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First published online 25 August 2004
doi: 10.1242/dev.01344
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1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, 240 Longwood Avenue Boston, MA 02115, USA
2 Cardiology Department, Children's Hospital Boston, 300 Longwood Avenue,
Boston, MA 02115, USA
* Author for correspondence (e-mail: andrew_lassar{at}hms.harvard.edu)
Accepted 8 July 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Nkx2.5, BMP, YY1, GATA, SMADs, Cardiogenesis, Chick
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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; Rawles,
1943; Rosenquist,
1966; Rosenquist and DeHaan,
1966). The tissues and signals that induce the specification of
cardiac progenitors have been the subject of intense investigation. Signals
from the anterior endoderm in both chick and amphibian embryos
(Jacobson and Duncan, 1968;
Sater and Jacobson, 1989;
Schneider and Mercola, 2001;
Schultheiss et al., 1995), and
from the organizer region in Xenopus embryos
(Schneider and Mercola, 2001)
have been shown to promote the formation of cardiac tissue. Signals that
promote the induction of cardiac mesoderm include BMP family members
(Andree et al., 1998;
Schlange et al., 2000;
Schultheiss et al., 1997;
Schultheiss and Lassar, 1997;
Shi et al., 2000); FGF8
(Alsan and Schultheiss, 2002;
Reifers et al., 2000); WNT11
(Eisenberg and Eisenberg,
1999; Pandur et al.,
2002); and Crescent, FrzB and Dikkopf
(Marvin et al., 2001;
Schneider and Mercola, 2001;
Tzahor and Lassar, 2001),
which act as antagonists of Wnt family members that are expressed in either
nascent mesoderm of the primitive streak or in the neural tube at
post-neurulation stages (Tzahor and
Lassar, 2001).
One of the earliest genes to be induced in cardiac precursor cells in
response to these various heart-inducing signals is the transcription factor
Nkx2.5. Nkx2.5 is a homolog of the Drosophila tinman gene,
and is first expressed in the anterior lateral regions of gastrula stage
vertebrate embryos termed the `cardiac crescent', which contains both cardiac
and foregut mesendoderm precursors. At later stages of development,
Nkx2.5 is expressed throughout the mature heart and in the pharyngeal
arches, spleen, thyroid, stomach and tongue
(Komuro and Izumo, 1993;
Lints et al., 1993).
Nkx2.5 works in concert with other transcription factors to regulate
early cardiac gene expression (Chen et
al., 1996; Chen and Schwartz,
1996; Sepulveda et al.,
1998; Sepulveda et al.,
2002). Given the role of Nkx2.5 in heart induction and
development, identification of the regulatory sequences and transcription
factors controlling the expression of this gene is of particular interest.
Several activating regions (ARs) that flank the mouse Nkx2.5 gene
have been described that are capable of driving transgene expression in both
the cardiac crescent and newly formed heart
(Lien et al., 1999;
Reecy et al., 1999;
Schwartz and Olson, 1999;
Searcy et al., 1998;
Tanaka et al., 1999).
Consistent with the finding that BMP signals promote Nkx2.5
expression and cardiac induction (Schlange
et al., 2000; Schultheiss et
al., 1997), several consensus binding sites for SMAD4-containing
complexes have been functionally implicated in driving expression of the
murine Nkx2.5 AR2 cardiac crescent enhancer
(Liberatore et al., 2002;
Lien et al., 2002). In
addition, consensus binding sites for the GATA zinc-finger transcription
factors have been found to be necessary for the activity of both AR1 and AR2,
and probably interact with GATA4, GATA5 or GATA6
(Lien et al., 1999;
Searcy et al., 1998).
