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J. Biol. Chem., Vol. 276, Issue 35, 32489-32494, August 31, 2001
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From the Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263
Received for publication, December 21, 2000, and in revised form, June 7, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Expression from the mouse
Ren-1c gene in As4.1 cells is dependent on a
proximal promoter element (PPE) located at approximately Renin is an aspartyl protease, which, as part of the
renin-angiotensin system, plays a critical role in the maintenance of blood pressure and electrolyte balance by converting angiotensinogen to
angiotensin I (1). Additionally, the renin-angiotensin system has been
implicated in aspects of renal development by the observation that pharmacological and genetic disruptions of renin-angiotensin system function result in aberrant renal morphology (for reviews, see
Refs. 2-4).
Renin gene expression is subject to complex developmental and
tissue-specific regulation (for review, see Ref. 5). In murine fetuses,
kidney renin transcripts can be detected as early as 14.5 days post
coitum in the newly developing arteries (6). On development of the
renal arterial tree, expression shifts to cells located in nascent
portions of the growing arteries until expression is restricted to a
small population of modified smooth muscle cells of the afferent
arteriole proximal to the glomerulus called juxtaglomerular cells. In
adult mice, renin is also expressed in adrenal gland, submandibular
gland, gonads, and coagulating gland.
Some mouse strains have only a single renin gene
(Ren-1c), whereas other strains have two copies,
a Ren-1 locus with allele Ren-1d and
a duplicated locus with Ren-2 (7, 8). Although these mouse
renin genes are approximately equivalently expressed in the adult
kidney, their expression patterns are different in some extra renal
tissues (9).
Identification of cis-acting sequences in the mouse renin
5'-flanking region has been accelerated by the isolation of a kidney tumor-derived As4.1 cell line from transgenic mice containing the mouse
Ren-2 5'-flanking sequence fused to SV40 T antigen (10). As4.1 cells are capable of expressing high levels of renin mRNA (10) and secreting active renin protein (11). By transiently transfecting these cells with wild-type or mutant
Ren-1c-chloramphenicol acetyltransferase
(Ren-CAT) constructs, two regions in the Ren-1c
5'-flanking sequence were found to be necessary for high level expression of mouse renin gene in addition to a TATA box, the PPE1 located at Hox genes are members of the homeobox family of
transcription factors and control many aspects of morphogenesis and
cell differentiation in animals (13). In vertebrates, there are 39 Hox genes organized in four clusters (A, B, C, and D) on
separate chromosomes with members of each cluster classified into as
many as 13 paralog groups based on sequence similarity (14).
Hox gene products can bind DNA as monomers or heterodimers
with three-amino acid loop extension (TALE) class homeodomain
proteins including PBX and MEIS on HOX·PBX or HOX·MEIS recognition
sequences (15-22). Interactions with PBX or MEIS proteins increase
both DNA binding affinity and specificity for HOX proteins. Although
the HOX·MEIS recognition sequences have not yet been identified in
any natural genes, the HOX·PBX binding sequences have been found in
genes such as Hox (23, 24), In this report we show that the renin gene contains a functional
HOX·PBX binding site in its promoter region. Homeodomain proteins
PBX1b and HOX family members (preferentially HOX9 and -10) can bind
this element in vitro and in As4.1 cells. Both PBX and HOX
binding sites are necessary for the expression of the Ren-1c gene. Moreover, we have demonstrated that
PREP1 can form a ternary complex with HOX and PBX1b on this site.
