(Received for publication, December 13, 1996)
From the Department of Medicine, University of
California San Diego, La Jolla, California 92093-0650 and
§ The Salk Institute, La Jolla, California 92037
Within the human ERBB-2 gene
promoter, a 100-base pair region 5 to the TATA box enhances basal
transcription 200-fold. Two palindromes present within this 100-base
pair region are important for transcription. The palindrome binding
protein was purified to homogeneity and found to be identical to
RBPJ
, the mammalian homolog of Drosophila Suppressor of Hairless
(Su(H)). Recombinant RBPJ
bound the ERBB-2 promoter with
affinity comparable with that seen with well characterized RBPJ
binding sites. RBPJ
activated an ERBB-2
palindrome-containing promoter in 293 cells. Because in
Drosophila Su(H) acts downstream of NOTCH and because
NOTCH·Su(H)/RBPJ
stimulates transcription from target promoters,
NOTCH-IC, a constitutively active form of NOTCH, was tested for effects
on the ERBB-2 palindrome. NOTCH-IC further increased
RBPJ
-mediated transcription on wild type but not mutant
ERBB-2 palindrome. Thus, RBPJ
can activate ERBB-2 transcription and serve as an anchor to mediate
NOTCH function on the ERBB-2 gene.
The human ERBB-2 gene, located on chromosome 17q21, encodes a 1255-amino acid protein-tyrosine kinase receptor (1, 2). This receptor is widely expressed at higher levels during fetal development than in the adult, where it is detected primarily in epithelial cells (3, 4). Amplification and overexpression is frequent in adenocarcinomas, especially in those arising in the breast and ovary where overexpression directly correlates with poorer patient outcomes (5, 6). Protein overexpression is frequently the result of gene amplification; however, tumors are reported to overexpress ERBB-2 mRNA and protein from single copy genes. Even with gene amplification, mRNA expression/gene is increased, indicating that transcriptional control mechanisms are important (6, 7).
To deduce transcriptional control mechanisms, the human
ERBB-2 gene promoter has been sequenced and found to contain
typical CAAT and TATA elements and four Alu sequences within 3.65 kilobases of the proximal promoter (8-10). A 100-bp region upstream of
the TATA box increases promoter activity 200-fold. This 100-bp enhancer contains an Sp1 site near its 5 end and a CAAT box near its 3
end
(11). It also contains two palindromic sequences that are conserved in
the rat and mouse ERBB-2 promoters (12). Using reporter
constructs, these palindromes were shown to exert both positive and
negative regulation of the human ERBB-2 promoter (11).
We previously described a palindrome binding protein
(PBP)1 that bound to the half-site of each
ERBB-2 palindrome with the core recognition sequence TGGGAG
(13). We now report that protein sequence analysis of purified PBP
identifies it as RBPJ (recombination signal
binding protein of immunoglobulin
J
gene). RBPJ
, which was initially cloned
by Matsunami et al. (14) based on recognition of
the J
recombination signal sequence, was subsequently isolated based on recognition of the Epstein-Barr virus (EBV) C
promoter (designated as CBF1) (15-17) and of the adenovirus
pIX gene promoter (18). RBPJ
is the mammalian homolog of
Drosophila Suppressor of Hairless (Su(H)) (19, 20). Further
analysis indicated that although RBPJ
contained a 40-amino acid
region of homology to integrase, it lacked such activity (21) and
recognized a composite sequence consisting of the heptamer
recombination recognition site and a BamHI linker (15, 22).
RBPJ
is widely expressed with two mouse and three human splicing
variants identified (23).
Genetic analyses indicate that Su(H) acts downstream of NOTCH in the
signaling pathway of sensory peripheral nervous system development in
Drosophila (19, 20). A similar pathway for lateral
inhibition in neuronal development exists in mammalian species (24).
The Drosophila Enhancer of split (E(spl)) and the mouse
Hairy enhancer of split (Hes-1) complexes are downstream of NOTCH and
Su(H)/RBPJ in this pathway (25, 26). In the best studied mechanism,
the intracellular domain (IC) of the transmembrane protein NOTCH is
translocated to the nucleus with RBPJ
that serves as the
site-specific DNA binding partner (26, 27). The NOTCH-IC·RBPJ
complex activates transcription of basic helix loop proteins encoded by
Hes-1. Events downstream of E(spl)/Hes-1 may be inhibitory (lateral
inhibition in neuronal development (28) and inhibition of myogenesis
(29)) or stimulatory. RBPJ
also acts in a protein complex with the
non-DNA-binding EBNA2 to regulate genes involved in EBV latency (15).
