From the Department of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway
Received for publication, September 28, 2000
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ABSTRACT |
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The Pax6 genes encode evolutionary
conserved transcription factors that act high up in the regulatory
hierarchy controlling development of central organs such as the eyes
and the central nervous system. These proteins contain two DNA-binding
domains. The N-terminal paired domain is separated from a paired-type
homeodomain by a linker region, and a transactivation domain is located
C-terminal to the homeodomain. Vertebrate Pax6 genes
express a paired-less isoform of Pax6 (Pax6 Pax6 is a member of the evolutionary conserved Pax family of
transcription factors, which plays pivotal roles during vertebrate development (1, 2). In addition to the paired domain
(PD),1 encoded by the paired
box, Pax6 contains another DNA binding domain, the paired-type
homeodomain (HD). The C-terminal part of the protein is rich in
proline, serine, and threonine (PST), and functions as a
transcriptional activation domain (3-7). The mouse and human Pax6
proteins have an overall amino acid sequence identity of 100%, with
zebrafish Pax6 being about 97% identical to the mammalian proteins
(reviewed in Ref. 8). The PD and the HD are more than 90% conserved in
the invertebrate proteins compared with the mammalian ones. In
vertebrates, Pax6 is expressed in the developing nervous
system, in the neuroretina and lens of the eye, in the nasal placode,
and in the pancreas (reviewed in Ref. 8). Due to a gene duplication
during insect evolution, Drosophila contains two
Pax6 homologs, eyeless (ey) and
twin of eyeless (toy). Toy regulates an
eye-specific enhancer of ey and thus acts above Ey in
activating the eye developmental program (9). Both these genes as well
as mouse and zebrafish Pax6 induce ectopic eyes when
overexpressed in the Drosophila embryo, indicating that the
Pax6 proteins have not only a conserved structure but also a conserved
function (9-11). Pax6 can also induce ectopic eyes in
Xenopus embryos in a cell-autonomous manner (12). Mutations in Pax6 cause the small eye phenotype in mice and
aniridia and Peters anomaly in humans (13-15). Studies of transgenic
mice with homozygous targeted disruption of either the LIM homeodomain
protein Lhx2 or the paired-type homeodomain proteins Rax/Rx and Hesx1 have revealed that, in addition to Pax6, also loss of
function of these homeobox genes lead to anophthalmic (eyeless) embryos (16-19). The Rax protein contains a paired-like HD but no other domains with sequence homology to Pax6. Rax is, similar to
Pax6, expressed in the developing forebrain and neuroretina
(17, 18, 20) of mouse, Xenopus, and zebrafish.
There are several reported isoforms of the Pax6 protein. One of these,
Pax6-5a, has a 14-amino acid insertion in the N-terminal subunit of the
PD generating an altered DNA-binding specificity (21). This isoform is
vertebrate-specific, since it is not found in invertebrates. Several
additional isoforms of Pax6 have been reported in quail and bovine
retina (22, 23). One of these is a 33/32-kDa isoform that lacks the PD
due to the use of an alternative start codon for translation in the
sequence between the PD and the HD (22). Interestingly, in C. elegans there are two transcripts of the Pax6 gene, one
that encodes the full-length protein and one initiated at an internal
promoter producing a transcript that does not encode the PD (24).
Together with the fact that there exist natural "P3" HD binding
sites in promoters of many retina genes (25), this indicates that the
paired-less Pax6 isoform may play an important role in vivo
during development and/or in the adult organism.
The paired-type HD in Pax proteins is distinguished from other HDs by
having a serine in position 50 instead of a glutamine or a lysine. The
paired-type HDs are able to bind cooperatively to palindromic DNA
sequences of the type TAATN2-3ATTA, named P2 or P3 after
the number of base pairs separating the two TAAT/ATTA palindromic core
sequences (26). The HD of Pax6 is shown to bind preferentially to P3
sites (4). In contrast to the cooperative DNA binding/dimerization of
other HDs (e.g. Hox), which relies on sequences extrinsic to
the HD, the paired class HDs can rely entirely on the 60-amino
acid-long HD to achieve cooperativity upon DNA binding (26).
Given the very conserved three-dimensional globular structure of HDs
(25, 27-30), it seems reasonable that this structure is evolutionarily
conserved not only for the purpose of DNA binding but also for
preserving protein-protein contacts. Hence, the PD and HD of
Drosophila Prd can bind cooperatively to DNA when present in
the same or different peptides (31). Moreover, upon binding to DNA, the
PD of Prd can interact with other paired-type HDs, and the same is true
for the Prd HD regarding interactions with other PDs (31). Engrailed-1
(En-1) interacts with the PD of Pax6 and prevents DNA binding (32).
En-1 is not dependent on binding DNA to exert this function. Pax5 is
shown to function as a cell type-specific docking protein for an ETS
family transcription factor at the B cell-specific mb-1
promoter (33). Recruitment of Net and Elk-1, but not SAP1a, by Pax5
defines a functional difference between closely related Ets proteins.
