(Received for publication, September 7, 1995; and in revised form, December 7, 1995)
From the
The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues. The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci.
The mammalian Pax gene family contains at least nine
members that are expressed in distinct spatiotemporal patterns during
embryogenesis and regulate the morphogenesis of a variety of tissues (1, 2, 3, 4) . As determined by the
analysis of both naturally occurring and genetically engineered
mutations, Pax proteins may function in the specification, adhesion,
migration, or proliferation of progenitor cells in the developing
nervous system(5, 6) , vertebral column(7) ,
kidney(8) , eye(9) , and immune system(10) .
The Pax genes encode transcription factors that bind as
monomers to specific DNA sequences via the 128-amino acid paired
domain. The paired domain, which is located at the amino terminus of
the Pax proteins, is composed of two structurally independent
subdomains, each of which contains a helix-turn-helix
motif(11) . The NH- and COOH-terminal subdomains of
the Pax-5 paired domain bind to adjacent major grooves of the DNA
helix(12) , while the Drosophila paired protein binds
to one major groove through its NH
-terminal paired
subdomain(11) . The NH
-terminal subdomain also
contains a
turn motif and COOH-terminal tail, which make contacts
in the minor groove and along the phosphate backbone of the DNA.
Alignment of the Pax binding sites indicates that the
NH
-terminal subdomain, the sequence of which is highly
conserved between the Pax proteins, contacts a conserved recognition
sequence whereas the more divergent COOH-terminal subdomain appears to
play a role in site-specific DNA recognition(12) .
The murine Pax-2 gene encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during brain, neural tube, kidney, eye, and ear development(13, 14, 15) . Partial loss of Pax-2 function in mice and humans results in kidney and retinal defects(16, 17) . In addition, it appears that the transition of kidney mesenchyme to epithelium, its proliferation, and subsequent terminal differentiation requires the properly timed activation and repression of Pax-2(8, 18) . The expression data also suggest that Pax-2 may be involved in the differentiation of specific neural cell types within the hindbrain and spinal cord(19, 20) . Several in vitro Pax-2A DNA recognition sequences have been identified based on PCR selection of Pax-2A bound random oligonucleotides(21) , revealing a fairly divergent consensus type sequence. Pax-2 binding sites have also been deduced by random nucleotide substitutions within a core paired domain binding sequence originally defined for the Pax-1 protein(22) . Although binding sequences and genes regulated by Pax-5(12, 23, 24, 25, 26) , Pax-6(27, 28) , and Pax-8(29) have been identified, there are currently no known in vivo DNA recognition sequences nor genes known to be regulated by Pax-2.
To better understand how Pax-2 exerts its morphologic function, it is necessary to identify in vivo DNA binding sites and genes activated or repressed by the Pax-2 protein. As a first step to isolate Pax-2 target genes in the developing spinal cord, DNA sequences bound to the Pax-2 protein were enriched from native chromatin, using Pax-2 specific antibodies, and cloned. Two unique genomic clones containing three specific Pax-2 binding sites were identified. The results demonstrate that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The report also demonstrates the feasibility of the chromatin immunoprecipitation method for the isolation of binding sites that can subsequently be used as sequence tags for the identification of loci regulated by transcription factors.
The IgG fractions of anti-Pax-2
antiserum were coupled to Aminolink-agarose beads (Pierce) according to
manufacturer's directions. Uncoupled and rabbit anti-Pax-2
IgG-coupled agarose beads were washed three times in 12 mM Tris, pH 7.5, 3 mM EDTA, 100 mM NaCl, and 1
mg/ml BSA. The chromatin supernatant was preabsorbed with the uncoupled
agarose beads for 30 min at room temperature with slow rotation. The
agarose was pelleted at 3000 g for 30 s, and the
supernatant was divided into two equal fractions. One half of
supernatant was incubated with uncoupled agarose beads, and the other
half was incubated with rabbit anti-Pax-2 IgG-coupled beads for 45 min
at room temperature with slow rotation. The agarose was pelleted and
washed three times with PBS containing 1 mM MgCl
and 1 mM CaCl
. The agarose was resuspended
in protease buffer (50 mM Tris, pH 8.0, 1% SDS, 100 mM NaCl) containing 1 mg/ml Pronase and incubated for 60 min at 50
°C. The DNA was extracted with phenol:chloroform (1:1),
ethanol-precipitated, and ligated into the BamHI site of
pBluescript (Bst) KS
(Stratagene Inc.).
