From the
The c- myb proto-oncogene product (c-Myb) is a
transcriptional activator that can bind to the specific DNA sequences.
Although c-Myb also represses an artificial promoter containing the Myb
binding sites, natural target genes transcriptionally repressed by
c-Myb have not been identified. We have found that the human
c- erbB-2 promoter activity is repressed by c-Myb or B-Myb in a
chloramphenicol acetyltransferase co-transfection assay. Domain
analyses of c-Myb suggested that Myb represses the c- erbB-2
promoter activity by competing with positive regulators of the
c- erbB-2 promoter. In in vitro transcription assays,
Myb proteins containing only the DNA binding domain could repress
c- erbB-2 promoter activity. Two Myb binding sites in the
c- erbB-2 promoter were critical for transcriptional repression
by c-Myb. One of the two Myb binding sites overlaps the TATA box, and
DNase I footprint analyses indicated that c-Myb can compete with TFIID.
These results suggest that Myb-induced trans-repression of the
c- erbB-2 promoter partly involves competition between Myb and
TFIID.
The c- myb proto-oncogene product (c-Myb)
All of the gene products of the myb gene
family (c-, A-, and B-Myb) are transcriptional regulators that bind to
the specific DNA sequence 5`-AACNG-3` through their DNA binding
domains, consisting of three imperfect tandem repeats of 51-52
amino acids (repeats 1, 2, and 3 from N terminus)
(10, 11, 12, 13, 14) . c-Myb has
three functional domains responsible for DNA binding, transcriptional
activation, and negative regulation, respectively
(15) .
Deletion of the negative regulatory domain results in increased DNA
binding and transforming potential
(16, 17) . This
domain contains a leucine zipper that seems to be an inhibitor binding
site
(17, 18) . c-Myb supports cellular proliferation by
activating the transcription of specific target genes. So far, only a
limited number of target genes for c-Myb have been identified: the
mim-1 gene encoding a secretable protein contained in the
granules of promyelocytes
(19) , c- myc (20, 21, 22) , cdc2 (23) ,
and c- myb itself
(24) . On the other hand, c-Myb can
also repress promoters containing its binding sites
(25) .
However, the natural target gene repressed by c-Myb has not been
identified.
The c- erbB-2 proto-oncogene (also called
neu or HER2) encodes a 185-kDa transmembrane glycoprotein that
has significant structural similarity to the epidermal growth factor
receptor
(26, 27, 28) . Although a 44-kDa
protein termed heregulin binds to a homologous receptor, the
c- erbB-4 protein, and activates the kinase activity of the
c- erbB-2 protein through transphosphorylation or receptor
heterodimerization
(29) , no ligands for the c- erbB-2
protein have yet been identified. The c- erbB-2 gene is
commonly expressed in fetal epithelium cells but is expressed at a low
level in normal postnatal tissues
(30, 31, 32) .
Apparently normal c- erbB-2 is frequently amplified in many
adenocarcinomas, especially in breast and stomach cancers
(33, 34) . Patients having breast cancers with
amplification of the c- erbB-2 gene have a shorter time to
relapse as well as a shorter overall survival
(34, 35) .
These findings indicate that control of the c- erbB-2 gene
transcription is critical for regulation of cellular proliferation and
transformation. As a first step to understand the transcriptional
control of the human c- erbB-2 proto-oncogene, the promoter
region was characterized
(36) . The c- erbB-2 promoter
has a TATA box, a CAAT box, and two GC boxes and initiates
transcription mainly at two sites, nucleotides +1 and -69.
Initiation at these two sites is controlled independently by the TATA
box and by the initiator (Inr)-like element located between -68
and -45
(37) .
Interestingly, it was reported that
c- myb expression was inversely correlated with
c- erbB-2 overexpression in noninflammatory breast cancer
(38) . Based on this observation, we have investigated whether
c-Myb directly regulates the c- erbB-2 promoter activity. Our
results suggest that c-Myb represses the transcription from the
c- erbB-2 promoter by directly binding to two sites in the
promoter. This implies that c- erbB-2 could be a natural target
gene repressed by c-Myb.
