| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 41, 28950-28957, October 8, 1999
From the Laboratory of Molecular Endocrinology, Massachusetts
General Hospital, Howard Hughes Medical Institute, and Harvard Medical
School, Boston, Massachusetts 02114
In the endocrine pancreas, The glucagon gene is expressed in the pancreatic endocrine
cAMP response element-binding protein
(CREB)1-binding protein (CBP)
and a closely related homologue p300 are known to integrate a number of
cell-signaling pathways and to serve as co-activators of various
transcription factors, including helix-loop-helix and homeodomain
proteins (11-14). Both CBP and p300 serve as adapter proteins linking
DNA binding transcription factors with the basal transcriptional
machinery. Furthermore, CBP and p300 are proposed to both activate
histone acetyltransferase and displace nucleosomes, as well as recruit
RNA polymerase II to the transcription complex (15).
In an attempt to understand the mechanisms underlying the
tissue-specific transcription of the proglucagon gene, we have examined the mediation of the transactivating properties of pax-6 and cdx-2. We
report here that the transactivating effects of both pax-6 and cdx-2
are mediated through their interaction with the transcriptional co-activator p300. Furthermore, our results suggest that the binding of
pax-6 to p300 is weak and that the presence of cdx-2 enhances the
physical interaction of pax-6 with p300. Whereas the transcriptional activity of pax-6 is dependent on its binding to its DNA recognition site within the G1 promoter element, binding of cdx-2 to the promoter is not absolutely required since pax-6 tethers cdx-2 to form a complex
with cdx-2 and p300.
Cell Culture and Transfection--
Baby hamster kidney (BHK)-21
cells were obtained from the American Tissue Culture Collection
(Manassas, VA). Expression and Transcriptional Reporter Plasmids--
Expression
plasmids were all driven by a cytomegalovirus (CMV) promoter with
exception of CREB (RSV-CREB, CREBm1 (RSV-CREBm1), E1A (pRSV-E1A), E1A
RG2 (pRSV-E1A RG2) expression vectors, and a In Vivo Labeling, Co-immunoprecipitation, and Glutathione
S-Transferase (GST) Pull-down Assays--
Recombinant GST proteins were synthesized in JM109 Escherichia
coli and purified on glutathione-Sepharose resin under
non-denaturing conditions. GST proteins were analyzed on SDS-PAGE
before use in the assay to ensure equivalence of preparations.
35S-Labeled proteins were generated with an in
vitro transcription/translation system (TNT, Promega Biotech,
Madison, WI) and exposed to the indicated GST protein. As a control, an
unprogrammed translation with [35S]methionine was
employed. Ten micrograms of GST or GST fusion proteins were bound to 25 µl of glutathione-Sepharose beads in a total volume of 250 µl of
incubation buffer containing 12 mM HEPES, pH 7.9, 4 mM Tris/HCl, pH 7.9, 50 mM NaCl, 10 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride for
1 h at room temperature. Beads were washed three times,
resuspended in 100 µl of incubation buffer, and incubated with 25 µl of L-[35S]methionine-labeled protein
(pax-6, cdx-2, p300, or CHOP) for 12 h at 4 °C. After extensive
(five times) washing with NEN (150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) at 4 °C, the proteins
trapped by the resin were resolved on SDS-PAGE and detected by
autoradiography. To analyze the facilitation of pax-6 interaction with
p300 through cdx-2, unlabeled recombinant cdx-2 protein was added to
the GST-pax-6-p300 pull-down assay in different amounts (see
"Results"). GST-cdx-2 and GST-pax-6 were generated by subcloning
the respective cDNAs into pGEX-KG by standard techniques (20).
GST-C/EBP Nuclear Protein Extraction and EMSA--
Nuclear proteins from
cell lines Analysis of the Protein-DNA Complexes of the G1 Element of the
Proglucagon Gene--
Electrophoretic mobility shift assays of Transient Transfection Experiments--
To further test the
functional interaction of pax-6 and cdx-2, transient co-transfection
studies with expression plasmids of pax-6 and cdx-2 with firefly
luciferase reporter constructs of the glucagon G1 element were
conducted. Both pax-6 and cdx-2 independently transactivate the
glucagon gene. When the binding 5' and 3' AT-rich sites of the promoter
are mutated, the activity of pax-6 and cdx-2 is markedly attenuated,
respectively (Fig. 2A). pax-6
and cdx-2 transfected in submaximal doses together elicit a synergistic
effect on the transactivation of the proglucagon gene promoter-reporter
construct. However, when the preferential DNA binding site for cdx-2
(the proximal 3' AT-rich site) is mutated, a synergistic
transactivating effect of pax-6 and cdx-2 together is (although
somewhat reduced) still preserved (Fig. 2B). These findings
are in line with observations recently reported (6, 7). In addition, in
a situation of mutated pax-6 DNA recognition site (
We hypothesized that pax-6 may be tethering cdx-2 to a transcriptional
complex, possibly involving a co-activator module. Therefore, we next
studied the transactivating properties of pax-6 and cdx-2 in
combination with p300. Indeed, addition of p300 expression plasmid
markedly increased transactivation through pax-6 and cdx-2 alone and in
combination (Fig. 2C; note the interrupted y axis in Fig. 2C). The synergistic effect of pax-6 and cdx-2 with
the co-activating effect of p300 was also present even when the 3' AT-rich site (binding site of cdx-2) was mutated (Fig. 2C).
This result further suggests that the cdx-2 DNA recognition site is not
necessary for cdx-2 to participate in the transcription of the glucagon
gene. In contrast, disruption of the pax-6 binding site did not allow
overexpressed pax-6 to further stimulate the transcription induced by
cdx-2 together with p300. Thus, it appears that the pax-6 DNA
recognition site (even in the presence of cdx-2) is required for pax-6
to participate in the transactivation of the glucagon gene.
Furthermore, disruption of the cdx-2 site leads to a substantial
reduction of the transactivation of pax-6 through interaction with p300
(Fig. 2C). This observation suggests that, for optimal
transactivation and interaction with p300, an optimal sequence of the
DNA within the promoter region is required. This may indicate the
importance of steric conformation of interacting proteins and DNA in
transcriptional control.