Analysis of Hox Gene Expression Using RT-PCR--
Total RNA from
As4.1 cells was isolated by TRIZOL reagent (Life Technologies, Inc.),
and first strand cDNA was then synthesized using
SUPERSCRIPTTM Preamplification System (Life Technologies,
Inc.). The homeodomain regions of Hox genes were amplified
from the first strand reaction product by PCR. The degenerate PCR
primers used were directed against the first
(5'-agctaaagcttCA(A/G)(A/G)(C/T)(G/C)(C/T)T(A/G)GA(A/G)(C/T)T(A/G)GA(A/G)AA(A/G)GA(A/G)TT-3') and third
(5'-agctatctagaCG(A/G)TT(C/T)TG(A/G)AACCA(A/G)AACA(A/G)AT(C/T)TT(C/G)A(C/T)(C/T)TG-3') Plasmid Constructions--
Plasmids
Full-length cDNA for HOXB6, -B7, -C8, -B9 or -D10 was isolated by
RT-PCR from As4.1 cells and cloned into
pcDNA3.1/myc-His(+)A vector (Invitrogen), which
contains a carboxyl-terminal Myc epitope. Full-length cDNA for
PREP1 was also cloned from As4.1 cells and inserted in
pcDNA3.1/V5-His(+)A (Invitrogen), which has a carboxyl-terminal V5
epitope. Full-length cDNA for PBX1b was cloned in the same vector
without incorporating any epitope. These plasmids were used in in
vitro transcription/translation and EMSAs. For expression in
mammalian cells, a stop codon was added immediately before the Myc or
V5 epitope sequence in the HOXD10 or PREP1 expression vector,
respectively, so that the carboxyl-terminal Myc or V5 epitope was not
translated. VP16-HOXD10 or VP16-PBX1b was constructed by
inserting HOXD10 or PBX1b full-length cDNA, respectively, in pVP16
(CLONTECH) so that a nuclear localization signal
and a VP16 activation domain were fused amino-terminally to HOXD10 or
PBX1b. VP16-PREP1 and VP16-HM were similarly constructed. VP16-PREP1 contained a truncated PREP1 with residues 1-75 deleted, whereas VP16-HM contains the HM region of PREP1 from residues 75 to 557.
Cell Culture and Transient Transfections--
As4.1 cells were
grown in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum and transfected using FuGENE 6 (Roche Molecular
Biochemicals). For each transfection in a 35-mm culture dish, 2.2 µg
of DNA including 0.5 µg of reporter plasmid, 0.5 µg of each
expression plasmid, nonspecific plasmid if necessary, and 0.2 µg of
plasmid containing the Rous sarcoma virus promoter driving
In Vitro Transcription and Translation--
HOX, PBX1b, and
PREP1 proteins were in vitro transcribed/translated by the
TNT Coupled Wheat Germ System (Promega). Parallel reactions containing
[35S]methionine were performed to correct differences in
translation efficiency.
EMSA--
The EMSAs were performed as described previously (12).
For each reaction (15 µl), about 0.2 ng of labeled DNA probe (20,000 cpm) were mixed with As4.1 cell nuclear extract (3-6 µg) or in vitro translated proteins (1-3 µl for each protein) and 1 µg
of poly(dI-dC) in 10 mM Hepes, pH 7.9, 10 mM
KCl, 50 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10% glycerol. The reaction mixture was
incubated on ice for 30 min and then run on a 6% polyacrylamide gel in
0.5× Tris borate-EDTA buffer. In a competition or supershift assay, an
excess amount of unlabeled DNA or 1 µl of antibody was added to the
reaction mixture 15 min or 1 h prior to the addition of labeled
DNA probe, respectively. Antibodies against PBX1/2/3, PBX1, PBX2, PBX3,
and PREP1 were purchased from Santa Cruz Biotechnology, Inc. The
antibody against the Myc tag was purchased from Invitrogen.
The Ren-1c PPE Is a HOX·PBX Protein Binding
Site--
The consensus sequence for HOX·PBX binding is NTGATTNATNN.
Using in vitro site selection assays, the preferred binding
site for PBX and HOX6-10 paralog members was shown to be NTGATTTATNN (or inverting N'N'ATAAATCAN') (18, 19). The sequence is a precise match
to the highly conserved PPE in mouse Ren-1 and
Ren-2 and rat and human renin promoters (Fig.
1). To test whether the PPE is a
HOX·PBX binding site, supershift EMSA was performed with antibodies
against PBX1/2/3, which recognizes long isoforms of PBX proteins
(PBX1a, PBX2, and PBX3a), PBX1, PBX2, and PBX3 (Fig. 2). Only PBX1
antiserum, which recognized both PBX1a and PBX1b, was able to
supershift the R1·As4.1 nuclear protein complex. The results
demonstrated that the PPE contains the PBX binding site and suggest
that PBX1b is the major PBX species binding at the PPE.