To clarify the mechanism of the Myb-induced
trans-repression of the c- erbB-2 promoter, we first
examined which functional domain(s) in c-Myb are required for
repression of the c- erbB-2 promoter activity by using some of
the c-Myb mutants for CAT co-transfection experiments (Fig. 2).
The NT2 mutant, which lacks the N-terminal 76 amino acids including
about one-half of repeat 1 in the DNA binding domain, had much weaker
activity than normal c-Myb to repress the c- erbB-2 promoter
activity. The
To further confirm that site I is really important
for the c-Myb-induced trans-repression, we did in vitro transcription experiments using the construct pEBCAT6 that
contains only one Myb binding site, I. Addition of 100 ng or 200 ng of
R123 decreased the level of transcription from both of two start sites
in pEBCAT6 54-57% or 18-19%. Disruption of site I in
pEBCAT6(pEBCAT6 mI) considerably impaired the trans-repression
by c-Myb. Addition of 200 ng of R123 only slightly repressed the
transcription from both sites to 67-75%. These results indicate
the importance of site I for trans-repression by c-Myb.
Among six Myb binding sites in the c- erbB-2 promoter
region, only two sites (sites II and IV) have sequences that matched
with the consensus sequence of the Myb binding sites. The sequences of
the four c-Myb binding sites other than sites II and IV are slightly
different from this consensus sequence, although all of them have the
sequence AAC. This indicates that these four Myb binding sites have the
greatly reduced binding affinity compared with sites II and IV.
However, the results of DNase I footprinting showed that 200 ng of the
c-Myb protein efficiently protected these four regions, two (sites I
and III) of which are responsible for the c-Myb-induced
trans-repression. These results may suggest that Myb binds
cooperatively to multiple sites in the c- erbB-2 promoter and
that these four sites have the higher binding affinity than that
obtained when they are individually used for binding assay.
Disruption of the two c-Myb-binding sites, I and III, almost
completely abrogated the c-Myb-induced trans-repression of the
c- erbB-2 promoter, indicating that binding of c-Myb to these
two sites causes transcriptional repression. Disruption of either site
I or III partly abolished the repression of transcription from both of
two start sites, -69 and +1. In addition, the amounts of
transcripts from both start sites were decreased by binding of c-Myb to
site I when pEBCAT6, which has only one c-Myb binding site, was used.
Thus, binding of c-Myb to either site I or III represses the
transcription from both start sites. Site I overlaps the TATA box, and
binding of c-Myb to site I blocks the binding of TFIID to the TATA box.
This should repress the transcription from +1, because
transcription initiation at +1 and -69 is controlled
independently by the TATA box and the Inr-like element
(37) .
Binding of c-Myb to site I could also block the elongation by RNA
polymerase started from -69. Site III overlaps not only one of
the two GC boxes but also one of the two dyad symmetry structures, both
of which contain the sequence 5`-TGGGAG-CTCCCA-3`. Binding of
c-Myb to site III may cause transcriptional repression by blocking the
binding of trans-acting factor(s) to these elements that
enhance the transcription from two start sites.
The c- myb proto-oncogene is mainly, but not exclusively, expressed in
immature cells of various hematopoietic lineages and is down-regulated
during terminal differentiation
(42, 43, 44) .
Recently, c- myb was also demonstrated to be involved in the
regulation of proliferation and/or differentiation of various types of
cells other than hematopoietic cells
(6, 8) . In
addition, it is now well established that expression of the c- myb gene is found in many types of tumors, such as breast cancer and
colon cancer
(5, 7) . On the other hand, the
c- erbB-2 gene is not expressed in hematopoietic cells but is
uniquely expressed in fetal epithelium such as transitional cells of
the renal pelvis and ureter
(30, 31, 32) . The
c- erbB-2 gene is frequently overexpressed in human
adenocarcinomas, especially in breast and stomach cancers
(33, 34) . Thus, both of two proto-oncogenes c- myb and c- erbB-2, are expressed in breast cancers.