Next, we attempted to inhibit the co-activation through p300/CBP by
overexpression of the adenovirus E1A protein, which specifically binds
the cystine/histidine-rich 3 (C/H3) domain of p300 and CBP and thereby
inhibits transcriptional co-activation (11-13). A mutated E1A protein
(E1A RG2), which is a much weaker inhibitor of transcriptional co-activation through p300/CBP, was used as a negative control (13). As
shown in Fig. 3A,
overexpression of E1A, but not of E1A RG2, reduced the basal expression
of a Analysis of Protein-Protein Interaction in Vitro and in
Vivo--
We confirmed that cdx-2 binds to GST-pax-6 (Fig.
4) (6, 7). Next, we tested whether cdx-2
and pax-6 bind to the CBP homologue p300. Indeed, GST-cdx-2 interacted
with in vitro translated p300 in a GST pull-down assay.
Furthermore, GST-pax-6 also interacted with p300. Furthermore, we could
demonstrate that GST-cdx-2 pulls down both in vitro
translated p300 and pax-6 simultaneously. Vice versa,
GST-pax-6 pulls down both in vitro translated p300 and cdx-2
simultaneously (Fig. 4C). Neither GST-pax-6 nor GST-cdx-2 interacted with in vitro translated labeled CHOP, suggesting
that nonspecific interactions of pax-6 and cdx-2 with p300 do not take place (Fig. 4B).
On the basis of the transient transfection experiments (Fig. 2), we
hypothesized that cdx-2 may be enhancing the interaction of pax-6 and
the basal transcription machinery through interaction with a
co-activator CBP/p300. To test whether cdx-2 may enhance the physical
interaction of pax-6 and p300, we added increasing amounts of unlabeled
cdx-2 to the GST-pax-6-p300 pull-down assay. As shown in Fig.
5, addition of increasing amounts of
recombinant cdx-2 (in contrast to in vitro translated oct3)
enhanced the recovery of labeled p300 protein by GST-pax-6. In
contrast, addition of unlabeled recombinant pax-6 failed to increase
recovery of labeled p300 through GST-cdx-2. These results, although not
directly quantitative, support the hypothesis that cdx-2 might
facilitate the protein-protein interaction of pax-6 with
p300.
In vivo labeling of overexpressed proteins in the mouse
glucagon-producing tumor cell line
To test whether pax-6 and cdx-2 associate with CBP/p300 co-activator at
native levels, we performed co-immunoprecipitation experiments with
nuclear extracts of
We next sought to determine the site of interaction between pax-6 and
cdx-2 with p300. GST pull-down experiments with GST constructs
encompassing different portions of p300 protein reveal that both pax-6
and cdx-2 interact with the cystine/histidine-rich domain 1 (C/H1) of
the p300 protein (Fig. 8).
This study provides evidence that both homeodomain proteins pax-6
and cdx-2 interact with co-activator p300 in the transcription of the
proglucagon gene. Transcriptional activation of pax-6 and cdx-2 is
increased by overexpression of p300 in the heterologous system of
BHK-21 fibroblasts. Furthermore, our studies confirm the observation
that pax-6 and cdx-2 interact synergistically on the proximal G1
promoter element of the glucagon gene (6, 7) and extend the finding by
demonstrating that this synergistic transcriptional activation is
mediated by the CBP homologue p300.
The proximal promoter element G1 ( It must be assumed that CBP/p300 would be present in all cells, and
further addition of p300 would not result in any substantial change in
transcription. However, CBP/p300 is shown to be a co-activator present
in limiting amounts such that different transcription factors compete
for the interaction with this co-activator (21). Thus, it is
conceivable that overexpression of p300 can lead to enhanced
transactivating effects as shown in the present study. However, the
synergistic effect of pax-6 and cdx-2 together with p300 on the
transcription of the proglucagon reporter gene at submaximal doses of
pax-6 and cdx-2 argues against the notion that CBP analogues may be
limiting in BHK-21 cells. The results of the present study rather
support the concept that pax-6 and cdx-2 facilitate or stabilize each
other's interaction with the co-activator p300 (Fig.
9). To this end, in vitro
binding studies show that both GST-pax-6 and GST-cdx-2 can pull down
in vitro labeled p300. However, the recovery of p300 through
GST-pax-6 was relatively low. By adding unlabeled in vitro
translated cdx-2 to the GST-pax-6-p300 pull-down assay, the recovery of
p300 increased. Although the data do not absolutely prove this idea,
they suggest, together with the results of the transient transfection
experiments that the interaction of pax-6 and p300 is relatively weak
and may be enhanced by the presence of cdx-2 (Fig. 2).
In addition, the facilitated interaction of pax-6 with p300 by cdx-2
does not absolutely require binding of cdx-2 to the proximal AT-rich
site of the proglucagon gene promoter. The transient transfection data
support the notion that, even in the absence of a cdx-2 DNA binding
site (Fig. 2, B and C), cdx-2 participates in the
transcriptional regulation of the proglucagon gene. Taken together with
the data that pax-6 binds cdx-2 in vitro and in
vivo, it is likely that pax-6 tethers cdx-2 to the complex formed
together with p300. On the other hand, in the presence of cdx-2,
overexpression of pax-6 shows a tendency (although not significant) to
further stimulate transcription even when its recognition site is
mutated (Fig. 2B). However, in Fig. 2C, this
property of pax-6 is not further recapitulated, suggesting that the DNA
binding site for pax-6 is necessary for pax-6 to fully participate in
the transcription of the glucagon gene through interaction with the G1
element. Another important observation is that mutation of the 3'
AT-rich site (cdx-2 binding site) significantly reduces the
transactivation capacity of pax-6 in the experiment with added p300
(Fig. 2C). It may be speculated that, even in the absence of
cdx-2, an optimal interaction of pax-6 with p300 requires a certain
optimal DNA sequence, which is no longer present when the 3' AT-rich
site is mutated. Alternatively, a cryptic pax-6 binding site within the
3' AT-rich site could be suspected, of which mutation leads to a
reduction of pax-6 transactivation. However, the EMSA experiments (Fig.
1B) do not suggest that pax-6 binds to the 3' AT-rich site.