The alteration of NOTCH that is necessary for nuclear translocation is
reported to occur upon ligand binding (27) via proteolysis (30), via
translocation that removes much of the ectodomain (31), and with
retroviral insertion (32).
Because RBPJ could bind to the two palindromes in the
ERBB-2 promoter, we examined its activity using reporter
gene constructs. We present evidence that RBPJ
stimulates promoter
activity via wild type but not mutant ERBB-2 palindromic
sequences; activity was markedly enhanced upon co-expression of the
intracellular domain of NOTCH but was not affected by co-expression of
EBNA2. These results suggest that ERBB-2 promoter activity
is regulated by a protein complex that contains RBPJ
.
293 cells were grown in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% calf serum. F9 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
Plasmid DNA was transfected as a calcium phosphate precipitate (33). A
-galactosidase expression vector under control of a cytomegalovirus
gene promoter was co-transfected with the luciferase reporter gene
constructs.
Luciferase activity was measured as described by de Wet et
al. (34). Aliquots of cell extract were added to an assay reaction containing 100 mM potassium phosphate (pH 7.8), 5 mM ATP, and 15 mM MgSO4 in a volume
of 0.35 ml. Reactions were initiated by the addition of 0.1 ml of 1 mM luciferin, and light readings were integrated over
10 s with a monolight 2110 luminometer. -Galactosidase activity
was measured as described by Norton and Coffin (35). Promoter activity
was expressed as light units of luciferase
activity/A420 unit of
-galactosidase
activity.
The intracellular region of human NOTCH
(NOTCH-IC) was a generous gift from Dr. David Baltimore, MIT. Viral
EBNA2 and human RBPJ cDNAs were generous gifts from Dr. Diane
Hayward, The Johns Hopkins University. The NOTCH-IC and RBPJ
cDNAs were regenerated by polymerase chain reaction and subcloned
into a modified pCDNA3 vector containing the luciferase gene
translation initiation codon and the hemagglutinin (HA) epitope
tag.
Eight copies of the wild type palindrome II (Pal II), four copies of
palindrome II mutated in both sites (Mb), or four copies of the
Hes-1 RBPJ binding site A were cloned in front of a
herpes simplex virus thymidine kinase promoter-driven luciferase
reporter construct to generate wtPal II-TK, mutPal II-TK, or Hes-TK,
respectively.
Binding reactions contained 2 × 104 cpm of DNA probe and varied amounts of protein with or without competitors or antibody in a final volume of 20 µl containing 100 mM KCl, 20 mM Tris (pH 7.5), 1 mM EDTA, 5 mM dithiothreitol, 5 mM MgCl2, 1 µg of poly(dI-dC), and 4% glycerol. The mixtures were incubated for 20 min at room temperature, loaded on a 4 or 6% acrylamide gel that had been run for 30 min at 4 °C in 0.5 × TBE (25 mM Tris borate, 0.5 mM EDTA), and electrophoresed at 10 mA for 2 h. Gels were dried and autoradiographed overnight.
Anti-HA antiserum was purchased from BABCO (Berkeley Antibody Co.,
Berkeley, CA). Oligonucleotides were synthesized using an Applied
Biosystems 380 DNA synthesizer. The oligonucleotides used in Figs. 1
and 3 have the following sequences, and the RBPJ consensus sequences
are underlined (references are indicated for each sequence): Pal
I, CTGGGAGTTGCCGACTCCCAG (13); Pal II,
CTGGGAGCGCGCTTGCTCCCAA (13); Mb,
CGAAACACGCGCTTGACAAAGA (13); M3, CTGGGAGCGCGCTTGACAAAGA (13);
596, GATGTGGTGGGAAAACCATTA (8); Cp,
ACACGCCGTGGGAAAAAATTT (36); Hes,
GTTACTGTGGGAAAGAAAGTC (26); LMP,
CAAGCTGTGGGAATGCGGTGG (36); pIX,
TTAAGGGTGGGAAAGAATATA (18); Sp1, GCTTGATCGGGGCGGGGCGATC (13).
