation product sophorose induce cellulase formation in pregrown mycelia (3, 9,
7). Despite the progress that has been made in the cloning of genes
encoding several H. jecorina cellulases (10-14), little
information is available about the transcriptional regulation of their
induction. More recently, two laboratories described a nucleotide
region, which is located closely upstream of the TATA box, to be
necessary to trigger sophorose induction of cbh1 (cellobiohydrolase I-encoding) gene transcription (15, 16), but neither
the binding motif nor the proteins binding to it have as yet been characterized.
We have previously identified the area between Microbial Strains and Plasmids--
T. reesei
(H. jecorina) QM9414 (ATCC 26921) was used throughout this
study. T. reesei (H. jecorina) TU-6 (18), a
pyr4 negative mutant of QM9414, was used as a recipient
strain for pyr4-mediated cotransformation experiments. The
strains were maintained on malt agar (containing 5 mM
uridine in the case of TU-6). Escherichia coli JM109 (19)
was used for the propagation of vector molecules. Plasmids pFG1 (18)
and pRLMex30 (20) were obtained from our department stock; plasmid
pCR2.1 was obtained from Invitrogen Corp. (San Diego, CA).
Fungal Growth and Induction of Cellulases--
H.
jecorina was grown in 250 ml of the medium described by Mandels
and Andreotti (21) in 1-liter Erlenmeyer flasks on a rotary shaker (250 rpm) at 28 °C using 108 conidia/liter (final concentration) as
inoculum. Carbon sources were added as indicated for the respective
experiments. For experiments with soluble inducers, the replacement
technique described by Sternberg and Mandels (4) was used. Mycelia,
pregrown for 24 h with 1% glycerol as the carbon source were
washed and resuspended in minimal medium lacking carbon source to give
a final density of 0.7-1.5 g dry weight/liter. Either sophorose (final
concentration 2 mM) or glucose (final concentration 1%,
w/v) were added, and incubation continued for a further 3 or 5 h, respectively.
Construction of pSMZ Reporter Plasmids--
The pSMZ reporter
plasmids were developed from plasmid pLMRex30 and contain the E. coli hph (hygromycin B-phosphotransferase-encoding) gene fused to the H. jecorina cbh2 5'- and
3'-noncoding regions. Primers used are specified in Table
I. To construct pSMZ1 (bearing the
cbh2 wild-type promoter), primers CKT 006 and P 91635 were used to amplify a 613-bp fragment from the cbh2 5'-noncoding
sequences, thereby also generating additional XhoI and
XbaI terminal sites. This polymerase chain reaction (PCR)
fragment was used to replace a XhoI/XbaI fragment
of the pki1 promoter in pRLMex30, thereby fusing the
cbh2 promoter fragment to the E. coli
hph gene, followed by the H. jecorina
cbh2 terminator. To yield pSMZ2, pSMZ4, and pSMZ5 (bearing
point-mutated cbh2 promoter sequences as shown in Fig.
4A), recombinant PCR as described previously by Higuchi (22)
was applied. For pSMZ2, primer pairs CKT 006/Pcbh2mutR2, and P
91635/Pcbh2mutF2, respectively, were used to amplify the primary PCR
products from plasmid pSMZ1 as template. To construct pSMZ4, primer
pairs CKT 006/Pcbh2mutR4 and P91635/Pcbh2mutF4, respectively, were used
for the primary PCR, and for construction of pSMZ5, primers CKT
006/Pcbh2mutR5 and P916357/Pcbh2mutF5, respectively, were used. For
construction of all plasmids (pSMZ2, pSMZ4, and pSMZ5) the secondary
PCRs were performed with primers CKT 006 and P 91635, resulting in
613-bp cbh2 promoter fragments bearing point mutations as
shown in Fig. 4A, which were directly cloned in vector pCR
2.1 using the TA Cloning Kit (Invitrogen). From the resulting plasmids,
the cbh2 promoter fragments were excised using restriction
enzymes XhoI/XbaI and inserted into pSMZ1,
thereby replacing the cbh2 wild-type promoter sequences. All
vectors were verified by double strand sequencing by the
dideoxynucleotide chain termination method (23), and the restriction
enzyme sites generated were proven via restriction enzyme analysis.
Isolation and Manipulation of Nucleic Acids--
Genomic DNA and
mRNA were isolated as described by Gruber et al. (18)
and Chomczynski and Sacchi (24), respectively. After electrophoretic
separation, RNA was blotted onto nylon membranes (Hybond-N, Amersham
Pharmacia Biotech) and hybridized according to standard protocols (25)
at 42 °C for 20 h. Washing was performed with 2× SSC(1×
SSC = 0.15 M NaCl and 0.015 M sodium
citrate) + 0.1% SDS at 42 °C(2 × 15 min). Standard methods
were used for plasmid isolation, restriction enzyme digestion, random
priming and Southern analysis (25). All PCR amplifications were
performed by the aid of Taq polymerase (Promega, Madison,
WI) in a Biometra thermocycler.