The interactions between the Pax5 PD and the ETS domain have not been
found in the absence of DNA. Nutt et al. (34) have recently
shown that the Pax5 PD alone is sufficient for transcriptional
activation from the mb-1 promoter. The PD of Pax5 also
interacts with PU.1, and Pax5 represses the immunoglobulin Here we describe an interaction between the Pax6 protein and the
paired-less Pax6 isoform, Pax6 Plasmid Constructs--
The pCI-Pax6 and pP6CON-LUC
plasmids have been described previously (11). pCI-Pax6 Transient Transfection Assays--
For transient transfection
assays HeLa cells (ATCC CCL 2) were grown in Eagle's minimum essential
medium supplemented with 10% fetal calf serum (Hyclone), nonessential
amino acids (Life Technologies, Inc.), 2 mM
L-glutamine, penicillin (100 units/ml) and streptomycin
(100 µg/ml). About 8 × 104 HeLa cells/well in
six-well tissue culture dishes were transfected by the calcium
phosphate coprecipitation procedure using from 1 to 2.8 µg of DNA
(see figure legends for details). To normalize for variations in
transfection efficiency, 0.05 µg of pCMV- GST Pull-down Assays--
GST fusion proteins were purified from
Escherichia coli LE392 extracts using glutathione-agarose
beads (Amersham Pharmacia Biotech). The proteins were not eluted but
were left on the beads and stored at 4 °C in phosphate-buffered
saline containing 1% Triton X-100 (PBT). The protocol for GST
pull-down assays was adapted from Nead et al. (47). One µg
of pCI-neo or pcDNA3-HA plasmids containing the genes of interest
was in vitro transcribed and translated using the TNT system
(Promega) according to the manufacturer's protocol in a total volume
of 50 µl. For each pull-down experiment, 10 µl of in
vitro translated 35S-labeled protein was diluted in
200 µl of ice-cold NET-N (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% Nonidet P-40
with one protease inhibitor mixture tablet (Roche Molecular Biochemicals) per 10 ml of buffer). GST-glutathione-agarose beads were
added, and the tubes were placed on a rotating wheel at 4 °C for
15-30 min and then centrifuged at 1,600 rpm for 2 min in a
microcentrifuge. Following this preincubation step, the supernatants were transferred to new tubes. About 2 µg of fusion protein
immobilized on glutathione-agarose beads were added, and the
interactions were allowed to proceed at 4 °C for 1 h on a
rotating wheel. Both the GST beads used for preincubation and the GST
fusion protein-containing beads were washed five times with NET-N, and
as much as possible of the buffer was removed after the last wash
before the beads were boiled in 25 µl of 2× SDS-polyacrylamide gel
loading buffer. The samples were run on a 10% SDS-polyacrylamide
gel, which was vacuum-dried before 35S-labeled
proteins were detected by autoradiography and/or by the use of a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Preparation of Nuclear Extracts and Coimmunoprecipitation
Experiments--
The protocol for harvesting cells was adapted from
Nead et al. (47). The cells were trypsinized and washed
twice with 5 ml of phosphate-buffered saline. They were counted in the
last wash with phosphate-buffered saline and washed once with 0.5 ml of
buffer A (20 mM Hepes, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol) containing the following protease and phosphatase inhibitors: 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 20 mM Western Blot Analyses--
Mighty Small (Hoefer) 10%
SDS-polyacrylamide gels were blotted onto Hybond nitrocellulose
membranes (Amersham Pharmacia Biotech), and the membranes were
incubated with blocking buffer containing 5% nonfat dried milk in TBST
(10 mM Tris, pH 8.0, 150 mM NaCl, 1% Tween 20)
for 1 h at room temperature or overnight at 4 °C. The primary
antibody was diluted 1:800 for the PC6 anti-Pax6 antibody or 1:50 for
the 12CA5 anti-HA antibody in blocking buffer and incubated with the
membrane as described above. The membrane was then washed in TBST 5-6
times during 30 min before the secondary antibody (diluted 1:5000 in
blocking buffer) was applied. The secondary antibody was incubated with
the membrane for 1 h at room temperature. Anti-rabbit horseradish
peroxidase-conjugated antibody (Transduction Laboratories) was used for
the Pax6 primary antibody, whereas anti-mouse horseradish
peroxidase-conjugated antibody (Transduction Laboratories) was used for
the HA primary antibody. The membrane was washed as described above and
then incubated for 1 min in ECL solutions (Amersham Pharmacia Biotech) (1 part of each of the solutions mixed with 2 parts of water, 1:1:2).
Signals were detected by autoradiography within the next 15-20 min.
Gel Mobility Shift Assay--
Nuclear extracts were prepared as
described above and used at concentrations specified in the legend to
Fig. 6. Binding of Pax6 to the 32P-labeled P6CON probe (50)
was performed as described by Carrière et al. (3) with
the exception that 4% Ficoll was used in the binding buffer instead of
10% glycerol. The binding reactions were incubated on ice for 20 min
and then run on a 5% polyacrylamide gel in 0.5× TBE (45 mM Tris borate, 1 mM EDTA). The gel was dried, and bound probe was detected by autoradiography and/or by a
PhosphorImager (Molecular Dynamics).
The Paired-less Pax6 Isoform (Pax6
The above mentioned results could be explained if 1) the Pax6
To discriminate between 1) titration of a negatively acting factor and
2) recruitment of an additional TAD to the promoter via HD interacting
with Pax6, we asked whether the HD alone was sufficient for
superactivation. As seen from Fig.
2A, cotransfection of the Pax6
expression vector with a vector expressing only the HD of Pax6 did not
cause any superactivation. Due to its small size and absence of a
nuclear localization signal, the HD was evenly distributed between the
nucleus and the cytoplasm of the transfected cells (data not shown).
Using fusions to GFP, we have found both the full-length and the The Pax6 HD Interacts with Pax6, Pax6 The C-terminal Subdomain of the Pax6 PD Can Also Interact with the
HDs of Pax6 Pax6 Neither Pax6 The Ability to Interact with Pax6 in Vitro and to Enhance
Transactivation Is Not Limited to Paired-type HD Proteins--
Pax6 is
coexpressed with various other HD-containing proteins during
development and in the adult. To see if Pax6 can interact with other
HDs as well as with the paired-type HDs of Rax and Pax6 We have shown that the Pax6 The most surprising finding was, however, that rather divergent HDs
were able to interact with Pax6 in vitro. Several of these HD proteins containing TADs could also superactivate Pax6-mediated transactivation, strongly indicating their ability to interact also
in vivo. Our results illustrate the potential influence that different coexpressed HD transcription factors may have on
Pax6-mediated gene regulation by interacting directly with DNA-bound
Pax6 without binding to a specific DNA sequence themselves.
During the last few years, evidence indicating important roles for
homeodomains in protein-protein interactions has accumulated. A number
of proteins interacting with different DNA-bound HD proteins have been
identified (55-62). In many cases, the HD itself is necessary and
sufficient for the interactions. Hence, the cardiogenic HD protein
Nkx-2.5 interacts both with the serum response factor and with the zinc
finger transcription factor GATA-4 via its HD (58, 63). The RAG1 HD
directly interacts with both HMG boxes of HMG1 and -2 (64). Smad1
interacts with the HD of HoxC8, leading to relief of HoxC8-mediated
repression of osteopontin gene transcription (65). There are also
examples of HD proteins interacting with each other independent of DNA
binding. Oct-1 and Pit-1 associate in solution through an interaction
mediated, in part, by the POU homeodomain of Pit-1 (66). HoxB8 and Pbx1
interact in the absence of DNA (67). The interaction was dependent on
the HD and a highly conserved hexapeptide for the Hox protein and was
dependent on the HD and a region immediately C-terminal of the HD for
Pbx1. Prep1 and Pbx1 dimerize efficiently in solution independent of DNA (68). Similar to the results that we report here for Pax6, the DBD
of the glucocorticoid receptor was found to bind to all HDs from the
seven different HD proteins tested in a GST pull-down assay (69).
Expression of the glucocorticoid receptor DBD in one-cell stage
zebrafish embryos severely affected their development, while expression
of a point mutant that could no longer bind HDs developed normally
(69). As with our findings with Pax6, the extent of glucocorticoid
receptor DBD-HD binding in vivo remains to be established.
If, and to what degree, interactions occur in different tissues where
Pax6 and other in vitro interacting HD proteins are
coexpressed must be evaluated carefully for each potential interaction.
We show for the first time that the HD and the PD of Pax6 can interact
off DNA. Several previous reports show that the PD and HD can interact
with each other both intermolecularly (31) and intramolecularly (70)
while bound to DNA. The PD/HD cooperativity reported by Jun and Desplan
is not specific for domains within the same protein, since the Prd PD
can cooperatively interact with the Engrailed HD to bind to DNA. The
Pax6 PD and Pax8 PD can likewise cooperatively bind together with the
Prd HD to DNA. DNA binding by the Pax3 PD and HD is reported to be
functionally interdependent, since certain mutations in the PD affect
the DNA binding by the HD, and vice versa (71-73).
