(Received for publication, August 25, 1995; and in revised form, December 5, 1995 )
From the
Increased expression of the protein-tyrosine kinase receptor
ErbB-2 occurs frequently in human breast and ovarian cancer and causes
transformation in experimental systems. Control of transcription of the erbB-2 gene is an important determinant of receptor
expression. Within the human erbB-2 promoter, a 100-base pair
(bp) region 5` to the TATA box enhances transcription 200-fold. Two
palindromes present in this 100-bp region are important for both
positive and negative transcriptional control. A nuclear palindrome
binding protein (PBP) has been purified to near homogeneity using
ion-exchange, DNA-affinity, and gel filtration chromatography. PBP is a
heterodimer consisting of a 69-kDa subunit that binds DNA and a
60-kDa
subunit that appears to enhance subunit binding. DNase I
footprinting and electrophoretic mobility shift assays indicate that
PBP binds to the half-site of each palindrome with the core recognition
sequence TGGGAG. By DNA binding specificity and lack of immunological
cross-reactivity, PBP is distinct from NF-
B and Ikaros, two
proteins with related DNA binding specificities. PBP is proposed to be
an important regulator of transcription of the erbB-2 gene.
The erbB-2 gene is the second identified member of the
EGF ()receptor subfamily of receptor tyrosine kinases that
also includes ErbB-3 and
ErbB-4(1, 2, 3, 4, 5) . erbB-2 was initially identified as the neu oncogene
in rat neuro/glioblastomas induced by transplacental mutagenesis with
ethylnitrosourea(6, 7, 8, 9, 10) .
It is widely expressed during fetal development but is low to absent in
normal adult tissues (11, 12, 13, 14) . Although the
activating point mutation in rat neu (Val
Glu) (15) has not been seen in human tumors (16, 17) , amplification and overexpression of the
human erbB-2 gene is frequent in adenocarcinomas, especially
those arising in breast and ovary, where overexpression directly
correlates with poorer patient outcomes(18, 19) . In
general, protein expression is the result of gene amplification;
however, many tumors overexpress erbB-2 mRNA and protein from
single copy genes(19, 20) . Even with gene
amplification, mRNA expression per gene is increased, indicating that
transcriptional control mechanisms are likely
important(20, 21, 22) .
We previously reported the sequence of 3.65 kilobases of the human erbB-2 gene promoter(23, 24) . This promoter has typical TATA and CAAT elements in the region proximal to the translation start site (+1) and 4 Alu sequences located in the upstream region(24) . A 100-bp region upstream of the TATA box increased basal promoter activity 200-fold(25) . This 100-bp region contains a strong Sp1 site near the 5` end and a functional CAAT box near the 3` end. Two palindromic sequences are a prominent feature of this 100-bp control region(23, 26, 27) ; the distal palindrome (Pal I) overlaps the major Sp1 site and the proximal palindrome (Pal II) overlaps the CAAT box (25) . An AP2 site which is located 5` to this 100-bp region increased expression in several breast cancer cell lines that exhibited elevated AP2 activity(21, 28) .
Similar palindromes are present
in rat, mouse, and human erbB-2 promoters(29) . Using
reporter constructions, these palindromes were shown to exert both
positive and negative regulation of the human erbB-2 promoter(25) . To investigate control of the erbB-2 promoter via these palindromic sites, we have purified and
characterized a protein complex that specifically interacts with active
but not with mutant palindromic sequences. This palindromic binding
protein (PBP) is a heterodimer consisting of 69- and 60-kDa subunits.
PBP interacts with the half sites of both palindromes I and II
primarily via the 69-kDa subunit. Although the palindrome DNA
half-sites resemble NF-B and Ikaros binding sites, nucleotide
competition and immunological studies indicate that PBP is distinct
from both.
