(Received for publication, October 24, 1995; and in revised form, December 21, 1995)
From the
The POU (Pit-Oct-Unc) family transcription factor Brn-3a
contains two distinct activation domains, one at the N terminus of the
molecule and one at the C terminus coincident with the DNA binding
domain. These different activation domains have been shown previously
to differ in their ability to activate an artificial test promoter
containing a Brn-3a binding site and the naturally occurring
-internexin gene promoter. Here we identify the target site for
Brn-3a in the
-internexin gene promoter and show that it can
confer responsiveness to Brn-3a on a heterologous promoter. One of the
single-stranded DNA sequences derived from either this novel Brn-3a
binding site or from the previously characterized site in the test
promoter are shown to bind Brn-3a preferentially compared with the
complementary single strand or the corresponding double-stranded
sequence. The pattern of responsiveness of these two sequences when
cloned upstream of the same test promoter and co-transfected with
constructs encoding various portions of Brn-3a indicates that the
activity of the two Brn-3a activation domains is dependent upon
differences in the context of the target sequence in each promoter
rather than on differences in the target sequence itself.
The POU (Pit-Oct-Unc) family of transcription factors play a critical role in the regulation of gene expression in neuronal cells acting by binding to sequences related to the consensus octamer motif ATGCAAAT in the promoters of their target genes (for review, see Verrijzer and van der Vliet(1993) and Wegner et al. (1993)). The Brn-3 family of mammalian POU factors are of particular interest as the mammalian factors most closely related to the nematode POU protein Unc-86 whose inactivation results in the failure to develop specific neuronal cell types particularly sensory neurons (Desai et al., 1988; Finney et al., 1988). Three mammalian Brn-3 factors encoded by distinct genes exist (Theil et al., 1993, 1994) and are known as Brn-3a (also known as Brn-3.0: Gerrero et al., 1993; Lillycrop et al., 1992), Brn-3b (also known as Brn-3.2: Lillycrop et al., 1992; Turner et al., 1994), and Brn-3c (also known as Brn-3.1: Gerrero et al., 1993; Ninkina et al., 1993).
In agreement with their similarity to Unc-86, all three of these factors are expressed in sensory neurons and exhibit distinct but related patterns of gene expression in the developing and adult nervous systems (Gerrero et al., 1993; He et al., 1989; Lillycrop et al., 1992; Ninkina et al., 1993; Theil et al. 1993; Turner et al., 1994; Xiang et al., 1993). In addition they exhibit distinct and often opposite changes in gene expression in cultured neuronal cells exposed to growth factors or stimuli inducing differentiation (Budhram-Mahadeo et al., 1995a, 1995b; Lillycrop et al., 1992).
These differences in expression
pattern are paralleled at the functional level. Thus Brn-3a can
activate artificial test promoters bearing octamer-related motifs
(Budhram-Mahadeo et al., 1994; Morris et al., 1994)
or a binding site derived from the pro-opiomelanocortin promoter
(Gerrero et al., 1993) and can also activate the naturally
occurring promoters of the pro-opiomelanocortin (Turner et
al., 1994) or -internexin (Budhram-Mahadeo et al.,
1995b) genes. In contrast Brn-3b has been shown to repress both the
test promoter bearing the octamer-related motif (Budhram-Mahadeo et
al., 1994) and the
-internexin promoter (Budhram-Mahadeo et al., 1995b) and can interfere with their activation by
Brn-3a (BudhramMahadeo et al., 1994, 1995b; Morris et
al., 1994).
In view of the activation of these two promoters by
Brn-3a and not Brn-3b, we have used chimeric constructs encoding
different regions of Brn-3a or Brn-3b to map the regions of Brn-3a
required for promoter activation. In these experiments, activation of
the octamer-containing test promoter is dependent on the DNA-binding
POU domain of Brn-3a (Morris et al., 1994). In contrast,
activation of the -internexin promoter requires a domain at the N
terminus of Brn-3a (BudhramMahadeo et al., 1995b). In
agreement with this difference observed using chimeric constructs, the
isolated POU-domain of Brn-3a when expressed in the absence of other
regions can activate the octamer-containing test promoter but not the
-internexin promoter (Budhram-Mahadeo et al., 1995b).