To determine which HOX protein might bind to the PPE, the
Hox gene complement in As4.1 cells was surveyed by
amplifying the highly conserved homeodomain region using RT-PCR. A
total of 45 clones was assessed, and the results from the analysis are
shown overlaid on an organizational map of the mouse Hox
gene clusters (Fig. 3). The results
showed that predominantly (33 of 45) Abd-B classes 9 and 10 are
represented, and specifically HOXD10, -A10, -A9, -B9, and -C9 are
expressed in As4.1 cells.
To test whether HOX and PBX proteins can bind to the PPE, EMSA was
performed with in vitro synthesized HOXB6, -B7, -C8, -B9, and -D10 proteins plus and minus in vitro synthesized PBX1b
(Fig. 4). Each HOX protein contained a
Myc carboxyl-terminal epitope so that the complex formed by the R1
probe and the HOX protein could be supershifted by antibody against the
Myc epitope. PBX1b alone was not capable of binding the PPE (Fig. 4,
lane 1). HOXB6 (Fig. 4, lane 2), -B7 (Fig.
4, lane 6), and -C8 (Fig. 4, lane 10) did not
bind to R1 detectably, however, in the presence of PBX1b they were able
to bind weakly (more apparent with longer exposure, Fig. 4, lanes
3, 7, and 11, and data not shown). However, HOXB9 and especially -D10 gave rise to prominent shifts with or without
PBX1b (Fig. 4, lanes 14, 15, 18, and
19). The HOX and HOX·PBX1b complexes were able to be
supershifted by Myc antiserum (Fig. 4, lanes 16 and
20), whereas the HOX·PBX1b complex was disrupted by
the addition of antibody against PBX1 (Fig. 4, lanes 17 and 21).
PREP1 Can Form a Ternary Complex with HOX·PBX on
Ren-1c PPE--
TALE class homeodomain proteins PREP1 and
MEIS have been shown to form ternary complexes with HOX·PBX (31-36).
We examined whether PREP1 is a component of the As4.1 cell nuclear
proteins that bind to the Ren-1c PPE. In
supershift assays, the antibody against PREP1 supershifted the complex
formed by Ren-1c PPE and As4.1 nuclear extracts
(Fig. 5A). In vitro
synthesized PREP1 was also tested for its ability to bind
Ren-1c PPE in the presence of HOXD10 and PBX1b
(Fig. 5B). The results indicate that, although PREP1 could
not bind to PPE alone or in combination with PBX1b (data not shown), it
forms a ternary complex with HOXD10 and PBX1b (Fig. 5B,
lane 3). The ternary complex could be supershifted or
disrupted by the addition of antibody against Myc, PBX1, or PREP1 (Fig.
5B, lanes 4-6). Moreover, the ternary complex
formed by HOXD10·PBX1b·PREP1 has almost the same electrophoretic mobility as the complex formed by As4.1 cell nuclear proteins (Fig.
5B, lane 3 versus lane 7) further
suggesting that the Ren-1c PPE binds a ternary
complex including HOX, PBX1b, and PREP1.
To further assess whether HOX is a member of As4.1 cell nuclear
proteins binding to PPE, we tested whether in vitro
translated HOXD10 can complex with PBX and PREP1 in As4.1 cell nuclear
extract on the PPE. One microliter of 10-fold diluted HOXD10 protein
synthesized in vitro was mixed with As4.1 cell nuclear
extracts in EMSA, and the presence of HOXD10 in the complex was
confirmed by supershift assays with Myc antiserum (Fig. 5C).
The results showed that the complex formed by this amount of HOXD10
alone was barely visible (Fig. 5C, lane 1).