DNA-affinity columns were prepared using established methods (30) in which multimerized double-stranded oligonucleotides were coupled to CNBr-activated Sepharose 4B. WT is the Pal II sequence flanked by TCGA (see Fig. 1). MT is Mb flanked by TCGA. The WT and MT oligonucleotides used for multimerization have the following sequences:
Figure 1:
Human erbB-2 promoter. A, diagram of the 100-bp enhancer region of the erbB-2 gene located between -329 and -230 bp relative to the
translation start site. The CCAAT box and major Sp1 binding site are boxed, and the palindromes are indicated with arrows. B, sequences of Pal I, Pal II, and mutant Pal II
oligonucleotides that correspond to the erbB-2 promoter and to
HIV NF-B and Sp1 consensus oligonucleotides. Nucleotide changes
are shown in lowercase. LSM, linker scanning
mutation.
The active fraction eluted from the second specific DNA-affinity column was diluted with an equal volume of buffer to give a final concentration of glycerol of 10% and KCl of 300 mM. A 500-µl aliquot was loaded onto a Superdex 200 FPLC column. The column was equilibrated and chromatographed in buffer containing 10% glycerol and 300 mM KCl. Individual fractions of 0.5 ml were collected and stored at -80 °C.
Figure 2:
Purification of the palindrome- binding
protein complex. A, aliquots of the indicated fractions from
each step of the purification were assayed by EMSA using the Pal II
oligonucleotide probe. NE, nuclear extract; FT,
flow-through; EL, eluate; NS, nonspecific; mt, Mb mutant DNA-affinity column; wt, wild-type Pal
II DNA-affinity column. B, elution of PBP from the Pal II
sequence-specific DNA-affinity column as a function of [KCl].
Aliquots of each fraction were assayed using a Pal II oligonucleotide
probe. C, Superdex 200 FPLC chromatography of PBP. Fractions
were assayed for PBP activity by EMSA using a Pal II oligonucleotide
probe. Arrows denote the elution positions of the molecular
mass markers (ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa),
ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNase A (14 kDa)). SM, starting material from second WT DNA-affinity column
before loading onto the Superdex 200 column. D, protein in the
peak of activity from the Superdex 200 column. The predominant 69-kDa
and 60-kDa bands are designated as and
,
respectively.
Figure 3: DNase I footprint of the erbB-2 enhancer region with purified PBP. The end-labeled 100-bp probe (-329 to -230 bp) was footprinted without(-) or with (+) PBP. Location of mapped PBP binding sites (A, B, C, and D), Sp1 binding sites (sites I, II, and III), CAAT box, and palindromes are indicated in the diagram at the left. The weak Sp1 binding sites II and III have not been shown to respond to Sp1(25) ; Sp1 site II corresponds to a region necessary for function of an alternate initiation site(35) .
Previous studies indicated that mutations in either the 5` or 3` half of the palindromes decreased only modestly the DNA binding and functional activity of the palindromes suggesting that the half-site was the functionally active response element(25) . At lower concentrations of PBP, a single complex (Ca) was observed in EMSA on both Pal I and Pal II (Fig. 4). With increasing amounts of PBP, a second slower migrating complex, Cb, was formed. These results support the concept that dimeric PBP binds principally to a half-site core sequence in either palindrome; with higher concentrations of PBP, both half-sites can be occupied simultaneously.
Figure 4: Formation of complexes on palindromes I and II. Increasing amounts of PBP (10 to 50 ng) were incubated with oligonucleotides corresponding to Pal I or Pal II, and the complexes were analyzed by EMSA. Ca and Cb denote PBP bound to one or two sites.
PBP bound to both
Pal I and Pal II and DNA binding specificity were confirmed using
oligonucleotide competitors in electrophoretic mobility shift assays.