Hence Brn-3a contains two distinct activation domains which differ
in their ability to activate different target promoters. Interestingly,
although the -internexin promoter contains several copies of the
octamer motif (Ching and Liem, 1991), these are not required for
activation by Brn-3a. Thus Brn-3a can activate a truncated
-internexin promoter containing only 77 bases of upstream sequence
and lacking any sequences closely related to the octamer motif
(Budhram-Mahadeo et al., 1995b). The difference in the effects
of different activation domains of Brn-3a on the two test promoters may
thus arise from a difference in sequence between the octamer motif and
the target sequence in the
-internexin promoter. Alternatively it
may be dependent on differences in the context of the Brn-3a target
site relative to other transcription factor binding sites in the two
promoters.
To distinguish these possibilities we have identified the
target site for Brn-3a in the -internexin promoter and have tested
the effect of different Brn-3a constructs upon this sequence when it is
placed in the same context as the octamer motif within a test promoter.
For the binding
assay, 10 fmol of [P]ATP-labeled oligonucleotide
probe was mixed with reticulocyte lysate in the presence of 20 mM Hepes, 5 mM MgCl
, 50 mM KCl, 0.5
mM dithiothreitol, 4% Ficoll, and 2 µg of
poly(dI-dC)/20-µl reaction volume. Competitor DNA was added at
appropriate molar excess at this stage, as required. The binding
reaction was incubated on ice for 40 min prior to electrophoresis on a
4% polyacrylamide gel in 0.25
TBE (1
TBE = 100
mM Tris/HCl, 100 mm-boric acid, 2 mM EDTA, pH 8.3)
for 2-3 h at 150 V and 4 °C. DNA-protein complexes were
visualized by autoradiography of the dried gel.
The double-stranded oligonucleotide was cloned into the BamHI site in the vector pBLCAT2 that contains the herpes simplex virus thymidine kinase promoter driving the cat gene (Luckow and Schutz, 1987). The response of this plasmid reporter was compared with that used in our previous experiments (Budhram-Mahadeo et al., 1994, 1995b), which contains the Brn-3 octamer-related binding site ATGCAATT cloned into the same site in the vector pBLCAT2. The Brn-3a and -3b expression vectors contain full-length cDNA or genomic clones for each of these proteins (Theil et al., 1993) cloned under the control of the Moloney murine leukemia virus promoter in the vector pLTR poly which has been modified by deletion of a cryptic splice site in the SV40 3`-untranslated region.
In our previous experiments, a construct containing
-internexin promoter sequences from -77 to +73 relative
to the transcriptional start site was strongly inducible by Brn-3a
(Budhram-Mahadeo et al., 1995b). To further delineate the
sequences in the promoter mediating this inducibility, this plasmid was
digested with the Bal-31 exonuclease to produce a construct which on
sequence analysis contained
-internexin promoter sequences from
-46 to +73. As indicated in Fig. 1, inducibility by
Brn-3a was completely abolished in the -46 construct, although
this construct retained detectable basal promoter activity, indicating
that the lack of inducibility was not due to the absence of a
functional promoter.
Figure 1:
Activation of -internexin promoter
constructs containing sequences from -77 or -46 to +73
relative to the transcriptional start site, upon co-transfection with
the Brn-3a transcription factor. In each case results are expressed as
-fold activation of promoter activity upon co-transfection of 5 µg
of target plasmid with 10 µg of a Brn-3a expression vector compared
with the activity observed with the expression vector lacking insert
alone.
These findings indicate that -internexin
promoter sequences located between -77 to -47 upstream of
the transcriptional start site are required for inducibility by Brn-3a.