Recently, it was reported that the R26G mutation in the N-terminal
subdomain of the PD of Pax6, which abolishes DNA binding by the PD,
lead to increased DNA binding by the HD. Furthermore, the I87R mutation
in the C-terminal subdomain of the PD resulted in loss of the ability
to bind DNA via either the PD or the HD (74). The first effect suggests interactions or at least conformational changes affecting both domains.
The latter effect may be related to our finding that it is the
C-terminal subdomain of the PD that interacts with the HD. Perhaps such
an interaction could be involved in stabilizing the DNA binding
conformation of both domains. Of other reported intermolecular
interactions involving paired-type HD proteins, Phox1 interacts with
the serum response factor, and this interaction seems to apply for
other paired-type HDs as well. The products of the Drosophila
paired and orthodenticle genes can bind serum response
factor, but the more distantly related homeobox genes fushi
tarazu and Deformed can not (39, 75). Rb was recently reported to interact directly with factors containing paired-like HDs,
including Pax3 (36). The residual HD homologous sequence of Pax5
interacts with Rb and TBP (38), and the HD of Pax6 does the same (37).
The repressor protein hDaxx and the WD40 repeat protein HIRA have both
been shown to interact with the HD of Pax3 and Pax7 (76, 77). The HD
protein Cdx-2/3 was recently found to interact with both the PD and the
HD of Pax6 (78). This is completely consistent with our results for
Rax, Chx10, Pbx1, and HoxB1. As mentioned in the introduction, En-1
interacts with the PD of Pax6 (32) and Pax5, via its PD, acts as a cell
type-specific docking protein for an ETS family transcription factor at
the B cell-specific mb-1 promoter (33). In fact, the Pax5 PD
alone is sufficient for transcriptional activation from this promoter (34). By interacting with another ETS family protein, PU.1, Pax5
mediates repression of the immunoglobulin What is the biological relevance of Pax6 interaction with Pax6PD) from an internal
start codon in the coding region between the paired domain and
homeodomain. We now provide evidence for an interaction between the
full-length isoform and Pax6
PD, which enhances the transactivation
activity of Pax6 from paired domain-binding sites. The paired-like
homeodomain protein Rax behaved similarly to Pax6
PD. Both Pax6
PD
and Rax bound to the homeodomain of Pax6 in vitro in the
absence of specific DNA binding. Coimmunoprecipitation experiments
following cotransfection confirmed the existence of complexes between
Pax6 and Pax6
PD, Pax6 and Rax, and Pax6
PD and Rax in
vivo. Interestingly, the C-terminal subdomain of the paired
domain and the homeodomain can interact with each other. The paired
domain can also interact with itself. Surprisingly, GST pull-down
assays revealed that the homeodomains of such diverse proteins as
Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 could all bind to
Pax6, and several of these enhanced Pax6-mediated transactivation upon
coexpression. Since many homeodomain proteins are coexpressed with Pax6
in several tissues during development, our results indicate the
existence of novel regulatory interactions that may be important for
fine tuning of gene regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3' light
chain enhancer by targeting PU.1 function (35). The retinoblastoma
protein, Rb, was shown to bind the HD of Pax3 (36). The HD of Pax6 and the partial HD of Pax5 can bind both Rb and TBP (37, 38). Finally, the
paired-like HDs of Prd and Phox1 interact with the serum response
factor (39).
PD, which enhanced the transactivation activity of Pax6 from PD-binding sites. Rax behaved similarly to
Pax6
PD, indicating a more general function of paired-like HD
interactions in modulation of transcriptional activation. Glutathione S-transferase (GST) pull-down experiments showed that
in vitro these interactions were mediated by the paired-type
HD of Pax6 by a DNA-independent mechanism. Coimmunoprecipitation
experiments performed using extracts from transfected cells confirmed
the existence of complexes between Pax6 and Pax6
PD, Pax6 and Rax, and Pax6
PD and Rax in vivo. Deletion of the HD indicated
that other parts of Pax6 also could contribute to the interaction, and
the C-terminal subdomain of the PD was identified as another interacting domain, whereas the C-terminal transactivation
domain (TAD) by itself was unable to interact with Pax6 in GST
pull-downs. Rax was also able to interact with the Pax6 PD.
Interestingly, the PD also interacted with itself. We were surprised to
find that HDs from various other HD proteins were also able to interact with Pax6 in GST pull-down assays, indicating a more general capability of protein-protein interactions involving the HD. Several of these HD
proteins also gave superactivation of Pax6-mediated transactivation upon cotransfection.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PD was made by
deleting a NheI fragment including the paired domain from
pCI-Pax6. To construct pCI-Rax, pBSK/mouse Rax (kindly
provided by C. L. Cepko) was cut with HindIII (made
blunt) and XhoI and cloned into the NheI (made
blunt) and XhoI sites of the pCI-neo expression plasmid
(Promega). pcDNA3-HA (kindly provided by J. Moscat) contains an
influenza virus hemaglutinin (HA) epitope tag inserted into the
HindIII-EcoRI sites of pcDNA3 (Invitrogen).
HA-Pax6 was constructed from a PCR product of full-length zebrafish
Pax6.1 cDNA (primers 6.HA.5 and 6.HA.3; Table I) made blunt at the 5'-end and cut with XbaI in the 3'-end before
cloning into the EcoRV and XbaI sites of
pcDNA3-HA. The HA-Pax6
PD construct was made exactly the same way
by use of the 6
PD.HA.5 and 6.HA.3 PCR primers (Table I). The PCR
product of the coding sequence from mouse Rax cDNA was
obtained using the HA-rax.5 and HA-rax.3 primers. The PCR product was
cut with EcoRI and XhoI and inserted into the
corresponding sites of pcDNA3-HA. The constructs of Pax6 lacking
all of the homeodomain (HA-Pax6
HD) were made by use of specific
primers containing XhoI, that would provide an open reading frame upon self-ligation (Table I). HA-Pax6 was used as a template, and
primers delA and delD were used to make HA-Pax6
HD. In
vitro mutagenesis was performed according to the instruction
manual for the QuickChange site-directed mutagenesis kit (Stratagene), but XhoI was added during the digestion with
DpnI, and the PCR fragments were self-ligated before they
were transformed into bacteria. HA-Pax6
HD was used as a template and
6
PD.HA.5 and 6.HA.3 were used as primers when the HA-Pax6
PD
HD
was constructed by PCR and cloned into the EcoRV and
XbaI sites of the pcDNA3-HA vector as described above
for the same primer set. The expression construct for the Pax6 HD was
made using PCR with primers HA-6HD.5 and HA-6HD.3 followed by digestion
with EcoRI and XbaI and insertion into the
EcoRI-XbaI sites of pcDNA3-HA and pGEX-4T-3
(Amersham Pharmacia Biotech). To make vectors for expression of fusions of Pax6 or Pax6
PD to green fluorescent protein (GFP), a 1.7-kilobase pair SmaI-XbaI fragment (GFP-Pax6) or a
1.3-kilobase pair PmlI-XbaI fragment
(GFP-Pax6
PD) from HA-Pax6 was inserted into
SmaI-XbaI-cut pEGFP-C1
(CLONTECH). The homeodomains from mouse Rax (17),
mouse Chx10 (40), mouse En-1 (41), mouse Six3 (42), mouse Lhx2 (43),
human Prep1 (44), human Prox1 (45), and human HoxB1 (46) were amplified
from cDNAs using the PCR primers described in Table I. The PCR
products were then cut with EcoRI and XhoI and
cloned into the corresponding sites of pGEX-4T-3. To express Chx10 and
Lhx2 in mammalian cells, the coding regions were cloned into
EcoRI-XhoI-cut pcDNA3-HA following PCR using
primer pairs Chx10.HA5/Chx10.HA3 and Lhx2.HA5/Lhx2.HA3 (Table
I) and digestion of the PCR products with
EcoRI and XhoI. All PCRs were performed with the
Pfu polymerase according to the instructions from the manufacturer (Stratagene), and all plasmid constructs were verified by
sequencing.