The information derived from Fig. 8 suggests that both pax-6 and cdx-2
interact with the same region (C/H1 domain) of p300. However, from the
result of transient transfection data, it cannot be assumed that the
interaction site with p300 would be absolutely identical, since it
pax-6 and cdx-2 do not appear to compete with the binding to p300, but
act synergistically at the functional level. Furthermore, the in
vitro observations indicate that cdx-2 may enhance binding of
pax-6 to p300, possibly by further stabilizing the interaction of these
proteins (Fig. 5). One possible explanation for our findings would be
that cdx-2 forms a bridge between pax-6 and p300, no longer requiring a
direct p300-pax-6 interaction, whereas in the absence of cdx-2, pax-6
has the capacity of interacting directly with p300. Alternatively, the
stronger functional and physical interaction of pax-6 with p300 in the
presence of cdx-2 may be due to a change in conformation of the
participating proteins when interacting with each other and thus
altering their capacity to associate with other proteins. Indeed,
altered conformation of interacting homeodomain transcription
factors has been described previously (22).
Taking all the above considerations into account, it appears that
although pax-6, cdx-2, and p300 can interact separately with each other
even in the absence of DNA, the optimal functional interaction of these
proteins requires an intact proximal G1 element of the proglucagon gene
(Fig. 2C). This notion is further supported by the EMSA
results, which demonstrate that both AT-rich sites of the G1 element
need be intact for the formation of a protein-DNA containing both pax-6
and cdx-2 (Fig. 1B). It is important to note at this point
that the mutations in the promoter region may also alter the geometric
association of the transcription factors, co-activator, and the basal
transcription machinery to such an extent that some of the results
generated by these methods may not only be accounted for just by the
presence or lack of DNA binding sites for transcription factors but
also by altered geometry in the region immediately upstream of the
transcriptional start site.
pax-6 has been shown to bind not only to the G1 element but also to the
upstream G3 enhancer element (8). Whether pax-6 bound to the G3 element
also interacts with cdx-2 and/or p300 cannot be addressed with the
present data.
cdx-2 has previously been reported to reduce the proliferation rate of
intestinal epithelial cells (10). In other systems, the inhibition of
progression through the cell cycle is reported to be mediated by
CBP/p300. Thus, the function of cdx-2 in pancreatic The in vivo relevance of our findings with regard to
glucagon gene expression needs to be addressed by further studies.
While the present studies show the importance of the interaction of the
transcription factors pax-6 and cdx-2 with the In conclusion, the present studies demonstrate that pax-6 and cdx-2
both interact with p300 at the protein-protein level. Further, the
interaction of pax-6 with p300 is enhanced by the presence of cdx-2.
Since cdx-2 participates in the transactivation of the proglucagon gene
even in absence of its DNA binding site in the G1 promoter element, it
is likely that pax-6 tethers cdx-2 to the complex formed by pax-6,
cdx-2, and the transcriptional co-activator p300. Both pax-6 and cdx-2
stimulate proglucagon gene expression in pancreatic We thank L. Dailey, D. J. Drucker, S. Efrat, R. Goodman, H. Lu, M. Montminy, M. G. Rosenfeld, and P. Traber for their generous contribution of material provided for the
presented studies. We thank the members of the Laboratory of Molecular
Endocrinology and M. Vallejo for valuable discussions and Townley Budde
and Richard Larraga for help in the preparation of the manuscript.
*
This work was supported in part by a Juvenile Diabetes
Foundation Career Development Award (to M. A. H.) and
National Institutes of Health Grant RO1 DK30834 (to J. F. H.).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.
2
M. A. Hussain and J. F. Habener,
manuscript in preparation.
The abbreviations used are:
CREB, cyclic AMP
response element-binding protein;
bp, base pair(s);
C/EBP
Glucagon Gene Transcription Activation Mediated by Synergistic
Interactions of pax-6 and cdx-2 with the p300 Co-activator*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell-specific
expression of the glucagon gene is mediated by DNA-binding proteins
that interact with the proximal G1 promoter element, which contains
several AT-rich domains. The homeodomain transcription factors brain-4, pax-6, and cdx-2 have been shown to bind to these sites and to transactivate glucagon gene expression. In the present study, we
investigated the interaction of cdx-2 and pax-6 with p300, a
co-activator coupled to the basal transcription machinery. In transient
transfection-expression experiments, we found that the transactivating
effects of cdx-2 and pax-6 on the glucagon gene were greatly enhanced
by the additional expression of p300. This enhancement was due to
direct protein-protein interactions of both pax-6 and cdx-2 with the
N-terminal C/H1 domain of p300. pax-6 and cdx-2 also directly
interacted with one another at the protein level. pax-6, bound to its
DNA recognition site in the glucagon G1 promoter element, tethered
cdx-2 to the molecular complex of pax-6 and p300. Further, we found
that the presence of cdx-2 enhanced the interaction of pax-6 with p300,
thus establishing a molecular complex of transcription factors
implicated in tissue-specific glucagon gene expression with the basal
transcriptional machinery.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells of the pancreatic islets, the L cells of the intestine, and
specific areas of the brain (1). The cell-specific expression of the
proglucagon gene is best studied in the pancreatic -cells, in which
expression is conferred by the proximal G1 promoter element (1). The G1
element contains two AT-rich motifs that are binding sites for
homeodomain transcription factors. Recently, the transcription factors
cdx-2, pax-6, and brain-4 have been shown to bind the AT-rich elements
and transactivate glucagon gene expression (2-4). Nuclear proteins of
glucagon-producing cell lines form three main protein complexes with
the G1 promoter element (5). One of these protein complexes contains
the POU domain transcription factor brain-4 (4). Of the two other
complexes, the lower molecular weight complex has been reported to
contain the paired-domain factor pax-6 as monomer, whereas the higher
molecular weight form contains pax-6 in a heterodimer with the
caudal-related factor cdx-2 (6, 7). Of these transcription factors,
brain-4 is expressed specifically in pancreatic -cells (4), and
pax-6 is expressed in all pancreatic endocrine cells implicated in
islet cell development, predominantly the pancreatic -cells (8, 9).
pax-6 also binds to the G3 element of the proglucagon gene enhancer at
a site that confers insulin inhibition of proglucagon gene expression
(8). cdx-2 is found in both pancreatic - and 
-cell lines (3), as
well as in intestinal epithelial cells (3, 4, 10). Recently, pax-6 and
cdx-2 have been shown to directly bind to each other and to
synergistically transactivate the proglucagon gene via
interaction with the G1 element of the proglucagon gene promoter (6,
7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC-1 cells were a generous gift from S. Efrat
(Albert Einstein College of Medicine, Bronx, NY). All cells were grown
at 37 °C in humidified 5% CO2, 95% O2 in
Dulbecco's modified Eagle's medium (DMEM, 4.5 g of glucose per liter
(Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal
calf serum (Life Technologies, Inc.) and 100 units of penicillin and
100 µg/ml streptomycin. Transfections were performed with LipofectAMINE reagent (Life Technologies, Inc.) according to the instructions of the manufacturer. Transfections for luciferase assays
were performed in 12-well dishes. One µg of the promoter-reporter construct was transfected per dish. Varying amounts of expression plasmid were co-transfected as indicated in the respective figures. A
RSV- galactosidase vector was co-transfected for normalization of
luciferase assays for transfection efficiency. Luciferase activity obtained following transfection was additionally normalized to the
background luciferase activity obtained following transfection of the
promoterless luciferase plasmid in the same experiment. Experiments
were done on three separate occasions. Mean ± S.D. of these
assays are given.
