From the Eppley Institute for Research in Cancer and
Allied Diseases and the § Department of Pathology and
Microbiology, University of Nebraska Medical Center, Omaha,
Nebraska 68198-6805
Received for publication, December 2, 2002, and in revised form, March 5, 2003
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ABSTRACT |
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Sox transcription factors play key
regulatory roles throughout development, binding DNA through a
consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox
proteins bind to this sequence, it has been observed that gene
regulatory elements are commonly responsive to only a small subset of
the entire family, implying that regulatory mechanisms exist to permit
selective DNA binding and/or transactivation by Sox family members. To
identify and explore the mechanisms modulating gene activation by Sox
proteins further, we compared the function of Sox-2 and Sox-11. This
led to the discovery that Sox proteins are regulated differentially at
multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the
transactivation domain of Sox-11 is primarily responsible for this
capability. Additionally, we demonstrate that the Sox-11 DNA binding
domain is responsible for selective cooperation with the POU factor
Brn-2. This requirement cannot be replaced by the DNA binding domain of
Sox-2, indicating that the DNA binding domain of Sox proteins is
critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to
activate gene expression in vivo. Our findings suggest that
the autoinhibitory domain can repress promiscuous binding of Sox-11 to
DNA and plays an important role in regulating the recruitment of Sox-11
to specific genes.
The Sox family of transcription factors is comprised of a
diverse group of proteins whose pattern of expression is regulated both
spatially and temporally (1). Sox factors are related by the homology
(usually >50%) found within their high mobility group
(HMG)1 DNA binding domains.
Sox HMG domains have been found to bind to the consensus sequence
5'-(A/T)(A/T)CAA(A/T)G-3' in the minor groove of DNA (2). As the number
of Sox proteins has grown to more than 20 members in multiple species,
including both vertebrates and invertebrates, this family has been
divided into seven subgroups, the members of which contain high
homology within both the HMG domain and flanking regions (1). In some
cases, subgroup members have similar patterns of expression during
development and perhaps redundant functions. For example, the
structurally similar group B members Sox-1, -2, and -3 show overlapping
expression in the fetal nervous system, and it has been suggested that
they perform similar functions (3, 4). Additionally, the group C
members Sox-4 and Sox-11 show high homology within their HMG domain and C-terminal tail, and they may also have complementary roles in the
developing nervous system (5).
The observation that the HMG DNA binding domains of many members of the
Sox family bind a consensus sequence has prompted the question of
whether there are selective mechanisms in place to recruit a specific
Sox protein to a gene regulated by an HMG binding site. A review of the
literature reveals that genes rely on a diverse array of mechanisms to
regulate DNA binding among members of other transcription factor
families, which may also apply to Sox proteins. A particularly
intriguing example of DNA binding regulation has been found within
multiple members of the Ets family. First characterized in Ets-1, the
affinity of these proteins for DNA is tightly regulated by
autoinhibitory regions flanking the DNA binding domain (6, 7).
Interestingly, the ubiquitous HMG-1 and HMG-2 proteins employ a similar
regulatory strategy whereby their highly acidic C-terminal tails are
capable of negatively affecting the ability of those proteins to bind DNA (8). Importantly, although the autoinhibitory domains of both Ets
and HMG proteins repress DNA binding, they can be neutralized by
post-translational modification or by interaction with partner proteins, which allows their recruitment to be exquisitely regulated in
a gene-specific manner (7, 9-12).
Although specific evidence of DNA binding autoregulation has not yet
been observed in Sox proteins, it is clear that cis-regulatory elements
are capable of selecting specific Sox transcription factors. Attempts
to uncover the mechanisms through which Sox specificity is achieved
have revealed that recruitment often requires proper interaction with
other transcription factors at adjacent binding sites on the DNA. This
is illustrated by one of the cis-regulatory elements that control
expression of the fibroblast growth factor 4 (FGF-4) gene in
embryonal carcinoma cells. FGF-4 expression depends on a
distal enhancer (13, 14), which contains an essential binding site for
Sox-2 (15-17). However, Sox-2 does not appear to be capable of
substantial activation of this gene on its own, but rather requires
cooperation with the POU transcription factor Oct-3 (18, 19). The
presence of Oct-3 at a site adjacent to Sox-2 promotes cooperative DNA
binding, resulting in a synergistic increase in transactivation (18,
19). The partnership of Sox-2 and Oct-3 is extremely specific for these
two proteins because it requires stereospecific alignment and
protein-protein interaction between the HMG and POU DNA binding domains
when Sox-2 and Oct-3 are present at their adjacent binding sites (18).
Additionally, the role of Oct-3 cannot be fulfilled by Oct-1 for the
FGF-4 enhancer, indicating that only certain Sox-POU
combinations can lead to productive regulation of a particular gene
(15). Specific partnerships have also been observed among other members
of the Sox and POU families. Utilizing the binding sequence found in
the FGF-4 enhancer, it was determined that Sox-10
specifically partners with Oct-6, and Sox-11 cooperates optimally with
the POU factors Brn-1 and Brn-2 (20, 21). These observations have led
to the hypothesis that a Sox-POU combinatorial code may exist as a
mechanism of transcriptional regulation (20).
Although significant progress has been made in understanding the
differential function of Sox proteins, both on their own and in
combination with other factors, little is known about the mechanisms
involved. To elucidate the mechanisms through which Sox proteins may be
regulated, we initiated a functional comparison of Sox-2 and Sox-11
using promoter/reporter gene constructs that contain HMG and POU
binding sites. Through this study, we determined that Sox-2 performs
very differently than Sox-11, being a much weaker transactivator, while
at the same time binding more strongly to DNA in vitro. To
understand the mechanisms involved in the functional differences
between Sox-2 and Sox-11, we addressed three questions. First, what
role does each of the domains of Sox-2 and Sox-11 play in differential
gene activation? Second, how specific are Sox-2 and Sox-11 partnerships
with POU proteins, and which regions of Sox-2 and Sox-11 are important
for Sox-POU partnerships? Third, which domain(s) are critical for the
decreased DNA binding of Sox-11 compared with Sox-2?
We have addressed these questions using Sox deletion mutants and
Sox-2/11 chimeric proteins created by interchanging the N terminus, HMG
DNA binding domain, or C terminus of both proteins. These studies led
to the discovery that the C-terminal transactivation domains play the
primary role in the differential capability of Sox-2 and Sox-11 to
transactivate. Additionally, we determined that Sox-11, but not Sox-2,
can cooperate with Brn-2 and that the Sox-11/Brn-2-specific cooperation
requires both adjacent binding of the two factors to DNA and the HMG
domain of Sox-11. Therefore, this study further supports the hypothesis
that a Sox-POU combinatorial code exists (20) and suggests that this
interaction code is dependent on the HMG domain of the Sox protein
involved. Finally, we also identified a novel domain within a conserved
region of Sox-11 which is capable of negatively regulating both its
ability to bind DNA in vitro and activate gene expression
in vivo. Together, this work significantly enhances our
understanding of the mechanisms that influence selective recruitment of
Sox proteins through partner specificity and DNA binding autoregulation.