Guérin et al. (38) demonstrated that the
c- myb expression is inversely correlated with
c- erbB-2 overexpression in noninflammatory breast cancer. In
addition to their results, our results shown here strongly suggest that
c-Myb represses the c- erbB-2 expression by directly binding to
the c- erbB-2 promoter. Compared with c- myb, the
B- myb mRNA is expressed in a variety of tissues including
spleen, placenta, and pancreas
(45) . Our results indicate that
B-Myb also represses the c- erbB-2 promoter activity.
Therefore, repression of the c- erbB-2 gene transcription by
the myb gene family could also occur in some types of tissues
or cancers other than breast cancers. Expression of both c- myb and B- myb is dependent on cell cycle. So, it is important
to examine whether c- erbB-2 transcription is regulated during
cell cycle.
(
)
is important in maintaining the proliferative state of
hematopoietic progenitor cells by regulating the transcription of some
target genes (for review, see Ref. 1). A block of c- myb expression reduces proliferation of hematopoietic precursor cells
(2) , and constitutive expression of c- myb blocks
differentiation of immature erythroid cells
(3) . Furthermore,
homozygous c- myb mutant mice are severely anemic and die
in utero due to defective fetal hematopoiesis
(4) .
Although c- myb expression was initially thought to be
restricted to the hematopoietic system, it has since been reported to
be expressed in nonhematopoietic tissues and cell lines such as colonic
tissues
(5) and smooth muscle cells
(6) . In these
cells, c- myb also appears to positively regulate proliferation
(6, 7, 8) . Two c- myb-related genes,
A- myb and B- myb, whose tissue specificities of
expression are different from c- myb, were identified
(9) .
Plasmid Construction
The plasmids pEBCAT1 and
pEBCAT4 and the Myb expression plasmids were described previously
(13, 15, 36) . To generate the plasmid pEBCAT6,
the 125-bp AccII- SmaI (nucleotides -86 to
+39) DNA fragment was inserted into the 5`-side of the CAT gene by
using a HindIII linker. The mutants of the c-Myb binding sites
in the c- erbB-2 promoter, in which the conserved sequence AAC
in the Myb binding sites was changed to CCC, were made by
oligonucleotide-directed mutagenesis
(39) or a polymerase chain
reaction-based method
(40) .
DNA Transfection and CAT Assay
CAT co-transfection
assays were done as described
(22) . Mixtures of these DNAs were
transfected into African monkey kidney cells (CV-1), and CAT assays
were done by using the cell extracts of which amounts were normalized
by the -galactosidase activities. The degree of conversion was
measured with a Bioimage analyzer (Fuji Photo Film Co., Ltd.). All CAT
co-transfection experiments were repeated at least twice, and typical
results are shown in the figures. The differences between each set of
experiments were within 30%.
DNase I Footprint Analysis
DNase I footprint
experiments were done with use of the bacterially expressed c-Myb
and/or human TFIID (-aminooctyl-agarose fraction)
(41) as
described
(37) . The full-length c-Myb expressed in
Escherichia coli was purified as described
(12) .
In Vitro Transcription
In vitro transcription experiments were done by using HeLa cell nuclear
extracts and supercoiled DNA template of pEBCAT4 or its derivatives,
and primer extension analysis was done as described
(37) . The
c-Myb R123 protein was expressed in E. coli and purified as
described
(12) .
Repression of c-erbB-2 Promoter Activity by
Myb
To examine whether c-Myb represses transcription of the
c- erbB-2 gene, we did a CAT co-transfection experiment using
the reporter plasmid pEBCAT1 in which the 530-bp promoter region of the
human c- erbB-2 gene was linked to the CAT gene (Fig.