PBP Purification and Peptide Sequencing
Purification of PBP
was described previously (Ref. 13; see Fig. 2). Briefly, nuclear
extract made from F9 cells was negatively absorbed to DE52 and to a DNA
affinity column prepared using a mutant oligonucleotide (Mb) followed
by two rounds of specific DNA affinity chromatography using palindrome
II (CTGGGAGCGCGCTTGCTCCCAA). The final step was
gel filtration chromatography. All active fractions eluting from the
Superdex 200 fast protein liquid chromatography column were pooled and
separated by SDS-polyacrylamide gel electrophoresis and then
transferred onto a nitrocellulose membrane. After visualization by
staining with Amido Black 10B, the protein band was excised and
subjected to in situ digestion with trypsin. The excised
band was treated with polyvinylpyrrolidone to prevent binding of the enzyme to the membrane. Digestion with trypsin (1 µg in 30 µl of
0.1 M TES, pH 8) was allowed to proceed overnight. The
supernatants were then fractionated by reversed-phase HPLC. Fractions
were collected manually, based on absorbance at 210 nm. Fractions
corresponding to symmetrical peaks in the chromatogram were then
subjected to chemical sequence analysis in an ABI470 (Applied
Biosystems Inc.) protein sequencer (37) and analyzed by matrix-assisted
laser desorption mass spectroscopy on a Bruker Reflex Time-of-Flight instrument. Two HPLC fractions of the protein digest were sequenced. One fraction yielded DGYIHYGQTVK (Fraction 29). The mass spectrum contained one strong signal at m/z = 1280. The other fraction was
Q(TP)(AV)(QL)(VL)(DP)(AV)(DT)(DL)(PV)XSQL (Fraction 46)
where residues given in parentheses were observed in the same
sequencing cycle, indicating the presence of two peptides in that
fraction. The mass spectrum contained a major signal at
m/z = 1235 and a weak signal at
m/z = 1750. A BLAST computer homology search
(38) revealed that all these sequences were identical to tryptic
fragments of mouse/Xenopus RBPJ. All molecular masses
determined were within 0.05% of the masses calculated for the
corresponding RBPJ
tryptic fragments.
Previous studies showed that a 100-bp region
upstream of the TATA box of the ERBB-2 gene promoter
increased basal promoter activity 200-fold (11). Two palindromic
sequences are a prominent feature of this 100-bp enhancer element in
addition to a strong Sp1 site near the 5 end and a CAAT box near the
3
end. The distal palindrome (Pal I) overlaps the Sp1 site, and the
proximal palindrome, Pal II, overlaps the CAAT box (8, 9). When this
100-bp region was used as a probe in EMSA, proteins in F9 nuclear
extract formed several complexes (Fig. 1). The complexes
that could be effectively competed by an Sp1 consensus sequence are
represented by Sp1 and proteins with a recognition motif similar to
Sp1. A complex designated as PBP could not be competed by the Sp1
consensus oligonucleotide but was effectively competed by the
palindrome sequence. F9 cells thus contain a specific palindrome
binding activity.
To determine the identity of PBP, it was
purified to homogeneity from F9 cells using ion exchange, DNA affinity,
and gel filtration chromatographies and SDS-polyacrylamide gel
electrophoresis. Fig. 2A summarizes the
purification steps. Fig. 2B shows purified PBP before
trypsin digestion, and the peptides identified by protein sequence
analysis and mass spectroscopy are shown in Fig. 2C. A
computer homology search revealed that all three sequences were identical to tryptic fragments of RBPJ, a protein originally identified through binding to the J
immunoglobulin
recombination signal sequence (14).
To verify that RBPJ had palindrome binding activity, the protein was
expressed as a HA-tagged fusion protein and transfected into 293 cells.
Transient expression of HA-RBPJ
resulted in a large increase of PBP
activity that could be competed by wild type Pal II but not by Mb
oligonucleotides. An antibody against the HA tag supershifted the
complex, supporting the sequence identification of PBP as RBPJ
(Fig.
