From the Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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The recently described Hmx family of homeodomain
proteins is predominately expressed in discrete regions of developing
sensory tissues. In this report, we have identified the preferred
DNA-binding site of the murine Hmx3 homeodomain protein by the
selection and amplification binding (SAAB) technique. The consensus
Hmx-binding site contained the sequence 5'-CAAGTG-3', which differs
from the 5'-TAAT-3' motif commonly associated with homeodomain
proteins. Instead, the Hmx consensus is similar to the
5'-CAAGTG-3'-binding sites of Nkx2.1 and Nkx2.5 homeodomain proteins.
Based on mutation studies, both the 5'-CAAG-3' core and the 3'-TG
dinucleotide are required for high affinity binding by Hmx3 and the
homologous Hmx1 protein. A critical determinant of this specificity is
the glutamine at position 50 in the third helix of the Hmx homeodomain. Hmx1 binds to the 5'-CAAGTG-3' element with an apparent dissociation constant of 20 nM. Unexpectedly, the human Hmx1
protein specifically repressed transcription from a luciferase reporter
gene containing 3 copies of the 5'-CAAGTG-3' sequence. In contrast, the
Nkx2.5 protein transactivated this luciferase reporter. Interestingly, co-expression of Hmx1 and Nkx2.5 attenuated each others activity, suggesting that genes containing the CAAGTG element can integrate signals from these proteins. Therefore, Hmx1 and Nkx2.5
proteins bind a unique DNA sequence and act as transcriptional antagonists.
Homeodomain genes are involved in a wide variety of developmental
pathways (1-4). Mutation studies and expression patterns of several
members of the homeodomain gene family have indicated a role in
controlling specification of cranial structures, including development
of neurons and sensory organs (2, 5-7). A novel homeodomain gene
family, Hmx, was first identified by Stadler et
al. (8) using low stringency screening of a human craniofacial cDNA library. Homologous Hmx genes have now been
identified in a diverse number of species (9). There are three closely
related members of the Hmx family in humans and mice, which
are designated Hmx1, Hmx2, and
Hmx3 (8, 9).1 The
murine Hmx2 and Hmx3 genes were origi-nally named
Nkx5.2 and Nkx5.1, respectively, based on limited
homology with the Nk homeodomain family of
Drosophila (10, 11). However, the Hmx family is
substantially different from the Nk family (9). Alignment of
the Hmx3 homeodomain protein sequence to the four Drosophila Nk genes yielded 50-61% amino acid sequence identity, which is not
substantially greater than seen when Hmx3 is compared with other
homeodomains, such as Drosophila Antp (47%). Furthermore, a
Drosophila Hmx homologue has been identified and found to
have 94% amino acid identity with the murine Hmx3 homeodomain (9). Finally, there is little or no homology between the Nkx and Hmx proteins outside the homeodomains. Hence, we have used the
Hmx nomenclature for these genes, as assigned by the human
gene mapping nomenclature committee (9).
The Hmx genes are believed to be important regulators of
development in sensory organs and neurons based on their expression patterns in the embryo (10, 12). The expression of murine Hmx3 is especially high in the otic vesicle, neuroectodermal
cells of the central nervous system, neuronal derivatives of the neural crest, including the dorsal root ganglia and myenteric ganglia, and
transiently in the second branchial arch. In particular, the predominant expression in the otic vesicle and postmitotic neurons has
suggested an involvement in the development of the inner ear and
specification of neuronal phenotype (13). The murine Hmx genes have very similar expression patterns with two chicken
homologues, GH6 and SOHo-1 gene (14, 15),
suggesting that the Hmx genes play an evolutionary conserved
role during development. The chicken homologue of Hmx1 (GH6) has been
reported to be expressed in the developing heart (14).
Nkx2.5 is expressed early during heart morphogenesis and
activates early cardiac gene expression (16-19). Hmx and
Nkx genes are found in overlapping regions in vertebrate embryos (10, 12, 14). In particular, Nkx2.5 and chicken Hmx1 are both
expressed in the developing heart myocardium (12, 14, 16). These
results suggest that Hmx and Nkx2.5 proteins both regulate early
transcription events in development.
To begin to address the functional role of Hmx homeodomain proteins, we
have identified the DNA-binding site of the Hmx1 and -3 proteins. We
show that these Hmx proteins prefer the core binding sequence
5'-CAAG-3' and not the typical 5'-TAAT-3' core found among most
homeodomain proteins (1-4). Instead, the Hmx-binding site resembles
the consensus site found for Nkx2.5 protein (also called Tinman) by
Chen and Schwartz (20) and Nkx2.1 protein (also called thyroid
transcription factor-1) by Damante et al. (21). We
demonstrate that Hmx1 represses transcription of a luciferase reporter
gene containing the Hmx preferred DNA-binding site. Since Nkx2.5
transactivates this reporter and the Nkx2.5 and Hmx proteins are
co-expressed in some tissues, we propose that Hmx1 and Nkx2.5 may
act as transcriptional antagonists.