1 A). When pEBCAT1 was co-transfected into CV-1 cells with the
effector plasmid to express the mouse c-Myb protein, the level of CAT
activity was decreased to 25% compared with the control effector
plasmid expressing no protein (Fig. 1 B). Similar repression of
CAT activity was also observed with the co-transfection of the B-Myb
expression plasmid.
DB mutant, which has no DNA-binding capacity, did
not repress the c- erbB-2 promoter activity at all. In
addition, the CT4 mutant, which has the whole DNA binding domain but
lacks the other two domains, had almost the same capacity to repress
the c- erbB-2 promoter activity as wild type. These results
indicate that the DNA binding domain of c-Myb is required and
sufficient for trans-repression of the c- erbB-2
promoter activity.
Figure 2:
DNA binding domain of c-Myb is sufficient
for the c-Myb-induced trans-repression of the
c- erbB-2 promoter activity. A, structure of normal
c-Myb is schematically shown on the top. Various deletion
mutants used for the CAT co-transfection assay are indicated. The
results of CAT co-transfection assays shown in B are
summarized on the right. The level of CAT activity are
expressed relative to that obtained without c-Myb. The mutants that can
repress the c- erbB-2 promoter activity are indicated by
shaded bars, and those that were inactive by open bars.
B, transient expression of CAT activity. The CAT co-transfection
experiments using the pEBCAT1 reporter plasmid and the effector plasmid
to express various c-Myb mutants indicated above each lane were done as described in Fig. 1 except that 4 µg of the
effector plasmid DNA was used. The relative CAT activity is shown by a
bar graph on the right.
Identification of Multiple Myb Binding Sites in the
c-erbB-2 Promoter
To examine whether Myb binds directly to the
c- erbB-2 promoter region, we did DNase I footprint analysis
using the bacterially expressed c-Myb (Fig. 3 A). Six sites
(sites I-VI) were almost completely protected by 200 ng of c-Myb. The
DNA sequences of the protected regions are shown in
Fig. 3B. All sites contained the sequence AAC in the
protected region as indicated for the consensus sequence of the Myb
binding site
(12) .
Figure 3:
Binding of c-Myb to the c- erbB-2
promoter region. A, DNase I footprinting of the
c- erbB-2 promoter region by using the recombinant c-Myb
protein. The HindIII- PstI (nucleotides +39 to
-213) fragment or the AccII- HindIII
(nucleotides -167 to -493) fragment of the
c- erbB-2 promoter region was P-labeled at the 5`
end of the lower strand. The fragment was incubated with or without
(-) 200 ng of the bacterially expressed c-Myb, followed by
partial digestion with DNase I. A + G refers to the adenine and
guanine marker obtained by the chemical cleavage of the same
end-labeled DNA fragment. Protected regions are shown on the
right. A schematic representation of the c-Myb binding sites
is indicated at the bottom. B, comparison of c-Myb binding
sites within the c- erbB-2 promoter. The DNA sequence of c-Myb
binding sites in the c- erbB-2 promoter region are indicated.
The c-Myb binding sites were aligned to set the consensus AAC sequence
at the center.
Specific Myb Binding Sites Are Responsible for the
c-Myb-induced trans-Repression
To efficiently analyze the
mechanism of the c-Myb-induced trans-repression of the
c- erbB-2 promoter, we needed to know the minimum length of the
c- erbB-2 promoter that has the full activity. To examine the
length of the functional promoter region, two additional constructs
(pEBCAT4 and pEBCAT6), that have 252- and 188-bp DNA fragments,
respectively, were made, and the abilities of these constructs to
express CAT activity were tested (Fig. 4, A and
B). CV-1 cells transfected with pEBCAT4 and pEBCAT6 expressed
the 12- and 6-fold higher CAT activity than cells transfected with
pEBCAT1. These results indicate that the region removed from pEBCAT1 to
generate pEBCAT4 contains the negative cis-element(s) and that
the 252-bp promoter region in pEBCAT4 has the full promoter activity.