In this study, we have characterized genomic flanking regions of the chick Nkx2.5 gene to identify three distinct cis-regulatory elements or cardiac activating regions (CAR 1, 2 and 3) that work in combination to drive transgene expression in both the cardiac crescent and in segment-specific compartments of the maturing heart. One enhancer, located 3' to the coding exons of chick Nkx2.5 (CAR3), confers BMP responsiveness to reporter genes in a heterologous assay system, and drives transgene expression in both the primary and secondary heart fields and throughout the outflow tract and right ventricle of the maturing heart. By a combination of deletion mapping and embryonic gel shift analysis, we have identified a 200 bp sequence capable of both conferring a BMP response and driving cardiac-specific transgene expression. This regulatory sequence contains a triad of functionally important binding sites for GATA4/GATA5/GATA6 and YY1 that act in conjunction with three closely associated SMAD-binding sites to regulate transgene expression in vivo and confer BMP responsiveness to CAR3 driven reporters in vitro. Detailed molecular analysis of this BMP response element (BMPRE) suggests that the transcriptional repressor, YY1, serves a primary role in both recruiting an activated SMAD complex to the BMPRE and becomes a transcriptional activator when bound adjacent to BMP-activated SMADs. These findings indicate that the combinatorial activity of both spatially restricted transcription factors such as GATA proteins work in concert with a more ubiquitously expressed transcription factor (i.e. YY1), the activity of which is modulated by SMAD association to drive a regionalized BMP response.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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), G. Sui and
Y. Shi (unpublished). PCMV-p21E1b was a kind gift from O. Kranenburg
(Kranenburg et al., 1995).
Transient and stable transgenic mouse assays
Nkx2.5-lacZ reporter constructs were introduced into a one-cell
stage FVB mouse embryo using standard methods. In the case of the
Nkx2.5-lacZ-BMPRE construct (Fig.
1R,S) an insulator sequence derived from the chick ß-globin
locus (Chung et al., 1993) was
appended immediately 3' to the reporter construct. F0 embryos were
collected at 7.5-10.5 days post-injection following maternal sacrifice, and
fixed and stained for ß-galactosidase activity according to previously
described methods (Zimmerman et al.,
1994). Transgenic status of individual embryos was determined by
PCR for the lacZ transgene from DNA derived from yolk sacs and embryo
fragments (Wassarman, 1993).
Stable lines were obtained by mating fully grown F0 to wild-type FVB mice.
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). Cells
were transfected with 0.1 µg luciferase or lacZ reporters, along
with 20 ng TK-renilla luciferase (Promega) and the indicated quantities of
expression vectors as per individual experiments. Individual wells were
assayed after 18-24 hours of incubation with or without the addition of 50
ng/ml recombinant human BMP2 (a kind gift from Genetics Institute/Wyeth
Pharmaceuticals) or conditioned medium from 293 cells programmed to
overexpress mouse BMP4 (D.-W. Kim and A.B.L., unpublished) using either a dual
luciferase assay system (Promega) or a chemiluminescent ß-galactosidase
assay (Galactolight; Tropix). Nkx2.5-driven luciferase activity was normalized
to that of TK-renilla luciferase. All transfections are representative of
three to five independent experiments with similar results.
Chick embryo extracts and gel shift assays
Anterior lateral plate, posterior primitive streak and heart explants were
dissected from staged chick embryos. Explants from 50-100 embryos were pooled
on ice in PBS, collected by mild centrifugation in a microfuge at 110
g, then resuspended and homogenized in an extraction buffer
containing 20 mM HEPES (pH 7.6), 20% glycerol, 500 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, protease inhibitors
(`C/Emplete, EDTA-free' protease inhibitor cocktail from Roche), 2 mM
Na4P2O7, 1 mM NaVO3, 10 mM NaF and
2.5 ng/ml calyculin B. Cellular debris was removed by centrifugation at 10,000
g for 10 minutes at 4°C. Shifts were performed with 1-2
µg total protein from the various extracts in 20 mM HEPES (pH 7.6), 100 mM
NaCl, 1 mM MgCl2, 10% glycerol and 0.5 µg dI/dC (Pharmacia),
resolved on 5% acrylamide/0.25x TBE at 4°C and autoradiographed. Gel
shifts were also performed using nuclear extracts prepared as above from COS-7
cells programmed to express either recombinant rat GATA4, chick GATA5, mouse
GATA6 or flag-tagged full-length and deletion mutants of human YY1. GST-SMAD1
and SMAD4 MH1 domain fusion proteins and control GST proteins were produced in
and purified from E. coli BL21 bacterial cells according to
manufacturer's instructions (Amersham). Gel shifts were performed as with
chick embryo extracts, using 250 ng purified proteins and 1 µg dA/dT
(Pharmacia). Supershifts were performed with the addition of 200 ng of control
Ig, or rabbit polyclonal anti-mouse GATA4 and GATA6 (Santa Cruz
Biotechnologies).