As shown in Fig. 5A, binding of PBP to P-labeled Pal I was competed by unlabeled Pal I or Pal II
oligonucleotides but not by an oligonucleotide containing the 8-bp
central region of Pal II, but with alterations in both of the
half-sites were identified by DNase I footprinting. Binding was not
competed by a consensus Sp1 binding site oligonucleotide or by an
NF-
B site oligonucleotide. These results indicate that, although
an Sp1 binding site partially overlaps Pal I, PBP does not recognize
the Sp1 binding site. While the PBP binding site closely resembles an
NF-
B site, PBP did not recognize an NF-
B site derived from
the HIV long terminal repeat (36) either by competition (Fig. 5A) or by direct binding assays (data not shown).
PBP binding exhibited similar binding specificity for Pal II (Fig. 5B). Binding was competed by mutations in either
palindrome half-site (M3, M5, LSM) but not by mutations in both (Mb).
These results confirm PBP binding to either half-site of the
palindromes.
Figure 5: Specificity of binding of PBP to the palindromes in the erbB-2 promoter. A, EMSA analysis of binding of PBP to 5-bromo-dUTP-substituted Pal I without (0) or with a 50-fold excess of the indicated oligonucleotides. comp, competitor. B, EMSA analysis of binding of PBP to 5-bromo-dUTP-substituted Pal II without (0) or with a 50-fold excess of the indicated oligonucleotides. C, UV-cross-linking analysis of binding of PBP to 5-bromo-dUTP-substituted Pal I without (0) or with a 50-fold excess of the indicated oligonucleotides. D, UV-cross-linking analysis of binding of PBP to 5-bromo-dUTP-substituted Pal II without (0) or with a 50-fold excess of the indicated oligonucleotides. Proteins were resolved on 10% SDS-PAGE gels and autoradiographed.
The
proteins present in the single site EMSA complex formed on Pal I and on
Pal II (Fig. 5, A and B) were eluted and
separated by SDS-PAGE. Approximately equal amounts of the 69- and
60-kDa species were detected in each complex (Fig. 6A).
Thus, although the 69-kDa subunit was the species cross-linked to
the DNA binding site, equal amounts of
and
subunits were
present in the DNA-bound complex providing evidence that PBP is a
heterodimer.
Figure 6:
Analysis of PBPDNA complexes. A, proteins in the PBP-palindrome complex. DNA
PBP
complexes in the gel-shifted complexes (Fig. 5, A and B) were identified by autoradiography of the wet gels and
excised. Gel slices were fragmented, suspended in 1% SDS heated at 68
°C, and the aqueous phase was recovered after centrifugation. After
trichloroacetic acid precipitation, the samples were suspended in water
and subjected to electrophoresis on a 10% SDS gel; proteins were
detected as described under ``Materials and Methods.'' B, detection of the DNA binding subunit of PBP. Southwestern
analysis was performed as described under ``Materials and
Methods.'' After protein transfer from SDS (left) and
native (right) polyacrylamide gels, nitrocellulose was
incubated with the
P-end-labeled Pal II oligonucleotide
without(-) or with (+) 20-fold excess of unlabeled probe.
Filters were autoradiographed at -80 °C with an intensifying
screen for 5 days (left) or 4 h (right).
Southwestern blotting confirmed specific binding of P-labeled Pal II to the 69-kDa
subunit of PBP (Fig. 6B). The higher molecular mass band was not
competed, consistent with it corresponding to the smaller amount of
higher molecular mass activity shown in Fig. 2C, and
the 87-kDa band was inconsistent. When Southwestern blotting was
carried out using nondenaturing polyacrylamide gel electrophoretic
separation of proteins, strong specific DNA binding was observed (Fig. 6B, right panel). This DNA binding
activity was at least 100-fold higher using native compared to
denaturing gel electrophoresis, a result that could reflect incomplete
protein renaturation or higher affinity of the heterodimer
PBP compared to the
subunit alone.
Figure 7:
Distinguishing PBP from NF-B and
Ikaros. A, NF-
B-binding proteins in
lipopolysaccharide-stimulated 70Z/3 cells. The HIV NF-
B
oligonucleotide was incubated with nuclear extract prepared from 70Z/3
cells stimulated for 1 h with lipopolysaccharide to induce formation of
Rel/p50 complexes in addition to p65/50 complexes present in these
cells. Competitor oligonucleotides (100-fold excess) or the indicated
antibodies were added, and complexes were analyzed by EMSA and
autoradiography. Ab, antibody. B, lack of effect of
anti-NF-
B/Rel and anti-Ikaros antibodies on PBP binding to the erbB-2 palindrome.