However, the addition of the same amount of HOXD10 to As4.1 cell
nuclear extract resulted in the formation of a complex that paralleled
in size the complex formed by As4.1 cell nuclear extract alone and was
more intense (Fig. 5C, lane 3 versus lane
2). The sharp increase in complex intensity by adding HOXD10
suggests the possibility that As4.1 cell nuclear extract can provide
factors that improve the ability of HOXD10 to bind the R1 PPE, and
these factors include PBX1 and PREP1. Myc antiserum was capable of
supershifting almost all the complex formed (Fig. 5C,
lane 4) suggesting that the exogenous HOXD10 was in excess of endogenous HOX proteins binding to the PPE. Antibodies against PBX1
and PREP1 also supershifted the complex (Fig. 5C,
lanes 5 and 6) indicating that the formation of
HOXD10·PBX·PREP1 ternary complex.
Both HOX and PBX Half-sites Are Crucial for Ren-1c Gene
Expression--
Based on HOX·PBX crystal structure (38, 39), crucial
contacts are made between PBX and the adenine at the nucleotide 4 position of the heterodimer recognition sequence and between HOX and
the nucleotide 8 position of the recognition sequence (see Fig.
6A). We examined the effects
of point mutations at these positions on the binding of As4.1 cell
nuclear extracts in EMSA (Fig. 6, A and B). A
single mutation of nucleotide 4 (Fig. 6A, R3) or
double mutations at nucleotides 3 and 4 (Fig. 6A,
R4) resulted in the formation of complexes with high
mobility equivalent to binding of the HOX monomer alone (Fig.
6B, lanes 2 and 3). The same results
were obtained using in vitro translated HOXD10·PBX1b (Fig.
6C, lanes 4 and 5). In contrast, the
single nucleotide mutation at nucleotide 8 (Fig. 6A,
R5) resulted in complete failure of complex formation with
either As4.1 cell nuclear extracts (Fig. 6B,
lane 4) or in vitro translated
HOXD10·PBX1b (Fig. 6C, lane 6). In competition
assays (Fig. 6B), the R1 oligonucleotide competed with
itself in the formation of the R1·As4.1 cell nuclear protein complex
very well at a 100-fold excess (Fig. 6B, lanes 5 and 6). However, oligonucleotides with PBX half-site
mutations exhibited only partial competition (Fig. 6B,
lanes 7 and 8), whereas the oligonucleotide with
the nucleotide 8 mutation failed to compete (Fig. 6B,
lane 9). These results are consistent with previous reports
that HOX can bind its recognition site as monomer; however, PBX cannot
bind to the HOX·PBX recognition sequence in isolation.
Effects of these point mutations on expression from the
Ren-1c promoter were also tested (Fig.
6D). The mutations were incorporated into plasmid HOXD10, PBX1b, and PREP1 Can Bind the Ren-1c PPE in
As4.1 Cells--
As4.1 cells were cotransfected with a reporter
construct containing three copies of the Ren-1c
PPE upstream of an E1b TATA box and expression vectors for HOXD10, PBX1b, PREP1, and various combinations. Overexpression of these proteins had little effect on the transcription of the reporter gene
(Fig. 7). However, fusion proteins
VP16-HOXD10, VP16-PBX1b, and VP16-PREP1, which have a VP16 activation
domain fused amino-terminally to HOXD10, PBX1b, and PREP1,
respectively, were capable of activating reporter gene expression by
11-, 15-, and 30-fold. However, they did not activate a similar
reporter gene with mutated Ren-1c PPE (data not
shown). These results suggest that HOXD10, PBX1b, and PREP1 are capable
of binding to the PPE in As4.1 cells. Moreover, the fusion protein
VP16-HM containing only the VP16 activation domain and HM domain, which
is the region in PREP1 interacting with PBX, was still capable of
activating the reporter gene by 30-fold. The results agree with
previous reports that the homeodomain of PREP1 is not necessary in the
ternary complex formation on a HOX·PBX site (31).
In this report, we have demonstrated that the PPE of the renin
gene is a HOX·PBX heterodimer binding site. First, the nucleotide sequences of PPE match perfectly to those of the HOX·PBX heterodimer recognition site. Second, Abd-B HOX9 and -10 members and PBX1b were
shown to bind this element with high affinity in vitro.
