©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
c-Myb Repression of c- erbB-2 Transcription by Direct Binding to the c- erbB-2 Promoter (*)

Gaku Mizuguchi (1), Chie Kanei-Ishii (1), Tomomi Takahashi (1) (2), Takashi Yasukawa (1) (3), Takahiro Nagase (1), Masami Horikoshi (4), Tadashi Yamamoto (2), Shunsuke Ishii (1)

From the (1) Laboratory of Molecular Genetics, Tsukuba Life Science Center, the Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan, the (2) Department of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan, the (3) Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan, and the (4) Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

The c- myb proto-oncogene product (c-Myb)() 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) .

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.


EXPERIMENTAL PROCEDURES

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) .


RESULTS

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.

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 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.

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.

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.




DISCUSSION

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: Myb, myb gene product; bp, base pair(s); Ad MLP, adenovirus major late promoter; CAT, chloramphenicol acetyltransferase.

G. Mizuguchi, C. Kanei-Ishii, and S. Ishii, unpublished results.


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