Sequences of primers used for PCR
-galactosidase expressing
-galactosidase from a cytomegalovirus (CMV) promoter was
included in each transfection. Extracts were prepared 24 h following transfection using a Dual-Light luciferase and
-galactosidase reporter gene assay system (Tropix) and analyzed in a
Labsystems Luminoskan RT dual injection luminometer.
Transfections were repeated with different DNA preparations.
-glycerophosphate, 25 mM NaF, 50 µM sodium vanadate, 0.7 µg/ml pepstatin A, and 200 µM phenylmethylsulfonyl fluoride. The cells were
resuspended in 80 µl of buffer A containing 0.1% Nonidet P-40 per
107 cells, placed on ice for 10 min, vortexed briefly, and
centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was
discarded, and the pellet containing the nuclei was resuspended in 28 µl of buffer C (20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) containing the same
protease and phosphatase inhibitors as buffer A. After 15 min on ice,
the samples were vortexed briefly and centrifuged as described above.
The supernatant was transferred to a new tube containing 140 µl of
buffer D (20 mM Hepes, pH 7.9, 20% glycerol, 0.1 mM KCl, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) with
protease and phosphatase inhibitors. The protein concentration was
determined using the Bio-Rad protein assay reagent, and 60-100 µg of
nuclear extracts were used for each immunoprecipitation. Protein
A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) were washed twice
in gel shift buffer (20 mM Hepes, pH 7.9, 60 mM
KCl, 1.5 mM MgCl2, 0.2 mM EDTA) and
saturated with bovine serum albumin in gel shift buffer for 0.5-1 h on
a rotator at 4 °C. The beads were washed twice in gel shift buffer
and resuspended to a 50% gel slurry in gel shift buffer containing the
protease and phosphatase inhibitors mentioned above. Thereafter,
120-200 µg of nuclear extracts were preincubated with the beads for
15-20 min at 4 °C, before they were split into two portions and
incubated at 4 °C with either 10 µl anti-HA hybridoma supernatant,
containing the monoclonal 12CA5 antibody, per 100 µg of nuclear
extracts or 4 µl of affinity-purified PC6 polyclonal antibody
directed against the C-terminal of Pax6 (48, 49) in a total volume of
500 µl in gel shift buffer. After 2 h, 20 µl of the bovine
serum albumin-saturated protein A-Sepharose beads were added, and the
samples were left at 4 °C for 1 h. The beads were then washed
three times in gel shift buffer containing 120 mM NaCl and
boiled in 25 µl of 2× SDS-polyacrylamide gel loading buffer. The
samples were run on a 10% SDS-polyacrylamide gel (Mighty Small,
Hoefer) in a Tris-glycine buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), and Western blots were performed as
described below.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PD) and the Paired-type
Homeodomain-containing Protein Rax Are Both Able to Enhance
Transactivation by Pax6 from a Minimal Promoter Containing Consensus
Binding Sites for the Pax6 Paired Domain--
In a careful study using
antisera raised against different parts of quail Pax6, Carrière
et al. (22) identified five Pax6 isoforms expressed in quail
neuroretina. The 33- and 32-kDa isoforms lack the PD but contain
the HD and the C-terminal TAD. This is due to the use of an internal
start codon located in the linker region between the PD and HD. The
presence of two proteins of 33 and 32 kDa was explained by alternative
splicing of a TAD exon (22). For simplicity, we will refer to these
paired-less isoforms as Pax6
PD and to the major full-length Pax6
isoform as Pax6. Consistent with previous findings for quail Pax6 (22),
we observed that the Pax6
PD isoform is coexpressed with the
full-length isoform in HeLa cells transiently transfected with an
expression vector for the zebrafish Pax6.1 cDNA. This
was also the case when the full-length Pax6 cDNA was
in vitro transcribed and translated (data not shown). Since
the Pax6 and Pax6
PD isoforms are coexpressed, we were interested in
determining if they would affect each other's activities. Knowing that
Pax6 proteins with and without the paired domain bind to different DNA
sequences (4), it seems logical to assume that they may transactivate
different target genes in vivo as well as sharing some
target genes. It is also likely that the isoforms may compete for the
same set of limiting coactivators or repressors and thereby indirectly
affect each other's transactivation potential. To see if Pax6
PD
could affect the transactivation exerted by Pax6 from a consensus Pax6
paired domain-binding site, HeLa cells were transfected with increasing
amounts of expression vector for Pax6
PD and a fixed amount of Pax6
expression vector together with a luciferase reporter
(pP6CON-LUC) containing six consensus Pax6 paired
domain-binding sites upstream of the adenovirus E1b minimal promoter
(11). Interestingly, coexpression of Pax6
PD caused an increase in
Pax6-mediated transactivation from paired domain-binding sites. Thus,
there seems not to be a competition for coactivator(s) or limiting
factors in the transcriptional apparatus (e.g. squelching).
While Pax6 by itself transactivated ~6-8-fold from the P6CON binding
sites, cotransfection with 2 µg of expression plasmid for Pax6
PD
resulted in a 3 times more efficient transcriptional activation with a
~20-fold increase above the background level defined by transfection
with the empty vector control (Fig.
1A). The increased
transcriptional activation was not caused by Pax6
PD activating the
reporter by itself, since transfection with this expression construct
alone gave no activation at all.
View larger version (32K):
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Fig. 1.
Both Pax6 PD and Rax
can enhance Pax6-mediated transactivation of a minimal promoter
containing consensus Pax6 paired domain-binding sites.