Expression and Purification of Hmx1 and -3 Proteins--
A
fragment of the murine Hmx3 gene was
PCR2 amplified from a genomic
clone provided by Dr. Tom Lufkin (Mt. Sinai Medical Center). The
primers were nucleotides 1232 to 1252 (5'-ggatccCCGGGCTCAGAGGACTGGAAG-3') and nucleotides 1675 to 1695 (5'-gaattcCCTGTCCCATCTCACACCGGC-3'), with BamHI and
EcoRI sites (lowercase) to facilitate subcloning into
pGEX4T-3 glutathione S-transferase (GST) vector (Pharmacia) and was confirmed by DNA sequencing. The resulting plasmid, pGST-Hmx3, encodes amino acids 309-458, which contains the homeodomain 20 amino-terminal flanking residues, and the entire COOH-terminal region.
To make plasmid pGST-Hmx3 Gln SAAB Assay--
SAABs were done essentially as described by
Blackwell and Weintraub (23), using affinity chromatography (24). The
"random" oligonucleotide contained 20 random nucleotides flanked by
known sequences "b" and "a" that could be recognized by PCR
primer b (5'-AGACGGATCCATTGCA-3') and PCR primer a
(5'-TCCGAATTCCTACAG-3'), respectively (sequences from Ref. 23).
Double-stranded random oligonucleotides were generated by annealing
primer a (0.7 µg) to single-stranded random oligonucleotide (1.5 µg), then filled using Klenow polymerase, with tracer amounts of
[32P]dATP to facilitate quantitation. The double-stranded
oligonucleotide (500 ng) and GST-Hmx3 (500 ng) attached to
glutathione-Sepharose beads were incubated in 10 volumes of binding
buffer (20 mM Tris, pH 8.0, 50 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol) with 20 µg/ml bovine serum albumin, 2 µg/ml poly(dI-dC) at 4 °C for 1 h. The beads were concentrated and washed twice with binding buffer, then resuspended in 30 µl of H2O. A 10-µl
aliquot was then PCR amplified in a 25-µl reaction for 30 cycles of
94 °C (1.5 min), 40 °C (1 min), and 72 °C (2 min). For
subsequent rounds, 10 µl (approximately 50 ng) of the PCR reactions
was incubated with 50 ng of protein. Products from the last round were
gel purified, cloned into pGEM-T vector (Promega), and sequenced on one
strand using an SP6 primer.
Electrophoretic Mobility Shift Assay (EMSA)--
Complementary
oligonucleotides containing a consensus Nkx2.1 site (see Fig.
1C) (21) or a Bicoid site (5'-TAATCC-3'), with flanking
partial BamHI ends were annealed and filled with Klenow polymerase to generate a 32P-labeled probe for EMSAs, as
described (25). For standard binding assays, the oligonucleotide (1 pmol) was incubated in a 20-µl reaction containing binding buffer (10 mM Tris, pH 7.5, 5% glycerol, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol), 0.1 µg of
poly(dI-dC), 120 ng of Hmx3 (thrombin-cleaved from GST or
glutathione-eluted), or 300 ng of Hmx1 (PreScission cleaved) on ice for
15 min. For competition assays, unlabeled double-stranded
oligonucleotides were preincubated with the protein for 15 min on ice
prior to addition of the probe. Sequences of the Nkx2.1 probe and
competitor oligonucleotides, all with flanking partial BamHI
ends (in lowercase), are given in the figures, with the exception of
Hmx Mu3, 5'-gatccCGGACCCCCAGTGGGAGCATCTggatc-3' and Hmx Mu11,
5'-gattccTTGTTAATAATCTAATTACCCTAGggatc-3'. The samples were
electrophoresed for 2 h at 250 V in an 8% polyacrylamide gel in
0.25 × TBE (22.5 mM Tris-HCl, pH 8.5, 28 mM boric acid, 0.7 mM EDTA) at 4 °C
following pre-electrophoresis of the gels for 1 h at 200 V. The
dried gels were visualized by exposure to autoradiographic film. For
quantitative analyses to establish binding constants and relative
competitions, the amount of bound and free radioactive probe was
measured from dried gels using an InstantImager (Packard). The binding
constants were calculated by Scatchard plots. For determination of the
amount of binding competition, the ratio of bound to free probe was
normalized to the absence of competitor DNA, which was set at 100%.