-galactosidase reporter
(RSV--galactosidase) vector, which are driven by a Rous sarcoma
virus (RSV) promoter. pCMV-oct3, pCMV-brain-4, pCMV-pax-6, pRC-cdx-2,
pCMV-p300, pRSVE1A and pRSVE1A RG2, and various GST-constructs of p300
fragments were generously provided by L. Dailey (Rockefeller
University, New York, NY), M. G. Rosenfeld (University of
California, San Diego, CA), M. Busslinger (University of Vienna,
Vienna, Austria), P. Traber (University of Pennsylvania, Philadelphia,
PA), and M. Montminy (Harvard Medical School, Boston, MA), R. Goodman
(Vollum Institute, Portland, OR), and H. Lu (Vollum Institute),
respectively. Rat proglucagon gene 5'-flanking sequences from bp 
60,
and 93 to +68 bp inserted adjacent to a firefly luciferase reporter
have previously been described (4, 16). Mutations in the G1 element were generated by polymerase chain reaction and are identical to those
of the oligonucleotides used for the electrophoretic mobility shift
assay (EMSA) experiments (Table I). The mutated constructs were checked
by sequencing. pRSV-CREB and pRSV-CREBm1, which carries a mutation of
serine 133 to alanine, have been described previously (17).
TC-1 cells were incubated
in DMEM in 100-mm plates and transfected with 10 µg of expression
plasmids as noted above. Twenty-four hours later, cells were washed
twice with DMEM without methionine. Cells were then incubated in 1 ml
of DMEM without methionine, with 500 mCi of
[35S]methionine added (Tran35S-label,
Amersham Life Sciences, Buckinghamshire, United Kingdom) for 2 h.
Cells were then washed twice with phosphate-buffered saline, and
nuclear proteins were prepared according to the Schreiber method (18).
Immunoprecipitation was then performed essentially as described in Ref.
19. Briefly, 50 µg of nuclear extracts were diluted to 200 mM NaCl with EDTA low salt buffer (ELB) (100 mM
NaCl, 0.1% Nonidet P-40, 50 mM HEPES, 1 mM
phenylmethylsulfonyl fluoride, 5 mM EDTA, 0.5 mM dithiothreitol) (19) and to a final volume of 500 µl.
The solution was precleared with 20 µl of Protein A-Sepharose from
nonspecific binding. After centrifugation the supernatant was incubated
with a specific antibody to cdx-2, pax-6, or p300 overnight at 4 °C
on a rocker. After adding 100 µl of a Protein A-Sepharose slurry and
an additional incubation for 1 h, the supernatant was discarded
and the pellet was extensively washed in ELB. The immunoprecipitated
proteins in the pellet were resolved by SDS-PAGE and detected by
autoradiography. In experiments with phosphorylation-dependent binding
of CREB with p300 cells were treated with 10 nM forskolin
and 0.5 mM isobutylmethylxanthine for 15 min before
harvesting. All reagents used subsequently contained a mixture of
phosphatase inhibitors (10 mM sodium pyrophosphate, 0.4 mM sodium vanadate, 10 mM sodium fluoride, 4 mM EDTA, 20 mM okadaic acid). For
co-immunoprecipitation experiments to detect protein-protein
interaction in native nuclear extracts of TC-1 cells,
immunoprecipitation was conducted as outlined above without prior
exposure of the cells to radioactive label. After SDS-PAGE fractionation of the immunoprecipitate, the proteins were
electrophoretically transferred to a nitrocellulose filter. The
immunoreactivity of p300, pax-6, and cdx-2 was detected with the ECL
Western analysis system (Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom) with peroxidase-linked anti-rabbit or anti-mouse
immunoglobulin as the second antibody.
and GST-CHOP have been previously described (21). Various
GST constructs of p300 fragments were a generous gift by H. Lu).
TC-1 and BHK-21 cells with transfected CMV-pax-6,
CMV-cdx-2 were prepared as described previously (18). The synthetic
oligonucleotides listed in Table I
corresponding to glucagon promoter G1 sequences were annealed,
end-labeled with 32P with T4 polynucleotide kinase, and
purified over a Sephadex G-50 spin column (Roche Molecular
Biochemicals). EMSA were performed by incubating approximately 5 × 104 cpm of end-labeled DNA probe (0.02-0.03 ng of DNA)
with nuclear protein (0.75-7.5 µg) in a binding buffer (10 mM Tris-HCl, pH 8.0, 40 mM KCl, 6% glycerol, 1 mM dithiothreitol, 0.05% Nonidet P-40) for 20-30 min at
room temperature. For antibody interference experiments, nuclear
proteins were premixed with diluted preimmune or specific antiserum in
the binding buffer at room temperature for 20 min prior to adding the
labeled DNA probe. Different dilutions of the immune sera had been
tested prior to conducting the EMSA to rule out any nonspecific effect.
The reaction mixture was then analyzed by electrophoresis on a 6%
nondenaturing polyacrylamide gel. Following electrophoresis, the gel
was dried and exposed to x-ray film for 6-72 h. cdx-2, and p300
(anti-KIX) antisera were generously provided by P. Traber and M. Montminy. Antisera directed against pax-6 were purchased from the
Developmental Hybridoma Bank (University of Iowa, Iowa City, IA).
Anti-CREB antiserum 338, which recognizes a C-terminal portion of the
protein, does not distinguish between wild-type and Ser-133 mutated
CREB (17).