Inspection of this sequence revealed a sequence at -68 to
-61 that shared a five out of eight base pair match to the
octamer-related motif we used in our previous studies (BudhramMahadeo et al., 1994; Morris et al., 1994) and to the
consensus octamer motif (Fig. 2). Moreover this motif retained
the ATG sequence previously shown to be of critical importance for
Brn-3a to bind to its response element in the pro-opiomelanocortin
promoter (Gerrero et al., 1993) and in various synthetic
oligonucleotides (Li et al., 1994). We therefore tested the
binding of Brn-3a to an oligonucleotide consisting of the
-internexin promoter sequence from -77 to -60 that
contained this motif.
Figure 2:
Sequence of the consensus octamer motif (A: Falkner et al., 1996), compared with the
octamer-related motif used in our previous studies (B: Ching
and Liem, 1991) or the sequence from -68 to -61 in the
-internexin promoter (C: Morris et al.,
1994).
As illustrated in Fig. 3, this
double-stranded oligonucleotide was able to bind in vitro transcribed and translated Brn-3a, but did so rather poorly in DNA
mobility shift assays. Surprisingly, however, as shown in Fig. 3, strong binding of Brn-3a was observed with one of the
two corresponding single-stranded oligonucleotides containing the
antisense strand of the -internexin gene sequence (complementary
to the sequence illustrated in Fig. 2). Some binding of Brn-3a
was also observed with the other single-stranded oligonucleotide
containing the sense strand of the
-internexin gene. This binding
was not observed with control reticulocyte lysates that had not been
stimulated with mRNA or that expressed other unrelated in vitro transcription/translation products (data not shown).
Figure 3:
DNA
mobility shift assay using in vitro transcribed and translated
Brn-3a protein and P-labeled probes derived from the
single-stranded antisense sequence of the
-internexin promoter
from -77 to -60 (A), the complementary sense
sequence (S), or the corresponding double-stranded
oligonucleotide (D). The positions of the single- and
double-stranded oligonucleotides are indicated by the horizontal
lines.
This
result, therefore, confirms that the region of the -internexin
promoter from -77 to -60 can indeed bind Brn-3a paralleling
the role of this region in the Brn-3a responsiveness of the promoter.
However, the binding occurs with much higher affinity to one of the two
corresponding single-stranded sequences compared with the
double-stranded sequence.
As binding of Brn-3a to single- rather
than double-stranded sequences had not been reported previously, we
wished to test whether it was a peculiarity of the -internexin
sequence or could also be observed with other target sites for Brn-3a.
We therefore tested the binding of Brn-3a to the octamer-related
sequences used in our previous studies (Morris et al., 1994)
(oligonucleotide B in Fig. 2). In accordance with our
previous data, Brn-3a bound to this double-stranded oligonucleotide (Fig. 4A). However, as with the
-internexin
sequence, much stronger sequence-specific binding was observed to the
antisense oligonucleotide (complementary to the sequence illustrated in Fig. 2) with somewhat weaker binding being observed with the
other single-stranded oligonucleotide (Fig. 4A), and
this was confirmed by competition analysis (Fig. 4B).
Most importantly, in these experiments, no competition for binding to
the single-stranded sequence was observed with unrelated competitor
oligonucleotides confirming that the binding was sequence specific (Fig. 4B). This sequence specificity was observed for
both complexes formed by Brn-3a that may represent the binding of
monomeric and dimeric Brn-3a.
Figure 4:
A, DNA mobility shift assay using in
vitro transcribed and translated Brn-3a protein and P
labeled probes derived from the double stranded octamer-related
sequence (track 1), the single-stranded sense sequence (track 2), or the complementary antisense sequence (track
3). B, DNA mobility shift assay using in vitro transcribed and translated Brn-3a protein and
P
labeled probe derived from the single-stranded antisense octamer either
alone (track 1), or in the presence of one hundredfold excess
of unlabeled homologous oligonucleotide (track 2), unlabeled sense
strand (track 3), unlabeled double strand (track 4),
unlabeled antisense sequence from the
-internexin promoter (track 5), or an unrelated oligonucleotide (track
6).