A, the paired-less Pax6 isoform, Pax6
PD, enhances the
ability of full-length Pax6 to transactivate a promoter containing
paired domain-binding sites. HeLa cells were cotransfected with 0.25 µg of pCI-Pax6 and increasing amounts (0.25-2.0 µg) of
pCI-Pax6
PD or empty expression vector (pCI-neo), together with 0.5 µg of the luciferase reporter plasmid pP6CON-LUC. The
pP6CON-LUC reporter contains six consensus binding sites for
the Pax6 paired domain upstream of the adenovirus E1b minimal promoter.
Control transfections with 0.25 µg of pCI-neo and 0.5 µg of
pCI-Pax6
PD or vector control show that the empty expression vector
and pCI-Pax6
PD alone are not able to transactivate the luciferase
reporter gene. B, the paired-type HD protein Rax is also
able to enhance Pax6 transactivation of a promoter with paired
domain-binding sites. The experiments were conducted as in A
except that an expression vector for Rax (pCI-Rax) was used instead of
pCI-Pax6
PD. As seen for Pax6
PD in A, Rax alone is not
able to transactivate the P6CON-LUC reporter. C,
Pax6
PD can enhance transactivation mediated by a fusion protein
containing the Pax6 HD and TAD (amino acids 175-437) fused to the
DNA-binding domain of yeast GAL4. HeLa cells were transfected with 2 µg of reporter vector pG5E1BLUC, 2 µg of expression
vectors for GAL4 fusions, and 2 µg of expression vector for
Pax6
PD. The GAL4 DBD vector pSG424 (51) without insert and
pcDNA3-HA served as vector controls. To normalize for variations in
transfection efficiencies, 0.05 µg of pCMV-
-galactosidase was
included in all transfections, allowing measurements of
-galactosidase activities. The data are expressed as the ratio
between the measured luciferase and
-galactosidase activities with
the mean ± S.E. from transfection experiments performed in
triplicate. The data shown in A, B, and
C are representative of two other independent experiments
yielding similar results. D, schematic drawing of the
full-length Pax6 isoform, the Pax6
PD isoform, and the Rax protein
with the PD, HD, and octapeptide (OP) indicated.
PD
protein titrates a negative cofactor that interacts with the HD and/or
TAD; 2) Pax6
PD can interact directly with the Pax6 protein, thus
recruiting an additional TAD to the promoter; or 3) a direct binding of
Pax6
PD to Pax6 may induce a conformational change relieving
intramolecular repression. If the enhanced activation is dependent on
the HD, another paired-type HD protein could possibly have the same
positive effect on transcriptional activation by Pax6. Rax (also known
as Rx) is a candidate for such a positive effector. It has a
paired-type HD but displays no other sequence homology to Pax6, and it
is coexpressed with Pax6 and Pax6
PD in the neuroretina (17, 18, 20).
Indeed, when cotransfected with Pax6 Rax strongly enhanced
transcriptional activation from the P6CON binding sites (Fig.
1B). Cotransfection of 2 µg of Rax expression vector with
0.25 µg of Pax6 expression vector resulted in a 10-15-fold increase
in transactivation compared with Pax6 alone and a 30-40-fold
activation above the basal level. Thus, Rax is even more efficient than
Pax6
PD in stimulating transactivation by Pax6. Since the HD is the
only homologous sequence shared by Rax and Pax6
PD, these results
indicate that the paired-type HD is necessary for the observed
superactivation. Indeed, Pax6
PD was able to superactivate a
construct containing amino acids 175-437, which includes part of the
linker region, the HD and the TAD, of Pax6 fused to the DNA-binding
domain of yeast GAL4 (Fig. 1C). In this experimental
setting, transactivation of a luciferase reporter gene from a promoter
containing five GAL4 binding sites upstream of the TATA box from the
promoter of the adenovirus E1b gene is measured (51). A construct
containing only the Pax6 TAD fused to the GAL4 DBD was not
superactivated upon coexpression of Pax6
PD (Fig. 1C).
Thus, when the PD of Pax6 is substituted with the GAL4 DBD, the
Pax6
PD isoform causes superactivation, provided the HD is present in
the GAL4 fusion protein. This also demonstrates that the
superactivation mediated by Pax6
PD is not dependent on the presence
of the PD as the DNA-binding domain. As previously reported (6), it is
also evident from the data in Fig. 1C that the HD has an
inhibiting effect on the TAD in GAL4-Pax6
PD.
PD
isoforms of Pax6 to be strictly nuclear proteins in living cells (Fig.
2B). This is consistent with the location of a nuclear
localization signal both in the PD and directly N-terminal to the HD
(3). However, Carrière et al. (3) found the
PD
isoform also in the cytoplasm. This could be due to leakage from the
nucleus upon fixation of the cells. Alternatively, there may be
cell-specific differences in subcellular localization. To be absolutely
certain that enough of the Pax6 HD colocalized with Pax6 in the nucleus
to observe a superactivation, we also coexpressed Pax6 with a GFP-HD
fusion equipped with a strong nuclear localization signal.
Cotransfections with different amounts of this construct did not cause
any superactivation either (data not shown). Thus, although necessary
for superactivation, the HD is clearly not sufficient, suggesting that
it needs to be linked to a TAD.
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Fig. 2.
The Pax6 HD alone lacking the C-terminal
transactivation domain is not able to enhance Pax6 transactivation from
paired domain-binding sites. A, HeLa cells were
transfected as described in the legend to Fig. 1 except that increasing
amounts of an expression vector for Pax6 HD were used instead
of pCI-Pax6 PD or pCI-Rax. The data shown represent the mean ± S.E. for two independent experiments performed in triplicate.
B, both the full-length isoform and the paired-less isoform
of Pax6 are exclusively nuclear proteins. HeLa cells were transfected
with full-length Pax6 (GFP-Pax6) or Pax6
PD (GFP-Pax6
PD) fused to
green fluorescent protein (GFP), and living cells were analyzed by
fluorescence microscopy (left panels) 24 h
post-transfection. The right panels represent
phase-contrast micrographs of the same cells with "bleed-through"
from the fluorescence filter showing that only the nuclei of the cells
display green fluorescence.
PD, and Rax in
Vitro--
Pax6, Pax6
PD, and Rax all contain a paired-type HD (Fig.
1D). Furthermore, the results of the cotransfection
experiments with Pax6 and the Pax6 HD suggested that the HD is not
acting by sequestering any inhibitory factors. Thus, we performed GST pull-down experiments to determine whether the Pax6 HD fused to GST
could interact directly with in vitro translated Pax6,
Pax6
PD, and Rax proteins labeled with [35S]methionine.
Fig. 3 shows that the HD of Pax6 is
indeed capable of interacting with the full-length Pax6 isoform,
Pax6
PD, and Rax. Paired-like HDs are the only HDs that can
cooperatively dimerize on consensus palindromic
TAATN2-3ATTA sites without the need for other parts of the
protein (26). The P6CON-LUC reporter used in our
transcriptional activation assays does not contain such a consensus
palindromic site, but to be sure that the observed interactions in GST
pull-down assays are not dependent on unspecific DNA binding, these
assays were also performed in the presence of 100 µg/ml EtBr. EtBr is
known to inhibit protein-DNA interactions (52). As can be seen from
Fig. 3, the observed interactions are direct and not dependent on DNA
binding.