Oligonucleotides used for EMSA
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC-1
nuclear extracts with bp 93/60 proximal element G1 of the glucagon
promoter reveal four major protein-DNA complexes. Of these, complex A, which is found at various intensities and is usually weaker than complexes B and C, is most likely formed by a heterodimer of pax-6 and
cdx-2 (Fig. 1A; Refs. 6 and
7). Complex B contains the POU domain transcription factor brain-4 (4),
and complex C most likely contains pax-6 homodimer (6, 7). Complex D
appears to contain all three homeoproteins, although an unspecific
effect of antibodies cannot be definitively ruled out with the present data. Of note, cdx-2 antiserum does not clearly disrupt but
attenuates the intensity of complex A. This suggests that complex A is
composed of a pax-6/cdx-2 heterodimers (Fig. 1A), as has
previously been reported by others (6, 7). In EMSA experiments with
nuclear extracts from BHK-21 cells overexpressing transfected pax-6 and cdx-2, a heterodimer of pax-6 and cdx-2 can form complex A only when
both AT-rich binding sites are intact. An interaction of pax-6 and
cdx-2 does not become apparent when the proximal 3' AT-rich site, the
preferential binding site for cdx-2 (3, 6, 7), is mutated (Fig.
1B). Complex C is formed by pax-6 alone (Fig.
1B). Fig. 1C depicts the preferential binding
sites of pax-6, cdx-2, and brain-4.
View larger version (37K):
[in a new window]
Fig. 1.
EMSA with islet TC-1
and BHK-21 cells overexpressing pax-6 or cdx-2 to identify G1-binding
proteins. Nuclear extracts (10 µg each) were preincubated with
labeled wild-type G1 probe 93/60 (Table I) either alone or with
nonspecific (NS) serum, or antiserum directed against brain4
(brn4), pax-6 (pax-6), or cdx2
(cdx2), following which DNA-protein complexes were
resolved by PAGE and autoradiography. The free-migrating DNA probe was
run off the gel. A, four major protein-DNA complexes A, B,
C, and D are formed. As can be deduced by immunointerference of the
complexes, brain-4 is present in complex B and pax-6 is in complex C. cdx-2 antiserum attenuates the intensity of complex A, suggesting that
complex A is formed by pax-6/cdx-2 heterodimers. Complex D is disrupted
by all three antibodies suggesting the presence of all three tested
homeoproteins. B, with the wild-type 93/60 G1 probe
pax-6 forms complex C as a monomer. Complex A is formed by pax-6 and
cdx-2 heterodimer. cdx-2 alone does not form a complex that is present
in TC-1 nuclear extracts. cdx-2 preferentially binds to the 3'
AT-rich site, but forms a complex with the G1 element when the 3' site
is mutated (93/60 m3'). pax-6 binds the 5' AT-rich site and not the
3'AT-rich site. No complex is formed with pax-6 when the 5' AT-rich
site is mutated (93/60 m5'). Formation of complex A requires that
both AT-rich sites are intact. C, schematic of the proposed
DNA binding sites of homeoproteins pax-6, cdx-2, and brain-4. pax-6
binds to the 5' AT-rich site within the G1 element of the proglucagon
gene, whereas cdx-2 binds preferentially to the 3' AT-rich site (3, 4,
6, 7). As previously reported, brain-4 preferentially binds to the 5'
AT-rich site (4).
93 m5' GLU-LUC,
distal 5' AT-rich site), overexpression of pax-6 showed a tendency
(although not significant) of enhancing the transactivation through
cdx-2 (Fig. 2B).
View larger version (37K):
[in a new window]
Fig. 2.
Proglucagon promoter G1 element
transactivation assays. A, both pax-6 and cdx-2
transactivate the proglucagon promoter-reporter constructs encompassing
the G1 element independently in a dose-dependent manner in
BHK-21 fibroblasts. Mutation of the 3' AT-rich site ( 93 m3')
abrogates the transactivation by cdx-2. Mutation of the 5' AT-rich site
(93 m5') abrogates the transactivation by pax-6. BHK-21 fibroblasts
were co-transfected with 1 µg of cells/well with the respective
proglucagon promoter-reporter construct together with varying amounts
(50-150 ng) of the transcription factor expression plasmid. Luciferase
activity was measured 18-48 h after transfection. Results were
normalized to -galactosidase activity derived from a co-transfected
RSV--galactosidase expression plasmid. Total amount of transfected
DNA was kept by adding empty expression vectors to the reaction.
B, synergistic activation of the wild-type proglucagon
promoter-reporter construct by submaximal doses of pax-6 and cdx-2. The
synergistic effect of pax-6 and cdx-2 on proglucagon gene transcription
is attenuated but still present after mutation of the cdx-2 DNA
recognition element (93 m3'). However, the synergistic effect is
lost, when the pax-6 binding site is mutated (93 m5'). C,
transactivation of the proglucagon gene by pax-6 and cdx-2 is enhanced
by p300. The synergistic effect of pax-6 and cdx-2 on the
transactivation of the proglucagon gene is greatly increased by
co-expression of p300. Even after mutation of the cdx-2 DNA binding
site (93 m3'), the enhancing effect of p300 is present. However, the
synergistic effect is largely lost, when the pax-6 binding site is
mutated (93 m5').
93 glucagon promoter-luciferase reporter in TC-1 cells.
Furthermore, overexpression of E1A, but not the mutant E1A RG2, in
heterologous BHK-21 fibroblasts inhibited the transactivation by cdx-2,
pax-6, and cdx-2 and pax-6 together, of the glucagon promoter-reporter
construct (Fig. 3B). These results suggest that an
endogenous co-activator homologue of CBP/p300 is involved in the
transcription of the glucagon gene in and that transactivation by pax-6
and cdx-2 involves the co-activator CBP/p300.
View larger version (21K):
[in a new window]
Fig. 3.
Adenovirus protein E1A interferes with
transcription of the glucagon promoter-reporter construct.
A, overexpression of E1A but not of the mutant E1A RG2
reduces basal activity of a 93 bp glucagon promoter-luciferase
reporter in TC-1 cells. Cells of the glucagon-producing pancreatic
tumor cell line TC-1 were transiently transfected with 5 µg of
93 bp glucagon promoter-reporter construct and increasing amounts of
E1A or E1A RG2 expression plasmid. Wild-type E1A, but not mutant E1A
RG2 mutant, which does not interact with p300 (11-13), reduces basal
activity of the glucagon-promoter reporter. TC-1 cells were
co-transfected with 5 µg of cells/well with the wild-type 93 bp
proglucagon promoter-reporter construct together increasing amounts of
E1A or E1A RG2 expression plasmids. Luciferase activity was measured
18-48 h after transfection. Results were normalized to
-galactosidase activity derived from a co-transfected
RSV--galactosidase expression plasmid. Total amount of transfected
DNA was kept by adding empty expression vectors to the reaction.
