| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
-Actin Promoter Activity
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-actin genes is
developmentally regulated during skeletal and cardiac muscle
development through fine-tuned control mechanisms involving multiple
cooperative and antagonistic transcription factors. Among the
cis-acting DNA elements recognized by these factors is the
sequence CC(A/T)6GG of the serum response
element (SRE), which is present in a number of growth factor-inducible
and myogenic specified genes. We recently showed that the cardiogenic
homeodomain factor, Nkx-2.5, served as a positive acting accessory
factor for serum response factor (SRF) and together provided strong
transcriptional activation of the cardiac -actin promoter. In
addition, Nkx-2.5 and SRF collaborated to activate the endogenous
murine cardiac -actin gene in 10T1/2 fibroblasts, by a mechanism
that involved coassociation of SRF and Nkx-2.5 on intact SREs of the
-actin promoter. Here, we show that the second SRE of the avian
cardiac -actin promoter served as a binding site for Nkx-2.5, SRF,
and zinc finger containing GLI-Krüppel-like factor, YY1.
Expression of YY1 inhibited cardiac -actin promoter activity,
whereas coexpression of Nkx-2.5 and SRF was able to partially reverse
YY1 repression. Displacement of YY1 binding by Nkx-2.5/SRF complex
occurs through mutually exclusive binding across the CaSRE2. The
interplay and functional antagonism between YY1 and Nkx-2.5/SRF might
constitute a developmental as well as a physiologically regulated
mechanism that modulates cardiac -actin gene expression during
cardiogenesis. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-actin genes of higher vertebrates
belong to a multigene family consisting of at least six different
actins that are differentially expressed in a tissue-specific and
developmentally regulated manner (1, 2, 3). Although the cardiac and
skeletal -actins are expressed predominantly in the respective
sarcomeric tissues of the mature animal (4), the two genes are
regulated independently: cardiac -actin is the major striated actin
isoform in embryonic skeletal muscle, as well as in the heart, and is
gradually replaced by skeletal -actin as skeletal muscle matures
(5). In warm-blooded vertebrates, cardiac -actin transcripts are
first detected in the presumptive ventricular myocardium of the heart
primordia (6). As cardiac morphogenesis proceeds, cardiac -actin
mRNA accumulates throughout the myocardium of the tubular heart. The
spread of cardiac -actin mRNA and the appearance of skeletal
-actin transcripts to other regions of the myocardium parallel the
regional progression of myofibril formation in the developing chick
heart (7). Thus, expression of -sacromeric actins begins during
fusion of the heart primordia, steadily increases during looping and
formation of the vascular trunks, and later is exclusively expressed in
the muscular walls of the cardiac chambers. Although the molecular
basis for actin isoform switching during development is being resolved
at the transcriptional level and is thought to involve the specific
interaction of regulatory proteins with various cis-acting
elements that control the expression of a particular actin isoform, the
mechanism(s) of interaction between trans-acting factors and
cis-acting elements mediating tissue-specific expression
of -actin genes are largely unknown.
Studies of muscle -actin gene expression in various species by gene
transfer methods have suggested that the mechanism of tissue-specific
actin gene expression is well conserved in higher vertebrates. In the
case of the skeletal -actin gene, the capacity for selective
expression of the chicken skeletal -actin resides approximately 200
bp upstream from the transcription initiation site (8). This 200-bp
promoter sequence is also sufficient for directing the expression of
chloramphenicol acetyltransferase gene in striated muscle of transgenic
mice (9). In the case of the cardiac -actin gene, studies have also
concentrated on the proximal promoter region. The first 117-bp upstream
sequences from the transcription initiation site of the human cardiac
-actin promoter are sufficient to confer muscle-specific expression
on a heterologous reporter gene (10). The cis-acting
sequence elements controlling tissue-restricted and
differentiation-dependent expression in the chicken cardiac -actin
promoter are also located predominantly within 300-bp upstream
sequences (11, 12, 13). Sequence comparison of the muscle-specific
-actin promoters has revealed several regions of sequence
conservation present in the chicken, mouse, rat, and human cardiac and
skeletal -actin genes (14). A highly conserved motif,
CC(A/T)6GG, termed CBAR, CArG box, or serum response
element (SRE), is found in multiple copies within the 5'-flanking
regions of the vertebrate -actin genes (8, 14). Mutagenesis studies
of vertebrate cardiac and skeletal -actin promoters demonstrate that
SREs play a positive role in myogenic induction (8, 10, 15, 16).