Finally, point mutation of a critical nucleotide either in the HOX or
PBX half-site showed the expected effects on HOX·PBX binding in EMSA. In addition, these mutations dramatically reduced the transcriptional activity of Ren-1c gene suggesting that both PBX
and HOX half-sites are necessary for renin gene expression.
The human renin PPE was previously identified as a Pit-1 site in
GC cells (40). However, our results have shown that the PPE in
the mouse renin promoter is not a Pit-1 binding site (12). The PPE in
mouse is a poor consensus for Pit-1. Moreover, the As4.1 cell nuclear
proteins were not capable of binding to a human Pit-1 consensus
oligonucleotide. Furthermore, the major nuclear protein complex
from As4.1 cells formed on mouse Ren-1c PPE
could not be competed by the Pit-1 oligonucleotide, although it could
be competed by the oligonucleotide containing the human PPE. These
observations suggest that in As4.1 cells the primary proteins binding
to the PPE are HOX and PBX, not Pit-1. Whether HOX and PBX proteins
bind to the human PPE in GC cells has not been examined.
A direct test of whether HOXD10 protein is involved in the formation of
the ternary complex on the mouse PPE in supershift assays has not been
possible because of the unavailability of specific antibody. However,
other evidence suggests that HOXD10 is a major HOX protein binding to
the PPE in As4.1 cells. HOXD10 is expressed in As4.1 cells. Moreover,
in the presence of PBX1b, HOXD10 binds to the PPE in EMSA with higher
affinity compared with other selected HOX paralog members, and the size
of the complex mimics the endogenous complex formed in As4.1 cell
nuclear extracts. Furthermore, a small amount of in vitro
synthesized HOXD10 was capable of forming the HOXD10·PBX1b·PREP1
complex with endogenous PBX and PREP1. However, we cannot rule out a
redundant role for other HOX family members such as HOX9 members, which
are also expressed in As4.1 cells and recognize the same sequence
motifs as HOXD10. Recent evidence suggests that although different
HOX·PBX heterodimers have preferred sites in vitro, this
binding specificity is not always utilized in vivo (41, 42).
It has been suggested that the specificity of a HOX·PBX site might be
affected by cofactor binding sites. Thus, the study of renin gene
expression in mice lacking different HOX proteins will be helpful in
identifying the specific HOX family members necessary for renin expression.
We showed that overexpression of HOXD10, PBX1b, PREP1, or various
combinations was not able to activate a promoter containing three
copies of Ren-1c PPE. These results are in
agreement with previous results reported by Shen et al. (32)
and Shanmugam et al. (33) that HOX·PBX·MEIS complex does
not exhibit transcriptional activity on artificial promoters containing
HOX·PBX binding sites. Thus, other cofactor binding sites may be
necessary in the natural targets for the function of
HOX·PBX·PREP1/MEIS. We also cotransfected How the PPE bound HOX·PBX1b·PREP1 complex interacts with other
required transcriptional factors to regulate the mouse renin gene
expression remains to be elucidated. It is possible that the
PPE-binding proteins directly interact with a factor binding to an
element located within or immediately outside the renal enhancer region
located at A recent report by Saleh et al. (43) suggests that the
HOX·PBX complex recruits the coactivator CREB-binding protein to activate gene expression. A CREB/cAMP-response element modulator protein binding site within the Ren-1c enhancer
located at These findings strongly imply that renin and in larger scope the
renin-angiotensin system are immediate downstream targets of the Class
I Hox developmental control genes. As such, the production of angiotensin II, a classical pressor substance that also exhibits growth factor activities as mediated through the AT1 and AT2 receptor system (44), is regulated by an important class of developmental patterning genes. The mammalian metanephric kidney is but one of three
phylogenetically and ontogenetically discernable kidneys that arise at
different points along the anterior-posterior body axis in vertebrates.
Intriguingly the preferred recognition sequence for the HOX paralogs
expressed at these points on the body axis is precisely that found in
the PPE of the renin genes, and renin gene expression has been noted in
vascular tissues of pronephric and mesonephric as well as metanephric
kidneys (45, 46).
