(Received for publication, December 9, 1994; and in revised form, January 26, 1995)
From the
The oncoprotein v-ErbA is a mutated version of thyroid hormone
receptor 1. Although the basis for the oncogenic action of v-ErbA
is unknown, expression of this protein is known to inhibit thyroid
hormone and retinoic acid induction of target genes. The DNA binding
domain of v-ErbA differs from that of thyroid hormone receptor
1
in two amino acids felt to be crucial for determining the specificity
of DNA binding. However, the DNA binding properties of v-ErbA have not
been examined independent of a comparison of binding to already known
thyroid hormone response elements. In the current studies a non-biased
strategy was used to select from a pool of random DNA those sequences
that bind v-ErbA with high affinity. The highest affinity binding
sequence was identified as the decamer 5`-T(A/G)AGGTCACG, which is
closely related to the optimal thyroid hormone receptor
1 binding
sequence, TAAGGTCA. Transfection studies demonstrate that among equal
thyroid hormone responsive elements, those that contain the optimal
v-ErbA consensus will be repressed by v-ErbA in preference to those
that do not. These studies indicate that v-ErbA and thyroid hormone
receptor
1 regulate overlapping sets of response elements, and
that all sequences that are highly responsive to thyroid hormone are
not necessarily responsive to v-ErbA.
The avian erythroblastosis virus causes acute erythroleukemia
and fibrosarcomas in chickens. Its genome contains two oncogenes
designated v-erbA and v-erbB(1, 2) .
The v-erbB product is a truncated form of the epidermal growth
factor receptor. The v-ErbA protein is a mutated form of the thyroid
hormone receptor 1 (TR
1) (
)and belongs to the
large family of zinc finger transcription factors which includes the
receptors for steroids, vitamin D, and retinoic acid
(RAR)(2, 3) . v-ErbA has little to no transforming
capacity of its own in vivo but cooperates with the
transforming properties of a variety of tyrosine kinase-encoding
oncogenes, including v-ErbB and sarcoma oncogenes, as well as with the
Ha-Ras oncogene(4, 5) . v-ErbA blocks spontaneous cell
differentiation and allows tolerance to a wide variation in the pH and
ionic strength of culture media(4, 5) .
A
comparison of the amino acid sequence of the chicken TR1 and
v-ErbA proteins reveals that v-ErbA has undergone 13 single amino acid
mutations(6) . Two of these are located in the P and D boxes of
the DNA binding domain, regions that are crucial for determining the
DNA sequence specificity of protein binding(6, 7) .
Additional mutations, including a 9-amino acid deletion, are found in
the carboxyl-terminal domain. As a consequence of these mutations,
v-ErbA expressed in mammalian cells is unable to bind thyroid hormone
(T
)(6, 8) . In mammalian and avian cells
v-ErbA acts as a constitutive dominant repressor of transcription
regulated by TR and RAR, suggesting that these activities may be
central to its oncogenic
activity(9, 10, 11, 12) . However,
the actual mechanisms underlying v-ErbA's oncogenic action are
unknown, as are the key target genes for v-ErbA induced oncogenesis.
There is evidence to suggest that the DNA recognition sequences of
v-ErbA and TR1 are related although not necessarily
identical(9, 10) . Therefore the goal of the present
work was to identify the optimal DNA sequence for v-ErbA binding and to
study its function as a v-ErbA response element. A non-biased approach
was taken similar to that employed by Blackwell et
al.(13) , to study the DNA binding of c-Myc. By using
random DNA pool selection, competition DNA binding assays, DNA
footprinting, mutational analysis, and transient transfections, we were
able to identify the optimal v-ErbA binding site as the decamer
5`-T(A/G)AGGTCACG.
Eight rounds of selection were performed in total. In the third EMSA
a v-ErbA-P-labeled DNA complex could be visualized. In the
first 2 rounds, the protein-DNA incubations used 50 mM KCl,
and in the first 6 rounds 5 µg of v-ErbA was employed. To select
for higher affinity binding sites, rounds 3-5 and 6-8 were
performed with 150 and 400 mM KCl, respectively, and the
amount of v-ErbA was reduced to 500 ng for rounds 7 and 8. The selected
v-ErbA-binding DNA pool was ligated into the BamHI site of the
plasmid vector pUTKAT3(14) . Individual bacterial clones were
isolated and used to generate plasmid DNA. The sequences of all clones
were determined by the dideoxynucleotide method using vector primers.