DNA Transformations--
E. coli transformations were
carried out according to standard techniques (25). Transformation of
T. reesei (H. jecorina) TU-6 was carried out as
described by Gruber et al. (26) using cotransformation of
pFG1 with linear 2.6-kb HindIII/EcoRI fragments of the pSMZ vectors (Fig. 4A).
Determination of Gene Copy Number and Integration Locus in
Transformants--
Southern hybridization was carried out as described
by Sambrook et al. (25). Chromosomal DNA was digested with
EcoRI, and hybridization was performed using a 862-bp
EcoRI fragment of the vector pSMZ1 bearing 613 bp of the
cbh2 promoter and 249 bp of the hph gene labeled
with [ Hygromycin B Resistance Assay--
Transformants were analyzed
for hygromycin B resistance by means of a plate assay essentially as
described previously (27).
Electrophoretic Mobility Shift Assay (EMSA)--
DNA fragments
used for EMSA were generated as follows. A cbh2 fragment
containing the regulatory nucleotide regions from In Vitro Footprinting Procedure--
In vitro
methylation protection and hydrazine interference footprinting was
performed with a 191-bp DNA fragment containing upstream regulatory
sequences of the cbh2 gene from
In vitro methylation protection footprinting was performed
by incubating 3 × 106 counts of end-labeled DNA
fragment with 100 µg of cell-free extract as described above,
methylating with dimethylsulfate, and subjecting the mixture to EMSA
analysis. After separation by native gel electrophoresis, complexes
were visualized by autoradiography, excised, eluted, purified, and
cleaved with piperidine (G) or 0.5 M HCl, 0.1 M NaOH, 1 mM EDTA (A/G) as described previously (29). After
resuspension in loading buffer, the samples were applied to denaturing
polyacrylamide gel electrophoresis on a 6% sequencing gel as described
by Strauss et al. (28).
In vitro hydrazine interference footprinting was performed
by incubating 300,000 counts of end-labeled, hydrazine-treated DNA
fragment (25) with 100 µg of cell-free extract and separating the
bound and free probes by native polyacrylamide gel electrophoresis. After elution and purification, the samples were cleaved with piperidine, suspended in loading buffer, and loaded on a 6% sequencing gel.
In Vivo Genomic Footprinting via Ligation-mediated
PCR--
T. reesei (H. jecorina) QM9414 was
cultivated as described above. Methylation of genomic DNA was performed
at 30 °C in a shaking water bath by transferring 18-ml aliquots of
the cultures to 100-ml Erlenmeyer flasks at the indicated time points
and incubating them with 40 µl of dimethyl sulfate in 2 ml of 200 mM MES buffer (pH 5.5) for 2 min. Methylation was stopped
by addition of 50 ml of ice-cold TLE
To cleave guanine as well as adenine bases, extracted methylated DNA
was incubated on ice for 2 h with 25 µl of 1 M HCl.
After precipitation with ethanol, the DNA was dissolved in 180 µl of bidistilled water and incubated at 90 °C for 30 min with 20 µl of
1 M NaOH, followed by a second precipitation with ethanol. Finally, DNA was dissolved in Tris-EDTA at a concentration of 0.4 mg/ml. In vitro methylation and cleavage of genomic DNA was performed as described by Mueller and Wold (30). Methylated and cleaved
DNA was analyzed by ligation-mediated PCR as described by Garrity and
Wold (31) and modified by Wolschek et al. (32) using Vent
polymerase (New England Biolabs, Beverly, MA) and primer sets P1c, P2c,
and P3c for visualizing the coding strand and P1n, P2n, and P3n for
visualizing the noncoding strand (cf. Table I).
Delimitation of the cbh2 Promoter Area, Which Binds a Protein
Complex Unique for Sophorose Induction--
When H. jecorina is pregrown on a carbon source not inducing cellulase
formation (glycerol) and then replaced to a medium containing the
inducer sophorose, a transient accumulation of cbh1 and
cbh2 mRNA is triggered (Fig.
1A). This pattern of
expression is also paralleled by that of transcript formation of other
cellulase-encoding genes (e.g. egl1 and
egl2; data not shown). Fusion of 613 bp of the upstream
regulatory region of the cbh2 gene to the E. coli hygromycin B phosphotransferase (hph) gene as a reporter
mimicked this induction (Fig. 1A), thus providing evidence
for a sophorose-responding element within these 613 bp. Within these
613 bp, a 191-bp fragment has previously been identified to form a
unique complex with proteins isolated from sophorose-induced or
glucose-grown mycelia (17). To further delimit this area, we prepared
extracts from cultures replaced on glucose or sophorose and subjected
them to EMSA with three smaller DNA fragments spanning this area.