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Fig. 3.
The HD of Pax6 interacts with full-length
Pax6, Pax6 PD, and Rax in vitro
in a DNA-independent manner. GST pull-down assays with
the Pax6 HD fused to GST and immobilized on glutathione-agarose beads
and full-length Pax6, Pax6
PD, and Rax proteins produced by in
vitro transcription and translation in the presence of
[35S]methionine. Twenty-µl portions of the in
vitro translation reactions (of a total of 50 µl) were
preincubated with GST immobilized on glutathione-agarose beads before
incubation with the GST fusion protein containing the HD of Pax6
(GST-Pax6 HD). Both the GST beads used for preincubation and the
GST-Pax6 HD beads were washed several times before they were boiled and
run on a 10% SDS-polyacrylamide gel. The experiments were performed
with (lanes 2, 5, and 8)
and without (lanes 1, 4, and
7) 100 µg/ml EtBr to determine whether the interactions
were dependent on DNA binding. Five µl of the in vitro
translated proteins were run on the same gel to visualize the signal
from 25% of the input (lanes 3, 6,
and 9). The interactions with Pax6, Pax6
PD and Rax are
specific, since no binding was observed to GST alone (not shown)
PD and Rax--
Since the results so far indicated that
the HD was responsible for the interaction, the HD was deleted from the
full-length Pax6 isoform. This construct, HA-Pax6
HD, was then
in vitro transcribed and translated and used in a GST
pull-down assay with the Pax6 HD fused to GST. Surprisingly,
HA-Pax6
HD was still able to interact with the Pax6 HD (Fig.
4A). Consistently, transient
transfections where HA-Pax6
HD was cotransfected with Pax6
PD and
the pP6CON-LUC reporter showed that the isoform of Pax6
lacking the PD was able to superactivate Pax6
HD (Fig.
4B). GST fusions containing the HD, PD, and TAD of Pax6 were
therefore used in a GST pull-down assay to see if domains other than
the HD would make contact with full-length Pax6. The results clearly
showed that the PD but not the TAD was able to do this (Fig.
4C). Within the PD, the C-terminal subdomain (PD-C)
displayed a significant interaction, while the N-terminal subdomain did
not (Fig. 4D). This was also the case for Pax6
HD and
Pax6
PD. Thus, interactions both between the PD (Pax6
HD) and
between the HD (Pax6
PD) with the C-terminal subdomain of the PD were
observed, suggesting that in full-length Pax6 both domains could in
principle contribute to this interaction. However, when both the PD and
the HD were missing from the Pax6 protein (HA-Pax6
PD
HD), this
construct was neither capable of superactivating Pax6 when
cotransfected nor able to interact with the Pax6 PD or HD in GST
pull-down assays (Fig. 4, E and F, respectively). We also found that the isolated Pax6 HD and PD fused to GST both interacted strongly with in vitro translated Rax (Fig.
4G). Taken together, these results show that the paired-type
HD of Pax6 and Rax can interact with each other/themselves and with the
C-terminal subdomain of the PD. In addition, the PD can also interact
with itself.
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Fig. 4.
Both the C-terminal subdomain of the Pax6
paired domain and the homeodomain are involved in protein-protein
interactions. A, the Pax6 HD can bind to a Pax6 protein
with the HD deleted. GST pull-down assay, performed as described in the
legend to Fig. 3, showing that in vitro translated Pax6 HD
binds to a GST-Pax6HD fusion protein immobilized on glutathione-agarose
beads. B, Pax6
PD can enhance transactivation by a Pax6
protein lacking the HD. HeLa cells were transfected with 0.25 µg of
pcDNA3-HA or HA-Pax6 or HA-Pax6
HD and 0.5 µg of pCI-Pax6
PD
or empty expression vector (pCI-neo), together with 0.5 µg of the
luciferase reporter plasmid pP6CON-LUC and 0.05 µg of
pCMV-
-galactosidase. C, the full-length isoform of Pax6
binds to both the PD and the HD but not to the TAD in vitro.
D, the C-terminal subdomain (PD-C), but not the N-terminal
subdomain (PD-N), of the paired domain can interact with full-length
Pax6 in a GST pull-down assay (upper panel). Such
an interaction can be mediated by both the PD (HA-Pax6
HD;
middle panel) and the HD (Ha-Pax6
PD;
lower panel) of Pax6. E, a deletion
mutant of Pax6 lacking both the PD and the HD (Pax6
PD
HD) cannot
enhance Pax6-mediated transactivation of the pP6CON-LUC
reporter. HeLa cells were transfected with 0.25 µg of pCI-neo or
pCI-Pax6 and 0.75 µg of HA-Pax6
PD, HA-Pax6
PD
HD, or empty
expression vector (pcDNA3-HA), together with 0.5 µg of the
luciferase reporter plasmid pP6CON-LUC and 0.05 µg of
pCMV-
-galactosidase. F, the deletion mutant of Pax6
lacking both the PD and the HD (Pax6
PD
HD) cannot bind to either
the HD or the PD. G, Rax binds to both the HD and the PD
in vitro. A GST pull-down assay is shown, in which
the HD, PD, and PD-linker-HD from Pax6 fused to GST on
glutathione-agarose beads are shown to bind in vitro
translated Rax. H, schematic drawing of the full-length and
Pax6
PD isoforms and the deletion mutants Pax6
HD and
Pax6
PD
HD. The latter retains only the linker region and the TAD.
The GST pull-down assays (A, C, D,
F, and G) were performed as described in the
legend to Fig. 3. Similar amounts of GST fusion proteins immobilized on
beads were used in all experiments. The reporter gene assays
(B and E) were performed essentially as described
in the legend to Fig. 1, and the data shown are expressed as the ratio
between the measured luciferase and
-galactosidase activities with
the mean ± S.E. from transfection experiments performed in
triplicate. The results are representative of two other independent
experiments yielding similar results.