Polymerase chain reaction with primers A and B was used to amplify the
contained 49-bp sequences, which were purified by PAGE and used in the
studies described below.
Co-transfections included 1 µg of a
human growth hormone (GH) expressing vector (pTKGH) per 60-mm Petri
dish to control for transfection efficiency. Cells were transfected in
the presence of 10% charcoal stripped fetal bovine serum and 100 nM dexamethasone. Cells were cultured ± 10 nM T for 2 days prior to harvest. CAT and hGH assays were performed as
described previously(17) . v-ErbA suppression of CAT reporter
gene expression was calculated as CAT/hGH for cells cultured with
v-ErbA divided by CAT/hGH for cells cultured without v-ErbA in either
the presence or absence of T
. Results are presented as the
mean ± S.E. for four independent transfections per assay
condition.
Figure 1:
EMSA selection of a DNA pool that binds
v-ErbA monomers. DNA pools were end-labeled with
[-
P]ATP, incubated with recombinant v-ErbA
or buffer, and subjected to PAGE. The v-ErbA-DNA complex is invisible
following incubation with the random pool DNA (lane 2), but is
clearly seen following 6 rounds of selection (lanes 3 and 4, indicated by the arrow). v-ErbA-DNA binding was
performed in the presence of 50 mM KCl except in lane
4, where 400 mM was used to select for higher affinity
protein-DNA interactions.
A faint slower migrating complex also was seen in the EMSAs ( Fig. 2and Fig. 3). The exact nature of this complex is not known. The band was supershifted by a specific v-ErbA antibody (gift of M. Privalsky; data not shown), indicating that it contains v-ErbA. The band was not affected by an anti-MBP antibody and did not co-migrate with an MBP-v-ErbA fusion protein-DNA complex, indicating that the band does not contain MBP or MBP-v-ErbA fusion protein. Footprinting studies were not possible due to the faint nature of the protein-DNA complex. This faint band was not present when the probe size was decreased to 20 bp. Overall the data suggest the faint band may be a v-ErbA homodimer that requires weak and perhaps relatively nonspecific interactions with other sequences in the 49-bp probe.
Figure 2:
Affinity of v-ErbA for individual clones
analyzed by competition EMSA. P-Labeled clone 5 was
incubated with 50 ng of recombinant v-ErbA plus various amounts of
non-radiolabeled competitor DNAs, and protein-DNA complexes were
analyzed by EMSA. The v-ErbA-DNA monomer complexes are indicated by the arrow. The dose of competitor DNA that competes 50% of the
v-ErbA-
P-labeled clone 5 complex (C
) was
determined by densitometry and used as a measure of relative affinity.
A competition assay using both a high affinity clone (clone 5; C
of 0.7 ng) and a low affinity clone (clone 35; C
of
3.4 ng) is shown. A faint slower migrating complex (indicated by the asterisk) was seen in these EMSA studies, and may represent a
v-ErbA homodimer interacting weakly with other sequences in the 49-bp
probe.
Figure 3:
Effect of mutations in the sequence
TGAGGTCACG on the binding of v-ErbA analyzed by competition EMSA. P-Labeled clone 5 was incubated with 50 ng of recombinant
v-ErbA plus the indicated amounts of non-radiolabeled competitor DNAs.
Competitors were synthetic 20-bp oligonucleotides; the top strand
sequences are shown, except that all also contain GATCT at the 5` end
and CGATC at the 3` end. The first competitor contains the optimal
decamer TGAGGTCACG, and it shows 50% competition at 0.1 ng (A). All other competitors contain mutations, shown with lower case letters in the sequence (B-D). Mutant
competitors are weaker than the consensus, thus confirming assignment
of the optimal sequence. The asterisk denotes a faint slower
migrating complex as described in Fig. 2.