In these experiments the binding
affinity of the octamer-related sequence for Brn-3a appeared to be
higher than that of the -internexin sequence when the amount of
either antisense single-stranded or double-stranded probe bound in each
case was compared. In agreement with this, unlabeled
-internexin
antisense sequence competed relatively poorly for Brn-3a bound to
labeled antisense octamer-related sequence compared to the degree of
competition observed with unlabeled homologous competitor (Fig. 4B, track 4).
In similar experiments, Brn-3b also exhibited preferential binding to the antisense strand of each sequence compared with its binding to the corresponding sense strand or double-stranded sequence, indicating that this preference is common to Brn-3a and Brn-3b (Fig. 5). Thus these observations indicate that Brn-3a and Brn-3b can bind to at least two specific sequences with higher affinity for one of the two single-stranded sequences than for the corresponding double-stranded sequence.
Figure 5:
DNA
mobility shift assay with in vitro transcribed and translated
Brn-3b protein and P labeled probes derived from the
antisense sequence of the
-internexin promoter (A), the
complementary sense strand sequence (S), or corresponding
double-stranded oligonucleotide.
In our previous
experiments (Budhram-Mahadeo et al., 1995b; Morris et
al., 1994) both the synthetic octamer-related sequence upstream of
the thymidine kinase promoter and the -internexin promoter were
activated by Brn-3a and not by Brn-3b. However these two promoters
responded differently to constructs encoding chimeric proteins
containing different regions derived from Brn-3a and Brn-3b (Fig. 6), indicating that the
-internexin promoter was
activated by an activation domain located at the N terminus of Brn-3a,
whereas activation of the synthetic octamer-related sequence was
dependent on the POU domain of Brn-3a. Such a difference might be due
to the difference in sequence between the two Brn-3a binding sites (Fig. 2) or to the different context of each sequence relative
to other sequences in the two different promoters.
Figure 6:
Summary of gene trans-activation data
obtained using either the tK promoter carrying a synthetic octamer
motif (tK-Oct) (data from Morris et al., 1994) or
carrying the response element from the -internexin promoter (tk-int) (Fig. 7) or with the intact
-internexin
promoter (Budhram-Mahadeo et al., 1995b) together with Brn-3a
or -b or with constructs encoding chimeric proteins with different
regions derived from Brn-3a or Brn-3b. The division of Brn-3a and -3b
subdomains I, II, III, and IV is as follows. For Brn-3a: domain I,
amino acids 1-40; domain II, amino acids 41-108; domain
III, amino acids 109-267; domain IV (POU domain), amino acids 268
to the end. For Brn-3b; subdomain II, amino acids 1-92; subdomain
III, amino acids 93-169; subdomain IV (POU domain), amino acids
170-end.
Figure 7:
Chloramphenicol acetyltransferase assay
following transfection of a construct in which the sequence from
-77 to -60 in the -internexin promoter has been cloned
upstream of the tK promoter in the plasmid pBLCAT2. 5 µg of this
construct was co-transfected with 10 µg of either expression vector
lacking any insert (V) or the same vector containing inserts
encoding Brn-3a (A), Brn-3b (B) or the chimeric
constructs (1-4) numbered as in Fig. 6. Values
are expressed relative to that obtained upon co-transfection with
expression vector alone and are the average of three independent
experiments whose standard deviation is shown by the bars.
To distinguish
between these possibilities, the double-stranded oligonucleotide
containing the -internexin sequence from -77 to -60
was cloned into the BamHI site in the vector pBLCAT2 (Luckow
and Schutz, 1987), thereby placing it at -115 relative to the
transcriptional start site of the thymidine kinase promoter the same
position as the octamer-related sequence in the pBCLCat2-derived
construct used in our previous experiments (Morris et al.,
1994).
In these experiments (Fig. 7) the thymidine kinase
promoter containing the -internexin sequence was activated by
Brn-3a, confirming that the binding of Brn-3a to the
-internexin
sequence from -77 to -60 was able to produce
transcriptional activation of a heterologous promoter. Moreover this
promoter construct was not activated by Brn-3b, allowing us to use the
chimeric constructs to determine the region of Brn-3a required for
activation. Interestingly, the ability of these constructs to activate
the thymidine kinase promoter containing the
-internexin sequence
was dependent on the POU domain being derived from Brn-3a and did not
depend on the N-terminal region. Thus, for example, the promoter was
activated by construct 4, which has the POU domain derived from Brn-3a
but lacks the N-terminal domain, whereas construct 3, which has the
N-terminal domain of Brn-3a but the POU domain of Brn-3b, did not
activate the promoter (Fig. 7).