GenBank (release 84.0) and EMBL (release 39.0) data base searches were
performed. The BESTFIT program was used for sequence alignments
(Genetics Computer Group Sequence Analysis software package).
Whole cell extracts containing full-length and truncated forms of
the Pax-2 protein were prepared from transiently transfected COS-7
cells. pS1-Pax-2 expression plasmids were a gift from Martyn Goulding.
COS-7 cells (5 10
) were transfected 4-6 h
after seeding by the calcium phosphate method with 10 µg of
pS1-Pax-2 expression plasmid and 10 µg of Bst DNA/100-mm
dish(32) . As a control, COS-7 cells were mock-transfected or
transfected with the Bst or pS1 expression plasmid containing
no insert. After a 16-h incubation with the calcium
phosphate-precipitated DNA, the cells were washed in PBS and refed with
fresh medium. Cells were harvested 72 h post-transfection and
resuspended in 200 µl of buffer (20 mM HEPES, pH 7.8, 150
mM NaCl, 25% glycerol, 0.5 mM DTT, 0.2 mM EDTA) plus protease inhibitors (0.5 mM APMSF, 0.5
µg/ml leupeptin, 2 µg/ml aprotinin). The cell lysate was
sonicated three times for 5 s at high intensity and microcentrifuged at
12,000
g for 10 min at 4 °C. Western blot analysis
using anti-Pax-2 antibodies of the cleared whole cell extracts using
detected immunoreactive bands corresponding to the predicted molecular
masses of 48 kDa for Pax-2A, 46 kDa for Pax-2B, 33 kDa for trPax-2A,
and 31 kDa for trPax-2B within their respective protein extract. No
immunoreactive bands were present in mock-, Bst-, or
pS1-transfected COS-7 cells. Pax-2 protein was immunoaffinity-purified
from embryonic day 11 whole mouse nuclear extracts as
described(15) . The presence and relative size of the Pax-2
protein within the purified sample was confirmed by Western blot
analysis using anti-Pax-2 antibodies(15) . Chloramphenicol
acetyltransferase (CAT) assays were performed as described by Gorman et al.(32) .
Double-stranded
H2A 2.2 and Tg oligonucleotides (25-100 ng) were end-labeled with
T4 polynucleotide kinase and [P]ATP (3000
Ci/mmol). Double-stranded P2BS1 and P2BS2 oligonucleotides
(25-100 ng) and gel-purified DNA fragments were labeled with
Klenow enzyme and [
P]dCTP or
[
P]dATP (3000 Ci/mmol). Binding reactions were
performed in a total volume of 10 µl at 4 °C for 30 min and
contained 2-3 µl of immunoaffinity-purified Pax-2 protein or
an empirically determined amount of purified bacterial Pax-2 fusion
protein, 100 ng of poly(dI
dC),
P-labeled probe
(10,000-40,000 cpm), 0.5 mg/ml BSA, and Z-buffer. Binding
reactions containing protein extracts prepared from transfected COS-7
cells were performed in a total volume of 15 µl at room temperature
for 30 min and contained 5 µl of protein extract, 1 µg of
poly(dI
dC), binding buffer (15 mM Tris, pH 7.5, 90
mM KCl, 0.7 mM EDTA, 0.2 mM DTT, 1 mg/ml
BSA, 6.5% glycerol), and
P-labeled probe. For competition
experiments, the purified bacterial Pax-2 protein was preincubated with
the indicated weight excess of unlabeled DNA or poly(dI
dC) for 10
min before adding the
P labeled probe. For antibody
supershift assays, protein A-purified rabbit anti-Pax-2 IgG or rabbit
anti-laminin IgG were incubated at 80 µg/ml with purified bacterial
Pax-2 protein, 500 ng of poly(dI
dC), and
P-labeled
probe. The laminin polyclonal antibody reacts with all three laminin
chains and was kindly provided by H. Kleinman. Free DNA and DNA-protein
complexes were resolved at room temperature on 6% neutral
polyacrylamide gels in 0.5
TBE at 150 V.