In the case of TR1, mutating the two nucleotides
immediately 5` to the hexamer AGGTCA from TA to GC decreased the
affinity of the site for TR
1 5-fold(15) . However,
mutations of the two nucleotides immediately 3` of the AGGTCA did not
affect the affinity for TR
1 (data not shown).
Figure 4:
Combined guanine methylation interference
and uracil interference footprinting of v-ErbA monomer binding to clone
5. A, the DNA strand of clone 5 containing the sequence
TAAGGTCACG was arbritrarily designated the top strand. F and F
represent the free DNAs cleaved either at guanines alone or
guanines plus uracils, respectively. B
represents v-ErbA-bound DNA cleaved at guanines plus
uracils. Footprinted bases are indicated by closed circles. B,
the DNA sequence of clone 5 is indicated, with footprinted bases
designated by closed circles.
Figure 5:
A DR+4 of the decamer TAAGGTCACG is a
more potent v-ErbA response element than a DR+4 of the idealized
hexamer AGGTCA in a transient transfection assay. JEG-3 cells were
transfected with the reporter plasmid pUTKAT3 containing a single copy
of either a DR+4 of TAAGGTCACG (RT3) or a DR+4 of AGGTCA
(6DR), along with the internal control plasmid pTKGH. All cells also
received the expression vector for TR1 ± v-ErbA (or empty
vector). Cells were cultured for 2 days in the presence or absence of
10 nM T
and then cell lysates were analyzed for
CAT activity and medium for hGH. v-ErbA suppression is defined as
CAT/hGH for cells cultured with v-ErbA divided by CAT/hGH for cells
cultured without v-ErbA.
v-ErbA
also caused 60% suppression of T
-mediated CAT activity
when a single copy of the optimal decamer was used as a monomer T
response element (data not shown). However, the hexamer AGGTCA
used as a single site is not T
responsive (15) ,
and therefore cannot be tested for v-ErbA suppression.
v-ErbA potentiates the ability of a variety of transmembrane
oncoproteins to transform erythroblasts and
fibroblasts(4, 5) . In animal cells v-ErbA behaves as
a repressor of transcriptional activation regulated by TR and
RAR(9, 10, 22) . v-ErbA arrests the
expression of at least three erythroid genes: carbonic anhydrase II,
erythrocyte anion transporter (band 3), and -aminolevulinate
synthase. However, the block in erythroid differentiation is not
related to the suppression of any of these three genes(23) .
Although evidence suggests that the oncogenic function of v-ErbA
correlates with its ability to interfere with RAR action on a synthetic
palindromic retinoic acid response element
(RARE)(22, 24) , naturally occurring RAREs appear to
be direct repeats, not
palindromes(25, 26, 27, 28) .
Despite major advances in our understanding of v-ErbA function, the
genetic targets that account for its oncogenic action remain unknown.
Thus, it is not clear if this oncogenic function is based on
suppression of RAR or TR function, or if it is mediated through
uncharacterized v-ErbA specific responsive genes.
v-ErbA is closely
related to its cellular counterpart TR1. In the DNA binding
domain, these proteins differ in two amino acids located in the P and D
boxes, regions known to be crucial for DNA specific
recognition(6, 7) . The importance of the unique P box
serine in v-ErbA has been addressed by mutating it to glycine as is
found in TR
1 (S61G)(22, 29) . v-ErbA carrying
this S61G mutation is unable to effect erythroid neoplasia. This result
is consistent with the notion that this serine is crucial to the
oncogenic function of v-ErbA, and suggests that this oncogenic function
requires different DNA binding properties than are found in TR
1.
However, interpretation of these data is somewhat complicated by the
observation that a v-ErbA in which both the P and D box amino acids are
mutated to those found in TR
1 (S61G, T78K) has partial restoration
of its transforming properties relative to the S61G
mutant(30) . Nevertheless, these studies suggest that v-ErbA
and TR
1 have distinct DNA binding properties, and that these
differences play a role in the function of v-ErbA. Thus,
characterization of the optimal binding sites for v-ErbA should provide
insight into the mechanism of its oncogenic action, and might suggest
potential target genes for this oncoprotein. With this as a rationale,
we utilized a non-biased random DNA selection strategy to identify the
optimal DNA binding site for v-ErbA as the decamer T(A/G)AGGTCACG.