Plasmids--
Constructs pCATSO3, pCMVSox-2F1-180,
pCMVSox-2, pCMVOct-3, pCMVOct-1, and CMV-
The plasmids pCMVSox-11 and pCMVBrn-2 were gifts from Michael Wegner
(20). Sox-11F was constructed using the primers FLAGSox-11 (5'-CGTGCTGGTACCGCCACCATGGACTACAAGGACGACGATGATATGGTGCAGCAGGCCGAGAGC-3') and Sox-11/395 (5'-GCTGGATCCGACCGCCACGACTGCCTCCCG-3') to amplify the coding region from CMV5-Sox-11. This PCR incorporated the FLAG
epitope sequence, preceded by a Kozak sequence and a KpnI site, onto the 5'-end of the coding region of Sox-11 and a
BamHI site at the 3'-end. The
KpnI/BamHI sites were then digested and the
Sox-11F sequence ligated into the KpnI/BamHI
sites of the CMV5 expression vector. The Sox-2F expression plasmid
referred to in this report was constructed by removing the Sox-2F
sequence from the pCEP4 vector (19) using the
KpnI/BamHI sites and religating the Sox-2F
sequence into the same sites present in CMV5.
Site-directed mutagenesis or deletions were carried out using a
modification of the QuikChange (Stratagene, La Jolla, CA) method. All
amino acid numbers are based on the wild-type Met being defined as 1, not the Met included with the FLAG tag. Briefly, two primers,
complementary to the sequence flanking the targeted region and to each
other, were used in PCR amplification of the entire plasmid, thus
modifying the indicated template. Unless otherwise noted, each of the
primers identified herein represents the sense strand of a perfectly
complementary pair of primers used during PCR. To begin the
construction of the Sox-2/11 chimeras, unique restriction sites were
incorporated at the N-terminal and C-terminal flanks of the HMG box of
both the Sox-2F and Sox-11F plasmid. Sites were chosen in such a way
that little alteration would be made to the wild-type coding sequence.
Mutagenesis to include a NheI site was performed with either
the sox2mut1 (5'-GGCAACCAGAAGAATGCTAGCCCGGAACGTGTCAAGAGGCCC-3') or
sox11mut1 (5'-CCGGACTGGTGCAAGACGGCTAGCGGCCACATCAAACG-3') primer pair. The NheI alteration added a Ser following Ala-39 of
Sox-2F but did not affect the coding sequence of Sox-11F. The products of these PCR reactions were then used in a second round of mutagenesis to include a NotI site using either the sox2mut2
(5'-GTACACGCTTCCCGCGGCCGCTTTGCTCGCCCCCGG-3') or sox11mut2
(5'-GCCAAGCCCAGCGCGGCCGCACAGAGCCCGGACAAGAGC-3') primer pair. This
replaced Gly-131 and Gly-132 of Sox-2F as well as Ala-134 and Gly-135
of Sox-11F with the amino acids Ala-Ala-Ala. This cloning resulted in
Sox-2F double mutant and Sox-11F double mutant, each of which contained
a KpnI and BamHI site at their extreme 5'- and
3'-ends, respectively, as well as an NheI and
NotI site at the 5'- and 3'- respective ends of their HMG
boxes. These sites made it possible to use standard restriction digests
and ligations to interchange either the N-terminal, HMG, or C-terminal
region of Sox-2F with the corresponding region of Sox-11F, resulting in
the creation of the chimeric plasmids Sox-11-2-2F, -11-11-2F, -2-11-2F,
-2-11-11F, -2-2-11F, and -11-2-11F.
To create Sox-11F Cell Culture and Transient Transfection--
HeLa cells were
maintained and transfected using the calcium phosphate precipitation
method as described previously (19). All transfections were performed
in triplicate with representative experiments shown. Plasmid DNA was
isolated and purified using Qiagen tip-500 columns.
Extract Preparation and Western Blotting--
HeLa cells were
transfected with 5 µg of the relevant plasmids as described above,
and extracts were prepared 2 days post-transfection. Whole cell protein
extracts were prepared as described previously (19). Nuclear extracts
were prepared using the NE-PER kit (Pierce) following the
manufacturer's protocol. The isolated nuclei from ~6-7 × 106 cells were lysed in 100 µl of nuclear extraction
reagent supplemented with various protease and phosphatase inhibitors
(22). When preparing Sox-11F, Sox-11F
Western blotting was performed using 10 µl of in vitro
translated lysate or nuclear lysate from 5 × 105
transfected HeLa cells as described (19) using the anti-FLAG M2
antibody (Sigma). Proteins were detected using the enhanced chemifluorescence (ECF) kit (Amersham Biosciences) and scanned on a
Storm PhosphorImager (Molecular Dynamics). Quantitation was performed
using the ImageQuant 5.0 analysis software (Molecular Dynamics).
Western blots were performed in duplicate or triplicate with
representative blots shown.
EMSA--
Gel mobility shift analysis was based on the method of
Fried and Crothers as modified by this laboratory (23). Complementary oligodeoxynucleotide probes were annealed for each probe or competitor, and the resulting double-stranded oligodeoxynucleotide probes were
labeled with [ Functional Comparison of Sox-2 and Sox-11--
The initial goal of
this study was to elucidate the mechanisms permitting selective
recruitment and activation by specific Sox proteins at a particular
gene. To address this issue, we compared the function of Sox-2 with the
related transcription factor Sox-11. These proteins were selected for
several reasons. First, the gross domain structure of each has been
studied, revealing the location of the DNA binding domain and
transactivation domain of each protein (4, 19, 20). Second, to
understand the function of a transcription factor in vivo,
it is best studied in a model system using a known binding sequence
from a regulated gene. The binding site for Sox-2 within the enhancer
of the FGF-4 gene has been determined and provides an
excellent model system (15, 16). Thus far, target genes of Sox-11 have
not been identified; however, the FGF-4 enhancer sequence
has also been utilized in a partial characterization of Sox-11 (20),
indicating that it may provide a relevant system in which to study this
transcription factor. Finally, the expression pattern of Sox-2 and
Sox-11 within the developing nervous system overlaps, leading to the
postulate that there are cell types in which some genes may be
regulated differentially depending on whether Sox-2 or Sox-11 binds to
a cis-regulatory element (24).
To compare Sox-2 and Sox-11 functionally, we assayed their ability to
activate the pCATSO3 promoter/reporter gene construct in HeLa cells,
which do not contain any known Sox-like activity (15, 19). The pCATSO3
plasmid contains six tandem repeats of a 24-bp region of the
FGF-4 enhancer upstream from an SV40 promoter driving the
expression of a chloramphenicol acetyltransferase (CAT) reporter gene.
A promoter/reporter construct containing multiple HMG binding sites was
necessary for these studies because gene activation by either Sox-2 or
Sox-11 via a single HMG site did not rise above the basal expression of
the promoter/reporter gene construct (data not shown). The 24-bp region
contains both the HMG binding site and POU binding site found in the
FGF-4 enhancer. The pCATSO3 construct was employed to
perform a functional comparison of Sox-2 and Sox-11 because it has been
used previously to identify the transactivation domain of Sox-2 and to
identify p300 as a potential coactivator of Sox-2 activity (19).
Furthermore, a very similar construct, also containing the HMG/POU
enhancer sequence of the FGF-4 gene, was utilized in the
initial characterization of Sox-11 (20).
For the functional comparison of Sox-2 and Sox-11, HeLa cells were
transiently transfected with pCATSO3 and increasing concentrations of
expression plasmid encoding either Sox-2F or Sox-11F, each tagged at
their N terminus with a FLAG epitope. This revealed that Sox-11F is a
much more potent activator of the pCATSO3 promoter/reporter gene
construct than Sox-2F (Fig.