Figure 1: Properties of bacterial and eukaryotic Pax-2 proteins. A, schematic of the Pax-2 proteins used for constituting bacterial fusion proteins and for transfection of COS-7 cells. Alternative splicing of the murine Pax-2 gene generates at least two proteins, Pax-2A and Pax2B, which differ by 23 amino acids. Both forms of the Pax-2 protein contain a paired domain (striped box), an octapeptide domain (solid box), the first helix of the paired homeodomain (checkered box), and a serine/threonine/tyrosine-rich carboxyl terminus. B and C, gel motility assays using the H2A 2.2 sequence (B) or the Tg sequence (C) as probes bound to recombinant Pax-2 proteins (as indicated) or immunopurified Pax-2 (mPax-2). D and E, gel motility assay using H2A 2.2 (D) or Tg (E) probes bound to whole cell extracts from transfected COS-7 cells as indicated.
As expected, recombinant Pax-2A, Pax-2B, and Pax-2 (PD) fusion proteins purified from E. coli bound to the H2A 2.2 and Tg sequences (Fig. 1, B and C). Consistent with the smaller size of the Pax-2 (PD) protein, the migration of the Pax-2 (PD)-DNA complex was faster than the migration of the Pax-2A and Pax-2B-DNA complexes. The presence of Pax-2A and Pax-2B protein within the retarded protein-DNA complexes was confirmed by gel mobility supershift assays using anti-Pax-2 antibodies (data not shown). There were no retarded protein-DNA complexes when the DNA probes were incubated with BSA or bacterial control proteins. To confirm these results, Pax-2A and Pax-2B proteins transiently expressed in COS-7 cells and immunoaffinity-purified mouse Pax-2 protein from nuclear extracts (mPax-2) also bound to the H2A 2.2 and Tg paired domain recognition sequences (Fig. 1, D and E). Consistent with the smaller size of the truncated Pax-2 proteins (trPax-2), the migration of the trPax-2 protein-DNA complexes were faster than the migration of the full-length Pax-2 protein-DNA complexes. Band shifts corresponding to Pax-2 protein-DNA complexes were not present when the probes were incubated with extracts prepared from mock- or Bst-transfected COS-7 cells. Finally, the bacterially expressed Pax-2A and Pax-2B protein-DNA complexes co-migrated with the mPax-2 shifted band (Fig. 1, B and C). These results demonstrate that the Pax-2 paired domain is sufficient for DNA binding, that both forms of the Pax-2 protein can independently bind DNA, and that bacterially expressed Pax-2 binds with similar properties as Pax-2 purified from embryonic nuclear extracts.
Immunopurified DNA clones were screened for the presence of a Pax-2 binding site by gel shift assays using recombinant Pax-2 proteins produced in E. coli. Plasmids containing DNA inserts were pooled together into groups of 3-5 plasmids such that the sizes of the DNA inserts within a group were different. The insert DNA was released from the Bst plasmid by endonuclease digestion and end-labeled, and the pooled clones were subjected to gel mobility shift assays in the presence of BSA or partially purified bacterially expressed Pax-2 protein (Fig. 2). Two Pax-2-enriched clones from the first experiment, four Pax-2-enriched clones from the second experiment, and one control clone displayed a retarded complex in the presence of Pax-2 protein. Further gel shift analysis of clone 2-10 and control clone 3-1, which were repetitive sequences, revealed that a bacterial protein present within the partially purified Pax-2 protein sample and not Pax-2 was binding to these sequences (data not shown). The binding of Pax-2 protein to four of the remaining Pax-2-enriched clones 2-4, 2-5, 2-6, and 2-17 was characterized in more detail.