B, E1A interferes with transactivation of glucagon promoter
by both cdx-2 and pax-6. BHK-21 cells were transfected with 1 mg of
93 bp glucagon promoter-reporter plasmid together with expression
plasmids for pax-6, cdx-2, or both. Overexpression of E1A protein
inhibits transactivation by either transcription factor or by both
together. E1A RG2 mutant, which does not interact with p300 (11, 13),
did not show this inhibition. BHK-21 fibroblasts were co-transfected
with 1 µg/well of cells with the wild-type 93 bp proglucagon
promoter-reporter construct together with 100 ng of pax-6 or cdx-2 and
50 ng of the E1A or E1A RG2 expression plasmids, respectively.
Luciferase activity was measured 18-48 h after transfection. Results
were normalized to -galactosidase activity derived from a
co-transfected RSV--galactosidase expression plasmid. Total amount
of transfected DNA was kept by adding empty expression vectors to the
reaction.
View larger version (22K):
[in a new window]
Fig. 4.
Protein-protein interaction GST pull-down
assays. GST-protein attached to Sepharose beads was incubated with
in vitro-translated [35S]methionine-labeled
protein. GST-protein and protein interacting with the GST-tagged
protein were recovered by centrifugation of the Sepharose beads. After
repeated washing procedures, the radioactively labeled protein trapped
by the GST fusion protein was subjected to SDS-PAGE and detected by
autoradiography. Input lane of in vitro-translated protein
corresponds to 50% of protein used for GST pull-down assay.
A, protein-protein interaction of pax-6 and cdx-2 is
demonstrable as opposed to lack of both of these proteins with GST only
or with GST-C/EBP fusion protein. B, protein-protein
interactions of both GST-pax-6 and GST-cdx-2 with p300. GST-pax-6 and
GST-cdx-2 (in contrast to GST-C/EBP) do not interact with CHOP.
GST-C/EBP pulls down CHOP protein, as has been described previously
(20). C, simultaneous in vitro protein-protein
interactions of GST-pax-6 with p300 and cdx-2, and GST-cdx-2 with pax-6
and p300. No interaction is found with GST only or with GST-CHOP.
View larger version (34K):
[in a new window]
Fig. 5.
cdx-2 enhances the in vitro interaction of pax-6 and p300. pax-6 does not further
enhance interaction of cdx-2 and p300. GST-pax-6 was co-incubated with
labeled in vitro translated p300, and varying amounts of
in vitro unlabeled cdx-2 were added to the incubation
mixture. Addition of cdx-2 increased recovery of labeled p300 through
GST-pax-6. In contrast, addition of oct3 did not have any effect on the
interaction of pax-6 and p300. Input lane of in vitro
translated protein corresponds to 50% of protein used for GST
pull-down assay.
TC-1 cells were in accordance with the in vitro GST pull-down assays (Fig.
6, A-C). As an internal control for the experiments, we used the known interaction of Ser-133-phosphorylated CREB with p300 (Fig. 6D).
View larger version (43K):
[in a new window]
Fig. 6.
Immunoprecipitation and
co-immunoprecipitation assays. TC-1 cells were co-transfected
with 10 µg of each of the indicated expression vectors. Twenty-four
hours later, cells were washed and incubated in
[35S]methionine for 1 h. Thereafter, nuclear
extracts were harvested for immunoprecipitation with the indicated
antisera. Recovered proteins were subjected to SDS-PAGE and subsequent
autoradiography. A, immunoprecipitation of in
vivo [35S]methionine-labeled cdx-2, pax-6, and p300.
There is no cross-reactivity among the different antisera. The
immunoprecipitation detects only the overexpressed proteins.
B, co-immunoprecipitation of in vivo
[35S]methionine-labeled cdx-2, pax-6 and p300. pax-6 and
cdx-2 interact in vivo. Both pax-6 and cdx-2 interact with
p300 in vivo. C, co-immunoprecipitation of
in vivo [35S]methionine-labeled cdx-2, pax-6
and p300. The results suggest that pax-6, cdx-2, and p300 proteins
interact with each other and are found together in a heteromeric
complex. D, co-immunoprecipitation of labeled p300 and
phosphorylated CREB. Wild-type phosphorylated CREB interacts with p300.
CREB with Ser-133 mutated to alanine is not phosphorylated and does not
interact with p300. TC-1 cells were transfected with 10 µg of
empty vector, pRSV-CREB, pRSV-CREBm1, or pCMV-p300 or the indicated
combinations. Twenty-four hours later, cells were washed and incubated
in [35S]methionine for 1 h. Before harvesting cells
were stimulated with forskolin and isobutylmethylxanthine to
phosphorylated CREB. Thereafter, nuclear extracts were harvested for
co-immunoprecipitation with anti-CREB antiserum of interacting
proteins, which were analyzed by SDS-PAGE and subsequent
autoradiography.
TC-1 cells. As shown in Fig.
7, immunoprecipitation with p300
antiserum and subsequent immunoblotting for pax-6 or cdx-2 also
revealed an interaction of the homeoproteins with CBP/p300.
Immunoprecipitation with cdx-2 or pax-6 antiserum and subsequent
immunoblotting allows detection of p300 immunoreactivity, suggesting
that both homeodomain transcription factors interact with p300 in
TC-1 cells.
View larger version (42K):
[in a new window]
Fig. 7.
Co-immunoprecipitation of nascent, unlabeled
proteins from TC-1 nuclear extracts. 40 µg of TC-1 nuclear extracts were immunoprecipitated with p300,
cdx-2, or pax-6, or nonspecific antibody (NS).
Antibody-protein complexes were precipitated with Protein A-Sepharose.
After washing, the protein precipitate was subjected to SDS-PAGE,
transblotting, and Western immunoblot with pax-6, cdx-2, or p300
antiserum. p300 antiserum co-immunoprecipitates both cdx-2
(A) and pax-6 (B). Both cdx-2 (C) and
pax-6 (D) antisera co-immunoprecipitate p300
immunoreactivity.