Expression and Reporter Constructs--
An expression plasmid
containing the cytomegalovirus (CMV) promoter linked to a human Hmx1
EcoRI-BamHI fragment was constructed in pcDNA
3.1 MycHisC (Invitrogen). Dr. Yutzey, Children's Hospital Medical
Center, Cincinnati, OH, kindly provided the murine Nkx 2.5 expression
plasmid (pEMSV-Nkx2.5). The Hmx-TK-luc reporter plasmid has Hmx-binding
sites (same oligonucleotides as used in the EMSAs
(5'-gatccCACTGCCCAGTCAAGTGTTCGGATg-3' annealed to
5'-gatccATCCGAACACTTGACTGGGCAGTGg-3')) ligated into the
BamHI site upstream of the thymidine kinase (TK) promoter in
the TK-luc plasmid (25). Hmx-TK-luc contains three inserts, 2 in the
sense and 1 in the antisense orientation. A CMV Cell Culture, Transient Transfections, Luciferase, and
Identification of the Hmx3 DNA-binding Site--
To determine the
DNA-binding site of Hmx3, the SAAB technique was used with bacterial
expressed GST-Hmx3 protein containing the homeodomain and COOH-terminal
region. Previous studies with other homeodomain proteins have shown
that the information necessary for DNA binding is contained within the
homeodomain region (3, 4). For example, a truncated
homeodomain-containing Nkx2.1 protein binds with the same apparent
KD as the full-length protein (21). The sequences of
oligonucleotides selected after 2, 3, and 5 sequential SAAB rounds are
shown in Fig. 1A. As early as
rounds 2 and 3, a consensus containing 5'-CAA(T/G)-3' was observed. Following the fifth round, there was a selection for oligonucleotides containing the consensus sequence 5'-CAAGTGCGTG-3'. In many cases, multiple copies of this sequence were found in the oligonucleotides. Each of the nucleotides within the consensus was highly conserved, with
frequencies ranging from 75 to 100% occurrence (Fig. 1B). In particular, the 5'-CAAG-3' core was very predominant, being present
in all of the selected oligonucleotides. These results indicate a
strong preference for this consensus sequence.
Comparison of Hmx3 Binding to Nkx and Hmx Consensus
Sites--
Because the Hmx and Nkx2.1 and 2.5 consensus elements both
contain a 5'-CAAGTG-3' motif, but the flanking sequences differed, we
tested whether Hmx3 could also bind the Nkx2.1 element. Using a
competitive EMSA DNA binding assay, we showed that the Hmx3 protein
bound to DNA containing the Nkx2.1/2.5 consensus site and was
efficiently competed by either the Nkx2.1/2.5 or the Hmx consensus
motifs (Fig. 1C). Thus, Hmx3 binds both Nkx2.1/2.5 and Hmx
consensus binding sites without preference for flanking sequences. As a
control for binding specificity, Hmx3 binding to the 5'-CAAGTG-3' consensus sequence was not competed by an oligonucleotide containing an
octamer consensus site recognized by the Oct-1 POU homeodomain. It
should be noted that the 5'-CAAGTG-3' consensus site for Hmx and Nkx
proteins also contains a consensus helix-loop-helix protein-binding site (5'-CANNTG-3') (27). While it was very unlikely that Hmx3 DNA
binding activity was similar to helix-loop-helix proteins, we ruled out
this formal possibility by demonstrating that Hmx3 did not bind to
oligonucleotides containing two other 5'-CANNTG-3' motifs. Both the
5'-CAGCTG-3' and 5'-CACCTG-3' elements recognized by the AP4 and muscle
creatine kinase helix-loop-helix proteins, respectively, did not bind
Hmx3 (data not shown). Thus, Hmx3 specifically binds the Hmx and
Nkx2.1/2.5 motifs with comparable affinities.
Mutations in the CAAG Core and Flanking TG Dinucleotide Reduce Hmx3
Binding--
The binding of Hmx3 to the CAAG core was compared with
the canonical TAAT sequence found in the Antennapedia class of
homeodomains (28). Hmx3 binding to the competitor was greatly reduced
when the 5'-CAAG-3' sequence was mutated to a typical
homeodomain-binding site that matches the Msx consensus
binding site, 5'-TAATTG-3' (Msx) (Fig.
2A) (28). An even greater
effect was seen when the competitor binding site was mutated at both
the core and flanking dinucleotide 5'-TAATCA-3'
(Mu10) (Fig. 2A). Similarly, mutation to
5'-TAATTA-3' (Hmx Mu11), which matches a
consensus Idx-binding site (29), greatly reduced Hmx3 binding to the
competitor (data not shown). Finally, complete removal of the CAAG core
(Hmx Mu3) completely eliminated binding to the competitor (Fig.