Figure 2: Primary screening of immunopurified DNA clones for Pax-2 binding. The 10 control clones from the second chromatin immunoprecipitation experiment were pooled together into three groups, Cl through C3. The 18 Pax-2-enriched clones from the first experiment and the 40 Pax-2-enriched clones from the second experiment were pooled together into 16 groups, A4-A15 and A44-A47. The DNA inserts were analyzed by gel mobility shift assay using either bacterially expressed Pax-2A (A) or Pax-2B (B) protein. As a negative control, the pooled DNA sequences were incubated with BSA(-). Free probe and protein-DNA complexes were resolved on a 40-cm 6% neutral polyacrylamide gel. DNA inserts that displayed a band shift are indicated. a, control clone 3-1; b, clone 2-10; c, clone 2-5; d, clone 2-6; e, clone 2-17; f, clone 2-4; g = clone 1-3.
The location and sequence of the Pax-2 binding site within clone 2-6 and 2-17 were determined by DNase I footprinting assays using recombinant Pax-2 proteins (Fig. 3). For 2-6, two protected regions spanning nucleotides 1-34 (box a) and 189-220 (box b) were evident at increasing concentrations of Pax-2B (Fig. 3A). These protected regions were not present when the DNA was incubated with BSA or control proteins. There was no evidence of Pax-2 binding in the region spanning nucleotides 250-620 (Fig. 3B). This was consistent with the observation that a restriction fragment spanning nucleotides 370-620 did not display a band shift in gel shift assays (data not shown). For clone 2-17, one protected region spanning nucleotides 76-103 was evident at increasing concentrations of Pax-2A protein (Fig. 3C). This protected region was not present when the DNA was incubated with BSA or control proteins.
Figure 3: DNase I footprinting analysis of clone 2-6 and 2-17 sequences. The 620-base pair insert of clone 2-6 was independently labeled at nucleotide 1 (A) or nucleotide 620 (B) and subjected to DNase I footprinting analysis using recombinant Pax-2B protein produced in E. coli. As a negative control, the DNA sequences were incubated with BSA and control proteins. A, two protected regions spanning nucleotides 1-34 (box a) and 189-220 (box b) were present at increasing concentrations of Pax-2B protein but not BSA or control proteins. B, there were no protected regions spanning nucleotides 250-620 of clone 2-6. C, clone 2-17 was end-labeled and subjected to DNase I footprinting analysis using Pax-2A protein. A protected region (box) spanning nucleotides 76-103 was present at increasing concentrations of Pax-2A but not BSA or control proteins. Guanine (G) marker is a Maxam and Gilbert sequencing ladder.
Figure 4: Gel mobility supershift assays of the P2BS1 and P2BS2 DNA-protein complexes. Recombinant Pax-2 fusion proteins made in E. coli were subjected to gel shift analysis using the P2BS1 (A) and P2BS2 (B) sequences in the presence of no antibody, anti-Pax-2 antibody, or anti-laminin antibody. As a negative control, the DNA sequences were incubated with BSA or control proteins. Free probe and Pax-2 protein-DNA complexes are indicated. Arrows highlight precipitation of antibody-protein-DNA complexes in the wells of the gel and supershifted complexes.
The ability of Pax-2 protein produced in eukaryotic cells to bind to the P2BS1 and P2BS2 sequences was also examined (Fig. 5). Gel mobility shifts were evident when P2BS1 (Fig. 5A) and P2BS2 (Fig. 5B) were incubated with full-length and truncated forms of the Pax-2 protein transiently expressed in COS-7 cells. These retarded complexes were not present when the probes were incubated with extracts prepared from mock- or Bst-transfected COS-7 cells. The same protein/DNA complex was also present when P2BS2 and a restriction fragment spanning nucleotides 189-276 of clone 2-6 (data not shown) were incubated with mPax-2.
Figure 5: Binding of Pax-2 protein expressed in eukaryotic cells to the P2BS1 and P2BS2 sequences. P2BS1 (A) and P2BS2 (B) sequences were analyzed by gel mobility shift assay using Pax-2 protein produced in COS-7 cells. As a control, the DNA sequences were incubated with extracts prepared from mock- and Bst-transfected COS-7 cells. Background complexes present when the DNA probes were incubated with the various pS1-Pax-2 extracts as well as the mock and Bst extracts did not contain Pax-2 protein, as their migration remained the same regardless of which COS-7 extract was used.