Co-immunoprecipitation and western blot analyses
For co-immunoprecipitation experiments, 5 µg of the indicated expression
plasmids were used per 100 mm plate. pCS2 empty vector was used to adjust
total DNA amounts where necessary. Total cell extracts were prepared in Co-IP
buffer containing 50 mM Tris (pH 7.8), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1 mM
Na3VO4, 1 mM Na4P2O7,
1.5 mM MgCl2, 1 mM DTT, 10% glycerol, 0.5% NP-40 and various
protease inhibitors (`Complete, EDTA-free' protease inhibitor cocktail from
Roche). The extracts were then centrifuged for 10 minutes at 10,000
g at 4°C and the supernatants were used for
immunoprecipitation assays as previously described
(Kim and Cochran, 2000).
Anti-Myc rabbit polyclonal antibody and anti-Myc monoclonal (9E10) antibody
were obtained from Upstate Biotechnology. Anti-YY1 polyclonal and monoclonal
antibodies were obtained from Santa Cruz Biotechnology. Protein G-sepharose
was obtained from Sigma.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Schultheiss et al., 1995), we
investigated whether sequences flanking the chick gene were capable of driving
cardiac-specific transgene expression in mouse embryos. Transient expression
of the hsp68-lacZ reporter
(Kothary et al., 1989) linked
to three non-overlapping regions of the chick Nkx2.5 gene drove
cardiac-specific gene expression in transgenic mice (see Fig. S1C in the
supplementary material). We termed these various regions of the chick
Nkx2.5 gene, cardiac activating regions (CARs) 1, 2 and 3. In an
effort to better define their distinct roles, we performed further transgenic
analysis of the various CARs in combination with the native Nkx2.5
promoter. When fused to the minimal promoter for the Nkx2.5 gene (see
Fig. S2B in the supplementary material), the various above-described CARs
drove several distinct patterns of cardiac-specific transgene expression in
developing mouse embryos (see Fig. S2C-O in the supplementary material). CAR2,
when appended to the Nkx2.5-lacZ reporter (see Fig. S2B in the
supplementary material) drives transgene expression in the posterior regions
of the early cardiac crescent at E7.5 (see Fig. S2C in the supplementary
material) and in the outflow tract at later looping heart stages (E10.5) (see
Fig. S2D,E in the supplementary material) (data not shown). CAR3, when
positioned downstream of the minimal Nkx2.5 promoter (as it is
located in this position relative to the endogenous promoter in the native
chick Nkx2.5 gene), drives transgene expression in the anterior
cardiac crescent at E7.5 (see Fig. S2F in the supplementary material) and in
the mesoendoderm in the pharyngeal region adjacent to the outflow tract at
E8.5 (see Fig. S2G in the supplementary material), in a pattern that is very
reminiscent of the Isl1-positive progenitors of the secondary heart field
(Cai et al., 2003). By E10.5,
robust Nkx2.5-lacZ-CAR3 transgene expression was observed in the
branchial arches and extended through the outflow tract and right ventricle up
to the interventricular septum (see Fig. S2H in the supplementary material). Although CAR1 failed to drive transgene expression in the early cardiac
crescent when linked to either the hsp68 or the minimal Nkx2.5
promoter (data not shown), the combination of CAR1, CAR2 and CAR3 (see Fig.
S2B in the supplementary material) drove exceptionally robust levels of
lacZ expression in the branchial arch region and in the myocytes of
the outflow tract, and both right and left ventricles at E10.5 (see Fig. S2M-O
in the supplementary material).
CAR3 contains a BMP response element capable of driving transgene expression in the cardiac crescent, the branchial arches, the outflow tract and the right ventricle
Prior work by ourselves (Schultheiss et
al., 1997) and others (Andree
et al., 1998; Schlange et al.,
2000) has indicated that BMP family members are necessary for
inducing cardiac mesoderm and Nkx2.5 gene expression in early chick
embryos. We therefore tested the CARs flanking the chick Nkx2.5 gene
for responsiveness to BMP signaling by employing P19 embryonal carcinoma
cells, which are known to be responsive to BMP signals
(Chen et al., 1998;
Hata et al., 2000).