PD and Rax Form Protein Complexes with Full-length Pax6 in
Vivo--
To see if the interaction observed in vitro in
GST pull-down assays could be observed in vivo, HeLa cells
were transfected with Pax6 alone or Pax6 in combination with either HA
epitope-tagged Pax6
PD or Rax. A polyclonal affinity-purified
antibody against the C terminus of Pax6 effectively
coimmunoprecipitated HA-Rax, as seen on a Western blot stained with the
anti-HA antibody (Fig. 5A,
lane 3). The anti-Pax6 antibody does not by itself
precipitate the HA-Rax protein (data not shown). The same nuclear
extracts were used for immunoprecipitations with the HA antibody, with the purpose of detecting coimmunoprecipitated Pax6 proteins on Western
blots using the anti-Pax6 antibody. However, a strong band of about 50 kDa was detected in all lanes of these blots, corresponding to the Ig
heavy chain of the HA antibody, making it impossible to detect the
48-kDa Pax6 protein (data not shown). To avoid this problem, we
transfected the paired-less isoform of Pax6, Pax6
PD, with HA-Pax6 or
HA-Rax. As seen in Fig. 5B, the anti-Pax6 antibody was
equally efficient this time in coimmunoprecipitating the HA
epitope-tagged Rax protein (lane 3). This proves
that the paired domain of Pax6 is not required for the interaction with Rax, since Rax is coimmunoprecipitated both with Pax6 and Pax6
PD. When the HA antibody was used for the immunoprecipitation, however, no
detectable amounts of Pax6
PD was coimmunoprecipitated. This can
possibly be explained by smaller amounts of the Pax6
PD protein in
this extract compared with the others (compare lanes
11 and 12, Fig. 5B). It is also likely
that the binding of the HA antibody to HA-Rax and the binding of
Pax6
PD to HA-Rax are mutually exclusive. HA-Pax6 and Pax6
PD
coimmunoprecipitated (Fig. 5B, lane
5), proving that Pax6 and Pax6
PD can interact in
vivo. Based on the in vitro interactions described
above, it seems reasonable to assume that the two proteins interact
directly with each other also in vivo.
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Fig. 5.
Pax6 PD and Rax
coimmunoprecipitate with the full-length Pax6 isoform from nuclear
extracts of transfected HeLa cells. A, full-length Pax6
expressed from a pCI-neo vector was cotransfected with expression
vectors for either HA-tagged Pax6
PD or HA-Rax. The PC6 polyclonal
affinity-purified antibody directed against the C terminus of the Pax6
protein (
-Pax6) was used to immunoprecipitate (IP) Pax6
from 100 µg of HeLa cell nuclear extracts, and the monoclonal 12CA5
anti-HA antibody (
-HA) was used to detect coimmunoprecipitated
HA-Rax (lane 3) by Western blotting
(WB). The HA- Pax6
PD protein is also detected by the
anti-HA antibody (lane 2), since this protein is
also precipitated by
-Pax6. Lanes 4-6 show a
Western blot of 5 µg of the nuclear extract used for
immunoprecipitation stained with the
-Pax6 antibody. The upper band
corresponds to full-length Pax6 (48 kDa); the lower band is Pax6
PD
(34 kDa) coexpressed from an internal start codon of the
Pax6 cDNA. The intermediate band observed in
lane 5 is the cotransfected HA epitope-tagged
Pax6
PD (36 kDa). B, to be able to detect
coimmunoprecipitated Pax6 with the
-Pax6 antibody, we cotransfected
HA-Pax6 and HA-Rax with the truncated version of Pax6, Pax6
PD. HeLa
cell nuclear extracts (60 µg) were either immunoprecipitated with
-Pax6 antibody (lanes 1-3) or
-HA antibody
(lanes 4-6), and then the
-HA antibody or
-Pax6 antibody was used to detect coimmunoprecipitated HA-tagged
Pax6 (lane 2), HA-Rax (lane
3), or Pax6
PD proteins (lanes
4-6), respectively. Lane 5 shows that
Pax6
PD is coimmunoprecipitated with HA-Pax6. No detectable amounts
of Pax6
PD is coimmunoprecipitated with HA-Rax (lane
6) in this experiment. However, HA-Rax is clearly
coimmunoprecipitated with Pax6
PD (lane 3).
Lanes 7-9 and lanes 10-12
show Western blots of 3 µg of nuclear extracts stained with
-HA or
-Pax6 antibodies, respectively. The upper band in lane
11 is HA epitope-tagged Pax6.
PD nor Rax Affects the Binding of Pax6 to a
Paired Domain-binding Site--
The nuclear extracts used for
coimmunoprecipitations were used in gel mobility shift assays to see if
the presence of Pax6
PD or Rax caused a shift in the migration of the
Pax6 complex binding to the P6CON probe. No shifted complexes were
detected, and the DNA binding was not affected at all upon
cotransfection with Pax6
PD or Rax (data not shown). To be able to
adjust the amounts of Pax6, Pax6
PD, and Rax individually, they were
separately transfected into HeLa cells, and nuclear extracts were made.
Gel mobility shift assays were performed with 1 µg of nuclear extract
containing Pax6 combined with 1 or 4 µg of nuclear extracts
containing Pax6
PD or Rax. As can be seen from Fig.
6, the extracts containing Pax6
PD or
Rax alone (lanes 11-14) display no binding to
the P6CON probe. Importantly, the binding of the Pax6 protein to the
P6CON probe is not affected in any way by the addition of extracts
containing either Pax6
PD (lanes 5 and
6) or Rax (lanes 9 and
10).
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Fig. 6.
Pax6 PD and Rax
cannot bind to the P6CON binding site and do not affect the binding of
Pax6 to this site. Nuclear extracts (N.E.) from HeLa
cells transfected with the indicated expression constructs were used in
gel mobility shift assays as described under "Materials and
Methods." One µg of nuclear extract containing Pax6 bound the P6CON
consensus Pax6 paired domain-binding site as seen in lane
2. This binding was neither affected by the addition of 1 or
4 µg of nuclear extract from HeLa cells transfected with Pax6
PD
(lanes 5 and 6) or HA-tagged Rax
(lanes 9 and 10) nor by the addition
of nuclear extract from cells transfected with the empty expression
vectors for these constructs (pCI-neo (lanes 3 and 4) and pcDNA3-HA (lanes 7 and
8), respectively). As can be seen from lanes
11-14, Pax6
PD and Rax are not able to bind the P6CON
probe.
PD, a
selection of various HDs were fused to GST and tested for their ability
to bind to in vitro translated Pax6 in a pull-down assay.
These isolated HDs were from Pax6 itself as a control, Rax, Six3, Lhx2,
Chx10, En-1, Prep1, Prox1, and HoxB1, and the pull-down experiments
were performed both in the presence and absence of EtBr. Surprisingly,
all of the different HDs were able to interact with Pax6, and this
interaction was not dependent on DNA (Fig.
7A). We also tested if the
full-length proteins of three very different HD proteins (Pbx-1, HoxB1,
and Chx10) could interact with the PD or the HD of Pax6. As seen from
Fig. 7B, these proteins could interact with both these
domains. All of the tested HDs come from transcription factors
coexpressed with Pax6 during development, and their HD sequences range
from 65 to only 13% sequence identity with the Pax6 HD (Fig.