1a). At all concentrations
tested, transfection of Sox-11F led to 40-75-fold more reporter gene
activity than Sox-2F. This experiment was also repeated with Sox-2 and Sox-11 expression constructs without FLAG epitopes, and no differences were observed between the ability of the tagged and untagged proteins to transactivate (data not shown). Additionally, to verify that the
functional differences observed between Sox-2F and Sox-11F were not
caused by variations in protein expression, Western blot analysis was
performed on nuclear extracts of transfected HeLa cells using the
anti-FLAG M2 antibody. This analysis revealed that there was little
difference between Sox-2F and Sox-11F expression in the nucleus (Fig.
1b). In fact, in multiple experiments, we found that Sox-11F
expression was consistently lower than Sox-2F. Thus, the difference in
transactivation capability between Sox-2F and Sox-11F (Fig.
1a) may be an underestimate.
Our observation that Sox-11F was such a potent activator on its own led
us to question whether an endogenous POU factor could be binding at the
adjacent site and assisting in Sox-11F-mediated activation. This is an
important question because Sox-2 requires the partner Oct-3 for
activation. Thus, if Sox-11 can activate significantly without a
partner, it would imply that a key functional difference exists between
these two proteins. To test this, we constructed the promoter/reporter
construct pCATS4, which is similar to pCATSO3 except that the POU site
has been scrambled, and there are only four HMG sites in the promoter.
When activation of pCATS4 by Sox-2F and Sox-11F is compared with
pCATSO3 it is clear that Sox-11F can activate strongly in a
partner-independent manner, whereas Sox-2F requires the adjacent POU
site and binding of Oct-3 for significant activation (Fig
1c).
To understand further the function of Sox-2 and Sox-11, we examined how
each of the individual domains of these two proteins contributes to
their different abilities to transactivate. For this purpose, we
divided Sox-2 and Sox-11 into three regions: the N-terminal region, the
HMG DNA binding domain, and the region C-terminal to the DNA binding
domain. The N-terminal regions of both proteins contain ~40 amino
acids, with no known function in either protein. The HMG DNA binding
domains of Sox-2 and Sox-11 are 65% identical and consist of 79 amino
acids. The C-terminal regions of both Sox-2 and Sox-11, which are known
to contain modular transactivation domains (4, 19, 20), consist of 189 and 265 amino acids, respectively. To determine the role of each of these regions, we created chimeric proteins by interchanging the N-terminal portion, the HMG box (including ~10 amino acids of the
C-terminal flanking sequence), or the C-terminal portion of Sox-2 and
Sox-11 in all possible combinations. We hypothesized that if one or
more of these regions of Sox-11F were responsible for its strong
transactivation, placing them into Sox-2F should convert Sox-2F into a
strong transactivator. These chimeras were created by first using PCR
mutagenesis to insert unique restriction enzyme sites on either side of
the plasmid sequence encoding the HMG domain of both proteins. These
novel sites were designed to allow for minimal perturbation of the
wild-type amino acid sequence, and we determined that they had no
effect, either functionally or in protein expression, on Sox-2F or
Sox-11F (data not shown). The presence of these unique sites within
Sox-2F and Sox-11F allowed us to interchange each domain by restriction
digestion and in-frame religation, resulting in six chimeras (Fig.
2). The expression level and expected
molecular mass of the chimeric proteins were determined by
Western blot analysis (Fig. 2). Some differences in the level of
chimeric protein expression were apparent; for example, the average
expression of the constructs containing the Sox-2 C terminus was
~3-fold higher than those containing the Sox-11 C terminus. However,
normalizing for these differences during our functional analysis does
not affect our conclusions (see below).
The ability of each of the chimeras to activate pCATSO3 was then
compared with that of Sox-2F and Sox-11F to determine the role of each
region in gene activation (Fig. 2). Clearly, there are striking
differences in transactivation capabilities between the chimeras
containing the C-terminal region of Sox-2F (Sox-11-2-2F, -11-11-2F, and
-2-11-2F) and those containing the C-terminal region of Sox-11F
(Sox-2-11-11F, -2-2-11F, and -11-2-11F). For example, when the C
terminus of Sox-2F is replaced with that of Sox-11F (Sox-2-2-11F) the
chimeric protein is capable of activating gene expression 20-fold
higher than Sox-2F. Moreover, in the reciprocal chimera, replacing the
C-terminal region of Sox-11F with that of Sox-2F (Sox-11-11-2F)
resulted in a 30-fold decrease in transactivation capability. Hence, we
conclude that the domain primarily responsible for the functional
difference between Sox-2F and Sox-11F lies in their C-terminal regions,
which are known to contain their respective transactivation domains
(19, 20). As mentioned above, because those chimeras containing the
Sox-11 C terminus are less abundant (as determined by Western blot
analysis) but exhibit stronger reporter gene activation, normalization
to relative protein levels would not alter this conclusion.
Next, we addressed the role of the HMG domain or N terminus in gene
activation. When either of these domains is interchanged within those
chimeras containing the Sox-2F C terminus, the reporter gene expression
is so low that no significant differences can be measured. However, it
is apparent that both of these regions play a functional role when the
chimeras containing the Sox-11F C terminus are considered (Fig. 2).
Comparison of either Sox-11F with Sox-11-2-11F or Sox-2-11-11F with
Sox-2-2-11F reveals a decrease in activation of ~2.5-fold in each
case, indicating that the HMG domain of Sox-11F contributes to stronger
potentiation compared with the HMG domain of Sox-2F. A similar
consideration of the chimeras containing the N-terminal rearrangements
demonstrates that the N terminus of Sox-2F enhances transactivation
~2-fold (compare Sox-11F and Sox-2-11-11F, or Sox-2-2-11F and
Sox-11-2-11F). These findings demonstrate that the N terminus and HMG
domains of these Sox proteins make a contribution to their ability to activate transcription.
Domain Requirements in Sox-POU Partnerships--
Although
understanding the properties of a transcription factor on its own is
important, it is also critical to understand its ability to work in
conjunction with other transcriptional regulators. Indeed, it is widely
recognized that many Sox proteins, including Sox-2 and Sox-11, can
activate transcription synergistically when bound to DNA adjacent to
specific members of the POU family of transcription factors (15, 19,
21, 25). The observation that the HMG and POU domains of Sox-2 and
Oct-3 are capable of direct interaction (18) suggests that functional
Sox-POU partnerships may depend on a selective interaction between
their DNA binding domains. To test this possibility, we compared
the ability of Sox-2, Sox-11, and the Sox-2/11 chimeras to cooperate
with various POU proteins in transcriptional activation and determined
which domains are required for cooperation.
To study the selective nature of Sox-POU cooperation, HeLa cells were
transfected with either Sox-2 or Sox-11 and the POU proteins Oct-3 or
Brn-2 and then assayed for the ability of each combination to activate
the pCATSO3 promoter/reporter gene construct. We first tested the
effect of escalating doses of Oct-3 on the ability of Sox-11 to
transactivate. Although Sox-11 is known to cooperate with Brn-2 (20),
it was unknown how it would respond in the presence of Oct-3. This
study demonstrated that Sox-11 is capable of synergistically activating
gene expression with Oct-3 (Fig.