Administration of BMP2 or BMP4 (50 ng/ml) to transfected P19 cells induced the
expression of reporter constructs containing CAR3 appended to either the hsp68
promoter or the minimal Nkx2.5 promoter three- to fourfold relative
to a co-transfected CMV-renilla luciferase control plasmid
(Fig. 1A,B,M). By contrast,
constructs containing these same promoters driven by either CAR1 or CAR2
failed to show any BMP response in transfected P19 cells
(Fig. 1A). By sequential
deletion analysis of the 2 kb CAR3 sequence, we were able to localize a 200 bp
BMP response element (BMPRE) to nucleotides +2150-2350 in CAR3
(Fig. 1B). Interestingly,
reporters driven by the 200 bp BMPRE displayed a greater response to BMP
signals than did reporters driven by the original 2 kb fragment containing
CAR3, suggesting that sequences outside the 200 bp BMPRE attenuate the BMP
responsiveness of CAR3 in P19 cells. Transgenic analysis revealed that only
reporter transgenes containing the 200 bp BMPRE from CAR3 were capable of
driving lacZ expression in the cardiac crescent, branchial arches and
outflow tract/right ventricle of the developing heart
(Fig. 1C-L; data not shown).
When appended to the endogenous chick Nkx2.5 promoter, we found that
either the 2 kb fragment containing CAR3 or the 200 bp subfragment containing
the BMPRE drove BMP-responsive luciferase reporter expression in transfected
P19 cells (Fig. 1M) and
tissue-restricted lacZ expression in the branchial arches, outflow
tract and right ventricle in transgenic mice
(Fig. 1N-S).
SMAD binding elements are necessary for BMP induced activity of a CAR3 BMPRE-driven reporter in vitro and for CAR3-driven cardiac-specific transgene expression in vivo
Because SMADs are known to transduce BMP signals into the nucleus by both
recognizing SMAD binding elements (SBEs) and associating with specific
transcription factors, we evaluated whether SMAD1/4 would interact with
sequences within the CAR3 BMPRE. The MH1 domain of SMAD proteins is known to
bind weakly to the sequence GTCT/AGAC
(Attisano and Wrana, 2000;
Massague and Chen, 2000;
Zawel et al., 1998). We found
three such putative SMAD-binding elements (SBE1-3; boxed in green in
Fig. 2A) located within the 200
bp BMPRE of CAR3. We found that the DNA-binding MH1 domain of SMAD4 could bind
to oligomers containing either of these potential SBEs in vitro
(Fig. 2B, lanes 3, 9 and 15),
and that disruption of the GTCT/AGAC consensus binding sequence at each of
these sites significantly diminished this interaction
(Fig. 2B, lanes 6, 12 and
18).
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Factors binding to sequences in the BMPRE are present in extracts made from the embryonic chick heart
To identify transcription factors that interact with the 200 bp CAR3 BMPRE
(shown in Fig. 3C), we employed
an electrophoretic mobility shift assay (EMSA) to determine whether factors in
whole cell extracts made from various embryonic chick tissues interact with
the BMPRE. As shown in Fig.
3A,B, endogenous Nkx2.5 is expressed in both stage 6-8 anterior
lateral plate (ALP) tissue and in hearts of day 3 (HH stage 24) chick embryos,
and is not expressed in posterior primitive streak (PPS) tissue from stage 6-8
chick embryos (Fig. 3A,B). We
systematically assayed the ability of components in extracts derived from
these dissected tissues (outlined in Fig.
3A,B) to bind to double stranded 30-40 bp oligomers representing
consecutive overlapping portions of the 200 bp BMPRE (diagrammed by numbered
lines in Fig. 3C). As shown in
Fig. 3D, we were able to detect
distinct DNA binding activities interacting with six different regions within
the BMPRE. Five of these binding activities (A1, C, C', D, E) were found
at approximately equal levels in extracts made from either stage 6-8 ALP or
PPS (Fig. 3D, lanes 1, 2, 9,
10, 13, 14, 15, 16, 19 and 20). By contrast, activity A2, although present to
some degree in ALP extracts, was relatively enriched in PPS extracts
(Fig. 3, lanes 1 and 2).
Conversely, activity B was relatively enriched in ALP extracts
(Fig. 3D, lanes 5, 6, 7, and
8). Whereas complexes B, C, C', D and E were all observed in extracts
made from 3-day-old hearts (Fig.
3D, lanes 21-30), a factor (A3) that bound to site A with a
distinct mobility and binding specificity compared with A1 and A2 (data not
shown) was additionally observed in 3-day-old heart extracts
(Fig. 3D, compare lane 21 with
lanes 1 and 2).
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We noted that complex B interacts with oligomers (#3 and #4,
Fig. 3C) containing a variant
GATA-binding site (AGATTG) (Molkentin,
2000) (boxed in orange, Fig.