2B). These results demonstrated that the 5'-CAAG-3' core is
critical for Hmx3 binding activity.
The relative difference between Msx (TAATTG)
and Hmx Mu10 (TAATCA) competitions indicated
that the 3'-TG dinucleotide contributes to Hmx3 binding. This
contribution was confirmed by the reduced Hmx3 binding to the
competitor seen upon mutation of the TG in the context of the
5'-CAAG-3' core sequence (Hmx Mu2, CAAGGT) (Fig.
2A). To further test the importance of the TG dinucleotide in the absence of the 5'-CAAG-3' core, the TG was mutated to a GG (Ftz)
or AT (Hmx Mu9) following a 5'-TAAT-3' core. Hmx3 did not bind either
of these DNAs in competition assays (Fig. 2A). Quantitation
of the effect of each competitor on Hmx3 binding is shown in
Fig. 2B.
We also confirmed that sequences outside the 5'-CAAG-3' and flanking TG
dinucleotide motif are not required for Hmx3 binding. This was
important to test since the SAAB consensus had contained an additional
four nucleotides (CGTG) downstream of the CAAGTG motif. Changes in
these nucleotides (Hmx Mu4) did not noticeably affect Hmx3 binding
activity within the parameters of the competition assay (10-50-fold
molar excess) (Fig. 2A). This is consistent with Hmx3
binding to the Nkx2.1/2.5 consensus sequence, which also lacks the
3'-CGTG (Fig. 1C). Taken together these results show that
both the 5'-CAAG-3' and the 3'-TG dinucleotide are the key
components of the DNA-binding site.
Mutation in the Recognition Helix of Hmx3 Reduces DNA Binding
Activity--
It has been shown that the 3'-dinucleotide of the
DNA-binding site can confer binding specificity that is determined by
the amino acid at position 50 of the classical TAAT binding homeodomain proteins (4, 30). In addition, Damante et al. (21)
demonstrated that the glutamine at position 50 is important for
recognition of the TG dinucleotide in the Nkx2.1 consensus DNA-binding
site, 5'-CAAGTG-3'. Hmx3 also contains this glutamine in the highly conserved third helix, suggesting that this residue may play a similar
role in Hmx3. We made the corresponding mutation in Hmx3 changing the
glutamine (Q) to a lysine (K). The glutamine to lysine mutation
decreased binding to undetectable levels (Fig.
3A). As a control, the mutant
protein could bind to a bicoid element (5'-TAATCC-3') (Fig.
3B). The lysine at position 50 is important for recognition of the CC dinucleotide in the bicoid element (4, 31). GST-Hmx3 Gln Hmx1 Binds the 5'-CAAGTG-3' Element with a Similar Specificity as
Hmx3--
To determine if the binding characteristics of the
homologous human Hmx1 protein were similar to Hmx3 we used a
competitive EMSA DNA binding assay as described above. Hmx1 was chosen
so as to extend our findings to another member of the Hmx family. Human
Hmx1 has 92% amino acid identity to murine Hmx3 in the homeodomain. We
demonstrate that the Hmx1 protein bound to DNA containing the Nkx2.1/2.5 site and was efficiently competed by the Nkx and Hmx consensus elements, but not other motifs (Fig.
4A). Hmx1 had the same DNA
binding specificity as shown for the truncated Hmx3 protein (Fig.
4B).
The binding affinity of Hmx1 to the 5'-CAAGTG-3' sequence was measured
by EMSA (Fig. 4C). The apparent dissociation constant (KD) was calculated using different protein and
probe concentrations by Scatchard analysis as 20 nM (Fig.
4C). This KD is higher than those
reported for Nkx2.1 (3 nM), Antennapedia (1.2 nM), and Engrailed (1-2 nM) (4, 20, 21). The
KD using the GST-Hmx3 fusion protein was 1.4 nM, which is very similar to the reported
KD values of other homeodomain proteins (Fig.
4D). We have seen that the GST moiety can affect binding of
the Pitx2 homeodomain protein. GST-Pitx2 has an apparent
KD of ~0.5 nM while the non-fusion
purified Pitx2 protein demonstrated a KD of 50 nM (26).
Hmx1 Specifically Represses Transcription from a Promoter
Containing the 5'-CAAGTG-3' sequence--
To determine if Hmx1 could
regulate a reporter gene containing the 5'-CAAGTG-3' sequence we
co-transfected an expression vector encoding human Hmx1 (CMV-Hmx1) with
a luciferase reporter containing three Hmx-binding sites (Hmx-TK-luc)
into HeLa cells. As a control for transfection efficiency, a CMV
For comparison, we asked if Nkx2.5 could transactivate the 5'-CAAGTG-3'
reporter under our conditions. We found that Nkx2.5 caused a 3-fold
stimulation of this reporter and had no effect on the reporter without
the 5'-CAAGTG-3' elements (Fig. 5B). This is consistent with
published reports that Nkx2.1 and Nkx2.5 are transcriptional activators
(20, 21). Thus, Hmx1 represses, while Nkx2.5 activates, transcription
via the 5'-CAAGTG-3' element.