3a), indicating that it
cooperates with this POU factor similarly to Sox-2 (18, 19). To
ascertain whether Oct-3 could be cooperating with Sox-11 by
up-regulating Sox-11 protein expression, we performed Western blot
analysis on duplicate samples of cells transfected with Sox-11F in the
presence or absence of Oct-3 and quantitated the expression of Sox-11F
(Fig. 3c). This demonstrated that Oct-3 had little effect on
Sox-11 expression. Finally, we examined whether Brn-2 could partner
with both Sox-11 and Sox-2. Interestingly, we determined that Sox-11,
but not Sox-2, cooperates with Brn-2 to activate transcription (Fig.
3b). This selective partnership further supports the
hypothesis that restrictive mechanisms are in place which allow only
specific Sox-POU combinations to cooperate in transcriptional
activation.
To begin to elucidate the mechanism underlying the Sox-11/Brn-2
selective partnership, we first examined whether Brn-2 needed a POU
binding site adjacent to the HMG site to cooperate with Sox-11 in gene
activation. This was addressed by assaying the ability of Sox-11 and
Brn-2 to activate pCATS4, in which the POU site is scrambled. In this
experiment, we found that removal of the POU site completely abrogates
Sox-11/Brn-2 cooperation (Fig. 4a). This indicates that the
enhancement of Sox-11 activation by Brn-2 requires that they be located
at adjacent sites on the DNA and is not the result of a general
up-regulation of Sox-11 protein expression. Next, we examined which
domains are needed for these partnerships. Previous studies using
Oct-3/1 chimeric proteins have determined that the Oct-3 POU domain is
necessary for its cooperation with Sox-2 on the FGF-4
enhancer sequence. When the POU domain of Oct-3 is exchanged with that
of Oct-1, the resulting Oct-3-1-3 protein can no longer synergize with
Sox-2 (26). The creation of the Sox-2/11 chimeric proteins provided us
with a similar system in which to isolate the domain of Sox-11 which
permits selective cooperation with Brn-2. To test this possibility, we
compared the ability of Sox-2-11-2F and Sox-11-2-11F to transactivate in the presence of Brn-2 (Fig. 4b). This study reveals that
by interchanging the HMG domain of Sox-2 with that of Sox-11, the chimeric protein Sox-2-11-2F is able to cooperate functionally with
Brn-2 in a dose-dependent manner. This is observed in the Brn-2 dependent increase in Sox-11F and Sox-2-11-2F activity above that
seen with each protein alone. The importance of the Sox-11 HMG domain
is supported further by the fact that when Sox-11-2-11F is transfected
in combination with Brn-2 no cooperative activation is detected above
the activity of Sox-11-2-11F alone. Thus, we demonstrated that the
cooperation of Sox-11 with Brn-2 appears to be dependent on the HMG
domain of Sox-11 as well as adjacent binding of the two proteins.
In Vitro DNA Binding of Sox-2 and Sox-11--
Binding of Sox
transcription factors to DNA has been demonstrated to be an essential
and often highly regulated step in their role as transcriptional
activators (18, 25, 27-29). With multiple examples of binding
regulation within the Sox family and an indication that the HMG DNA
binding domains of Sox-2 and Sox-11 have variable effects on selective
POU partnerships as shown by our chimeric studies, we examined whether
there are differences in the in vitro DNA binding
capabilities of Sox-2 and Sox-11. To accomplish this goal, HeLa cells
were transfected with either Sox-2F or Sox-11F, and nuclear extracts
were prepared for use in EMSA to measure the ability of each Sox
protein to bind a radiolabeled probe (hmg1) containing a single HMG
binding sequence. In each EMSA, the concentration of Sox protein
included was first normalized after quantitation by Western blot
analysis. When in vitro DNA binding of Sox-2F is compared
with Sox-11F, a single complex was observed with Sox-2F, whereas little
or no binding was detected with Sox-11F (Fig
5a). The Sox-2F complex was
partially supershifted specifically with the M2 antibody and competed
by excess, unlabeled wild-type probe, but not a probe in which the HMG
site has been scrambled (Fig. 5a). In contrast, we detected
little or no binding of full-length Sox-11F despite our attempts to use
multiple binding buffers, different nonspecific competitors, or lower
ionic strength electrophoresis conditions (data not shown). To examine
roles of the HMG domains of Sox-2F and Sox-11F in mediating their
differences in DNA binding, an EMSA of the Sox chimeras in which the
HMG domains were interchanged was also performed (Fig. 5b).
Surprisingly, Sox-2-11-2F was capable of binding the DNA probe with an
intensity similar to that of Sox-2F, whereas DNA binding by
Sox-11-2-11F was not detected. Thus, the region(s) responsible for
differential DNA binding between Sox-2F and Sox-11F lies outside of the
HMG domain.
Investigation of the DNA Binding Autoregulation of Sox-11--
The
observation that region(s) of Sox-11 beyond its HMG domains may repress
DNA binding led us to hypothesize that a domain capable of
autoinhibiting Sox-11 exists in this protein. To determine whether an
autoinhibitory region exists within Sox-11, deletion constructs were
made and tested for their ability to bind DNA. Our deletion strategy
focused on two acidic regions within the C terminus, which were
particularly intriguing because of the acidic nature of autoinhibitory
regions isolated in other proteins (8, 30-33). The distal region
(amino acids 283-395) contains an acidic/hydrophobic region known to
contain the transactivation domain
(TAD) (20), while the central region (amino acids 189-224) contains a
highly acid-rich (AR) set of amino acids (20/27
consecutive residues being Asp or Glu). The AR region of Sox-11 cannot
act as a TAD (20),2 nor does
it have any other known function.
To study each of these regions, three Sox-11F deletion constructs (Fig.
6a) were placed in vectors
allowing protein production either through in vitro
translation (Fig. 6b) or in HeLa cells (Fig. 6c).
For these studies, protein production was determined by Western blot
analysis (Fig. 6b, top), and a similar amount of
each Sox-11F deletion construct was included in the binding reaction
(Fig. 6b, bottom). After protein normalization,
the DNA binding capability of the Sox-11F deletion mutants was
determined by EMSA. This was accomplished using either the hmg1 or the
hmg2 probe, which contains two HMG sites separated by 14 bp. The
experiments performed with hmg2 revealed the same trend in binding
intensity between the Sox-11 mutants as the hmg1 probe (data not
shown). Our examination of the DNA binding ability of in
vitro translated Sox proteins revealed that identical to our
observations from HeLa cell extracts, Sox-2F binding was much stronger
that Sox-11F (Fig. 6b, compare lanes 2 and
3) despite similar protein expression. Interestingly, when
the AR region is removed from Sox-11F, the protein binds well to the
radiolabeled probe (lane 4). Furthermore, although some
binding by Sox-11F
To determine whether the AR region is able to repress DNA binding of
in vivo produced protein, we also compared binding of proteins expressed in HeLa cells (Fig. 6c). This was an
important question because Sox-11 may have post-translational
modifications in vivo which affect DNA binding and are not
present on in vitro translated protein. As described
previously, in this DNA binding analysis, we normalized for any
differences in protein expression by including equal amounts in the
binding reactions, based on prior Western blot analysis (data not
shown). Using extract containing Sox-11F, a faint DNA-protein complex
was observed (indicated by *), which could be supershifted specifically
by the anti-FLAG M2 antibody (lanes 4 and 5). In
comparison, Sox-11F Influence of the Autoinhibitory Domain on Sox-11 Gene
Activation--
Autoinhibitory domains present in other transcription
factor families are thought to act as regulatory switches capable of repressing recruitment to a gene unless inhibition is relieved. For
example, the Ets family member PEA3 contains two domains that autoinhibit DNA binding to an optimized PEA3 binding site in
vitro. Evidence that these autoinhibitory domains also regulate
in vivo recruitment of PEA3 was observed when the ability of
full-length PEA3 to activate a reporter gene was compared with a
deletion construct in which one autoinhibitory domain was removed. In
these studies, removal of the autoinhibitory domain increased
activation of the reporter gene ~2-fold, which indicates that the
optimal capability of full-length PEA3 to activate gene expression was reduced (34).