To test for specificity
of Pax-2 binding, the P2BS1 and P2BS2 sequences were incubated in the
presence of increasing amounts of unlabeled competitor DNA (Fig. 6). In the absence of competitor DNA, band shifts were
present when Pax-2A and Pax-2B protein were incubated with the P2BS1 (Fig. 6A) and P2BS2 (Fig. 6B)
sequences. The observed Pax-2A and Pax-2B protein-DNA complexes were
competed for completely by the addition of a 100-fold molar excess of
unlabeled P2BS1 and P2BS2 oligonucleotides. In contrast, increasing
amounts of the nonspecific competitor poly(dIdC) did not reduce
the amount of Pax-2 protein-DNA complex formation. These results
confirmed that Pax-2A and Pax-2B were specifically binding to the P2BS1
and P2BS2 sequences.
Figure 6: Competition experiments with P2BS1 and P2BS2. Recombinant Pax-2 proteins produced in E. coli were subjected to gel shift analysis using the P2BS1 (A) and P2BS2 (B) sequences in the absence(-) or presence of the indicated fold excess of cold competitor. Pax-2 protein-DNA complexes are indicated.
To confirm that a Pax-2 DNA recognition
sequence was present within clone 2-17, a restriction fragment spanning
nucleotides 55-106 (P2BS3) was subjected to gel shift assays (Fig. 7). Band shifts were present when this DNA probe was
incubated with full-length Pax-2 proteins expressed in bacteria (Fig. 7A) and eukaryotic cells (Fig. 7B). Full-length Pax-2 protein-DNA complexes were
not present when the DNA probe was incubated with BSA, control, mock,
or Bst protein samples. pS1-trPax-2A and pS1-trPax-2B
protein-DNA complexes could not be definitively identified, most likely
due to their comigration with other protein-DNA complexes (Fig. 7B, arrow). Protein-DNA complex I did
not contain Pax-2 protein, as it was not supershifted with anti-Pax-2
antibodies (data not shown), the addition of the H2A 2.2
oligonucleotide to the binding reaction did not reduce complex I
formation (Fig. 7C), and the addition of cold clone
2-10 sequences to the binding reaction reduced the formation of complex
I but did not affect the binding of Pax-2B (Fig. 7).
Furthermore, following the addition of cold clone 2-10 sequences to the
binding reaction, the Pax-2 (PD) protein-DNA complex was more visible
(data not shown). The specificity of Pax-2 binding to this sequence was
examined by competition gel mobility shift assays using bacterially
expressed Pax-2B protein that was preincubated with a 300-fold molar
excess of cold clone 2-10 sequences. A 100-fold excess of the
nonspecific competitor (poly(dIdC)) did not interfere with the
binding of Pax-2B to the DNA probe, whereas a 100-fold excess of the
specific (2-17 or H2A 2.2) dramatically decreased Pax-2B protein-DNA
complex formation (Fig. 7C). These results confirmed
that at least one specific Pax-2 binding site was present within clone
2-17 between nucleotides 55 and 106.
Figure 7: Sequence-specific binding of Pax-2 protein to clone 2-17 sequences. A gel-purified restriction fragment spanning nucleotides 55-106 of clone 2-17 was subjected to gel mobility shift analysis with bacterially expressed Pax-2 proteins (A and C), mPax-2 (A), and Pax-2 protein produced in COS-7 cells (B). Arrow in B highlights the proposed migration of trPax-2 protein-DNA complexes with another non Pax-2-containing protein-DNA complex. C, competition experiment with 100-fold molar excess of 2-17 or H2A 2.2. All lanes contain a 300-fold molar excess of unlabeled clone 2-10 sequences to compete out non-Pax-2 proteins that may also react with 2-17.
The nucleotide sequences of the three Pax-2 binding sites characterized in this report are compared in Fig. 8. There is significant homology over a 23-nucleotide stretch that spans the binding region. The previously reported consensus sequence derived by PCR amplification of random oligonucleotides bound to the Pax-2 paired domain also exhibits homology to the P2BS site, particularly the TCA nucleotide motif at the 5` end and the AC nucleotides at the 3` end. The Pax-5 binding sequence is more loosely defined (12) does share features with the Pax-2 consensus consistent with the high level of homology between the Pax-2 and Pax-5 paired domains.