60 and a
241-base pair enhancer region located at 2625 relative to the
transcription start site. The PPE (TAATAAATCAA) is identical to a
consensus HOX·PBX binding sequence. Further, PBX1b has been shown to
be a component of a PPE-specific binding complex present in nuclear
extracts from As4.1 cells. The binding affinities of different paralog
HOX members to the PPE were examined in the absence or presence of
PBX1b. HOXB6, -B7, and -C8 failed to bind the PPE alone but showed weak
affinity in the presence of PBX1b. In contrast, HOXD10 and to a lesser
degree HOXB9 bound the PPE with high affinities regardless of whether
PBX1b was present. Abd-B HOX members, including HOXD10, -A10, -A9, -B9,
and -C9, are expressed in As4.1 cells. The ability of HOX and PBX1b to form a ternary complex with PREP1 on the PPE is also demonstrated both
in vivo and in vitro. Point mutations in either
the HOX or PBX half-site of the PPE disrupted the formation of the
HOX·PBX complex and dramatically decreased transcriptional activity
of the Ren-1c gene demonstrating that both the
HOX and PBX half-sites are critical for mouse renin gene expression.
These results strongly implicate Abd-B class Hox genes and
their cofactors as major determinants of the sites of renin expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60 bp and a
241-bp enhancer located 2.6 kb upstream of the transcription start site
(12). The PPE was shown to bind As4.1 cell nuclear proteins in
electrophoretic mobility shift assays (EMSAs). Further competition
assays indicated that the minimal sequence required for protein binding
included N(1-3)TAATAAATCA. Mutation of the PPE in the
Ren-CAT construct containing a 4.1-kb mouse renin sequence dramatically
reduced the chloramphenicol acetyltransferase activity in transfection
assays suggesting a critical role of this element in the regulation of
mouse renin gene expression.
2(V) collagen (25), Eph
receptor EphA2 (26), and fork head (fkh) (27).
Moreover, MEIS or its homolog PREP1 interacts with PBX through its
amino-terminally located HM domain (28-30). This interaction is
essential for PBX nuclear translocation. Several studies have also
demonstrated that MEIS or PREP1 can form ternary complexes with HOX and
PBX (31-36), and this trimeric complex has been shown to play an
important role in regulating several HOX-responsive genes (31,
34-36).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices. The 150-bp fragment resulting from RT-PCR was
isolated and subcloned. Sequences of individual clones were then determined.
4.1R1, 4.1M,
4.1mh, 4.1mp, and 4.1mp2 were constructed by inserting the
wild-type or mutant Ren-1c sequences from
4100 to +6 bp into pGL2-basic (Promega). The reporter
construct 3XR1-TA was made by inserting three copies of
Ren-1c PPE into a pGL2-basic-derived plasmid
containing the adenovirus E1b TATA box (37).
-galactosidase were mixed with 4.4 µl of FuGENE reagent. Forty-eight hours after transfection cells were harvested and measured
for luciferase and -galactosidase activities using the Luciferase
Assay System (Promega) and Galacto-Light PlusTM
chemiluminescent reporter assay (Tropix), respectively. The luciferase activity is normalized with -galactosidase activity to correct differences in transfection efficiency between experiments. All transfection results represent the average ± S.D. of at least three separate experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the proximal promoter regions of
renin genes. Sequences of mouse Ren-1c and
Ren-2 and human and rat renin gene promoter regions are
aligned using Multiple Sequence Alignment (InforMax, Inc.). Positions
1, 60, and 117 and the TATA box corresponding to those on
Ren-1c or Ren-2 promoter are
indicated. Two highly conserved regions including the TATA box and the
PPE located at 60 are shown in boldface.
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Fig. 2.
Binding of PBX1b in As4.1 cell nuclear
extract to the PPE of Ren-1c gene.
EMSA was performed on the R1 probe (5'-CTGGGGTAATAAATCAAAGCAGA-3')
using As4.1 cell nuclear extract. Antibodies -PBX1/2/3, -PBX1,
-PBX2, and -PBX3 were used to supershift the DNA·protein
complexes. The supershifted complex (SS) is only observed in
lane 3, which is overlapped with the bottom of the sample
loading well indicated by an asterisk. The retarded complex
(C) and free probe (FP) are indicated.