To examine whether the AR region can regulate the ability of Sox-11 to
activate gene expression from transfected promoter/reporter gene
constructs, we compared Sox-11F and Sox-11F In this study, we compared the ability of Sox-2 and Sox-11 to
activate expression of promoter/reporter gene constructs
via the HMG binding site found in the FGF-4 distal enhancer,
which is a well characterized regulatory element for studying the
action of Sox proteins (15, 19, 20). This comparison has led to the
identification of three Sox regulatory mechanisms, which together can
regulate gene expression at multiple levels. First, we demonstrate that
the transactivation domains of Sox-2 and Sox-11 are key determinants of
their differential effects on transcription. Second, our study of
Sox-POU partnerships demonstrates that the selection of Brn-2 by Sox-11
is determined by its HMG domain, highlighting the importance of this
domain not only in DNA-protein interactions, but in protein-protein interactions as well. Third, the identification of a novel
autoinhibitory region within Sox-11 is an important advance in
understanding how its binding to DNA may be regulated. These studies
point to a model of Sox function in which DNA binding is not controlled by a single domain, but rather may be modulated by interplay with another region of the protein, leading to finely tuned gene activation.
Function of Sox Proteins Is Highly Dependent on Their
TADs--
Using a panel of Sox-2/11 chimeric proteins, we demonstrate
that Sox-11 is a much more potent transcriptional activator than Sox-2
in the model system used in this study. A functional comparison of
these chimeras demonstrates that although the N-terminal and HMG
domains modulate gene activation to a small extent, the primary region
responsible for the potency of Sox-11 lies within its C-terminal TAD.
In several cases, the potency of a TAD has been linked to its primary
amino acid sequence. Domains that are rich in acidic and hydrophobic
residues function differently in some contexts compared with those
composed of glutamine-, serine-, or proline-rich regions (37). We
demonstrate that this pattern is also observed in our comparison of
Sox-2, which has a serine/proline TAD, and Sox-11, which has a C
terminus consisting of acidic and hydrophobic amino acids.
Interestingly, Sox-4, Sox-22, and rainbow trout Sox-24 show high
homology to the acidic TAD of Sox-11 at their C terminus (38-40). This
homology suggests that these proteins are also very strong
transcriptional activators. In fact, we have determined that this is
true of Sox-4, which we determined to be capable of 8-10-fold greater
activation of the pCATSO3 promoter/reporter gene construct than
Sox-2.2 These differences in transactivation potency within
the Sox family provide a mechanism for altering gene expression through
selective recruitment of one Sox protein over another.
Sox-POU Cooperation Is Selective and Dependent on the HMG
Domain--
Although the study of a Sox factor on its own yields
important insight into protein function, many Sox family members are influenced by other factors that bind DNA at adjacent sites.
Specifically, Sox-2 and Sox-11 have been found to cooperate with the
POU transcription factors Oct-3 and Brn-2, respectively (15, 19, 20).
The observation that partnerships between Sox and POU factors are not
promiscuous, but rather are partner-specific has led to the hypothesis
that a Sox-POU "code" exists which allows only some Sox-POU
pairings to form productive complexes (20). Our studies give further
credence to this hypothesis because Sox-2 is capable of cooperating
only with Oct-3 and not Brn-2. Additionally, Sox-11 cooperates with
Brn-2 (also shown in previous studies (20)) and Oct-3.
The cooperative nature of Sox proteins has been found to require the
HMG domain in multiple cases. Selective HMG-mediated interaction with
another transcription factor has been observed on the FGF-4
enhancer where Sox-2 and Oct-3 have been shown to partner via their DNA
binding domains (15, 16). The importance of Sox HMG domains has also
been shown in the case of the A Novel Autoinhibitory Region Is Present in Sox-11--
To expand
our study of Sox-2 and Sox-11, we compared their ability to bind DNA
in vitro. Using both in vivo and in
vitro produced proteins, we demonstrate the formation of an
intense Sox-2·DNA complex, whereas little or no binding of Sox-11 was
detected, despite our use of the same amount of protein in the binding
reaction. We also observed that Sox-2-11-2F bound DNA with intensity
similar to Sox-2, whereas in contrast Sox-11-2-11F was incapable of
binding under these conditions. This suggests that the minimal HMG
domains of the two proteins are likely to bind DNA to a similar extent. The similar binding of Sox-2F and Sox-2-11-2F indicates that regions of
Sox-11 beyond the HMG domain must influence DNA binding in vitro. Using a series of deletions constructs, we determined that a highly AR region within Sox-11 is capable of sharply repressing DNA
binding by the Sox-11 HMG domain in vitro, identifying it as
an autoinhibitory region. Importantly, the repression of DNA binding by
the AR region is seen with in vitro produced protein as well
as HeLa-derived proteins. This suggests that inhibition is an intrinsic
property of the protein, rather than the result of post-translational
modification or interaction with a repressive factor present in HeLa
extract. The AR region is particularly interesting because of its
homology to Glu/Asp acid-rich stretches present in the HMG-1/2
proteins, which have been found to act as powerful negative regulators
of DNA binding in those proteins as well (8, 41). Furthermore, the
existence of acidic autoinhibitory regions has been reported in a wide
range of transcription factors including RXR, Rfx1, E12, and Nkx6.1,
although never before in a Sox protein (30-33). The importance of the
acidic region within Sox-11 is highlighted by the fact that long acidic
stretches are also present within other Sox family members, such as
Sox-22 and Sox-24 (39, 40). Therefore, other Sox proteins may also
contain binding autoinhibitory regions.
There are several mechanisms through which binding autoregulatory
regions may act. The functional and sequence similarities between the
autoinhibitory region of Sox-11 and the extensively characterized
acidic tails of chromosomal HMG proteins suggest that the two protein
groups may be regulated by related mechanisms involving negative
charge. This possibility is supported by a study of the repressive
acidic tails of chromosomal HMG-1 and HMG-2. It has been shown that the
longer acidic tail of HMG-1 results in decreased ability to bind DNA
compared with HMG-2 (41). These negatively charged autoinhibitory
domains may be repulsed by the negative charge on the phosphate
backbone of DNA or directly contact the positively charged HMG domain,
resulting in a decreased affinity of the HMG domain for DNA. Indeed, in
the case of HMG-1/2, it has been found that the negative acidic tail
can interact directly with its HMG domain (42). It is possible that the
autoinhibitory domain of Sox-11 acts similarly, perhaps by directly
interacting with other domains of Sox-11 or by inducing conformation
changes, resulting in decreased DNA binding as postulated in Fig.
8.