Figure 8: Nucleotide sequence alignment of Pax-2 binding sites. Capital letters highlight nucleotides common to at least two of the Pax-2 binding sites identified in this report. The Pax-2 consensus DNA recognition sequence derived from P2BS1, P2BS2, and P2BS3 is aligned with the Pax-2 paired domain consensus sequence derived by PCR screening(21) , the Pax-5 consensus sequence derived from several in vivo DNA recognition sequences(12) , and the consensus DNA recognition sequence for the paired domain of Drosophila Prd(38) . Capital letters highlight nucleotides common to these three consensus sequences. Y = T or C, S = C or G, R = A or G, and K = G or T.
Figure 9: Relative binding of Pax-2, Pax-5, and Pax-8 to P2BS1 and P2BS2. A, Western blot of whole cell lysates from transfected cells probed with antibodies against Pax-2. Equal amounts of protein were loaded and transfection efficiencies were similar, based on CAT assays of an RSVCAT internal standard. B, gel mobility shift assays using the Pax-2 DNA binding sites P2BS1 and P2BS2 and the Pax-5 binding site H2A 2.2(33) . Proteins are whole cell lysates from cells transfected with the indicated expression plasmids.
The transfected cell lysates were also used to determine if Pax-5 and Pax-8 bound the Pax-2 binding sites. Both Pax-2 and Pax-5 bind the H2A 2.2 sequence with near-equal affinity, while Pax-8 binding is detectable but reduced (Fig. 9B). However, both the P2BS1 and P2BS2 sites bind preferentially to Pax-2, with less Pax-5 binding and no detectable Pax-8 binding activity (Fig. 9B). The homology within the paired domains of Pax-2 and Pax-5 is 97%. Nevertheless, the 3 amino acid substitutions appear to reduce the binding efficiency of Pax-5 to P2BS1 and P2BS2. The Pax-8 paired domain is 92% homologous to Pax-2 and binding is severely reduced, with little detectable complexes observed in the gel shift assay using P2BS1 and P2BS2. Thus, the binding sites identified in this report bind more efficiently to Pax-2 than the closely related proteins Pax-5 and Pax-8.
The identification of target DNA binding sites and associated genes is a necessary step toward elucidating the molecular mechanisms underlying Pax-2 function during development. We have demonstrated the feasibility of using specific antibodies and a functional DNA binding assay to screen for Pax-2 binding sites within the genome of expressing cells. Chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and the enriched DNA cloned. Two unique immunoprecipitated DNA clones containing three Pax-2 binding sites were identified and confirmed by gel mobility shift assays using immunopurified Pax-2 and Pax-2 transiently produced in COS-7 cells. Specificity of binding was further confirmed by competition gel mobility shift assays and supershift assays using antibodies specific for the Pax-2 protein. The in vitro DNA binding data strongly suggest that these binding sites represent biologically relevant Pax-2 recognition sequences. A comparison of the three Pax-2 binding sites revealed a 23-base pair region that displays extensive homology. The Pax-2 consensus sequence derived from these three binding sites is similar to the previous Pax-2 (21) and Pax-5 (12) consensus recognition sequences. It is striking that Pax binding sites reflect an unusually high level of divergence making consensus site calculation difficult. This may be in part due to the large region of contact. The presence of a helix-turn-helix homeo domain at the COOH-terminal end of some Pax proteins (Pax-3, Pax-7, Prd) may also introduce additional specificity by stabilizing protein-DNA interactions through contact of neighboring homeodomain recognition sites(35, 36) .