It is not completely understood which regions of the v-ErbA protein account for its specific high affinity interaction with this 10-bp DNA sequence. It has been suggested for several members of the erbA superfamily, including v-ErbA, that the P box is responsible for the recognition of the hexamer AGGTCA. There also is evidence that the A and T boxes located 3` to the zinc fingers are important in DNA-protein interaction(31, 32) . In particular, the A box is critical for recognition of the 5` end of the core DNA motif for the orphan receptor NGFI-B/nur 77(31) . By analogy the A box in v-ErbA could determine the specific recognition of the two nucleotides T(A/G) at the 5` end of the v-ErbA consensus site. It also has been shown that amino acid sequences outside the DNA binding domain of v-ErbA play a role in half-site specificity. Thus, the specific recognition for thymine at position 4 of the hexamer AGGTCA is determined by two amino-terminal amino acids, His-12 and Cys-32, interacting with Ser-61 in the P box of the DNA binding domain(33, 34) .
A comparison of the optimal DNA
binding sites of v-ErbA and TR1 (15) shows that they are
highly related but not identical (T(A/G)AGGTCACG versus TAAGGTCA, respectively). In general v-ErbA appears to have a
stricter specificity for binding at the 3` end of the recognition
sequence, and TR
1 at the 5` end. This is true not only based upon
the sequences selected from the random pools and their mutagenesis, but
is supported by the footprinting data. Thus, v-ErbA but not TR
1
shows footprints on the bottom strand opposite the top strand eight and
ninth nucleotides (AC) of the recognition sequence. In contrast,
TR
1 (15) but not v-ErbA shows footprints of the top strand
thymine at position 1 and the bottom strand thymine at position 2.
These results suggest that v-ErbA should bind to and repress certain
TREs more than others. Previous data support this concept in that it
was shown that TR1 can bind to the hexamer AGGACA whereas v-ErbA
cannot(33) . Consistent with this binding data, v-ErbA was not
able to repress T
induction from a TRE comprised of two
copies of the AGGACA hexamer(33) . However, TREs containing the
sequence AGGACA are functionally weak relative to those containing
AGGTCA. We find that TR
1 is 6 times more potent in activating CAT
expression on a reporter containing a thymine than on a reporter
containing an adenine at position 4 of the hexamer (TRE's,
TAAGGTCACGTAAGGTCACG versus TAAGG ACACGTAAGGACACG; data not
shown). Thus, TREs comprised of the hexamer AGGACA are weak T
inducers but even weaker at allowing v-ErbA suppression.
In
contrast with the above, the random selection strategy allowed us to
identify two sequences that are essentially equally strong as TREs, yet
v-ErbA differs dramatically in its ability to inhibit the T response on these two elements. Furthermore, this function of
v-ErbA correlates directly with its relative binding affinities for
these DNA sequences. This suggests that among equally T
responsive genes, some will be repressed by v-ErbA substantially
more than others. Thus, one can use DNA sequence to begin to predict
which genes are likely to be repressed by v-ErbA to the greatest
degree. In addition, one may speculate that cells may contain an
endogenous repressor with activity similar to v-ErbA, and that this,
too, might regulate a specific subset of TREs based upon sequence. In
this regard it is interesting to note that the orphan receptors COUP-TF (35) and ARP-1 (36) share identical P boxes with
v-ErbA. Furthermore, COUP-TF has been shown to repress T
action(37) , although a detailed analysis of its DNA
binding specificity and function has not been reported.
In
conclusion, our results indicate that the optimal binding site for
v-ErbA is the decamer T(A/G)AGGTCACG. This sequence binds v-ErbA with
higher affinity and also gives stronger suppression of TR1
mediated activation than does the hexamer AGGTCA. Despite the amino
acid differences in the P and D boxes, the optimal binding sites for
TR
1 and v-ErbA are very similar. The identification of new,
naturally occurring v-ErbA binding sites will be an essential component
of understanding its oncogenic action.