When pEBCAT4 was used for a co-transfection assay, c-Myb repressed the
CAT activity expressed from pEBCAT4 to about 20%
(Fig. 4 C). In contrast, c-Myb did not repress the CAT
activity expressed from the reporter plasmid pEBCAT4 m(I + II
+ III), in which all three Myb binding sites were mutated.
Furthermore, c-Myb repressed the CAT activity expressed from pEBCAT6,
but did not repress CAT activity from pEBCAT6 mI in which one Myb
binding site was disrupted (Fig. 4 D). Similar results
were obtained by using the B-Myb expression plasmid (Fig. 4 C and D). Although these results appears to indicate that
the most proximal Myb binding site I is critical for repression by Myb,
disruption of only this site in pEBCAT4 did not completely abolish the
Myb-induced trans-repression.(
)
These
results indicate that Myb represses the c- erbB-2 promoter
activity through binding to the three Myb binding sites, I, II, and
III.
Figure 4:
The 252-bp c- erbB-2 promoter
region containing three Myb binding sites is responsible for the
c-Myb-induced trans-repression. A, structures of the
CAT reporter plasmids used. In the construct pEBCAT4 m(I + II
+ III), three Myb binding sites are disrupted. B,
deletion analysis of the c- erbB-2 promoter. Plasmid DNA shown
above each lane (10 µg) was transfected into CV-1
cells with the internal control plasmid pRSV--gal DNA (2 µg),
and the CAT activity was assayed for 5 h. The relative CAT activity is
shown by a bar graph. C, CAT co-transfection assay using
pEBCAT4 or pEBCAT4 m(I + II + III). CAT co-transfection assay
was performed as described in Fig. 1, and the relative CAT activity is
shown by a bar graph. D, CAT co-transfection using pEBCAT6 or
pEBCAT6 mI. CAT co-transfection assay was similarly
performed.
A CAT co-transfection assay is not suitable to accurately
estimate the degree of contribution of each Myb binding site to the
c-Myb-induced trans-repression, because the differences
between sets of CAT co-transfection assays are high. Instead of that,
we used the in vitro transcription assay (Fig. 5).
Transcription of the c- erbB-2 gene starts mainly at two sites,
nucleotides +1 and -69, and initiation at these sites is
controlled independently by the TATA box and the Inr-like element,
respectively
(37) . The in vitro transcription assay is
suitable also for analysis of the transcription started from each of
two sites. Since the CT4 mutant can repress the c- erbB-2
promoter activity in the CAT co-transfection assay (see Fig. 2),
the bacterially expressed R123 protein that contains only the whole DNA
binding domain of c-Myb is expected to repress the transcription from
the c- erbB-2 promoter in vitro. Addition of 100 ng of
R123 to the in vitro transcription reaction decreased in the
amount of transcripts started from -69 and +1 in pEBCAT4 to
41% and 63%, respectively (Fig. 5, pEBCAT4 template).
More R123 (200 ng) resulted in the severe reduction of the amount of
transcripts started from -69 and +1 to 15% and 29%,
respectively. Thus, R123 can repress the transcription from two sites
in the c- erbB-2 promoter in vitro.
Figure 5:
Repression of the c- erbB-2
promoter activity by c-Myb in vitro. A, structures of
the c- erbB-2 promoter-CAT constructs used as DNA templates.
The mutants in which a single or multiple Myb binding sites are mutated
are named according to the position of the mutation. The results of
in vitro transcription assays shown in B are
indicated below. The relative amounts of the transcripts
initiated at positions -69 ( open bar) or +1
( shaded bar) in the presence of c-Myb are shown in comparison
with that obtained in the absence of c-Myb. B, in vitro transcription assays. A mixture of the closed circular DNAs of the
c- erbB-2 promoter-CAT construct shown above each
lane and the control template, Ad MLP-CAT, was transcribed in
HeLa nuclear extracts. In lanes indicated by + and ++,
100 and 200 ng of c-Myb R123 protein was added, respectively. The
transcribed CAT RNAs were analyzed by primer extension analysis as
described (37). The extended primers corresponding to the
c- erbB-2-CAT mRNA started from -69 and +1, and that
corresponding to the Ad MLP-CAT mRNA are shown by arrows.