Hereby, the binding ability was retained by a 94-bp fragment, spanning
from Identification of the CAE--
To precisely identify the protein
binding sequence, we performed in vitro footprinting with
cell-free extracts of H. jecorina: methylation protection
demonstrated protein contact with G
To investigate whether the observed binding in vitro
actually reflects the binding conditions in vivo, we
performed in vivo genomic footprinting by the aid of
ligation-mediated PCR of the respective area of the cbh2
upstream regulatory region. To this end, methylated DNA was isolated
from mycelia, treated with HCl, and cleaved with NaOH (29) before the
respective PCR amplification steps. Thereby, several purine bases on
the coding strand within and in the direct neighborhood of the region
previously identified in vitro were identified to be
involved in protein-DNA contact (Fig. 2B); on the template
strand, no protection of As within the identified motif could be
detected. Consistent with the results from in vitro
footprinting, essentially the same protection pattern was observed
under all conditions tested.
Summarizing the in vitro and in vivo data, we
therefore propose that the nucleotide area between CAE Consists of Two Adjacent, Cooperating Protein Binding
Motifs--
Visual inspection of the identified nucleotide region
revealed no major similarities to known protein binding motifs with two
exceptions: one is a CCAAT box on the template strand (=ATTGG, at Mutations Impairing Protein Binding in Vitro Lead to a Loss of cbh2
Gene Transcription in Vivo--
To analyze whether the identified
motifs actually confer inducibility of cbh2 transcription
in vivo, a set of reporter constructs consisting of the
613-bp of the 5'-noncoding region of the cbh2 promoter fused
to the hph structural gene from E. coli were
made. The different constructs were essentially identical with the
exception of the point mutations introduced, which exactly resembled
those used for the analysis of protein-DNA complex formation in
vitro (pSMZ2 = CKT 065; pSMZ4 = CKT 083; pSMZ5 = CKT 063; cf. Figs. 3D and 4A).
Reporter cassettes derived from plasmids pSMZ1-pSMZ5 were introduced
into H. jecorina by cotransformation with pFG1 (26). For
each construct, 30 pyr+ transformants were purified to
mitotic stability, and their DNA was isolated and examined for
integration and copy number of the respective reporter cassette by
Southern analysis. Cotransformation frequency varied between 30 to
50%, leading to 9 (pSMZ1), 10 (pSMZ2), 13 (pSMZ4), and 15 (pSMZ5)
stable co-transformants per construct, respectively. All obtained
transformants exhibited copy numbers from 1 to 4 copies, where the
reporter cassette had integrated into ectopic loci. They were examined
for the expression of hygromycin B resistance on plates containing
cellulose or glucose as carbon sources, respectively. The observed
results, taking into account the copy number of the integrated reporter
cassette, pointed to a weak influence of mutations present in pSMZ2 and
pSMZ5 (less than 20%), whereas the mutation of both motifs in pSMZ4
led to a complete loss of inducibility of the hph reporter
gene (data not shown).
To directly demonstrate the effect of these mutations on the induction
of transcription from the cbh2 promoter, selected
transformants were induced by sophorose and subjected to Northern
analysis. Consistent with the results from hygromycin B resistance
formation, strains SMZ2 and SMZ5 exhibited only a partial reduction in
hph gene transcription compared with transformants SMZ1
bearing the wild-type promoter (about 80% and 62% to 44% residual
growth in relation to copy number, respectively), whereas strains SMZ4
showed no transcript formation under inducing conditions (Fig.
4, B and C).
These data are in perfect agreement with those from EMSA analysis and
stress that mutations in one of the two deduced binding areas alone
(strains SMZ2 and SMZ5) cause only a partial reduction in gene
transcription, whereas mutation of both results in a complete loss of transcription.
The CCAAT Box in CAE Binds a Hap-like Protein Complex--
In view
of the CCAAT box on the template strand of CAE, we reasoned whether
this box may in fact bind a H. jecorina homologue of the
yeast and A. nidulans Hap complex (35-37). Because the
hap genes of H. jecorina have not yet been
cloned, and defective strains are therefore not yet available, we used
two different indirect approaches to answer this question. First, we
made use of an antibody against A. nidulans HapC (38). This
antibody was shown to recognize one major protein of H. jecorina on Western blots, which exhibited a similar, yet not
identical, size as A. nidulans HapC (data not shown). When
this antibody was applied in EMSA analysis with cell-free extracts of
induced and noninduced mycelia of H. jecorina, a supershift was observed in both cases, indicating the binding of the antibody to
components of the DNA binding complex of both induced and noninduced cells (Fig. 5A).
In a second approach, we made use of a fragment of the A. nidulans amdS promoter, for which HapC binding has been
demonstrated in vivo and in vitro (37, 39).