3).
RBPJ was originally isolated as a protein that bound to
the J
recombination signal sequence consisting of a
heptamer and a nonamer with a 23-bp spacer (14). RBPJ
protein is
highly conserved from Caenorhabditis elegans to Homo
sapiens (14, 15, 19, 20, 39). The consensus recognition sequence
for the RBPJ
family has subsequently been determined as GTGGGAA,
which is present in all RBPJ
binding sites characterized so far
(22). The PBP core site CTGGGAGC is close but not identical to the
consensus sequence (13). To compare the affinity of ERBB-2
palindromes with other RBPJ
binding sites, palindromes as well as
other characterized binding sites were used as unlabeled competitors in
EMSA using partially purified F9 nuclear extract and Pal II as a probe.
As shown in Fig. 4, the EBV Cp sequence is the optimal
binding site. Pal I, Pal II, and half-site mutation M3 have affinity
similar to that seen with the Hes-1 and pIX gene
promoters. A perfect matching consensus site is located at
596 of the
ERBB-2 gene promoter (8). It is, however, a poor competitor,
indicating that flanking sequences significantly affect RBPJ
binding
affinity for the core consensus sequence.
RBPJ
To study the function of
RBPJ on the ERBB-2 promoter palindrome, RBPJ
was
transfected into 293 cells along with reporter constructs containing
wild type or mutant palindrome sequences (Fig. 5). The
control reporter TK was induced 3-fold by RBPJ
. RBPJ
activated
wtPal II-TK 16-fold, whereas mutPal II-TK was induced no more than the
background activation seen with the vector TK. Constitutively active
forms of mammalian NOTCH stimulated transcription of the
Hes-1 gene presumably through RBPJ
and its binding site
on the Hes-1 promoter (26). The intracellular domain of
NOTCH was transfected to determine whether an active form of human
NOTCH (NOTCH-IC) had any effect on the ERBB-2 gene (Fig. 5).
NOTCH-IC alone did not increase reporter activity above background. However, co-transfection of NOTCH-IC with RBPJ
activated wtPal II-TK
activity 82-fold. NOTCH-IC failed to affect the basal stimulation of
RBPJ
seen with TK and mutPal II-TK. The effect of NOTCH-IC was thus
specific to the wild type ERBB-2 palindrome-containing gene
and was dependent on RBPJ
.
The effects of RBPJ and NOTCH-IC on ERBB-2 palindromes
were compared with effects on the Hes-1 promoter. As shown
in Fig. 6, NOTCH-IC alone was sufficient to induce
activation of the Hes-1 promoter, whereas RBPJ
alone did
not stimulate the Hes-1 reporter. Co-transfection of RBPJ
and NOTCH-IC caused no change in activity beyond that observed with
NOTCH-IC alone. EBNA2 is a well described activator of some RBPJ
response genes (15, 36). It up-regulates Cp and CD23 genes utilizing
RBPJ
and its binding sites on extended promoter elements. When EBNA2
was transfected into 293 cells, it had no effect on ERBB-2
or Hes-1 promoter activity. EBNA2 also had no effect on
activation mediated by RBPJ
and/or NOTCH-IC on either the
ERBB-2 palindromes or the Hes-1 site. Thus
EBNA2 requires interactions additional to those mediated via
RBPJ
(40), whereas effects of NOTCH·RBPJ
occur on
promoters containing only RBPJ
response elements.
The present studies identify the protein (PBP) that binds to the
two palindromes of the proximal enhancer of ERBB-2 as
RBPJ. Peptide sequences of tryptic fragments of PBP were identical
to those of RBPJ
(14-16). Although the ERBB-2 palindrome
half-sites do not perfectly match the reported RBPJ
consensus
binding sequence (22), PBP/RBPJ
bound to the palindromic sites with
an affinity similar to that observed for the consensus sites.
NF
B/Rel binding sites, which also resemble the palindrome
half-sites, do not compete for PBP binding (13). Additionally, nuclear
extracts containing epitope-tagged RBPJ
formed a specific complex
with ERBB-2 palindromes. Previous studies identified two
components of PBP (13). We suggest that these likely represent splicing
variants of RBPJ
with varying N termini (23).