4A), while complexes C and C' interact with oligomers [5 (C)
and 7 (C'), Fig. 3C]
containing sequences similar to the consensus GATA-binding site WGATAR
(Molkentin, 2000) (boxed in
red in Fig. 4A). Because GATA4,
GATA5 and GATA6 are all expressed in lateral plate mesoderm and embryonic
cardiac tissue (Arceci et al.,
1993; Heikenheimo et al.,
1994; Laverriere et al.,
1994; Morrisey et al.,
1996; Morrisey et al.,
1997), we examined whether these GATA factors could interact with
binding sites B, C or C'. As shown in
Fig. 4B, nuclear extracts from
COS cells programmed to express either GATA4, GATA5 or GATA6 specifically
shifted oligomers 3 (site B), 5 (site C) and 7 (site C') by EMSA
(Fig. 4B, lanes 2-4, 10-12 and
18-20). These shifts were significantly diminished by specific mutation of the
GATA consensus binding sites (Fig.
4A,B, lanes 6-8, 14-16 and 22-24).
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Site D binds an activity common to ALP, PPS and day 3 heart extracts. We observed that a DNA-binding complex on site D was abrogated by a linker scan mutation at nucleotides 127-137 in the 200 bp BMPRE that strongly inhibited the BMP responsiveness of a BMPRE-driven reporter in P19 cells (Fig. 4A; data not shown). Mutation of these residues abrogated interaction of a protein present in both day 3 heart extracts and P19 cells with oligo #8 spanning residues 120 to 160 of the BMPRE (Fig. 4C, lanes 19 and 20; data not shown).
To ascertain the importance of either GATA-binding sites (B, C or C') or site D for BMP responsiveness in vitro or cardiac gene expression in vivo, mutations that blocked protein-DNA complex formation in vitro (described above) were built into the 2 kb CAR3. Mutations that eliminate either the binding of GATA6 to site B, the binding of GATA4/GATA5 to both sites C and C', or the binding of factor D to site D, each blocked BMP-mediated induction of a luciferase reporter driven by the Nkx2.5 minimal promoter appended to CAR3 (Nkx2.5-lux-CAR3) (Fig. 4D). In addition, mutation of each of these binding sites significantly diminished lacZ expression of cognate Nkx2.5-lacZ-CAR3 constructs in transient transgenic mouse embryos (Fig. 4E-L). Thus, binding sites for GATA4, GATA5, GATA6 and site D are all necessary for BMP-mediated induction and cardiac-specific gene expression of CAR3-driven transgenes.
Site D and its associated SMAD binding site comprise a minimal BMP responsive module
Examination of the 200 bp BMPRE in CAR3 revealed that binding sites for
SMAD4 MH1 were located adjacent to a combination of GATA6 and
GATA4/GATA5-binding sites [located in SMAD region 1 (SR1)], a single
GATA4/GATA5-binding site (located in SR2) or site D (located in SR3) (SR1, SR2
and SR3 are shown in Fig. 5A). As SMADs often bind to DNA adjacent to transcription factors whose activity
they modulate (Attisano and Wrana,
2000; Whitman,
1998), we evaluated whether reiterated versions of the various
SMAD regions would constitute a minimal BMP inducible regulatory element. We
constructed Nkx2.5 promoter-luciferase reporters driven by either five copies
of SR1 (5xSR1, containing SBE1 and the adjacent GATA6 and
GATA4/GATA5-binding sites), five copies of SR2 (5xSR2, containing SBE2
and the adjacent GATA4/GATA5-binding site), or five copies of SR3
(5xSR3, containing SBE3 and the adjacent factor D binding site)
(Fig. 5A,B). Although
reiteration of either SBE1 with its associated GATA4/GATA5 and GATA6-binding
sites (5xSR1), or SBE2 with its associated GATA4/GATA5-binding site
(5xSR2) failed to constitute a BMP inducible regulatory element,
reiteration of SBE3 with the adjacent factor D-binding site (5xSR3 or
4xSR3) resulted in a BMP inducible regulatory element
(Fig. 5B,C).
|
),
unless these cells were supplemented with exogenous SMAD1 and SMAD4
(Fig. 5D). Together, these
results suggest that BMP signaling activates the CAR3 response element, at
least in part, by regulating the transcriptional activity of the site
D-binding factor, and that this modulation requires the presence of a
BMP-activated SMAD complex on the adjacent SBE3 site.
Site D-binding factor is the zinc-finger transcription factor YY1
A detailed examination of nucleotides 115-155 of the BMPRE encompassed by
the SR3 construct revealed the presence of two similar motifs of the sequence
CCATC, present as inverted repeats in nucleotides 120-124 and 135-139.