Except for the CCAAT box, this oligonucleotide shares no further
sequence identity with the investigated area ( Does a XlnR homologue Bind to the 3' Motif of CAE?--
The
demonstration of binding of a HapC homologue to the 5' part of CAE
supports the assumption that the observed binding involves two
different proteins rather than a single one contacting the whole area.
As the 3' area, as explained before, bears some resemblance to the
proposed binding sequence for the xylanase regulator XlnR (33), we also
tested whether a respective H. jecorina homologue may be
involved in this binding. To this end, we tested whether the binding of
protein extracts to oligonucleotide CKT 057 could be competed by a
respective fragment of the A. niger xlnD promoter (xlnD(wt)), for which XlnR binding has been demonstrated in
vivo and in vitro (33) and which, with exception of the
GGCTAAA sequence, does not contain any nucleotide similarity to CKT
057. However, even at a high excess (50-fold) of xlnD(wt), only a low
degree of competition was observed (Fig.
6). To test whether this low degree of
competition was specific at all, we repeated the experiment with a
mutated version of the competing oligonucleotide, for which a loss of
XlnR binding had been demonstrated (33). In our case, we again observed
a weak competition at a 50-fold excess of the competitor xlnD(mut),
(Fig. 6) and thus, the mutation has no effect. From these results we
conclude that binding to the 3' area of CAE is not because of a protein
with binding characteristics similar to XlnR.
In this study, we have used in vitro and in
vivo techniques to identify the nucleotide area in the
cbh2 promoter with the sequence 5'-ATTGGCAATTA-3' (CAE),
which is responsible for binding of protein complexes from
cellulase-forming (induced) and nonforming (not-induced) mycelia of
H. jecorina and which is essential for induction of gene
expression by cellulose and sophorose in vivo. Results from
the application of different in vitro and in vivo techniques point to this region to be essential for this process. To
the best of our knowledge, this is the first time that a
cellulose-responsive nucleotide region has been verified in any
cellulase-encoding gene, and these results could therefore become a
basis for deeper investigations into the molecular biology of cellulase induction.
Results from mutational analysis of CAE are consistent with the
assumption that it consists of two adjacent and functionally cooperating binding sites for transcriptional activators. Indirect evidence suggests that one of these involves a H. jecorina
homologue of the CCAAT box binding protein HapC. The CCAAT motif is a
common cis-acting element found in the promoter and enhancer
regions of a large number of genes from eukaryotes, including
filamentous fungi, and a number of different nuclear proteins have been
found to interact with the CCAAT sequence (40). However, with respect to fungi, the only CCAAT box binding protein characterized in detail is
the Saccharomyces cerevisiae Hap complex, which consists of
at least three subunits: Hap2p, Hap3p, and Hap5p (41, 42). In
filamentous fungi, A. nidulans hapC and
Neurospora crassa aab-1, which exhibit
significant similarity to the S. cerevisiae HAP3 and HAP5 genes, respectively, are so far the only
functionally homologous genes known (37, 43). HapC has been shown to
bind to CCAAT boxes present in the promoters of genes as
amdS, gatA, taaG2, yA,
pcbAB, and pcbC (37-39, 44, 45). It is
interesting to note that functional inactivation of the CCAAT motif in
these promoters has been leading to an altered efficacy of expression of the respective genes but not to a loss of their regulation. HapC has
therefore been considered to be only responsible for basal expression
(37). On a first glance, this compares well with the results from the
present study, in which a mutation of the CCAAT motif alone only
slightly decreased transcription from the cbh2 promoter.
However, our data with H. jecorina show that the presence of
the CCAAT box becomes essential for transcription if the adjacent,
3'-located protein binding motif is mutated, and vice versa,
a mutation in the CCAAT element is balanced by binding via the adjacent
protein binding motif. The fact that a mutation in the CCAAT box of
H. jecorina cbh2 does not lead to a change in the
size of the bound proteins indicates an interaction between the
proteins binding to the CCAAT box and the GTAAT motif, stressing that
binding to either of these motifs is apparently sufficient for
functional complex formation. Whether this mechanism may be a general
feature of fungal CCAAT boxes remains a matter of speculation so far.
However, the presence of a binding site for a transcriptional regulator
adjacent to the CCAAT box as observed in this study has been shown in
the A. nidulans amdS, gatA, and pcbAB/pcbC promoters (34, 45). In higher
eukaryotes, the Hap homologue NF-Y was in some cases shown to promote
transcription by stabilizing or recruiting the binding of additional
factors to adjacent promoter or enhancer elements. This function seems to be brought about by NF-Y-induced DNA distortions at the CCAAT box
(46). Such a mechanism would also be considered possible for the
cbh2 promoter.