P-Labeled Pal I
oligonucleotides were incubated with PBP without (0) or with the
indicated antibodies and protein complexes detected by EMSA and
autoradiography.
Excessive mitogenic signaling via the ErbB-2 receptor
tyrosine kinase may result from mutational activation as occurs with
rat neu(15) or from gene amplification and enhanced
transcription of wild-type erbB-2(18, 19, 20) . Regulation of
transcription of the erbB-2 gene is one important determinant
of the extent of ErbB-2 expression. It is thus important to identify
elements that control transcription of the erbB-2 gene. As
assayed using reporter gene constructions in HeLa and CV1 cells, full
promoter activity of the proximal 1500 bp of human erbB-2 was retained in the -330 bp proximal to the translation
start site(23) . Several breast cancer cell lines are reported
to have strong AP2 activity which increased expression via a response
element located at -397 bp(21, 28) . A 100-bp
strong enhancer region is located proximally at -329 to
-230 bp(25) . This region contains a 5` Sp1 site and a 3`
CAAT box. There are 2 dyad symmetries within this 100-bp region that
are highly conserved among human, rat, and mouse
promoters(29) . When placed in front of a minimal TATA box
promoter, palindromes enhanced activity. Whereas deletion of Pal I in
the context of the erbB-2 promoter reduced activity consistent
with an enhancing effect, deletion of the 3`-half of Pal II that
overlaps the CAAT box increased promoter activity. Function of the
palindromes was thus complex and dependent on their context within the
promoter.
A dimeric protein complex that specifically binds these erbB-2 palindromes has been purified more than 17,000-fold.
The first indication that PBP exists as a complex was its aberrant
migration on gel filtration chromatography. The excessive retention of
the 69- and 60-kDa proteins on the Superdex 200 column is, perhaps, due
to hydrophobic interaction with the column material, and the
co-migration of both proteins with the peak of EMSA activity provided
evidence that both participated in the active PBP complex.
Approximately equal amounts of the 2 subunits were isolated from the
DNA-bound complex separated on EMSA. The 69-kDa subunit contacted
the palindrome half-sites. The amount of the 60-kDa
subunit
cross-linked to Pal I was small, less specific, and binding to Pal II
was not detected. Only binding to the
subunit was detected by
Southwestern analysis. Although we cannot exclude that the
subunit is a proteolytic product of
, this appears unlikely.
Approximately equal amounts of
and
were present in the
protein complexes bound to both Pal I and Pal II. Moreover, material
purified through 2 site-specific DNA-affinity columns and resolved on
Superdex 200 contained approximately equal amounts of
and
.
Although
did not bind DNA, Southwestern analysis revealed
100-fold higher DNA binding to the native complex than to the
subunit resolved on denaturing gels. While this could be due to
incomplete renaturation of the
subunit, the results taken
together suggest that PBP is a heterodimeric complex in which binding
of the
subunit to DNA is enhanced by the
subunit.
DNase I footprinting confirmed binding to 4 sites within the 100-bp region that correspond to the halves in each palindrome. Each half of the two palindromes appears to be an independent binding site with the sequence TGGGAG. Increasing amounts of PBP resulted in formation of a slower migrating complex on EMSA consistent with occupancy of both halves of the two palindromes.
Cloning will be required to further
characterize PBP and its relation to other transcription factors. DNA
binding specificity and lack of immunological cross-reactivity indicate
PBP is distinct from NF-B and Ikaros, two proteins with related
DNA binding specificity. We suggest that PBP will prove an important
regulator of erbB-2 transcription and thus biological
responses to this tyrosine kinase growth factor receptor.