View larger version (28K):
[in a new window]
Fig. 8.
Mapping of the interaction site of p300 with
cdx2 or pax-6. Both homeoproteins interact with C/H1 domain of
p300. GST fusion proteins with different fragments of the p300 protein
(generously provided by H. Lu) attached to Sepharose beads were
incubated with in vitro translated
[35S]methionine-labeled cdx2 or pax-6. GST-protein and
protein interacting with the GST-tagged protein were recovered by
centrifugation of the Sepharose beads. After repeated washing
procedures, the radioactively labeled protein trapped by the GST fusion
protein was subjected to SDS-PAGE and detected by autoradiography.
Input lane of in vitro translated protein corresponds to
50% of protein used for GST pull-down assay. A, schematic
of p300 protein with defined domains and of GST fusion constructs.
C/H, cystine-rich domains 1-3; C/H3 is site of interaction
of p300 with TFIIb and the adenovirus oncoprotein E1A; KIX,
domain interacting with phosphorylated CREB; Q-rich,
glutamine-rich domain. B, cdx-2 interacts with the C/H1
domain p300. No interaction is detectable with any other domain.
C, pax-6 interacts with the C/H1 domain p300. No interaction
is detectable with any other domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 to 52 bp) of the proglucagon
gene is sufficient for cell-specific expression of the proglucagon gene
in pancreatic -cells (5). Nuclear extracts form pancreatic -cell
lines contain brain-4, pax-6, and cdx-2, all homeoproteins, which bind
to the G1 element of the proglucagon gene (3, 4, 6, 8). Three major
protein-DNA complexes are formed between -cell line nuclear extracts
and the G1 element (Fig. 1A; Refs. 6 and 7). A high
molecular weight complex (A) is formed by a heterodimer of
pax-6 and cdx-2. The formation of this complex in EMSA is dependent on
the presence of both AT-rich sites present in the G1 promoter element
(Fig. 1B). The protein-DNA complex B contains the
homeodomain protein brain-4 (Fig. 1A; Ref. 4), and complex C
contains pax-6 homodimer (6, 7). The elucidation of the identity of
complexes A and C are largely in agreement with references (6, 7). The
protein complex B cannot be accounted for binding by brain-4 alone,
since nuclear extracts of BHK-21 cells overexpressing brain-4 form a
protein-DNA complex, which is smaller in size than complex B (data not
shown). In addition, a combination of brain-4 and pax-6 or cdx-2
incubated with the G1 element fail to form complex B in EMSA
experiments (data not shown). Therefore, a protein in addition to
brain-4 must be assumed to be involved in the formation of complex B
(4). Further studies are required to elucidate the identity of this additional factor.
View larger version (19K):
[in a new window]
Fig. 9.
Proposed model of the interaction of pax-6
and cdx-2 with the co-activator p300. Binding of pax-6 to 5'
AT-rich site of the G1 promoter element of the proglucagon gene is
strong and required for the interaction to take place. Interaction of
cdx-2 to the 3' AT-rich site in the promoter is weaker. pax-6 and cdx-2
interact with each other. Both pax-6 and cdx-2 interact with p300.
Interaction of pax-6 with p300 is enhanced by the presence of
cdx-2.
-cells may not
only be the expression of the glucagon gene but also inhibition of
-cell proliferation. Little information is available on the
pancreatic phenotype in cdx-2 knock-out mice. pax-6, on the other hand,
has been implicated in pancreatic islet cell development and phenotype
determination (8, 9). pax-6 mutant and knock-out mice have reduced
pancreatic endocrine cell mass, with markedly reduced
glucagon-producing cells (8, 9). The present results, together with
previous reports, further support the notion that the transcription
factors implicated in islet cell development are also involved in gene
expression of terminally differentiated cells (8, 9, 23).
-cell-specific G1
element of the glucagon gene promoter and with the co-activator p300,
it should be stated that expression of neither pax-6 nor cdx-2 is
restricted to pancreatic -cells. Of the transcription factors known
to be expressed in pancreatic -cells, brain-4 appears to be
restricted to the glucagon-producing cells (4). It is conceivable that
the interaction of cell-specific with non-cell-specific transcription
factors leads to an optimal expression of a certain cell-type
restricted gene. To this end, in preliminary studies, we have found
that overexpression in transgenic mice of brain-4 in the context of
other transcription factors required of pancreatic endocrine cell
phenotype can lead to ectopic expression of the glucagon
gene.2
-cells through
interaction with the cell-specific G1 element. Thus, pax-6, cdx-2, and
p300 form a functional complex in the -cell-specific expression of
the proglucagon gene. However, it should be mentioned, as noted above, that additional protein complexes also bind to the G1 element of the
proglucagon gene promoter, which confers -cell-specific expression
of the glucagon gene. The elucidation of these additional transcription
factors will lead to a better understanding of the complex nature of
the tissue-specific expression of the glucagon gene.
ACKNOWLEDGEMENTS
FOOTNOTES
Investigator with the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit St., WEL320, Boston, MA 02114. Tel.: 617-726-5190; Fax: 617-726-6954; E-mail: jhabener@ partners.org.
ABBREVIATIONS
, CAAT
element-binding protein ;
CHOP, C/EBP homologous protein;
CBP, CREB-binding protein;
EMSA, electrophoretic mobility shift assay;
ELB, EDTA low salt buffer;
GST, glutathione S-transferase;
NEN, NaCl-EDTA-Nonidet P-40, p300, protein homologous to CBP;
RSV, Rous
sarcoma virus;
PAGE, polyacrylamide gel electrophoresis;
DMEM, Dulbecco's modified Eagle's medium.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Habener, J. F.
(1996)
in
The Insulinotropic Gut Hormone Glucagon-like Peptide-I
(Fehmann, H. C.
, and Goeke, B., eds), Vol. 13
, Karger, Basel, Switzerland
2.
Laser, B.,
Meda, P.,
Constant, I.,
and Philippe, J.
(1996)
J. Biol. Chem.
271,
28984-28994 3.
Jin, T.,
Trinh, D. K. Y.,
Wang, F.,
and Drucker, D. J.
(1997)
Mol. Endocrinol.
11,
203-209 4.
Hussain, M. A.,
Lee, J.,
Miller, C. P.,
and Habener, J. F.