7C). To test whether coexpression of these HD proteins with
Pax6 could affect Pax6-mediated transactivation from paired
domain-binding sites, we carried out cotransfection experiments in HeLa
cells with pP6CON-LUC as the reporter. This assay functions
as a mammalian cell one-hybrid assay for protein-protein interactions
in vivo if the protein interacting with Pax6 contains a
transactivating domain. Completely consistent with this notion, Pbx1,
HoxB1, Lhx2, and Chx10 were able to superactivate Pax6-mediated
transactivation, while Prep1 and Prox1 did not give any significant
effects. Pbx1, HoxB1, and Lhx2 contain experimentally proven TADs (53,
54). In addition, we find that both the paired-type HD proteins Rax and
Chx10 act as transactivators using a reporter containing Pax6
HD-binding sites (data not shown). Prep1 definitely lacks a
conventional TAD (54), while for Prox1 this is not known. Thus, these
results are consistent with in vivo interactions between
Pax6 and these HD proteins.
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Fig. 7.
Homeodomains from divergent homeodomain
proteins interact with Pax6 in vitro, and several of
these induce superactivation of Pax6-mediated transactivation.
A, the HDs of Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and
HoxB1 all interact with in vitro translated Pax6 in a
DNA-independent manner. The GST pull-down assays were performed as
described in the legend to Fig. 3. The experiments were performed with
(middle panel) and without (upper
panel) 100 µg/ml EtBr to determine whether the
interactions were dependent on DNA binding. The asterisk
indicates the Pax6 PD isoform, which also is translated from the
full-length cDNA. The lower panel shows a
Coomassie-stained SDS-polyacrylamide gel of the same amounts of GST-HD
fusion proteins immobilized on beads as used in the pull-down
experiments. B, full-length versions of the HD proteins
Pbx1, HoxB1, and Chx10 can interact with both the PD and the HD of Pax6
in vitro. The GST-pull downs were done as described in the
legend to Fig. 3. C, phylogram of the HD sequences used in
the GST pull-down assay in A. The sequence identity between
the Pax6 HD and the individual HDs are given in parentheses. The
phylogram was made using the Growtree program of the GCG software
package with Jukes-Cantor distances and the UPGMA method. D,
Pbx1, HoxB1, Lhx2, and Chx10 enhance Pax6-mediated transactivation of a
minimal promoter containing consensus Pax6 paired domain-binding sites.
HeLa cells were cotransfected with 0.25 µg of pCI-Pax6 or empty
expression vector (pCI-neo) and 0.75 µg of expression vector for the
different homeodomain proteins together with 0.5 µg of
pP6CON-LUC and 50 ng of pCMV-
-galactosidase. Pbx1, Prep1,
and HoxB1 were expressed from the pSG5 expression vector, while Prox1,
Lhx2, Chx10, and Pax6
PD were expressed from pcDNA3-HA. The data
shown are representative of at least three other independent
experiments with each experiment performed in triplicate. The
numbers above the gray
columns indicate -fold activation relative to the background
(white columns) level observed for transfections
with expression vectors for the different homeodomain proteins alone
together with empty expression vector for Pax6 (vector control).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PD isoform and Rax both enhance the
transcriptional activation mediated by the full-length Pax6 isoform
using a reporter containing P6CON consensus paired domain-binding sites
upstream of a minimal promoter. The
PD isoform and Rax are unable to
bind the P6CON binding site and do not by themselves cause
transactivation from the P6CON reporter; nor do they seem to work by
enhancing the DNA binding of the Pax6 PD to the reporter. In
vitro the PD and HD of Pax6 are able to bind full-length Pax6, Pax6
PD, and Rax proteins in GST pull-down assays, and in
vivo Pax6 is coimmunoprecipitated with Pax6
PD or Rax from HeLa
cell nuclear extracts. The in vitro interactions also occur
in the presence of EtBr, suggesting that they are DNA-independent. Our results show that the HDs interact with each other and with the PD and
that the PD can also interact with itself. The C-terminal subdomain of
the PD was found to be important as a protein-protein interaction surface.
3' light chain enhancer
(35). The Pax5 PD has also been found to interact with the runt DBD of
AML1 (79). Several observations of Pax gene behavior can be
attributed to the PD and/or HD being involved in protein-protein
interactions. First, the Pax3 HD alone fused to a GAL4 binding site can
work as a repressor of transcription (80). Second, Pax3 can repress
activity from the N-CAM promoter through a mechanism independent of DNA
binding (81). Third, Pax6 has recently been found to be a repressor of
the
B1-crystallin promoter (82). The repression activity was
dependent on the PD and the HD but not the TAD. Pax6-5a or the PD alone
could not repress transcription. Fourth, Pax5 has been reported to act
as both an activator and a repressor of genes in B cells dependent on
the promoter context (83). Together, these results indicate that Pax
protein interactions with other proteins on the promoter is important
in determining the function of the Pax proteins as activators or
repressors. This has also been reported for some Hox proteins (see Ref.
84 and references therein).
PD,
Rax, other paired-type HD proteins, and even very divergent HD
proteins? All of the proteins we have tested in this work are coexpressed with Pax6 in some tissue(s) during development. Except for
Hox1, Pbx1, and Prep1, the other six HD proteins are all expressed in
the developing eye and crucial for normal development of the eyes. For
example, Pax6 and Rax are expressed in overlapping areas of the
neuroretina. They are both expressed uniformly in the early retina and
then restricted to the inner nuclear layer (8, 17). The distinct and
overlapping expression patterns of Pax6 and Rax and/or other HD
proteins could possibly cause a fine tuning of the transcriptional
regulation of target genes. It is known that the eyes are especially
dosage-sensitive to the expression of Pax6 protein (13-15). If Pax6
function is dependent upon specific interactions with other proteins
through its PD or HD, this could contribute to the observed dosage
sensitivity. In this scenario, both loss-of-function and
gain-of-function of Pax6 gene expression would disturb the delicate
balance between factors participating in these interactions. In
conclusion, our studies suggest that both the HD and the PD of Pax6
should be viewed not only as DNA-binding domains but also as
protein-protein interaction domains capable of both intramolecular and
intermolecular interactions.
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ACKNOWLEDGEMENTS |
---|
We are grateful to J. Botas, C. L. Cepko, P. Gruss, A. Joyner, R. R. McInnes, S. Tomarev, and V. Zappavigna for the generous gift of cDNA clones for murine Lhx2, Rax, Six3, En-1, Chx10, human Prox1, Prep1, and HoxB1, respectively.
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FOOTNOTES |
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* This work was supported by grants from the Norwegian Cancer Society, the Norwegian Research Council, the Aakre Foundation, and the Blix Foundation (to T. J.).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.
Fellow of the Norwegian Research Council.
§ These authors contributed equally to this work.
¶ Fellow of the Norwegian Cancer Society.
To whom correspondence should be addressed: Dept. of
Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. Tel.: 47-776-44720; Fax: 47-776-45350; E-mail: terjej@fagmed.uit.no.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008882200
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ABBREVIATIONS |
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The abbreviations used are: PD, paired domain; GST, glutathione S-transferase; DBD, DNA-binding domain; GFP, green fluorescent protein; HD, homeodomain; PCR, polymerase chain reaction; TAD, transactivation domain; HA, hemagglutinin; CMV, cytomegalovirus.
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REFERENCES |
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