P-labeled HinfI-digested pBR322 was used as a
size marker. The amount of each extended primer corresponding to the
c- erbB-2-CAT mRNA was measured and normalized with respect to
that generated from the Ad MLP-CAT mRNA and is expressed by a bar
graph relative to that obtained in the absence of c-Myb in
A.
To examine
which Myb binding site among three sites in pEBCAT4 is important for
the trans-repression by c-Myb, three CAT constructs in which
one of three Myb binding sites was mutated were generated and used as
the DNA templates for in vitro transcription (Fig. 5).
Disruption of site I (pEBCAT4 mI) or III (pEBCAT4 mIII) partly impaired
the c-Myb-induced trans-repression. Compared with the case of
pEBCAT4, addition of 200 ng of R123 reduced the amount of transcripts
from both of two sites in pEBCAT4 mI or pEBCAT4 mIII less, to
47-50% or 43-44%, whereas the amount of transcripts from
both sites in the site II mutant (pEBCAT4 mII) was similarly reduced to
24-32% by 200 ng of R123. The importance of the two Myb binding
sites (I and III) was also confirmed by use of the CAT construct
pEBCAT4 m(I + III) in which both sites were disrupted. Addition of
100 ng of R123 did not repress the transcription from both sites in
this construct at all, while 200 ng of R123 slightly reduced the amount
of transcripts from either site to 75-88%. Thus, c-Myb represses
the c- erbB-2 promoter activity through binding to the two
sites, I and III.
Competition between c-Myb and TFIID for Binding to the
c-erbB-2 Promoter
The Myb binding site I overlaps the TATA box
to which TFIID binds. This allowed us to speculate that the
c-Myb-induced trans-repression is partly due to the
competition between c-Myb and TFIID for binding to this promoter. We
examined this possibility by DNase I footprinting analysis using
purified human TFIID and the c-Myb R123 protein (Fig. 6). The
TFIID purified from HeLa cells protected a region corresponding to
nucleotides -18 to -32 that contained the TATA sequence at
the center. On the other hand, c-Myb R123 protected a region between
nucleotides -11 and -25 that contained the AAC (GTT in the
opposite strand) sequence. Thus, the two protected regions partly
overlaps. Addition of both 600 ng of TFIID and 220 ng of R123 protected
the Myb binding region, indicating that c-Myb competes with TFIID for
binding to this region. This could explain at least partly the
mechanism of trans-repression of the c- erbB-2
promoter by c-Myb.
Figure 6:
Competition between c-Myb and TFIID for
binding to the c- erbB-2 promoter. TFIID binding to the TATA
element and Myb binding to the site I in the c- erbB-2 promoter
were analyzed by DNase I footprinting. The
HindIII- HaeII fragment (nucleotides +39 to
-195) prepared from the plasmid pEBCAT1 was
P-labeled at the 5` end of the HindIII site. The
labeled DNA fragment was incubated with 600 ng of purified human TFIID
( lane 3), 220 ng of recombinant c-Myb protein ( lane
5), both TFIID and recombinant c-Myb protein ( lane 4), or
no protein ( lane 2). DNase I digestions were done with 3
( lane 2 and 3) or 3.5 ( lanes 4 and
5) µg/ml DNase I. The positions of the protected regions
by TFIID and c-Myb are shown on the right. A+G ( lane 1) refers to adenine and guanine markers obtained
by the chemical cleavage of the same end-labeled DNA fragment. At the
bottom, the DNA sequence of the region protected by TFIID or
c-Myb is shown, where the TATA element and the AAC sequence (GTT in the
opposite strand) conserved in many Myb binding sites are shown by
open letters.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.