Hmx1 and Nkx2.5 Act as Transcriptional Antagonists--
Since
Nkx2.5 (Tinman) and Hmx1 are expressed in the developing heart and both
bind the same core DNA element (5'-CAAGTG-3') we then asked if these
factors had an antagonistic effect on transcription. The Nkx2.5 and
Hmx1 expression vectors were co-transfected along with the 5'-CAAGTG-3'
TK-luc reporter plasmid. Co-transfection of equal amounts of each
vector resulted in an overall 2-fold repression of transcription (Fig.
6). This is an intermediate value between
the 3-fold activation by Nkx2.5 alone and the 4-fold repression by Hmx1
alone. These results indicate that Hmx1 can antagonize Nkx2.5
activation of the reporter plasmid, and conversely Nkx2.5 can attenuate
Hmx1 repression. To vary the relative amounts of Hmx1 protein compared
with Nkx2.5 in the transient transfection assay, we varied the amount
of expression vector DNA. Antagonism was observed even with lower
levels of Hmx1 expression vector DNA (Fig. 6). These results suggest
that the relative levels of Nkx2.5 and Hmx1 proteins may regulate the
activity of genes containing the CAAGTG element.
This study represents the first molecular/biochemical
characterization of members of the Hmx homeodomain family. Hmx 1 and Hmx 3 strongly prefer the 5'-CAAGTG-3' DNA-binding site in contrast to
the 5'-TAAT-3' motif preferred by nearly all other metazoan homeodomain
proteins. The usual bias among homeodomain proteins for a 5'-TAAT-3'
core was recently confirmed by Wilson et al. (30) using the
SAAB selection strategy similar to the one used in this study. For Hmx
proteins, both the 5'-CAAG-3' core and the 3'-flanking TG dinucleotide
contribute to binding specificity. This binding site has a striking
similarity to the consensus sites identified for the Nkx2.5 and Nkx2.1
proteins. Chen and Schwartz (20) used a similar SAAB methodology with
Nkx2.5 to identify a high affinity 5'-TNNAGTG-3' motif and lower
affinity 5'-TAAT-3' containing motifs. Similarly, Damante et
al. (21) have shown that Nkx2.1 bound the core consensus sequence
5'-CAAGTG-3', which is also recognized by the Nkx2.5 protein (20).
Thus, Hmx binds to the same 5'-CAAGTG-3' sequence as Nkx2.1 and 2.5 proteins.
DNA binding specificity of homeodomains is dictated mostly by residues
in the recognition helix and the NH2-terminal arm (21, 30,
32-34). We have shown that the glutamine at position 50 of the Hmx
recognition helix is essential for binding. The position 50 residue has
been shown to be critical for recognizing the 3'-dinucleotide of the
DNA binding sequence of both TAAT-binding and CAAG-binding proteins (4,
21, 30). For example, conversion of a glutamine to lysine at position
50 in the Ftz homeodomain changed the recognition sequence from
5'-TAATGG-3' to 5'-TAATCC-3' (32-34). The
latter sequence is bound by the Bicoid protein, which contains a lysine at position 50. Consequently, Hmx requires the same residue that has
also been identified as a critical determinant in other groups of
homeodomain proteins. Recently, a detailed set of experiments was
performed to determine the amino acids required for binding to the
5'-CAAGTG-3' sequence by Nkx2.1 (35). This study demonstrated that the
amino acid in position 54 of the homeodomain is involved in the
recognition of the guanosine at the 3' end of the core sequence
5'-CAAG-3'. The authors further demonstrated that the 5' cytosine is
recognized by the amino acids located in positions 6, 7, and 8 of the
NH2-terminal arm. Comparison of the Nkx and Hmx sequences
supports and extends the conclusions that these residues contribute to
binding to the 5'-CAAGTG-3' sequence. Specifically, Nkx2.1 has a
tyrosine at position 54 that is conserved among Nkx2 family members. In
contrast, all the Hmx family members contain an asparagine at this
position. These residues are similar in that both have bulky polar side
chains. The Nkx2.1 residues at positions 6, 7, and 8 are valine,
leucine, phenylalanine, while Hmx proteins contain threonine, valine
(isoleucine in one case), phenylalanine at these positions. Between the
two families, positions 7 and 8 are identical or homologous, while the
residues at position 6 are structurally quite different. Hence the
difference in position 6, together with the different, albeit similar,
residues at position 54, suggests that there is some flexibility in the
binding determinants of the CAAG-binding group of homeodomain proteins.