To study the DNA binding properties of the Pax-2 protein, recombinant Pax-2A, Pax-2B, and Pax-2 paired domain proteins, purified from bacterial cells, were compared to Pax-2 proteins synthesized by transfected eukaryotic cells and to immunoaffinity-purified Pax-2 from neural tube nuclear extracts. Regardless of the source, all Pax-2 proteins, including the paired domain only truncation, specifically bound to the H2A 2.2 and Tg recognition sequences. A lack of post-translational modifications and the low renaturation efficiency of denatured proteins to a biologically active form are problems associated with the production of recombinant proteins in E. coli(37) . The isolation of Pax-2 protein from an endogenous source ensures that post-translational modification and folding of the protein is correct. In addition, any associated proteins that are needed for or enhance binding could co-purify along with the Pax-2 protein. The different isoforms and truncated forms of Pax-2 were also expressed in the COS-7 eukaryotic cell line. The binding of all three protein sources to the H2A 2.2 and Tg paired domain DNA recognition sequences demonstrates that Pax-2 does not require specific post-translational modifications or cofactors for binding in vitro. In addition, the comigration of immunoaffinity-purified Pax-2 protein-DNA complexes with the bacterially expressed Pax-2 protein-DNA complexes suggest that Pax-2 is not binding to these sequences as a complex of proteins. Finally, the DNA binding ability of the bacterially expressed Pax-2 proteins demonstrates that the DNA binding domain of the proteins can properly refold following purification under denaturing conditions. However, the large molar excess of bacterially expressed proteins within the DNA binding assays suggest that only a small fraction of the solubilized protein is biologically active and/or that the lack of post-translational modifications affects the DNA binding affinity of these proteins. Despite these problems, the bacterially expressed Pax-2 proteins served as an abundant source of purified biologically active Pax-2 protein.
Alternative splicing of transcription factor gene transcripts can generate protein variants with diverse function(38, 39) . For example, alternative splicing of the Pax-6 gene in vertebrates gives rise to a Pax-6 isoform with a 14-amino acid insertion in the paired domain that results in a dramatically altered DNA binding specificity (40) . This alternatively spliced Pax-6 exon is lacking in the Drosophila(41) and sea urchin Pax-6 genes(42) . In addition, alternative splicing of the murine and human Pax-8 gene gives rise to several Pax-8 isoforms that differ in their carboxyl-terminal regions downstream of the paired domain(43, 44) . The DNA binding and transactivation properties of the Pax-8a and Pax-8b isoforms are similar to each other but differ from Pax-8c and Pax-8d. The results presented in this paper demonstrate that the Pax-2A and Pax-2B proteins have similar DNA binding specificity in vitro. In addition, truncated forms of the Pax-2 protein did not exhibit an altered DNA binding specificity compared to full-length Pax-2 proteins, which suggests that binding specificity is determined by the paired domain and that the 23-amino acid insertion does not affect the DNA binding properties of the Pax-2 protein. In addition, similar to Pax-8a and Pax-8b, there is evidence that Pax-2A and Pax-2B are transcriptional activators in cell culture assays(22) . Moreover, in zebrafish both the length and sequence of the insertion are different from that of mouse Pax-2(45) . The analysis of this alternatively spliced Pax-2 exon from other species may help define the functional properties of the Pax-2A and Pax-2B proteins. Finally, a second alternative splice site has been identified in the murine and human Pax-2 gene that generates a protein with a serine/threonine/proline-rich COOH terminus(46) . The DNA binding and transactivation properties of the resulting proteins are currently unknown.
Several lines of evidence suggest that Pax-2, Pax-5, and Pax-8 subfamily may serve the same redundant function in the neural tube where they are coexpressed in similar dorso-ventral patterns(3) . The binding of the Pax-2 protein to Pax-5 (H2A 2.2) and Pax-8 (Tg) DNA recognition sequence and the ability of Pax-2 and Pax-8 to activate transcription from the same reporter construct in transient assays (43) support this hypothesis. Alternative splicing of Pax-2 and Pax-8 generates two proteins, Pax-2B and Pax-8b, which are collinear with Pax-5 along the less conserved COOH-terminal region of the proteins. Finally, Pax-5 mutant mice do not display any obvious neural tube abnormalities posterior to the hindbrain(10) . It would be interesting to determine if Pax-2 or Pax-8, which are expressed at the midbrain-hindbrain junction, can rescue the abnormal development of the posterior midbrain and anterior cerebellum of Pax-5 mutant mice. Despite the high degree of homology within their paired domains, the binding sites identified in this report were able to distinguish among the Pax-2, 5 and 8 proteins. Pax-2 bound the P2BS1 and P2BS2 sequences with higher efficiency, compared to Pax-5 and Pax-8.