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Fig. 3.
Analysis of Hox gene
expression in As4.1 cells. RNA from As4.1 cells was isolated, and
the Hox gene complement was amplified by RT-PCR using
homeodomain-specific degenerate primers directed against the first and
third -helices. The resulting DNA fragments from RT-PCR were
subcloned and sequenced to profile Hox expression in As4.1
cells. A total of 45 clones were assessed, and results from this
analysis are shown overlaid on an organizational map of the mouse
Hox gene cluster. The plain text numbers 1-13
designate Hox paralog groups. The bold text
numbers indicate the numbers of clones isolated for each Hox
member.
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Fig. 4.
Binding of in vitro
synthesized HOX and PBX1b to the PPE. EMSA was performed on
the R1 probe using in vitro synthesized PBX1b, HOXB6, HOXB7,
HOXC8, HOXB9, and HOXD10. All synthesized HOX proteins have a
carboxyl-terminal Myc epitope. Antibodies against PBX1
( -PBX1) and the Myc epitope (-Myc) were
used to supershift or attenuate the DNA·protein complexes. Free probe
(FP) is indicated.
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Fig. 5.
Formation of a HOXD10·PBX1b·PREP1 ternary
complex on the PPE of Ren-1c gene.
A, EMSA was performed on the R1 probe using As4.1 cell
nuclear extract. Antibody against PREP1 ( -PREP1) was used
in EMSA to supershift the DNA·protein complex. The supershifted
complex (SS) in lane 3 is overlapped with the
bottom of the sample loading well indicated by an asterisk.
The retarded complex (C) and free probe (FP) are
indicated. B, EMSA was performed to access the binding of
in vitro translated HOXD10, PBX1b, and PREP1 to the R1
probe. As4.1 cell nuclear extracts (As4.1 NE) were also
examined by EMSA. Antibodies against PBX1 (-PBX1), PREP1
(-PREP1), and the Myc epitope (-Myc) were
used to supershift or attenuate the DNA·protein complexes. The
retarded bands representing HOX monomer, HOX·PBX heterodimer,
HOX·PBX·PREP1 ternary and supershifted complex (SS) are
indicated. Free probe is not shown. C, EMSA was performed
using R1 as probe. One microliter of 10-fold diluted in
vitro synthesized HOXD10 protein containing a carboxyl-terminal
Myc epitope was used in EMSA in the absence or presence of As4.1 cell
nuclear extract. Antibodies against PBX1 (-PBX1), PREP1
(-PREP1), and the Myc epitope (-Myc) were
used to supershift the DNA·protein complexes. The retarded bands
representing HOXD10, As4.1 nuclear protein complex (C), and
supershifted complex (SS) are indicated. Free probe
(FP) is also indicated.
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Fig. 6.
Effects of point mutations in the PPE on its
ability to bind As4.1 cell nuclear proteins and in vitro
translated HOXD10·PBX1b and on Ren-1c
gene expression. A, shown are the sequences of
double-stranded oligonucleotides of the Ren-1c
PPE (R1) and its mutants containing mutations
(underlined) either in the PBX half-site (R3 and
R4) or in the HOX half-site (R5). The HOX·PBX
binding site in the R1 is indicated by a solid line. The
positions of nucleotides 3, 4, and 8 are also indicated. B,
R1, R3, R4, and R5 were used as probes in EMSA with nuclear proteins
from As4.1 cells. Competition of the R1 complex with 100-fold excess of
R1, R3, R4, and R5 was also performed. Samples in lanes did not
contain competitors. The retarded complex (C) and free probe
(FP) are indicated. C, R1, R3, R4, and R5 were
used as probes in EMSA with in vitro translated HOXD10
and/or PBX1b. Mock sample contained the lysate without any DNA added.
D, As4.1 cells were transfected with the indicated plasmids
shown at the top. The nucleotide substitutions in plasmids
4.1mh, 4.1mp, and 4.1mp2 are underlined. The
luciferase (Luc) activity is expressed relative to that of
plasmid 4.1R1.