With respect to the nature of the protein contacting the 3' adjacent
area within CAE, a sequence comparison showed identity in 5 of the 7 nucleotides with a box identified to bind the A. niger
transcriptional regulator of xylanase gene expression, XlnR (33), hence
raising the possibility that a H. jecorina homologue of XlnR
could bind to the 3' motif in CAE. However, the results obtained did
not support this hypothesis. While this paper was prepared for
submission, we became aware of the cloning of two putative regulatory
genes (ace1 and ace2) of cbh1 from
H. jecorina.2 The
ace2 gene product was thereby reported to bind to the DNA sequence 5'-GGCTAATAA-3', which matches the motif identified by us more
closely than that recognized by A. niger XlnR
(5'-GGCTAAA-3'). As our footprinting results show the protection of the
sequence GTAATA, we consider the involvement of ACE II not only in
cbh1 but also in cbh2 regulation plausible,
because both genes are coregulated by cellulose/sophorose. We therefore
speculate that ACE II and a complex containing Hap3p are involved in
cbh2 gene transcription.
If our hypothesis is true, how does the resulting protein complex bring
about induction of cbh2 gene transcription in the presence
of sophorose? Footprinting in vitro and in vivo
unequivocally showed that the same bases within CAE are protected under
induced as well as noninduced conditions. Hence both the Hap3p
homologue as well as ACE II appear to be formed constitutively, are not inducible by cellulose, and seem to bind in a similar fashion under
induced and noninduced conditions. In S. cerevisiae, Hap4p, which is not a DNA-binding protein but provides the activation domain
for signaling transcription (47) by binding to the
Hap2p-Hap3p-Hap5p-DNA complex, is responsible for activation of genes
regulated via this system. In analogy, a mechanism could be proposed in
which a cellulose-inducible protein binds to the CAE binding complex and hence promotes cbh2 transcription. However, looking at
the results from EMSA analysis, we see that cell-free extracts from induced mycelia form a protein-DNA complex of increased mobility, indicating that either its size has been decreased or its charge become
more negative. Unless this hypothetical protein has a very low
isoelectric point, the results from EMSA therefore do not support the
theory of binding of a further protein. An alternative explanation
would be modification of the DNA-protein complex by phosphorylation.
Phosphorylation of components of the C/EBP family of CCAAT-binding
proteins is well known (48-50) but has not yet been reported for the
Hap homologues. However, we have recently observed that treatment of
cell-free extracts of H. jecorina with alkaline phosphatase
inhibits protein binding to
CAE,3 which adds some support
to this possibility. Whether or not phosphorylation indeed plays a role
in CAE-protein binding and which of the protein components are
involved, therefore, is the subject of further studies.
We thank Dr. Y. Tsukagoshi (38) for the gift
of the anti-HapC antiserum.
*
This study was supported by a grant of the Austrian Science
Fund (project P11701-MOB) (to C. P. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
CAE, cbh2-activating element; bp, base pair(s); PCR, polymerase
chain reaction; EMSA, electrophoretic mobility shift assay; MES, 4-morpholineethanesulfonic acid.
2
A. Saloheimo, M. Ilmen, and M. Penttilä, a
contribution at European Conference on Fungal Genetics 4, April 5, 1998, Leon, Spain
3
M. Haller, S. Zeilinger, and C.P. Kubicek,
unpublished data.
Two Adjacent Protein Binding Motifs in the cbh2
(Cellobiohydrolase II-encoding) Promoter of the Fungus Hypocrea
jecorina (Trichoderma reesei) Cooperate in the
Induction by Cellulose*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glucosidases,
encoded by separate genes, which are coordinately expressed in the
presence of cellulose or the disaccharide sophorose. Using cell-free
extracts from sophorose-induced and noninduced mycelia and various
fragments of the cbh2 promoter of H. jecorina
in electrophoretic mobility shift assay (EMSA) analysis and performing
in vitro and in vivo footprinting analysis, we
detected the nucleotide sequence 5'-ATTGGGTAATA-3' (consequently named
cbh2-activating element (CAE)) to bind a protein complex with different migration in EMSA of induced and noninduced cell-free extracts. EMSA analysis, employing oligonucleotide fragments containing specifically mutated versions of CAE, revealed that protein binding requires the presence of an intact copy of either one of two adjacent motifs: a CCAAT (=ATTGG) box on the template strand and a GTAATA box on
the coding strand, whereas a simultaneous mutation in both completely
abolished binding. H. jecorina transformants, containing correspondingly mutated versions of the cbh2 promoter fused
to the Escherichia coli hph gene as a reporter,
expressed hph in a manner paralleling the efficacy of
CAE-protein complex formation in EMSA, suggesting that the presence of
either of both motifs is required for induction of cbh2
gene transcription. Antibody supershift experiments with anti-HapC
antiserum as well as EMSA competition experiments with CCAAT binding
promoter fragments of the Aspergillus nidulans amdS
promoter suggest that the H. jecorina CCAAT box binding
complex contains a homologue of HapC. The nature of the adjacent,
GTAATA-binding protein(s) and its cooperation with the HapC homologue
in cbh2 gene induction is discussed.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-D-Glucan cellobiohydrolases (CBH I and II; EC
3.2.1.91), which cleave cellobiosyl units from the nonreducing end of
cellulose chains; endo--1,4-glucanases (EG I, EG II, EG III, and EG
V; EC 3.2.1.4), which cleave internal glucosidic bonds; and
1,4--D-glucosidases (BG I, BG II; EC 3.2.1.21), which
cleave cellooligosaccharides to produce glucose are the most prominent members.