The sites identified in this report probably represent only a
small fraction of total available Pax-2 binding sites, and their
biological relevance remains to be determined. The Pax-2 binding sites
present in clones 2-6 and 2-17 were not able to confer Pax-2-dependent transcription activation in a transient
transfection assay of P19 embryonal carcinoma cells. The Pax-2 binding
regions were cloned upstream of the herpes simplex virus thymidine
kinase promoter and the CAT gene as a reporter construct. The reporter
was co-transfected with increasing amounts of Pax-2 expressing plasmid.
However, increasing in CAT activity was not observed using either DNAs
from clone 2-6 or 2-17. One possible explanation for these results is
that specific cofactors required for Pax-2 trans-regulation of the
immunopurified binding sites are lacking in P19 cells, as
transactivation is dependent upon the cell
line(22, 43) . Furthermore, Pax-2-dependent
transactivation in tissue culture requires multimerized binding sites,
usually six, upstream of a basal level promoter. These multiple Pax
binding sites are not found in genes known to be regulated by Pax-8(29) or Pax-6(27, 28) . However,
it cannot be ruled out that the orientation and/or accessibility of the
binding site(s) required for Pax-2 transregulation in these
assays is sub-optimal. Pax protein function may also depend on the
context of the recognition sequence. For example, in developing
B-lymphocytes, the Pax-5 protein acts as a transcriptional activator of
the CD19 and VpreBl genes, but is a repressor of the
Ig 3`
enhancer(23, 24, 25, 26) . These
results suggest that transcriptional regulation by the Pax proteins is
complex and may be modulated by interactions with other proteins. It is
also possible that Pax-2 may be acting as a repressor when bound to
these sites by sterically hindering activating factors from binding
either directly or by changing the conformation of adjacent DNA
sequences(47) . That the Pax-5 binding sites within the switch
regions of the immunoglobulin heavy chain gene locus appear to function
in heavy chain switch recombination suggest additional functions for
Pax proteins independent of transcription
regulation(48, 49, 50, 51) . In
addition, the Drosophila homeodomain-containing proteins, Even
Skipped and Fushi Tarazu, bind at low but significant levels to genes
that they are not expected to regulate(52) . Future studies
aimed at the identification of adjacent genes may elucidate the
biological significance of these Pax-2 binding sites.
Several methods have been used for the isolation and identification of downstream target genes for transcription factors, one of which is the chromatin immunoprecipitation technique. This technique can enrich for genomic fragments that are bound by the protein of interest in vivo. The restriction enzyme, Sau3A, was used to digest the chromatin in intact nuclei. Thus, only accessible chromatin will be cut leaving much of the transcriptionally inactive chromatin too large to diffuse out of the nuclei. The low salt elution conditions favor the maintenance of protein-DNA complexes during immunoprecipitation with specific antibodies(30) . Target genes for the Drosophila Ultrabithorax homeodomain-containing protein (53, 54) and the thyroid hormone receptor have been identified through this technique(55) . While the immunoprecipitation conditions favor the maintenance of protein-DNA complex associations, the stability of the complexes during the procedure has not been directly examined. To preserve protein-DNA complexes during immunopurification, the proteins can be cross-linked to the DNA with ultraviolet light or chemically before immunoprecipitation. Using cross-linked protein-DNA complexes, in vivo target genes have been immunopurified for the Ultrabithorax protein (56) and the mammalian Hox-C8 homeodomain-containing protein (57) and in vivo DNA target sequences have been immunopurified for the Drosophila Polycomb protein(58) . Approximately 10 fold enrichment for specific in vivo binding sites have been obtained through the noncross-linked procedure compared to over 100 fold enrichment for the chemically cross-linked technique. However, the cloning efficiency of the cross-linked immunopurified DNA is poor. In conclusion, through the identification of Pax-2 DNA recognition sequences, these studies have begun to address the function of Pax-2 during spinal cord development. Genomic DNA clones 2-6 and 2-17, which contain Pax-2 binding sites, can ultimately be used as molecular tags to identify nearby genes that may be regulated by Pax-2.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U46547 [GenBank]and U46548[GenBank].