4.1R1.
The nucleotide 8 mutation (4.1mh) in the HOX protein binding site
reduced the expression level of 4.1R1 by ~80%. The nucleotide 4 single mutation (4.1mp) or nucleotides 3 and 4 double mutations
(4.1mp2) in the PBX half-site also resulted in an ~85% reduction
in expression from the Ren-1c promoter. These
results suggest that the binding of PBX and HOX to PPE is crucial for
mouse renin gene expression.
View larger version (29K):
[in a new window]
Fig. 7.
HOXD10 (D10), PBX1b, and
PREP1 can bind to the Ren-1c PPE in As4.1
cells. As4.1 cells were cotransfected with reporter 3XR1-TA and
various expression vectors in combinations. The luciferase
(Luc) activity is expressed relative to that of plasmid
3XR1-TA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4.1R1 with HOXD10,
PBX1b, PREP1, or various combinations into As4.1 cells. The results
showed that the transcriptional activity of the reporter gene was
not affected by co-expression of these proteins (data not shown). It is
possible that a cofactor, which is necessary for
HOX·PBX·PREP1-mediated expression from 4.1R1, is limiting. Therefore, overexpression of HOX· PBX·PREP1 would not further result in an increase in transcriptional activity. When the VP16 activation domain is attached to either HOXD10, PBX1b, or PREP1, it can
substitute for the limiting cofactor to activate expression from
construct 3XR1-TA.
2.6 kb to bring the enhancer closer to the TATA box by
looping out. Recent findings by Ryoo et al. (35), Ferretti
et al. (36), and Jacob et al. (34) have
demonstrated that the vertebrate HoxB2 r4 or
Drosophila labial (lab) gene enhancer contains a
MEIS/PREP1 (or Drosophila homolog HTH) binding site located
almost one compete turn of the helix away from a HOX·PBX site. The
MEIS/PREP1/HTH site is required for the HOX·PBX·MEIS/PREP1 or HTH
ternary complex formation. However, there is no PREP1/MEIS site found
adjacent to the PPE in the Ren-1c gene.
Interestingly, surveys of the 4.1-kb Ren-1c
flanking sequences for PREP1/MEIS or PBX·PREP1/MEIS sites have revealed two such sites in proximity to the enhancer region. One of
these MEIS/PREP1 sites may interact with the HOX·PBX site in the
promoter region through the formation of the HOX·PBX·PREP1/MEIS ternary complex to bring the enhancer close to the transcriptional apparatus.
2.6 kb is critical for the enhancer
function.2 Deletion or
mutation of the CREB/cAMP-response element modulator protein binding
site results in complete loss of enhancer activity. It is thus possible
that the PPE bound HOX·PBX complex indirectly interacts with the CREB
site within the enhancer through a common CREB-binding protein
coactivator to activate Ren-1c gene expression.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL48459 (to K. W. G.), CA16056, and HD36416 (to S. C. P.).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.
This manuscript is offered belatedly in honor of Prof. Max L. Birnstiel's retirement as Director of the Institute of Molecular Pathology, Vienna, Austria.
Supported by a postdoctoral fellowship from the National
Institutes of Health.
§ Present address: Mondogen GmbH, Am Klopferspitz 19a, D-82152 Martinsried, Germany.
¶ Present address: School of Medicine and Biomedical Sciences, 40 CFS Bldg., 3435 Main St., Buffalo, NY 14214.
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton
Sts., Buffalo, NY 14263-0001. Tel.: 716-845-4572; Fax: 716-845-8169;
E-mail: gross@acsu.buffalo.edu.
Published, JBC Papers in Press, June 29, 2001, DOI 10.1074/jbc.M011541200
2 L. Pan and K. W. Gross, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: PPE, proximal promoter element; EMSA, electrophoretic mobility shift assay; bp, base pair(s); kb, kilobase pair(s); RT-PCR, reverse transcription-polymerase chain reaction; CREB, cAMP-response element-binding protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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