Nuclear proteins have been shown to interact with -actin CArG
sequences (17, 18, 19). A CArG box-binding factor reported to regulate the
transcription of -actin genes has been shown to be indistinguishable
from the serum response factor (SRF) that binds to the c-fos
SRE (20, 21, 22). Additionally, it has been reported that the proximal CArG
box from a cardiac -actin promoter is functionally interchangeable
with the SRE from the c-fos promoter (23). Subsequently, a
zinc finger protein described variously as YY1 (24), NF-E1 (25), 
(26), or UCRBP (27), which is a member of the GLI-Krüppel family,
was shown to bind to a subset of SREs (28, 20). YY1 is a
multifunctional transcription factor that can act as a transcriptional
repressor (24, 25, 27), a transcriptional activator (29), or a
transcriptional initiator (30). We and others previously showed that
its binding to an SRE competed with SRF for binding to the
c-fos and skeletal -actin promoters (28, 31). We proposed
that the skeletal -actin promoter could be repressed or activated by
two functionally opposite SRE-binding proteins, YY1 and SRF, depending
on the outcome of their competitive interactions with the most proximal
SRE (31). It is not clear, however, to what extent the two seemingly
ubiquitous DNA-binding factors may contribute to muscle-specific
expression of the actin genes. In a transfection assay, SRF was shown
to induce modest activation of the skeletal -actin promoter in
differentiation-blocked myoblasts (3)1, and like the closely related
cardiac -actin promoter, SRF- binding activity was also required for
the cardiac -actin gene transcription (10, 13).
It was proposed that additional factors might be required to optimally
drive these actin promoters, as well as to specify cell type-restricted
expression (37). Indeed, we recently showed that a murine
cardiac-specific homeodomain gene Nkx-2.5/CSX (32, 37), which has
significant homology to the Drosophila NK-2 class of
homeobox genes and was identified as a homolog of Drosophila
tinman, an obligatory mesoderm determination factor required for
insect heart formation (34), recognized a variety of SREs including the
four cardiac -actin SREs (35). Nkx-2.5 was also shown to interact
with SRF in the absence of SRE and, together with SRF, both factors
transactivated the cardiac -actin promoter and increased
transcription of the endogenous cardiac -actin gene in nonmuscle
cells (36). Thus, we proposed that SREs do not all share equivalent
roles, e.g. subtle differences in sequences that embed each
SRE may influence binding of a host of transcription factors, such as
YY1, SRF, and Nkx-2.5, and that Nkx-2.5 is one of the
cardiac-restricted SRF accessory factors that elicit cardiac-specific
transcription of SRE-containing promoters (35, 36). Although the
multiple CArG boxes act as positive regulatory elements in the
transcriptional control of the sarcomeric -actin genes and are
required for muscle-specific expression of these genes, the
contribution of these diverse elements to promoter function during
myogenic differentiation has not been completely resolved, and their
precise roles in gene regulation and expression regarding serum
inducibility and myogenic induction remain to be elucidated. Thus,
understanding the protein factors and their mechanistic interactions
with CArG boxes governing myogenic induction of -actin genes may
provide insights of how the actin isoform switch during development is
controlled.
Here, we defined positive and negative regulation of the avian
cardiac -actin promoter activity in chicken primary cardiomyocytes.
YY1 was found to repress the promoter by binding to cardiac actin SRE2
(CaSRE2). By contrast, Nkx-2.5 and SRF, which appeared to compete with
YY1 for binding to CaSRE2, were positive regulators of the promoter,
and coexpression of both factors was able to overcome the inhibitory
effect of YY1. Reduced YY1 binding to CaSRE2 by site-directed
mutagenesis stimulated cardiac -actin promoter activity at least
2-fold either in cotransfected 10T1/2 fibroblasts or in primary
cardiomyocytes. Thus, YY1, a preferentially enriched transcription
factor in replicating myoblasts and nonmuscle cells, appears to be a
transcriptional repressor responsible for inhibiting the cardiac
-actin promoter in these cell types. Our study suggest that both
increased Nkx-2.5 and SRF synthesis lead to displacement of YY1 binding
to cardiac -actin SREs, which is thought to result in
transcriptional activition of the cardiac -actin promoter during
cardiogenesis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-Actin Promoter in 10T1/2 Fibroblasts-actin
promoter in differentiation-blocked myoblasts (31) and, like the
closely related cardiac -actin promoter, both promoters require
intact SREs for activity (13, 16, 19). It was proposed (31) that
additional factors might be required to optimally drive these actin
promoters, as well as to specify cell type-restricted expression. We
asked whether ectopic expression of Nkx-2.5, driven by the MSV
long-terminal repeat, in a fibroblastic C3H10T1/2 (10T1/2) cell line
could stimulate the cardiac -actin promoter activity as assayed by a
luciferase reporter gene. In addition, 10T1/2 cells were also
transfected with an SRF expression vector driven by the cytomegalovirus
(CMV) promoter (Fig. 1
). Expression of either Nkx-2.5 or
SRF stimulated the cardiac -actin promoter activity about 3- to
5-fold, which is in the range of SRF-stimulated -actin promoter
activities previously reported by Lee et al. (31). The
combined transfection of Nkx-2.5 and SRF expression vectors led to
robust reporter gene activity, which was at least 15-fold greater than
controls (Fig. 1A) and as shown previously (35, 36). Nkx-2.5 and SRF
also weakly activated the -skeletal actin promoter (Fig. 1D).