We have demonstrated that Hmx1 can specifically repress transcription
of a reporter gene containing 3 copies of the 5'-CAAGTG-3' sequence.
Furthermore, we have shown that Hmx1 and Nkx2.5 act as transcriptional
antagonists. The degree of Nkx2.5 transactivation that we observed was
similar to previous results with the Nkx2.5 protein on multimers of its
binding site (20). Since both Nkx2.5 and chicken Hmx1 (GH6) have
overlapping expression patterns during heart development (12, 14),
these proteins may also differentially regulate genes containing the
5'-CAAGTG-3' element. There is precedence for the regulation of
transcription during differentiation by transcriptional antagonists. A
well studied example is the transcriptional antagonism between the
homeodomain proteins Ftz and Engrailed, where Engrailed represses or
quenches the activation of Ftz (36, 37). Recently, it has also been
shown that the winged helix HNF-3 In addition to our experimental system, there are now at least three
reports of 5'-CAAGTG-3' type elements controlling gene transcription in
response to Nkx2.1 and Nkx2.5. A 5'-CAAGTG-3' response element for
Nkx2.1 has been identified in the rat thyroglobulin promoter near the
TATA box (21). Recently, targets for Nkx2.5 have been identified in the
atrial natriuretic factor promoter (39) and the cardiac
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lys, an antisense primer containing
the point mutation (underlined), nucleotides 1423-1448, (5'-GGCGATTCTTGAACCAGATCTTGACC-3') was used with the
previous 5' primer (nucleotides 1232-1252) to make a PCR megaprimer.
In the second PCR step the megaprimer was used with the previous 3'
primer (nucleotides 1675-1695). The PCR profile was 94 °C, 2 min;
70 °C, 2 min, 72 °C, 3.5 min for 30 cycles using Pfu DNA polymerase (Stratagene). Hmx3 Gln
Lys DNA was cloned into pGEX4T-3 and confirmed by sequencing. Plasmid pGST-Hmx1 was made by a series of
sequential subcloning of the Hmx1 coding region from pBSK II Hmx1
cDNA plasmid (9) into pGEX 6P-1 (Pharmacia). The plasmids were
transformed into Escherichia coli JT4000 5-IV95, a
lon
protease-deficient strain, or BL21 cells.
Protein was isolated as described (22), with some modifications.
Cultures (500 ml) were induced with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside for 4 h at
30 °C. The bacteria were lysed in 10 ml of 10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5% low fat
dry milk, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin, 100 mg/ml phenylmethylsulfonyl fluoride, 5 mM dithiothreitol,
100 µg/ml lysozyme, on ice for 15 min, followed by addition of 1.5%
Sarkosyl and sonication for 1 min. In later experiments, protease
inhibitor mixture (Sigma) was used and 4% Triton was added prior to
sonication. After removal of debris, the supernatant was incubated with
100 µl of a 1:1 (v/v) slurry of glutathione-Sepharose (Pharmacia) in
phosphate-buffered saline overnight at 4 °C, then washed in
phosphate-buffered saline. Hmx3 was released by cleavage with 10 units
of thrombin (Sigma) at room temperature for 30 min. Hmx1 was released
by cleavage with 30 units of PreScission protease (Pharmacia) for
1 h at 4 °C. All proteins were analyzed following SDS-gel
electrophoresis by silver stain. In some experiments, GST-Hmx3 and
GST-Hmx3 Gln
Lys were eluted using 10 mM glutathione in
50 mM Tris, pH 7.6, for 5 min at room temperature. For
Western blots, the proteins were resolved by 12.5% SDS-polyacrylamide
gel electrophoresis, transferred to polyvinylidene difluoride filters
(Millipore), immunoblotted, and detected using ECL reagents from
Amersham. The antibody directed against Hmx3 (number 19902; used at
1/1000 dilution) was raised in rabbits to a MAP-conjugated peptide in the COOH-terminal region (IVRVPILYHENSAAEGAAAA) (Research Genetics Inc., Huntsville, AL). In some experiments, immunoblots were also done
using a GST antibody (used at 1/2000 dilution) (Pharmacia) (data not shown).
-galactosidase
reporter plasmid (CLONTECH) was co-transfected in
all experiments as a control for transfection efficiency.