Comparison of this sequence to known transcription factor binding consensus
sites revealed a similarity between this motif and the binding site for the
Gli-Kruppel zinc-finger transcription factor YY1, CCATNT(A/T) (shown
schematically in Fig. 6A). YY1
is a ubiquitously expressed, multifunctional transcription factor
(Shi et al., 1991), and has
been implicated in the positive or negative regulation of cardiac genes,
including BNP (Bhalla et al.,
2001) and cardiac myofibrillar genes
(Chen and Schwartz, 1997;
Latinkic et al., 2004;
MacLellan et al., 1994;
Sucharov et al., 2003). To
evaluate if YY1 binds to either of the CCATC repeats, we compared the
SR3-binding activities in extracts made from either day 3 chick heart extracts
or P19 cells with that of purified YY1. Mutation of the most 5' putative
YY1 binding site (mutation A) in SR3 failed to significantly affect the
interaction of the SR3 oligomer with either purified YY1 or the binding
activities in either P19 cells or day 3 heart extracts
(Fig. 6B, lane 2). By contrast,
mutation of the 3' most putative YY1-binding site (mutation B) in SR3
abrogated the interaction of the SR3 oligomer with purified YY1 and
significantly decreased interaction of this oligomer with binding activities
present in both embryonic chick heart or P19 cell extracts
(Fig. 6B, compare lanes 1 and
3). In addition, the interaction of the SR3 oligomer with factors present in
either embryonic chick heart extracts or P19 cells was extinguished by
incubation with an anti-YY1 antisera (Fig.
6B, lane 5), supporting the notion that YY1 in such extracts is a
component of the site D binding complex. Consistent with the gel shift
analyses, we found that mutation of YY1 site B eliminated BMP induction of
CAR3 driven reporters in P19 cells (Fig.
6C) and cardiac-specific expression of such reporters in
transgenic mice at both days 7.5 and 10.5
(Fig. 6D,E-G). Because mutation
of the YY1-binding site (mut B) did not alter interaction of the SR3 oligomer
with the SMAD4 MH1 domain in vitro (data not shown), we think it is most
likely that the loss of both BMP responsiveness and heart-specific transgene
expression following mutation of the YY1-binding site (mut B) in CAR3-driven
reporters reflects the loss of YY1 interaction with these sequences.
Conversely, because mutation of SMAD-binding element 3 (SBE3), which lies
adjacent to the YY1 binding site did not alter the interaction of YY1 with the
SR3 oligomer in vitro (data not shown), it seems most likely that the loss of
both BMP responsiveness and heart-specific transgene expression following
mutation of SBE3 in CAR3-driven reporters reflects the loss of SMAD
interaction with these sequences.
|
|
), which could potentially result following loss of YY1
activity (Y. Shi, personal communication). Although co-expression of
increasing amounts of wild-type YY1(1-414) did not significantly affect the
ability of BMP4 to induce the expression of this CAR3-driven reporter
construct (Fig. 7D, right
lanes), co-transfection with increasing amounts of YY1(261-414) markedly
attenuated induction of this reporter by BMP signals
(Fig. 7D, left lanes).
Together, these finding suggest that YY1 recruitment of SMAD1/4, which is
mediated by interaction with the N terminus of YY1, is necessary to coordinate
a BMP-mediated induction of the chick Nkx2.5 CAR3 enhancer. |
Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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), and
like its murine counterpart [termed AR2
(Schwartz and Olson, 1999)] is
capable of driving transgene expression in both the cardiac crescent and in
the outflow tract. Although the more 5' chick Nkx2.5 CAR1 does
not drive transgene expression in the primary heart field, when linked to CAR2
and CAR3 it extends the maintenance of transgene expression in the maturing
heart to include the left ventricle. Notably, CAR1, CAR2 and CAR3 alone or in
combination fail to drive transgene expression in the atria, where endogenous
Nkx2.5 is also expressed.
Although these enhancers drive accurate cardiac expression of
ß-galactosidase expression in transgenic mouse assays, only CAR2 is well
conserved between mammalian and avian species
(Liberatore et al., 2002;
Lien et al., 2002). The
absence of a similarly conserved CAR3-like element in genomic regions flanking
the mouse or human Nkx2.5 genes suggests that different species
employ a variety of regulatory sequences to drive expression of
Nkx2.5 homologs in the heart. However, in all cases examined to date,
GATA- and SMAD-binding sites play an essential role in the activity of the
regulatory sequences that drive Nkx2.5 expression in the forming heart.