RBPJ is reported to function as a site-specific DNA binding protein
that recruits additional transcription factors to target genes (17, 36,
41). RBPJ
represses transcription from its cognate site in the
adenovirus pIX gene promoter (18) and from a Gal4 binding
site when expressed as a Gal4 fusion (42). A repression domain in
RBPJ
was identified that coincided with that required for activation
by EBNA2 (42). EBNA2 was thus deduced to activate transcription by both
masking the repression domain of RBPJ
and by bringing a strong
transactivation domain to the RBPJ
binding site.
RBPJ is the mammalian homolog of Drosophila Su(H), which
functions in the NOTCH pathway and regulates development of the peripheral nervous system (19, 20). Su(H) interacts positively with
NOTCH (43, 44) and negatively with Hairless (45). Fortini and
Artavanis-Tsakonas (27) observed that Su(H) was translocated to the
nucleus when the transmembrane receptor NOTCH was activated by binding
the ligand Delta. Interestingly, an oncogene from a human T
lymphoblastic leukemia (TAN-1) was identified as a truncated NOTCH
lacking much of the extracellular domain (31). Kopan et al.
(30) presented evidence that proteolytic processing of NOTCH could
generate NOTCH-IC sufficient to act via a complex with RBPJ
in the
nucleus. RBPJ
binds to the intracellular domain of NOTCH, and
expression of RBPJ
with ectodomain-deleted forms of NOTCH (
EC-NOTCH), both with and without retention of the transmembrane domain, activates transcription from Hes-1 and EBV
C promoters (26, 46).
EC-NOTCH·RBPJ
and EBNA2·RBPJ
complexes exhibited similar transcriptional enhancement on EBV
C promoter sites (46).
RBPJ stimulated transcription from a reporter gene containing wild
type but not mutant ERBB-2 palindrome sequences. This activity was greatly enhanced by NOTCH-IC. Using immunofluorescence, we
found that NOTCH-IC and RBPJ
localize in the nuclei of 293 cells
(data not shown). These observations support models of
RBPJ
·NOTCH-IC heterodimers that act to enhance transcription from
RBPJ
response elements. Comparison of ERBB-2 palindrome
and Hes-1 elements in reporter gene assays indicates a
stronger effect of transfected RBPJ
alone on ERBB-2 and a
stronger effect of transfected NOTCH-IC alone on Hes-1. This
may reflect a higher affinity in vivo of endogenous RBPJ
for the Hes-1 site. RBPJ
was not inhibitory on either
site. On these sites, EBNA2 had no effect. This suggests that
stimulatory effects of EBNA2 require interaction with additional proteins such as Spi-1/Spi-B, which bind to a more extended EBV C element (40).
Several recognition elements have been identified within the extended
ERBB-2 promoter. Two AP2 and two Sp1 consensus binding sequences are found at 397,
359,
369, and
314, respectively, relative to the translation start site (8). Several breast cancer cell
lines are reported to have a strong AP2 activity that increases
expression via the response element located at
397 bp (47). A
stimulatory factor that binds to the promoter region 3
of the TATA box
has also been described (48). Analyses of the rat ERBB-2
promoter have identified a transacting factor that binds at
466 to
456 bp (49) and inhibition by co-expression of Rb (50), adenovirus
E1A (51), and c-Myc (52). However, deletional analysis of the human
ERBB-2 gene identified the region between
329 and
230 bp
as the major strong enhancer region; it contains two functionally
important palindromes (11). The activity of RBPJ
, which binds to
each palindrome half-site (13), is strongly augmented by NOTCH-IC.
Interestingly, mice that contain homozygous deletions of
ERBB-2 exhibit defects in development of cardiac trabeculae
(53), but development of cranial neural crest-derived sensory ganglia
and motor neurons was also impaired, implying important roles for
ERBB-2 in both cardiac and neural development. Control of
ERBB-2 expression by NOTCH signaling pathways that are known
to be important in neuronal development in transgenic mice thus appears
plausible.
We thank Dr. Diane Hayward, The Johns
Hopkins University, for human RBPJ and EBNA2 expression
plasmids and Dr. David Baltimore, MIT, for the intracellular region
of human NOTCH cDNA.