-Galactosidase Assays--
COS-7 and HeLa cells were cultured and
transfected as described (26) by a modification of the calcium
phosphate method or electroporation. For calcium phosphate
transfection, 5-10 µg of plasmid DNA was used. For electroporation,
HeLa cells were mixed with 2.5 µg (or as indicated) of expression
plasmids, 2.5 µg of reporter plasmid, and 0.5 µg of CMV
-galactosidase plasmid. HeLa cells were electroporated at 220 mV and
960 microfarads (Bio-Rad) plated in 60-mm culture dishes and fed with
5% fetal calf serum and Dulbecco's modified Eagle's medium. Cells
were then lysed and assayed for reporter activities and protein content
by Bradford assay (Bio-Rad). Luciferase was measured using reagents
from Promega.
-Galactosidase was measured using the Galacto-Light
Plus reagents (Tropix Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of Hmx3 binding sites.
A, alignment of the PCR product sequences after 2, 3, and 5 successive SAAB rounds (number of rounds is denoted by the prefix,
e.g. 2-1 is from round 2). The best match and other
conserved regions are indicated in bold. The common
sequences contributed from the PCR primers are indicated by
a and b. B, sequence of the consensus Hmx3
DNA-binding site determined from the PCR products after 5 SAAB rounds.
The frequencies of each consensus nucleotide within the best match of
each oligonucleotide, and from all the sites indicated in
bold are shown. C, Hmx3 protein was incubated
with the Nkx2.1 consensus sequence as the radioactive probe in the
absence or presence of unlabeled oligonucleotides as competitor DNAs.
Competitor oligonucleotides were used at 10-, 25-, and 50-fold molar
excess concentrations. The free probe and bound complex are indicated.
The sequences of the DNAs are shown at the bottom of the
figure with the terminal partial BamHI sites in
lowercase, and the CAAGTG motif separated by vertical
lines.
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Fig. 2.
Both the 5'-CAAG-3' core and 3'-TG
dinucleotide are required for Hmx3 binding. A, Hmx3 DNA
binding activity was measured by EMSA with the Nkx2.1 probe in the
presence or absence of competitor DNAs at 10-, 25-, and 50-fold molar
excess. The free probe and bound complexes are indicated. The
oligonucleotide sequences are shown below the autoradiogram,
with differences from the Hmx3 oligonucleotide underlined.
The terminal partial BamHI sites are in
lowercase. B, quantitation of the binding
efficiency of Hmx3 from the EMSA experiments. Dried gels were
quantitated using the InstantImager (Packard). The bound DNA
radioactivity was measured and the inhibition of bound complex from
50-fold excess of each competitor DNA was determined. The values are
normalized to 100% binding without competitor DNA, with the means and
standard deviations from two to six independent shown.
Lys mutant protein binding to the bicoid probe was competed by the
bicoid competitor DNA but not by Hmx DNA. For comparison, the wild type
Hmx3 protein bound very poorly to the bicoid probe and was effectively
competed by the Hmx element (Fig. 3B). Expression of the
GST-Hmx3 Gln
Lys protein in bacteria was confirmed by Western blots
with an antibody raised against a COOH-terminal peptide conserved in
the Hmx family (Fig. 3C). Varying concentrations of both
GST-Hmx3 and GST-Hmx3 Gln
Lys proteins were analyzed for binding to
the 5'-CAAGTG-3' sequence. Thus, Hmx3 binding to the 5'-CAAGTG-3' motif
requires the glutamine at position 50 of the homeodomain.
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Fig. 3.
The glutamine at position 50 in the
homeodomain is required for Hmx3 binding to the 5'-CAAGTG-3'
sequence. A, GST-Hmx3 and GST-Hmx3 Gln Lys DNA
binding activity was measured by EMSA with the Nkx2.1 probe. Several
concentrations of each protein were used for binding: 0.1, 0.5, 1 µl
of GST-Hmx3, and 1, 5, 10 µl of GST-Hmx3 Gln
Lys. The free probe
and bound complexes are indicated. B, GST-Hmx3 Gln
Lys
DNA binding activity was measured by EMSA with the bicoid probe
(5'-TAATCC-3'). Approximately 120 ng (0.1 µl) of GST-Hmx3 and
GST-Hmx3 Gln
Lys (1.0 µl) proteins were used to determine binding
activity. Binding activity was measured in the presence and absence of
competitor DNA at 50-fold molar excess (Bic, bicoid competitor).
C, Western blot of bacterial expressed GST-Hmx3 and GST-Hmx3
Gln
Lys proteins. Equal volumes of GST affinity purified fusion
proteins (4 µl) were resolved on a 12.5% SDS-polyacrylamide gel,
transferred to a polyvinylidene difluoride filter, and immunoblotted
using an antibody against a COOH-terminal peptide of Hmx3.
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Fig. 4.