Indeed, Bob Schwartz and colleagues have recently characterized a regulatory
element (termed G-S) located 
6 kb upstream of the mouse Nkx2.5
gene, which contains 27 consensus and five interspersed non-consensus
GATA-binding sites followed by nine consensus SMAD-binding sites, that
responds to BMP signals in transfected P19 cells and is capable of driving
transgene expression in both the cardiac crescent and lateral plate mesoderm
(Brown et al., 2003). Thus, it
is possible that BMP-mediated induction of Nkx2.5 expression relies upon
regulatory sequences that either contain a minimal number of GATA- and
SMAD-binding sites positioned adjacent to another SMAD-regulated transcription
factor, such as YY1 (as in chick Nkx2.5 CAR3), or relies upon a
highly reiterated number of GATA-and SMAD-binding sites (as in the murine
Nkx2.5 G-S sequence), which can respond to BMP signals in the absence
of other associated SMAD-regulated transcription factors.
BMP signals modulate Nkx2.5 expression by several synergistic pathways
Detailed mutagenesis of a 200 bp BMPRE within CAR3 revealed that binding
sites for GATA4, GATA5, GATA6, SMAD1/4 and YY1 are all necessary for both
BMP-mediated activation and cardiac-specific expression of reporter constructs
driven by this regulatory region. Our findings indicate that BMP signaling
engages several pathways to induce the expression of the Nkx2.5 gene
in cardiac progenitor tissue (Fig.
8). BMP signals are known to be transduced via BMP
receptor-activated SMAD1, SMAD5 or SMAD8, which bind to DNA in complex with
SMAD4. SMAD proteins have been documented to bind to consensus SBEs in both
distal and proximal regulatory elements in the murine Nkx2.5 gene
[i.e. AR1 and AR2 (Liberatore et al.,
2002; Lien et al.,
2002)], and in the chick Nkx2.5 CAR3 enhancer (this
work). Interestingly, while SBE1, SBE2 and SBE3 in CAR3 are all required to
maintain expression of transgenes in day 10.5 mouse hearts, the initiation of
CAR3-driven transgene expression in the cardiac crescent of day 7.5 mouse
embryos requires only SBE3, suggesting that the transcription factors required
to induce versus maintain CAR3-driven Nkx2.5 gene expression may be
distinct.
|
; Schlange et al.,
2000; Schultheiss and Lassar,
1997) and are sufficient to induce expression of GATA4, GATA5 and
GATA6 in explants of chick paraxial mesoderm (R. Reshef, H. Kempf and A.L.,
unpublished). Interaction of these GATA factors with both distal and proximal
regulatory elements in the mouse Nkx2.5 enhancer and with the chicken
CAR3 enhancer are also essential for cardiac-specific activity of these
regulatory elements. It is interesting that all of the well-characterized
murine and chick Nkx2.5 sequences which drive expression in the
primary heart field contain essential GATA-binding sites
(Brown et al., 2003;
Lien et al., 1999;
Searcy et al., 1998),
consistent with the finding in zebrafish that GATA5/Faust is essential for
Nkx2.5 expression in this species (Reiter
et al., 1999) and underline the importance of GATA proteins in
either the induction or maintenance of Nkx2.5 gene expression. Finally, we have documented that SMAD1/SMAD4 interaction with YY1 modulates the activity of this transcription factor when bound to an adjacent SMAD-binding site in the chick Nkx2.5 CAR3 enhancer and have found that this interaction is essential for the BMP-responsiveness of this regulatory element. Thus, BMP signals modulate the activity of the chick Nkx2.5 CAR3 enhancer by: (1) enabling SMAD complexes to directly bind to this regulatory element; (2) inducing the expression of GATA4, GATA5 and GATA6, which also bind to this regulatory region; and (3) modulating the activity of YY1 when bound to an adjacent SMAD-binding site in CAR3 (see Fig. 8).