To permit DNA binding in vivo, autoinhibitory domains must
be neutralized. This has been shown to occur through post-translational modifications and/or direct interactions with partner proteins. Specifically, in the study of cooperative DNA binding between HMG-1 and
the TATA-box binding protein, cooperation has been found to require the
acidic tail of HMG-1 (11). This is not surprising because the
hydrophilic nature of acidic autoinhibitory domains increases the
likelihood that they are exposed at the surface of the protein, which
places them in an ideal position to interact with other proteins.
Additionally, DNA binding of the maize chromosomal HMGB family can be
regulated by phosphorylation of the acidic tail, indicating that acidic
autoinhibitory regions can be regulated through multiple mechanisms
(12). Other examples for such a model of transcription factor
cooperation have also been found during the study of the autoinhibitory
domains of Ets-1 and PEA3, which are neutralized upon interaction with
the transcription factors AML1 and USF-1, respectively, thus enhancing
DNA binding (9, 43). Similarly, the transcription factor LEF-1 contains an autoinhibitory domain, which is neutralized upon binding of the
coactivator
In conclusion, this study makes several important contributions to the
understanding of how Sox-2 and Sox-11 are regulated functionally, both
on their own and in cooperation with other transcriptional regulators.
We demonstrate that when the two proteins are compared on their own,
their transactivation domains play the primary role in determining
their functional differences. However, in studying the selective
partnership of Sox-11 and Brn-2, the HMG DNA binding domain was found
to play a critical role. Building on the example observed in the
Sox-2/Oct-3 partnership, this study provides further evidence that the
Sox-POU code may depend on the DNA binding domains of these
transcription factors. Finally, we have identified a novel
autoinhibitory region within Sox-11 capable of significantly
influencing both DNA binding in vitro and gene expression
in vivo. This observation places Sox-11 on an expanding list
of transcription factors whose DNA binding appears to be autoregulated
by regions of the protein beyond the DNA binding domain. Furthermore,
as the autoinhibitory region identified within Sox-11 shows homology to
regions of other Sox proteins, this study suggests a similar mechanism
may influence the function of several other Sox family members.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal have been described
previously (19). The promoter/reporter construct pCATDC3 was
constructed similarly to pCATSO3 as follows. Complementary
oligonucleotides (synthesized by the Eppley Cancer Institute Molecular
Biology Core Facility) containing two sets of the HMG site from the DC5
enhancer site and the POU binding sites (in boldface) from the FGF-4
enhancer region and BamHI sites at both ends
(5'-GATCCTCATTGTTGTGATGCTAATGGCTCATTGTTGTGATGCTAATGGAG-3') were annealed and ligated to generate multimers. Differences in the DC5
and FGF-4 HMG binding sequence are underlined. These multimers were
then ligated into a BglII site upstream from the SV40
promoter of the pCAT vector (Promega) to generate pCATDC3, which
contains a total of six tandem copies of the HMG and POU sites.
Similarly, pCATS4 contains a total of four tandem copies of the FGF-4
HMG site and was made using identical primers as pCATSO3, with the exception of a scrambled POU site. The upper primer is as follows with
the HMG and scrambled POU sites in bold:
(5'-GATCTCTTTGTTTGGCGGATCAT GGCTCTTTGTTTGGCGGATCATGGA-3').
TAD, the primer pair sox11
tad
(5'-CTCTACTACAGCTTCAAGTGAGCGGCCGCAAACATCACCAAGCAGCAG-3') was used to
incorporate a stop codon following Lys-282 of Sox-11F. The Sox-11F and
Sox-11F
TAD expression constructs were then used as the templates to
create Sox-11F
AR and Sox-11F
AR
TAD, respectively, using the
primer pairs of sox11
ar
(5'-GCCAAGGTGGTCTTCCTGGACGCGCAGCAGCAACCCCCTCAG-3') to delete amino
acids 190-223. To facilitate in vitro translation of the
various Sox proteins, the coding regions from each of the above
CMV5-derived vectors were cloned into the
KpnI/BamHI sites of pCRScript (Stratagene),
downstream from the T7 promoter. The identity of all plasmids modified
by PCR-based mutagenesis as well as pCATS4 and pCATDC3 was verified by
sequence analysis at the Eppley Cancer Institute Molecular Biology Core Facility.
TAD, and Sox-11F
AR
TAD for
the nuclear extracts used in the electrophoretic gel mobility shift
assay (EMSA) shown in Fig. 6c, calpain inhibitor I was added
to the growth media 4 h before lysis and was included in the
extraction buffer to minimize degradation caused by potential
proteasome activity. Extracts were stored at
80 °C. Sox proteins
produced through in vitro transcription and translation were
made using the T7 Quick Coupled Rabbit Reticulocyte Lysate (Promega)
transcription/translation system.
-32P]dCTP by Klenow fill-in reaction of
the single-stranded regions at the end of each double-stranded
oligodeoxynucleotide probe (lowercase bases). The wild-type Sox probe
(hmg1) contained a single HMG binding site (bold), which was based on
the sequence present in the FGF-4 enhancer, whereas in the
mutant Sox probe (HMGmut) this sequence is scrambled (underlined). The
sequence of hmg1 (sense strand) is
5'-tagaAAACTCTTTGTTTGCCATGTCG-3', and the sequence of
HMGmut (sense strand) is 5'-tagaAAATTAGTCGAATGCCATGTC-3'. The hmg2 probe was created to mimic the pCATSO3 reporter construct in
that it contains multiple HMG binding sites separated by 14bp. The
sequence of hmg2 (sense strand) is
5'-tagaTCTTTGTTTGGCGGATCATGGCTCTTTGTTTGGCGGATCATGGA-3'. For gel mobility shift and competition assays, 1-2 µl of nuclear lysate or 3-4 µl of in vitro translated protein was
incubated (at room temperature) for 30 min in a 20-µl volume
containing 20 mM HEPES pH 7.6, 1 mM EDTA, 2 mM MgCl2, 20% glycerol, 50 mM NaCl, 5 µg of bovine serum albumin, 5 µg of p(dGdC)p(dGdC), and 20,000 cpm of labeled probe (final concentration of 0.5-1
nM) with or without competitor. The exact volume of nuclear
lysate used in each assay was determined by first normalizing for
differences in Sox protein concentration based on Western blot
analysis. In gel mobility supershift assays, reaction mixtures were
incubated for 1 h at 4 °C with the indicated antibody before
addition of the probe, after which the reaction was continued at room
temperature for 30 min. Immediately after the completion of binding,
the reactions were electrophoresed on a nondenaturing 4% Tris-glycine
polyacrylamide gel for 4 h at 150V. The gels were then dried and
exposed to a PhosphorImager screen for 1-7 days before scanning on a
Storm PhosphorImager. Quantitation of band intensities was performed using the ImageQuant 5.0 analysis software. Experiments using hmg2 (see
Fig. 6, b and c) were also performed with hmg1
with the same trend in binding intensity between the Sox-11 mutants. All aspects of each mobility shift assay were repeated and similar results were obtained.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Transactivation potency and protein
expression of Sox-2F and Sox-11F. a and c,
duplicate plates of HeLa cells were transfected with 5 µg of pCATSO3
and increasing amounts of plasmid expressing Sox-2F, Sox-11F, or Oct-3
as indicated. Results are shown as the relative CAT expression assayed
in each transfection compared with the pCATSO3 reporter alone. The
error bars represent the S.D. between the CAT activity of
the duplicate plates assayed. All transfections included empty CMV5
plasmid to ensure that the final amount of DNA was equivalent.