Hmx1 and Hmx3 have similar DNA binding
specificities. A, Hmx1 DNA binding activity was
measured by EMSA with the Nkx2.1 probe in the presence or absence of
competitor DNA at 50-fold molar excess. The free probe and bound
complex detected in the autoradiograms are indicated. B,
quantitation of the binding efficiency of Hmx1 from the EMSA
experiments. Binding was measured as described in the legend to Fig. 2.
The means and standard deviations from two to six independent
experiments are shown. C, Scatchard plot of Hmx1 protein
binding to increasing amounts of Nkx2.1 probe. D, Scatchard
plot of GST-Hmx3 protein binding to increasing amounts of bicoid probe.
The free and bound forms of DNA were quantitated using the
InstantImager.
-galactosidase reporter was also included. Unexpectedly, Hmx1
repressed transcription from the Hmx-TK-luc reporter approximately
4-fold compared with control cells transfected with the CMV vector
without Hmx1 (Fig. 5A). In the
absence of the Hmx sites, there was only marginal repression of the
reporter by Hmx1 (Fig. 5A). Thus, Hmx1 specifically
represses promoter activity of a reporter containing the 5'-CAAGTG-3'
sequence.
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Fig. 5.
Transcriptional repression of a 5'-CAAGTG-3'
containing luciferase reporter by Hmx1. A, HeLa cells
were transfected with either Hmx-TK-luciferase reporter gene containing
three copies of the Hmx binding sequence (striped boxes) or
the parental TK-luciferase reporter without the Hmx sites. The cells
were co-transfected with either the CMV-Hmx1 expression plasmid (+) or
the CMV plasmid without Hmx1 ( ). To control for transfection
efficiency, all transfections included the CMV
-galactosidase
reporter. Cells were incubated for 24 h, then assayed for
luciferase and
-galactosidase activities. The activities are shown
relative to the TK-luc without Hmx1 control (mean ± S.E.
(n = 8) from four independent experiments). All
luciferase activities were normalized to
-galactosidase activity.
The mean TK-luciferase activity without Hmx1 expression was about 1600 light units per 20 µg of protein, and the
-galactosidase activity
was about 15,000 light units per 20 µg of protein. B, HeLa
cells were transfected with either Hmx-TK-luciferase reporter gene
containing three copies of the Hmx binding sequence (striped
boxes) or the parental TK-luciferase reporter without the Hmx
sites. The cells were co-transfected with either the Nkx2.5 expression
plasmid (+) or a CMV plasmid without Nkx2.5 (
). Activities were
normalized as described in panel A, from three independent
experiments (n = 6).
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Fig. 6.
Transcriptional antagonism of the
5'-CAAGTG-3'-containing luciferase reporter by Nkx2.5 and Hmx1.
HeLa cells were co-transfected with the Hmx-TK-luciferase reporter gene
and either the Nkx2.5 expression plasmid (7.5 µg), the CMV-Hmx1
expression plasmid (0-7.5 µg). The total amount of DNA was held
constant by addition of the empty CMV vector ( ). To control for
transfection efficiency, all transfections were normalized to
-galactosidase from a co-transfected CMV
-galactosidase reporter.
Cells were incubated for 24 h, then assayed for luciferase and
-galactosidase activities. The activities are shown relative to
Hmx-TK-luc without Hmx1 or Nkx2.5 expression (mean ± S.E.
(n = 6) from three independent experiments).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HNF-3
proteins have
antagonistic transcriptional regulatory functions (38). HNF-3
activates transcription and HNF-3
represses activity by competing
for HNF-3
binding to a shared DNA element.
-actin gene
(40). The observation that natural Nkx-target genes can be regulated by
5'-CAAGTG-3' elements strengthens the likelihood that Hmx proteins will
also regulate genes containing this element. Furthermore, the shared
DNA binding specificity and overlapping expression patterns suggests
that the Hmx and Nkx gene families may recognize
and coordinately regulate overlapping sets of target genes during
specification of cardiac and neuronal phenotypes.
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ACKNOWLEDGEMENTS |
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We thank Drs. Scott Stadler, Tom Lufkin, and Jeffrey Murray for helpful discussions and kindly providing Hmx clones. We thank Dr. Katherine Yutzey for generously providing the Nkx2.5 expression vector.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD25969 and DE09170 (to A. F. R.), with tissue culture support from DK25295, and National Institutes of Health Postdoctoral Training Fellowship DK07018 (to B. A. A.).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.
Contributed equally to the results of this report.
§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242. Tel.: 319-335-7873; Fax: 319-335-7330; E-mail: brad-amendt{at}uiowa.edu.
1 J. Murray, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; SAAB, selection and amplification binding; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; CMV, cytomegalovirus; luc, luciferase; HNF, hepatocyte nuclear factor.
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REFERENCES |
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