Nkx-2.5 and SRF did not cotransactivate a minimal c-fos SRE
construct as well as a solitary skeletal -actin proximal SRE1
construct (Fig. 1, E and F), demonstrating that productive interactions
between these transfactors might be restricted to specific SREs. These
transcription factors did not stimulate the SRE-deficient promoters of
the herpes simplex thymidine kinase gene and the SV40-t-early gene
(Fig. 1, B and C). These results suggest that Nkx 2.5 and SRF
cotransactivation was restricted to the cardiac -actin promoter.
|
-actin gene activity? We recently conducted electrophoretic mobility
shift assays (EMSAs) with cardiac cell nuclear extracts to examine the
distribution of cardiac nuclear protein complexes that bound cardiac
actin SREs. SRF-binding complexes were previously detected on four
SREs, in which endogenous SRF bound more efficiently to the proximal
SRE1 and the distal SRE4, which adhered to the consensus SRE sequence
(CC(A/T)6GG). The most rapid migratory complex was detected
on the second and third SREs, which correlated with YY1-binding
activity. An intermediate migrating doublet, identified as Nkx-2-like
factors, were detected with the cardiac actin SRE2 oligonucleotide. In
fact, comparison of the well documented NK-2 consensus binding sequence
5'-CCACTCAAGT-3' (37, 38) was quite similar to SRE2 5'-CCATTCATGG-3'
(35). Thus, CaSRE2 served as a binding site for at least three distinct
nuclear factors, namely SRF, Nkx-2.5, and YY1, from primary cardiac
myocytes.
To determine whether the interaction of these three protein factors
with their target DNA (CaSRE2) was either mutually inclusive or
exclusive, EMSAs were conducted using bacterially expressed Nkx-2.5 and
SRF proteins and nuclear extracts prepared from 10T1/2 cells
transfected with a YY1 expression vector as a source of YY1 DNA-binding
activity. Whether Nkx-2.5 could compete with YY1 for DNA binding was
first determined by incubating a labeled CaSRE2 probe with
YY1-transfected 10T1/2 nuclear extracts mixed with increasing
concentrations of purified bacterial Nkx-2.5 protein. The YY1-binding
complex was significantly reduced in the presence of Nkx-2.5 (Fig. 2A), indicating that their SRE binding was indeed
mutually exclusive. We previously showed that bacterial expressed
MPB-Nkx-2.5 migrated as a doublet in EMSAs (37), and the Nkx-2.5
doublet observed in Fig. 2 was not due to any associations with YY1. In
contrast to Nkx-2.5, an Nkx-2.5 mutant (Nkx-2.5 pm) that lacks
DNA-binding activity due to a point mutation (Asn to Gln) at the
position 10 of the DNA recognition helix did not compete for YY1
binding to the oligonucleotide, indicating that the decrease in the
binding of YY1 to the oligonucleotide by Nkx-2.5 was dependent upon its
DNA-binding activity. In a similar assay, YY1 binding to CaSRE2 was
significantly decreased by increasing SRF binding activity (Fig. 2B),
suggesting their mutually exclusive binding to the CaSRE2 site.
|
-Actin Promoter Sequence Between -150 and -100
Contains a Strong Negative Element-actin promoter
(44), we wanted to determine whether YY1 could also repress the cardiac
-actin promoter. We asked whether YY1-binding sites in the cardiac
-actin promoter acted as negative regulatory elements. Several
cardiac -actin promoter deletion mutants (Fig. 3A)
linked to the luciferase reporter gene were constructed and were
transfected into primary chicken cardiomyocytes to assay their promoter
activities (Fig. 3B). The wild type promoter activity in these
cardiomyocytes was about 12-fold higher than that of a construct,
Del-58, which contains only the TATA box. Removal of the promoter
sequence (-310 to -200), which includes the distal SRE4, caused a
decrease in promoter activity to 60% of the wild type value. Further
removal of an additional 50-bp promoter sequence, construct Del-150,
which consists of SRE2 and SRE1, totally abolished the promoter
activity to a level even lower than that of Del-58 (
10% of the
Del-58 promoter activity). However, removal of sequence from -150 to
-100, which deleted SRE2, rescued promoter activity to a level 5-fold
of that observed with Del-58. We showed in Fig. 1
and in Chen et
al. (35) that coexpression of Nkx-2.5 and SRF was able to
transactivate the cardiac -actin promoter in 10T1/2 fibroblasts.