361 and 170 in the
5' regulatory sequences of the cbh2 (cellobiohydrolase II-encoding) gene to be able in vitro to form a
sophorose-specific DNA-protein complex (17). Starting from these
findings, we will show in this paper that within this area, a
5'-ATTGGGTAATA-3' undecamer (that we will call
"cbh2-activating element"
(CAE)1) is responsible for
the observed complex formation in vitro and essential for
cbh2 gene expression in vivo. Evidence will also be presented that a CCAAT box-binding protein (complex) and another yet
unknown protein bind to adjacent sites within this motif.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Oligonucleotides used throughout this study.
-32P]dCTP. Signals observed upon fluorography
for the single-copy gene cbh2 and for intact, integrated
vector copies, respectively, were calculated by imager analysis. Values
were corrected by including the length of the labeled probe. The copy
numbers were calculated by dividing the values observed by imager
analyses through the length of the labeled probe. To confirm the point
mutations in the cbh2 promoter (generating a DraI
restriction enzyme site) in transformants bearing the pSMZ2 and pSMZ4
plasmids, chromosomal DNA was digested with
DraI/XhoII/ClaI, and hybridization was
performed with a 2.6-kb XhoI/HindIII fragment of
the vector pSMZ1 bearing the cbh2 promoter and terminator
sequences and the hph structural gene labeled with
[-32P]dCTP.
263 to 170 was
prepared by PCR amplification of a 613-bp fragment of the
cbh2 upstream regulatory sequences using primers pcbh2F and
pcbh2R and subsequent digestion with DdeI, HhaI,
and HpaII. A 94-bp fragment was isolated and end-labeled
with [-32P]dCTP using Sequenase version 2.0 (Amersham
Pharmacia Biotech). After purification by nondenaturing polyacrylamide
gel electrophoresis, binding was achieved by incubating 100 µg of
cell-free extracts with 5 ng of labeled fragment on ice for 10 min.
Preparation of cell-free H. jecorina extracts following
growth on various carbon sources was carried out as described
previously (17). Because maximal accumulation of cbh1- and
cbh2-mRNA was observed after 5 h of incubation with
sophorose, we prepared protein extracts from mycelia harvested after
3 h. The binding assay and acrylamide gel electrophoresis have
been described previously (17). For competition experiments, synthetic
oligonucleotides were used in a 10-, 50-, or 150-fold molar excess. The
oligonucleotides were annealed with the complementary synthetic
oligonucleotide as described by Strauss et al. (28). After
annealing, double strands were filled in using Sequenase version 2.0 (Amersham Pharmacia Biotech). Oligonucleotides used for EMSA were also
annealed with their complementary oligonucleotides and end-labeled
using Sequenase version 2.0 and [-32P]dCTP as
described above. The resulting double-stranded oligonucleotides were
purified by nondenaturing polyacrylamide gel electrophoresis, and
binding was achieved by incubating 100 µg of cell-free extracts with
25 ng of labeled oligonucleotide. For supershift assays 5 µl of
polyclonal anti-HapC antiserum were added to the binding reaction.
361 to 171. The fragment
was generated by digestion of a 613-bp PCR product with
HpaII, and the resulting 233-bp fragment was isolated and end-labeled on the coding strand using Sequenase version 2.0 (Amersham Pharmacia Biotech) and [-32P]dCTP. After labeling, the
probe was digested with DdeI, and the resulting 191-bp
fragment was purified by nondenaturing polyacrylamide gel
electrophoresis. Labeling on the noncoding strand was performed by
cleaving the 233-bp HpaII fragment with DdeI and
labeling the digestion reaction with [-32P]dATP using
Sequenase version 2.0, and purification of the resulting 191-bp
fragment by nondenaturing gel electrophoresis.
buffer (10 mM Tris
(pH = 8), 1 mM EDTA, 300 mM LiCl, 2%
(v/v) -mercaptoethanol). Mycelial samples were filtered and washed
twice with 50 ml of TLE buffer, and genomic DNA was extracted
following a standard protocol. The extracted methylated DNA was cleaved
at guanine bases by incubation with 20 µl of piperidine for 30 min at
90 °C followed by three sequential ethanol precipitations and was
finally dissolved in Tris-EDTA at a concentration of 0.4 mg/ml.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
263 to 170 (Fig. 1B and C).