Hence the response of the
-internexin sequence to the chimeric constructs is identical to
that of the octamer-related sequence when the two are cloned into the
same context in the thymidine kinase promoter, although this sequence
responds differently when in its natural context in the
-internexin promoter. We showed previously (Budhram-Mahadeo et
al., 1995b) that the octamer-related sequence could also be
activated by a construct expressing the Brn-3a POU domain in isolation,
indicating that the POU domain of Brn-3a was not only necessary but
also sufficient for activation of this construct as well as for DNA
binding. In contrast, the natural
-internexin promoter was, as
expected, not activated by this construct (Budhram-Mahadeo et
al., 1995b). As shown in Fig. 8, however, when placed
upstream of the thymidine kinase promoter, the
-internexin
sequence was able to confer a response to the POU domain of Brn-3a
exactly as occurred with the octamer-related sequence in the same
promoter context. Moreover, as with the octamer-related sequence, the
-internexin sequence was not activated by Brn-3b.
Figure 8:
Chloromphenicol acetyltransferase assay
following transfection of 5 µg of the construct containing the
-77 to -60 -internexin sequence in pBLCAT2 together
with 10 µg of either expression vector lacking any insert (V) or the same vector containing inserts encoding the
isolated POU domains of either Brn-3a (A) or Brn-3b (B).
In the work presented here, we have extended out previous
studies of the -internexin promoter (Budhram-Mahadeo et
al., 1995b) to show that its responsiveness to the Brn-3a
transcription factor is dependent on a region from -77 to
-47 relative to the transcriptional start site. Moreover, a
portion of this region from -77 to -60 can bind Brn-3a or
Brn-3b and confer responsiveness to activation by Brn-3a upon a
heterologous promoter. The best match to the octamer motif (ATGCAAAT),
which binds many POU factors in this region, is the sequence ATGAAGCT,
which shows only a five out of eight match. This therefore confirms and
extends previous findings that Brn-3a and Brn-3b can regulate promoter
activity by binding to sequences either closely (Lillycrop et
al., 1995; Morris et al., 1994) or more distantly
(Gerrero et al., 1993; Li et al., 1994; Turner et
al., 1994) related to the octamer motif.
More interestingly,
however, we have shown for the first time that both Brn-3a and Brn-3b
bind strongly and in a sequence-specific manner to one single strand of
both the -internexin promoter sequence and a sequence more closely
related to the octamer motif. Moreover, this binding is considerably
stronger than that observed to either the corresponding double-stranded
sequence or the complementary single strand. Such behavior has never,
to our knowledge, previously been described for a POU family
transcription factor. Similar preferential binding to one single strand
compared with the corresponding double-stranded sequence or the
complementary single strand has been reported for proteins binding to
specific regulatory sites in a number of other cases. These include
proteins binding to the sterol regulatory element in the
hydroxymethylglutaryl-CoA reductase and synthase genes (Stark et
al., 1991), an inhibitory element in the growth hormone gene
promoter (Pan et al., 1990), a regulatory region of the
adipsin gene promoter (Wilkison et al., 1990) and an
inhibitory region in the androgen receptor gene promoter (Grossman and
Tindall, 1995). Similar preferential binding to one single strand has
also been reported for the transcription factor Myef-2 (Haas et
al., 1995) and for the estrogen receptor either alone or in
association with another factor (Mukherjee and Chambon, 1990).