BMP-activated SMADs modulate YY1 transcriptional activity
A SMAD1/SMAD4-binding site that lies immediately adjacent to the
YY1-binding site in CAR3 was found to be crucial for both BMP-mediated
activation of this regulatory sequence and expression of CAR3-driven
transgenes in the early cardiac crescent. Reiteration of this YY1 binding site
and the adjacent SMAD1/SMAD4-binding site was sufficient to constitute a
BMP-responsive element. We think it is most likely that BMP signals are
modulating the transcriptional activity of YY1 bound to CAR3 as opposed to
modulating YY1 expression or DNA interaction as we have not observed a change
in YY1-binding affinity for the CAR3 YY1-binding site in gel shift experiments
using nuclear extracts from either SMAD overexpressing or BMP-stimulated P19
cells, nor do these conditions result in a change in YY1 protein expression
levels, as assayed by western blot (data not shown). However, we observed by
co-immunoprecipitation assay that the N-terminal region of YY1 interacts with
the SMAD1/SMAD4 complex. Furthermore, we found that an N-terminal truncation
mutant of YY1 lacking this SMAD-interacting domain but capable of binding to
DNA acts in a dominant-negative fashion to inhibit BMP-mediated induction of
the chick Nkx2.5 CAR3 enhancer. Although we mapped the SMAD1/SMAD4
interaction domain of YY1 to the N terminus, others have documented
interaction of the C-terminal zinc-finger domain of YY1 with SMAD4
(Kurisaki et al., 2003), which
was not evident in our analysis. At present, we cannot account for this
difference; however, this discrepancy may reflect the different experimental
assays employed to map the SMAD interaction domains of YY1 in these two
studies, co-immunoprecipitation (present study) versus GST-pull down
(Kurisaki et al., 2003).
Interestingly, Kurisaki and colleagues have implicated YY1 as a repressor of
SMAD-mediated TGFß responses in fibroblast cell lines
(Kurisaki et al., 2003), and
presented evidence that YY1 overexpression correspondingly attenuated the
association of activated SMAD complexes with multiply reiterated SBEs. These
results are not inconsistent with our finding that YY1 and SMADs
synergistically activate the Nkx2.5 CAR3 enhancer, as it is possible
that YY1 recruits SMAD complexes to regions of the genome containing both YY1
and adjacent SMAD-binding sites at the expense of other SMAD targets that lack
adjacent YY1-binding sites.
How might the interaction of SMADs with YY1 modulate the activity of this
transcription factor when bound to CAR3? Because YY1 can function as either a
transcriptional activator or repressor
(Shi et al., 1991;
Thomas and Seto, 1999), SMAD
association with YY1 may serve to recruit co-activators that modulate the
activity of this transcription factor to become an efficient transcriptional
activator. Indeed, recruitment of co-activators such as p300 by TGFß
activated SMADs is a well-characterized mechanism for SMAD target gene
activation (Attisano and Wrana,
2000; Whitman,
1998). Similarly, known interacting partners of YY1 also include
several members of the histone deacetylase family
(Galvin and Shi, 1997;
Thomas and Seto, 1999;
Yao et al., 2001) as well as a
histone H4 methylase (Rezai-Zadeh et al.,
2003), which have been implicated in either transcriptional
repression or activation of YY1 regulated target genes, respectively. It will
be interesting to determine if SMAD association with YY1 alters the
interaction of this transcription factor with either of these families of
histone modifying enzymes, and to what extent chromatin modification is
responsible for appropriate regulation of Nkx2.5.
SMAD-mediated modulation of YY1 activity adds an interesting new facet to
the repertoire of functions of YY1 during heart development, which also
includes direct recruitment of transcriptional co-activators to promote the
expression of cardiac B-type natriuretic peptide
(Bhalla et al., 2001),
inhibition of the expression of the cardiac 
-actin gene
(Chen and Schwartz, 1997), and
both activation and inhibition of the expression of the cardiac-specific
Mlc2 gene (Latinkic et al.,
2004). Clearly, the context within which YY1 functions is of great
importance, and it is likely that transcription factors such as GATA and SMAD
proteins, when bound to neighboring cognate binding sites, modulate either the
association of co-factors with adjacently bound YY1 or the activity of such
co-factors. In addition to the GATA, YY1- and SMAD-binding sites, linker
scanning mutational analysis of the chick Nkx2.5 CAR3 BMPRE has
revealed other sites yet to be characterized that also have a significant
impact on the BMP response of this regulatory element (K.-H.L. and A.B.L.,
unpublished). A complete understanding of complex enhancers such as
Nkx2.5 CAR3 will require not only the identification of the
transcription factors that regulate their expression but also elucidation of
the transcriptional co-factors that are recruited to such regulatory elements
in a combinatorial fashion.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/19/4709/DC1
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