Consequently, this fragment was further analyzed by competition
experiments using three synthetic oligonucleotides spanning overlapping
parts of this region (Fig. 1, B and C).
Competition was only achieved with CKT 057, whereas oligonucleotides
CKT 067 and CKT 069 were unable to specifically compete (Fig. 1,
B and C), suggesting that the area between 250
and 243 was essential for complex formation. Hence sophorose-specific
protein binding seems to be confined to a relatively short motif within
the 613 bp.
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Fig. 1.
A, induction of cellulase formation by
sophorose in T. reesei (H. jecorina) wild-type
strain QM 9414 and a transformant bearing a fusion of a 613-bp 5'
upstream regulatory region of the cbh2 gene to the
hph reporter system. Northern analysis of cbh1
and cbh2 transcript accumulation upon replacement to 2 mM sophorose is shown. 20 µg of RNA was loaded, and
hybridizations were performed with a 1.4-kb BglI fragment of
the cbh1 gene, a 1.3-kb HaeII fragment of the
cbh2 gene, a 1-kilobase XbaI/NsiI
fragment of the hph gene, and a 1.9-kb KpnI
fragment of the act1 (actin-encoding) gene of H. jecorina. B, schematic drawing of the cbh2
5'-noncoding region including the 94-bp fragment (shown as full
bar) and the position of the oligonucleotides used for EMSA. The
putative TATA box and the transcription start points at 32 and 52
(51) are given. C, identification of a region with a CCAAT
motif as responsible for DNA-protein complex formation in EMSA. EMSA
analysis with labeled 94-bp cbh2 promoter fragment using
cell-free extracts of H. jecorina cultures grown in the
presence of glucose for 5 h (G) or sophorose for 3 h (S). F, free probe (without addition of
cell-free extract). In competition experiments using unlabeled
oligonucleotides CKT 057, CKT 067, and CKT 069, a 150-fold molar excess
of the competitor was added to the binding assay containing 100 µg of
protein and 5 ng of labeled fragment in a total volume of 20 µl.
241 of the coding
strand (Fig. 2C). In addition,
the involvement of T238, T239, and
T246 on the template strand was shown by hydrazine
interference (Fig. 2, A and C). Interestingly,
despite the different mobility of the protein-DNA complexes from
induced and noninduced mycelia, essentially the same
protection/interference pattern was observed under both conditions.
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Fig. 2.
Identification of nucleotides contacted by
DNA-binding proteins using in vitro and in vivo
footprinting techniques. A, in vitro
footprinting analysis using hydrazine interference technique of the
template strand with cell-free extracts from cultures grown in the
presence of glucose (Glu) or sophorose (Sop) and harvested 3 h
after the addition of sophorose and 5 h after the addition of
glucose. F (C/T) indicates DNA not incubated with cell-free
extract. Bases involved in protein/DNA contact are indicated by +.
B, in vivo genomic footprinting of the coding
strand with DNA methylated in vivo and treated with HCl and
cleaved with NaOH from cultures grown in the presence of glycerol
(Gly), glucose (Glu), or sophorose
(Sop). Control DNA methylated in vitro and
cleaved with piperidine or HCl/NaOH are given as F(G) and
F(A/G), respectively. Protected bases involved in
protein/DNA contact are indicated by +. C, summary of
contacted bases on both strands. Bases found by in vitro
footprinting techniques to be involved in protein/DNA contact are
indicated by * (hydrazine interference) and 
(methylation
protection), whereas bases found to be protected by in vivo
footprinting technique are indicated by +.
246 and 236 is
responsible for the binding of the protein complex.
246
to 242); the other is the sequence GGGTAAT (at 243 to 237), which
bears some similarity to the 5'-GGCTAAA-3' motif recently shown to bind
the xylanase regulator XlnR of Aspergillus niger (33). To
test whether any of these two motifs may be involved in protein
binding, we introduced point mutations into these two motifs and
compared the effect of these mutations on protein binding by EMSA (Fig.
3, A-D). To this end, we
replaced the two As in the CCAAT box by Ts, because this mutation has
been shown to impair binding of the Hap complex to the CCAAT motif in
the A. nidulans amdS promoter (34). The GGGTAAT
motif, on the other hand, was mutated to GGGTTTT because of the strong
protection of the two As in footprinting experiments. Interestingly,
competition experiments with either of the single mutated
oligonucleotides alone (CKT 063 and CKT 065) still exerted a very
strong effect on binding of cell-free extracts to the wild-type 94-bp
promoter fragment. A significant reduction in competition could only be
observed when both motifs were simultaneously mutated (CKT 083; Fig.