Several of these factors thus appear to bind to sequences that have been shown to have an inhibitory effect on promoter activity (Grossman and Tindall, 1995; Pan et al., 1990; Wilkison et al., 1990) and binding of the Myef-2 factor to its recognition element in the myelin basic protein gene promoter has also been shown to inhibit promoter activity (Haas et al., 1995). Indeed it has been suggested that binding of such factors could promote or maintain the DNA in a single-stranded form incapable of binding other factors necessary for transcription (Grossmann and Tudall, 1995). Interestingly, Brn-3b can also inhibit promoter activity and interfere with activation by Brn-3a (Budhram-Mahadeo et al., 1994, 1995b; Morris et al., 1994). It is possible, therefore, that binding of Brn-3a and Brn-3b may both produce structural alterations in the DNA, although in the case of Brn-3a, such changes activate transcription, whereas Brn-3b has the opposite effect.
In this
regard it is obviously of interest that in some cases the isolated DNA
binding domain of Brn-3a can act as an activation domain and stimulate
transcription (Morris et al., 1994). In contrast, at least in
the case of the -internexin promoter, other mechanisms must
operate which require the activation domain at the N terminus
(Budhram-Mahadeo et al., 1995b). In other situations, where
the activation domain of a specific factor can activate some promoters
but not others, this effect has been shown to depend on either the
nature of the binding site in each promoter or its context relative to
other binding sites. Thus, for example, the glucocorticoid receptor can
activate gene expression by binding to a glucocorticoid response
element, but represses rather than activates following binding to a
distinct sequence known as the nGRE (Sakai et al., 1988). In
contrast the YY1 factor activates the human papilloma virus 18 promoter
when an adjacent switching sequence is also present but represses the
promoter by binding to the same target sequence when the switching
element is absent (Bauknecht et al., 1995). Similarly, the
activity of the Drosophila dorsal protein is affected by the
presence or absence of adjacent binding sites for the dorsal switch
protein (Lehming et al., 1994), while the Ubx protein binds to
some DNA binding sites only when a binding site for the extradenticle
protein is present (Van Dijk and Murre, 1994).
In the case of Brn-3a, we show here that the different effects of the two activation domains which we described previously (Budhram-Mahadeo et al., 1994, 1995b) are dependent on differences in the context of the binding site rather than its sequence the first time this effect has been deonstrated for two activation domains in the same molecule. Such an effect might depend on the ability of the Brn-3a DNA binding POU domain to open its binding site to a single-stranded form in the presence of different flanking sequences. Alternatively it could depend upon its ability to produce alterations in the structure of the different sequences adjacent to its binding sites and/or on whether an activating effect was produced by disrupting such sequences. In situations where DNA binding by the POU domain did not produce activation, the N-terminal activation domain would be required and would presumably act in a conventional manner by interacting with other promoter-bound factors.
Alternatively both the POU and N-terminal activation
domains may act by interacting with other promoter-bound factors with
the two activation domains differing in the factors with which they
could interact. Thus promoter-specific activation by the two activation
domains would depend upon differences between promoters in the nature
of the transcription factors binding to the specific sequences in each
promoter or in the composition of the basal transcriptional complex
that binds at the TATA box in each promoter. In agreement with this
latter possibility the TATA-binding protein-associated factors (TAFs) ()have been shown recently to play a role in promoter
selectivity with different complexes assembling at different promoters
(Verrijzer et al., 1995). Moreover the two activation domains
located within either the estrogen receptor or the glucocorticoid
receptor have each been suggested to interact with different TAFs
(Tasset et al., 1990) reflecting the general finding that
different classes of activation domain can interact with different TAFs
(Chen et al., 1994).
Although further studies will
evidently be required to clarify the precise mechanism, it is clear
that the context of the binding site regulates activation by the two
activation domains of Brn-3a. Interestingly the Brn-3a primary
transcript can be alternatively spliced to yield mRNAs encoding
proteins with and without the N-terminal activation domain, but both
containing the POU domain (Gerrero et al., 1993), and the
outcome of this process can be regulated by exposure of neuronal cells
to specific stimuli. ()Thus the existence of two activation
domains in Brn-3a may allow complex patterns of gene regulation in
response to specific stimuli with some promoters being activated by
both forms of Brn-3a and others only when the full size form is made,
with such differential activation being dependent on the context of the
Brn-3a binding site in the target promoter.