CMV- -gal was also included to normalize for potential differences in
transfection efficiency. Each experiment was repeated at least three
times with a representative example shown. b, expression of
Sox-2F and Sox-11F was determined by Western blot analysis of nuclear
extracts obtained from transfected HeLa cells. The presence of each Sox
protein was detected using the anti-FLAG M2 antibody, visualized using
ECF, and quantitated using a Storm PhosphorImager and ImageQuant 5.0 software. The molecular masses are shown on the left of the
blot.
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Fig. 2.
Transactivation potency and protein
expression of Sox-2/11 chimeras. Top panel,
duplicate plates of HeLa cells were transfected with 5 µg of pCATSO3
and 100 ng of plasmid expressing the indicated Sox chimera. Results are
shown as the relative CAT expression assayed in each transfection
compared with the pCATSO3 reporter alone. The error bars
represent the S.D. between the CAT activity of the duplicate plates
assayed. Bottom panel, expression of chimeras was determined
by Western blot analysis of whole cell extracts obtained from
transfected HeLa cells. The presence of each Sox protein was detected
using the anti-FLAG M2 antibody, visualized using ECF, and quantitated
using a Storm PhosphorImager and ImageQuant 5.0 software. The molecular
mass is shown on the left of the blot. All transfections
included empty CMV5 plasmid to ensure that the final amount of DNA was
equivalent. CMV- -gal was also included to normalize for potential
differences in transfection efficiency. Each experiment was repeated at
least three times with a representative experiment shown.
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Fig. 3.
Sox-2 and -11 cooperate with Oct-3, but only
Sox-11 partners with Brn-2. HeLa cells were transfected with 5 µg of pCATSO3 and the indicated amounts of expression plasmids.
a, Sox-11F was transfected with increasing doses of Oct-3.
b, Sox-2F or Sox-11F was transfected with increasing doses
of Brn-2. Results are shown as the relative CAT expression assayed in
each transfection compared with the pCATSO3 reporter alone. The
error bars represent the S.D. between the CAT activity of
the duplicate plates assayed. All transfections included empty CMV5
plasmid to bring the final amount of DNA to 10 µg. CMV- -gal was
also included to normalize for potential differences in transfection
efficiency. Each experiment was repeated at least three times with a
representative example shown. c, expression of Sox-11F was
determined by Western blot analysis of whole cell extracts obtained
from two separate transfections of HeLa cells with 1 µg of each of
the indicated expression plasmids. Sox-11F protein was detected using
the anti-FLAG M2 antibody, visualized using ECF, and quantitated using
a Storm PhosphorImager and ImageQuant 5.0 software. Molecular mass is
shown at the left of the blot, and the relative intensities
of Sox-11F bands are shown below. All transfections included empty CMV5
plasmid to ensure that the final amount of DNA was equivalent.
CMV-
-gal was also included to normalize for potential differences in
transfection efficiency. This experiment was repeated three times with
a representative example shown.
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Fig. 4.
Adjacent binding sites and the Sox-11 HMG
domain are required for cooperation with Brn-2. a,
duplicate plates of HeLa cells were transfected with 5 µg of pCATS4
and 100 ng of Sox-11F, Brn-2, or 100 ng of both as indicated.
b, duplicate plates of HeLa cells were transfected with 5 µg of pCATSO3 and 100 ng of Sox chimera expression vector with 0, 100, or 500 ng of Brn-2 expression vector. Results are shown as the
relative CAT expression assayed in each transfection compared with the
pCATS4 or pCATSO3 reporter alone. The error bars represent
the S.D. between the CAT activity of the duplicate plates assayed. All
transfections included empty CMV5 plasmid to bring the final amount of
DNA to 10 µg. CMV- -gal was also included to normalize for
potential differences in transfection efficiency. Each experiment was
repeated at least three times with a representative experiment
shown.
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Fig. 5.
In vitro DNA binding of Sox
proteins. a, equal amounts of Sox-2F or Sox-11F protein
obtained from transfected HeLa cells were studied by EMSA with
radiolabeled hmg1 probe. To determine binding specificity, excess
unlabeled wild-type or mutant probe was included in the binding
reaction as indicated. Supershift analysis was performed by incubating
the binding reaction for 1 h at 4 °C with either anti-FLAG M2
antibody or an equal amount of nonspecific mouse IgG antibody before
adding the probe. b, equal amounts of the indicated Sox
protein obtained from transfected HeLa cells were studied by EMSA with
radiolabeled hmg1 probe. The EMSA gel was exposed to a PhosphorImager
cassette for detection and quantification using a Storm PhosphorImager
and ImageQuant 5.0 software. Each aspect of these experiments was
repeated at least twice using multiple preparations of nuclear extract.
The position of the free probe in the gel is indicated by
F.
TAD is observed (lane 5), it is also
enhanced upon removal of the central AR region (lane 6). These results indicate that the AR region of Sox-11 can inhibit DNA
binding of in vitro translated protein.
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Fig. 6.
Identification of autoregulatory regions
within Sox-11. a, diagram of Sox-11F deletion
contructs. Sox-11F represents the full-length protein (amino acids
1-395). Amino acids 190-223 were deleted from Sox-11F AR. Amino
acids 283-395 were deleted from Sox-11F
TAD. Amino acids 190-223
and 283-395 were deleted from Sox-11F
AR
TAD. b,
top, Western blot analysis of the in vitro
translated Sox proteins. The presence of each Sox protein was detected
using the M2 antibody, visualized using ECF detected by a Storm
PhosphorImager, and quantified using ImageQuant 5.0 software. The
molecular mass is shown on the left of the blot.
Bottom, equal amounts of in vitro translated
Sox-2F, Sox-11F protein, or the indicated deletion construct were
studied by EMSA with radiolabeled hmg2 probe. c, equal
amounts of Sox-11F protein or the indicated deletion construct produced
in HeLa cells were studied by EMSA with radiolabeled hmg2 probe.
Supershift analysis was performed by incubating the binding reaction
for 1 h at 4 °C with either anti-FLAG M2 antibody or an equal
amount of nonspecific mouse IgG antibody before adding the probe. The
EMSA gels were exposed to a PhosphorImager cassette for detection using
a Storm PhosphorImager and quantified using ImageQuant 5.0 software. Each
aspect of these experiments was repeated at least twice using multiple
preparations of extract. The position of the free probe in the gel is
indicated by F.
TAD binding resulted in two complexes (lane
6, single and double arrowheads), each of
which was shifted specifically by the M2 antibody (lanes 7 and 8). The finding that the intensities of the Sox-11F and Sox-11F
TAD complexes were relatively similar suggests that the TAD
of Sox-11F does little on its own to affect binding to DNA in
vitro. Next, the role of the AR region of Sox-11 in binding to DNA
was examined. Importantly, removal of this region resulted in the
formation of an intense, faster migrating complex (lane 9,
double arrowhead) as well as a less intense, slower
migrating complex (lane 9, single arrowhead).