(1997)
Mol. Cell. Biol.
17,
7186-7194[Abstract]
5.
Philippe, J.,
Drucker, D. J.,
Knepel, W.,
Jepeal, L.,
Misulovin, Z.,
and Habener, J. F.
(1988)
Mol. Cell. Biol.
8,
4877-4888 6.
Andersen, F.,
Heller, R.,
Petersen, H.,
Jensen, J.,
Madsen, O.,
and Serup, P.
(1999)
FEBS Lett.
445,
306-310[CrossRef][Medline]
[Order article via Infotrieve]
7.
Ritz-Laser, B.,
Estreicher, A.,
Klages, N.,
Saule, S.,
and Philippe, J.
(1999)
J. Biol. Chem.
274,
4124-4132 8.
Sander, M.,
Neubüser, A.,
Kalamaras, J.,
Ee, H. C.,
Martin, G. R.,
and German, M. S.
(1997)
Genes Dev.
11,
1662-1673 9.
St.-Onge, L.,
and Sosa-Pineda, B.
(1997)
Nature
387,
406-409[CrossRef][Medline]
[Order article via Infotrieve]
10.
Suh, E.,
Chen, L.,
Taylor, J.,
and Traber, P. G.
(1994)
Mol. Cell. Biol.
14,
7340-7351 11.
Eckner, R.,
Ewen, M. E.,
Newsome, D.,
Gerdes, M.,
DeCaprio, J. A.,
Lawrence, J. E.,
and Livingston, D. M.
(1994)
Genes Dev.
8,
869-884 12.
Arany, Z.,
Sellers, W.,
Livingston, D.,
and Eckner, R.
(1994)
Cell
77,
799-800[CrossRef][Medline]
[Order article via Infotrieve]
13.
Lundblad, J. R.,
Kwok, R. P.,
Laurence, M. E.,
Harter, M. L.,
and Goodman, R. H.
(1995)
Nature
374,
85-88[CrossRef][Medline]
[Order article via Infotrieve]
14.
Xu, L.,
Lavinsky, R. M.,
Dasen, J. S.,
Flynn, S. E.,
McInerney, E. M.,
Mullen, T.-M.,
Heinzel, T.,
Szeto, D.,
Korzus, E.,
Kurokawal, R.,
Aggarwal, A. K.,
Rose, D. W.,
Glass, C. K.,
and Rosenfeld, M. G.
(1998)
Nature
395,
301-306[CrossRef][Medline]
[Order article via Infotrieve]
15.
Ogryzko, V. V.,
Schiltz, L.,
Russanova, V.,
Howard, B. H.,
and Nakatni, Y.
(1996)
Cell
87,
953-959[CrossRef][Medline]
[Order article via Infotrieve]
16.
Jin, T.,
and Drucker, D. J.
(1995)
Mol. Endocrinol.
9,
1306-1320[Abstract]
17.
Walker, W. H.,
Sanborn, B. M.,
and Habener, J. F.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12423-12427 18.
Schreiber, E.,
Matthias, P.,
Müller, M. M.,
and Schaffner, W.
(1989)
Nucleic Acids Res.
17,
6419 19.
Lassar, A. B.,
Davis, R. L.,
Wright, W. E.,
Kadesh, T.,
Murre, C.,
Voronova, A.,
Baltimore, D.,
and Weintraub, H.
(1991)
Cell
66,
305-315[CrossRef][Medline]
[Order article via Infotrieve]
20.
Ausubel, F. M.,
Brent, R.,
Kingston, R. E.,
Moore, D. D.,
Seidman, J. G.,
Smith, J. A.,
and Struhl, K.
(1995)
Short Protocols in Molecular Biology
, John Wiley & Sons, Inc., Boston
21.
Ron, D.,
and Habener, J. F.
(1992)
Genes Dev.
6,
439-453 22.
Passner, J. M.,
Ryoo, H. D.,
Shen, L.,
Mann, R. S.,
and Aggarwal, A. K.
(1999)
Nature
397,
714-719[CrossRef][Medline]
[Order article via Infotrieve]
23.
Sosa-Pineda, B.,
Chowdhury, K.,
Torres, M.,
Oliver, G.,
and Gruss, P.
(1997)
Nature
386,
399-402[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Jin Mechanisms underlying proglucagon gene expression J. Endocrinol., July 1, 2008; 198(1): 17 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Davis, D. Davis, B. Norman, and J. Piatigorsky Gene Expression of the Mouse Corneal Crystallin Aldh3a1: Activation by Pax6, Oct1, and p300 Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 1814 - 1826. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Kratzner, F. Frohlich, K. Lepler, M. Schroder, K. Roher, C. Dickel, M. V. Tzvetkov, T. Quentin, E. Oetjen, and W. Knepel A Peroxisome Proliferator-Activated Receptor {gamma}-Retinoid X Receptor Heterodimer Physically Interacts with the Transcriptional Activator PAX6 to Inhibit Glucagon Gene Transcription Mol. Pharmacol., February 1, 2008; 73(2): 509 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Gosmain, I. Avril, A. Mamin, and J. Philippe Pax-6 and c-Maf Functionally Interact with the {alpha}-Cell-specific DNA Element G1 in Vivo to Promote Glucagon Gene Expression J. Biol. Chem., November 30, 2007; 282(48): 35024 - 35034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Wang, T. Liu, Z. Li, X. Ma, and T. Jin Redundant and Synergistic Effect of Cdx-2 and Brn-4 on Regulating Proglucagon Gene Expression Endocrinology, April 1, 2006; 147(4): 1950 - 1958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Kim, Y. T. Noh, M.-J. Ryu, H.-T. Kim, S.-E. Lee, C.-H. Kim, C. Lee, Y. H. Kim, and C. Y. Choi Phosphorylation and Transactivation of Pax6 by Homeodomain-interacting Protein Kinase 2 J. Biol. Chem., March 17, 2006; 281(11): 7489 - 7497. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Kasper, T. Fukuyama, M. A. Biesen, F. Boussouar, C. Tong, A. de Pauw, P. J. Murray, J. M. A. van Deursen, and P. K. Brindle Conditional Knockout Mice Reveal Distinct Functions for the Global Transcriptional Coactivators CBP and p300 in T-Cell Development Mol. Cell. Biol., February 1, 2006; 26(3): 789 - 809. [Abstract] [Full Text] [PDF] |