3A). Consistent results were also obtained when
oligonucleotide CKT 057 was used as a labeled probe in EMSA with the
same competitors as in Fig. 3A (Fig. 3B). To
directly compare the effect of these mutations on protein binding, all
four oligonucleotides were labeled and directly used as probes for EMSA
(Fig. 3C). In these experiments, two specific DNA-protein
complexes were detected with the wild-type promoter (CKT 057).
Substitution of CCTTT for CCAAT (CKT 065) or of GGTTTT for GGTAAT (CKT
063) completely eliminated one of the two protein-DNA complexes, and a
mutation in both motifs (CKT 083) resulted in a complete loss of
specific protein-DNA complex formation (Fig. 3C). It is
intriguing that the separation into two defined complexes, which is
because of the shorter DNA fragment used, was not observed with the
94-bp promoter fragment. Combining the results from Fig. 3,
A, B, and C, we conclude that CAE is simultaneously contacted by several DNA-binding proteins binding to two
different cooperating motifs.
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Fig. 3.
Characterization of protein binding to the
cbh2-activating element using various mutated
oligonucleotides. A, EMSA analysis with labeled 94-bp
cbh2 promoter fragment (see Fig. 1) and 100 µg of
cell-free extracts. In competition experiments, unlabeled
oligonucleotides CKT 057, CKT 063, CKT 065, and CKT 083 were added to
the binding assay in a 150-fold molar excess where indicated.
B, EMSA analysis using labeled oligonucleotide CKT 057 as a
probe and 100 µg of cell-free extract. As competitors,
oligonucleotides CKT 057, CKT 083, CKT 063, and CKT 065 were applied in
a 10- and 50-fold molar excess where indicated. C, EMSA
analysis using labeled oligonucleotides CKT 057, CKT 063, CKT 065, and
CKT 083 as probes and 100 µg of cell-free extracts. In competition
experiments (indicated by +), a 50-fold molar excess of unlabeled CKT
057 was added as competitor. D, sequences of annealed
oligonucleotides CKT 057 (bearing the wild-type CCAAT motif
(boxed)), CKT 063 (bearing two point mutations downstream of
the CCAAT element), CKT 065 (CCAAT motif altered to CCTTT), and CKT 083 (bearing four point mutations within and downstream of the CCAAT box),
that were used in EMSA as labeled probes and competitors, are given.
F, free probe; S and G, cell-free
extracts derived from cultures grown in the presence of glucose
(G) or Sepharose.
View larger version (40K):
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Fig. 4.
Effect of mutations in the cbh2
activating element on induction of cbh2 gene
expression. A, schematic representation of the reporter
cassette used for transformation. Point mutations are indicated by
arrows. B, Northern analysis of hygromycin B
phosphotransferase (hph) gene transcript formation in SMZ1,
SMZ2, SMZ4, and SMZ5 strains grown in the presence of glucose
(G) or sophorose (S); C, densidometric
calculation of formed transcript of the hph reporter gene in
relation to act1. 20 µg of total RNA were loaded.
Hybridization with the actin gene (act1) and the ethidium
bromide-stained gel are given as loading controls. The same blot was
used for both hybridizations after stripping the membrane.
View larger version (102K):
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Fig. 5.
The CCAAT motif is bound by a protein complex
containing a HapC-like component. A, effect of
anti-HapC antiserum (38) on the mobility of protein-DNA complexes
obtained with the labeled 94-bp cbh2 promoter fragment and
cell-free extracts from cultures grown in the presence of glucose
(G) or sophorose (S). The addition of 5 µl of
antiserum is indicated by +. B, EMSA analysis using labeled
oligonucleotide CKT 057 and cell-free extracts are as described in
panel A. In competition experiments, unlabeled
oligonucleotide OamdS (Table I) derived from the promoter region of the
amdS gene of A. nidulans, containing a CCAAT box
that was shown to bind an Aspergillus Hap-like complex (34,
37), was added to the binding assay in a 50-fold molar excess.
236 to 246) of the
cbh2 upstream regulatory region. Fig. 5B shows
that the amdS promoter fragment clearly competed the specific complex formed with oligonucleotide CKT 057 (Fig.
5B). From these data we conclude that a HapC homologue of
H. jecorina is part of the protein complex binding to the
CCAAT box in CAE of cbh2.
View larger version (121K):
[in a new window]
Fig. 6.
Analysis of the involvement of a XlnR-like
factor in binding to the cbh2 activating element. EMSA
analysis with labeled oligonucleotide CKT 057 as a probe and 100 µg
of cell-free extract of H. jecorina cultures grown in the
presence of glucose (G) or sophorose (S) is
shown. In competition experiments a 10- or 50-fold molar excess of
unlabeled oligonucleotide xlnD(wt) and xlnD(mut), respectively, was
added to the binding reaction where indicated. F, free
probe.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.: +43 1 58801 17252; Fax: +43 1581 62 66; E-mail:
szeiling{at}mail.zserv.tuwien.ac.at.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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