Both complexes were shifted specifically by the M2 antibody
(lanes 10 and 11), indicating they each contain
Sox-11F
AR
TAD, although the supershift of the faster complex
resulted in a smaller change in migration. This smaller shift was
observed in multiple experiments and may indicate that either this
antibody-Sox complex adopts a compact conformation, or perhaps it is
destabilized during electrophoresis, resulting in the release of the
Sox protein from the antibody. The binding intensity of
Sox-11F
AR
TAD was estimated to be 10-fold greater than Sox-11F (as
described under "Experimental Procedures"), indicating that this
region acts strongly as a autoinhibitory domain. The significance of
multiple DNA-protein complexes with Sox-11F
AR
TAD and
Sox-11F
TAD is not clear at this time, but they do not alter the
conclusion that a strong autoinhibitory region exists in Sox-11F. It is
also interesting to note that in the case of both Sox-11F and
Sox-11F
TAD, the addition of the M2 antibody led to an increase in
the intensity of the DNA-protein complex (compare lanes 3 and 4 or lanes 6 and 7), indicating
that antibody binding may in some way neutralize the autoinhibitory
region. We believe this further supports the hypothesis that an
autoinhibitory region exists within Sox-11F because antibody binding
has been found to release autoinhibition in both PEA3 and p53 (34, 35).
Taken together our findings suggest that a novel autoregulatory domain
is present in Sox-11, which we suspect plays an important role in its
function as a transcription factor in vivo. Furthermore,
because autoinhibition occurs with both in vitro and
in vivo produced protein it is most likely an intrinsic
property of the protein rather than the result of post-translational
modification or interaction with another factor.
AR activation of reporter
gene constructs. If the AR region of Sox-11F represses its DNA binding
in vivo, its removal may enhance gene activation. Alternatively, because Sox-11F can clearly activate pCATSO3 in HeLa
cells, autoinhibition may already be relieved in vivo, and thus removal of the AR region may have no effect on Sox-11 activation. In these studies we examined the effect of the AR region on Sox-11 activation via two bona fide Sox target sequences, the
FGF-4 HMG site as well as the HMG site present in the
-crystallin gene, hereafter referred to as the DC5 site. This site
was chosen because it is less strongly activated by Sox-11 compared
with the FGF-4 HMG site (36).2 The reporter construct
pCATDC3 was made to be identical to pCATSO3 with the exception of 3 bp
within each of the six HMG binding sites to conform to the DC5
sequence. When observing the activation of pCATSO3 by Sox-11F
AR
compared with Sox-11F, we found that there was a reproducible
1.4-2-fold increase in activation when the AR region was absent (Fig.
7a). Furthermore, the
activation by Sox-11F
AR compared with Sox-11F on pCATDC3 revealed
that the difference in gene activation was even more pronounced with
this construct, increasing in a dose-dependent manner to an
almost 5-fold enhancement in gene activation upon removal of the AR
region (Fig. 7b). Finally, because both Sox-11F
AR and
Sox-11F are expressed at almost identical levels in HeLa cells (Fig.
7c), expression differences do not explain the functional
differences we are observing. Therefore, these studies support a model
in which the ability of Sox-11 to bind to a gene and regulate its
expression in vivo may be modulated by a acidic
autoinhibitory domain.
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Fig. 7.
The Sox-11 autoinhibitory domain represses
gene activation in vivo. a and
b, duplicate plates of HeLa cells were transfected with 5 µg of either pCATSO3 (a) or pCATDC3 (b) along
with the indicated amounts of either Sox-11F or Sox-11F AR expression
vector. The differences in amounts of Sox plasmid transfected with
pCATSO3 compared with pCATDC3 were necessary because of the differing
sensitivities of the reporter constructs to Sox-11 and were selected as
the minimal amounts needed to achieve activation above the basal
reporter gene expression. The number to the right
of each bar represents the -fold difference in activation by
Sox-11F
AR compared with full-length Sox-11F at the same amount of
transfected expression plasmid. All transfections included the empty
CMV5 plasmid to bring the final amount of DNA to 10 µg. CMV-
-gal
was also included to normalize for potential differences in
transfection efficiency. Each experiment was repeated at least three
times with a representative experiment shown. c, expression
of Sox-11F and Sox-11F
AR was determined by Western blot analysis of
nuclear extracts obtained from transfected HeLa cells. The presence of
each Sox protein was detected using the anti-FLAG M2 antibody,
visualized using ECF, and quantitated using a Storm PhosphorImager and
ImageQuant 5.0 software. The molecular mass is shown on the
left of the blot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-crystallin (DC5) enhancer, where
either Sox-1 or Sox-2 can partner with the paired domain transcription
factor Pax6 (29). On the DC5 enhancer, the partnership of Sox-1 or
Sox-2 with Pax6 is specific to their HMG domains and cannot be
satisfied by the HMG domain of Sox-9 (36). These examples indicate that
although HMG domains exhibit the highest homology of any Sox domain,
the minor differences that exist in this domain are capable of
generating specific partnership interactions. Consistent with these
precedents of HMG-mediated selection, we show here that the HMG domain
of Sox-11 is responsible for its ability to cooperate with Brn-2
because only Sox-11F and Sox-2-11-2F can partner with Brn-2, whereas
Sox-2F and Sox-11-2-11F cannot. Thus, there is growing support for a
model in which the HMG domain serves two functions, DNA binding and
partner selection, which may permit selective recruitment of Sox
proteins only to specific genes.
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Fig. 8.
Multiple levels of regulation impact Sox
function. This model illustrates the regulatory mechanisms
examined in this study which may influence Sox protein function. These
include autoinhibition as a mechanism for controlling DNA binding,
which we hypothesize is the result of Sox-11 existing in either an
active conformation or a repressed conformation that requires the AR
region of Sox-11. Additionally, selective partner interactions through
the HMG domains of Sox-2 and Sox-11 can influence which POU proteins
cooperate with Sox proteins in gene activation. Finally, if multiple
Sox proteins are able to bind the same site, gene activation may be
modulated through their differing transactivation capabilities.
-catenin, resulting in enhanced DNA binding (44). Interestingly, our promoter/reporter gene activation studies indicate that the Sox-11 autoinhibitory domain may be at least partially neutralized in vivo because Sox-11 is able to bind DNA and
transactivate when expressed in HeLa cells. Importantly, deletion of
the AR inhibitory region leads to an ~2-5-fold boost in gene
activation via two different promoter/reporter gene constructs,
indicating that its autoinhibitory capacity is present in
vivo and is capable of regulating gene expression. Based on the
conservation of sequence and function between chromosomal HMG proteins
and Sox-11, it is plausible that the neutralization of the Sox-11
acidic autoinhibitory domain in the full-length protein may also occur
through a number of complex mechanisms, including post-translational
modification or interaction with other proteins.
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FOOTNOTES |
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* This work was supported in part by Grant CA 74771 from the NCI, National Institutes of Health, and by Cancer Center Support Grant CA 36727 (to the core facilities of the University of Nebraska Medical Center Eppley Cancer Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by Training Grant CA 09476 from the NCI, National Institutes of Health.
Supported by an Emley graduate fellowship.
** To whom correspondence should be addressed: Eppley Cancer Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Tel.: 402-559-6338; E-mail: arizzino@unmc.edu.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M212211200
2 M. Wiebe and A. Rizzino, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
HMG, high mobility
group;
AR, acid-rich;
-gal,
-galactosidase;
CAT, chloramphenicol
acetyltransferase;
CMV, cytomegalovirus;
DC5,
-crystallin enhancer;
ECF, enhanced chemifluorescence;
EMSA, electrophoretic gel mobility
shift analysis;
FGF-4, fibroblast growth factor 4;
POU, Pit-Oct-Unc;
TAD, transactivation domain.
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