Therefore, we wanted to determine whether coexpression of both factors
could restore the promoter activity of Del-150 in these cells. As shown
in Fig. 3C, the wild type promoter activity was stimulated to a level
roughly 20-fold above the background by cotransfection with Nkx-2.5 and
SRF expression vectors. In contrast to the wild type promoter, the
Del-150 construct could not be activated by the combined transfection
with Nkx-2.5 and SRF. These results suggest that a negative regulatory
element between -150 and -100 is likely the CaSRE2, and that
negative-acting YY1 binding to this element might be dominant over the
positive regulators, Nkx-2.5 and SRF.
|
-Cardiac Actin
Promoter-actin gene, the wild type cardiac -actin
promoter construct was transfected into 10T1/2 cells together with
various combinations of protein expression vectors. As shown in Fig. 4, YY1 did not repress the basal activity of cardiac
-actin promoter possibly due to silence of the promoter in 10T1/2
cells. However, exogenous YY1 repressed the Nkx-2.5- and SRF-activated
promoter activities by 55% and 45%, respectively (compare the third
and fifth with fourth and sixth cotransfection assays in Fig. 4).
Repression of the SRF-stimulated cardiac -actin promoter activity by
transfected YY1 could be restored by Nkx-2.5 to a level that was higher
than that transfected with either Nkx-2.5 or SRF alone (compare the
eighth with third and fifth cotransfection assays). In
contradistinction to the wild type Nkx-2.5, Nkx-2.5 pm failed to
overcome the repression by YY1 (compare the seventh with the eighth
transfection assay). Additionally, consistent with the previous
observation that inhibition of the skeletal -actin promoter function
caused by transfected YY1 DNA could only be partially relieved by
cotransfected SRF (39), overexpression of Nkx-2.5 and SRF could only
partially restore the YY1-repressed cardiac -actin promoter activity
to a level about 60% of that obtained with cotransfection of Nkx-2.5
and SRF expression vectors without exogenous YY1 (compare the eighth
with the ninth cotransfection assay), which is consistent with results
shown in Fig. 3, in which the YY1 binding site in construct Del-150
acted as a negative site, and with the idea that suggested an ascendant
negative effect of YY1 on the sarcomeric -actin promoters.
|
-Actin Promoter
Activity-actin promoter activity, we wanted to eliminate YY1-binding
activity to CaSRE2, by generating a cardiac -actin SRE2 mutant
promoter (SRE2m), in which the SRE2 sequence was converted from
5'-CCATTCATGGCC-3' to 5'-CCATTCAGATCT-3'. Based on the sequence
alignment, this SRE2 mutation should eliminate SRF and YY1 DNA-binding
activities, but retain Nkx-2.5-binding activity. A band shift assay
using the wild type or mutated SRE2 oligonucleotide probe confirmed
that this SRE2 mutant selectively eliminated binding of SRF and YY1
DNA-binding activity, without disrupting Nkx-2.5-DNA interaction (Fig. 5A). An EMSA competition assay using Nkx-2.5-transfected
fibroblast nuclear extracts with a CaSRE2 probe against a wild type
CaSRE2 or a mutated CaSRE2 (SRE2m) oligonucleotide confirmed that SRF-
and YY1-binding complexes were not competed by the SRE2m
oligonucleotide, whereas these complexes were efficiently competed by a
self-competitor (Fig. 5B). In addition, SRE2m competed for Nkx-2.5
binding as well as SRE2 oligonucleotide. This observation suggested
that the SRE core of CaSRE2 was indeed the major contact site between
these factors and DNA. The effect of the SRE2 mutation on promoter
activity was then examined by a transient cotransfection assay (Fig. 6). The SRE2 mutation caused about an overall 2-fold
increase of promoter activity over the wild type promoter in 10T1/2
cells cotransfected with Nkx-2.5 or SRF expression construct alone or
both [compare wild type (WT) with SRE2m in Fig. 6B]). The promoter
activities of these two reporter genes were also examined in primary
chicken cardiomyocytes. As shown in Fig. 6C, transcriptional activity
of SRE2-mutated promoter was elevated by at least 2-fold over the wild
type promoter. Thus, YY1, by binding to the cardiac actin SRE2, acts as
a repressor responsible for inhibiting the cardiac -actin promoter,
and elimination of YY1 binding enhanced transcription of the cardiac
-actin promoter in transfected cells.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-actin SRE2 and
SRE3, which by themselves are highly conserved during evolution (14),
with the YY1 consensus sequence, 5'-AANATGGNG/C-3' (31), revealed that
both SREs contain a homologous motif, 5'-NATGGNC-3', to the 3'-half of
the YY1 consensus (Fig. 7). Interestingly, the
Xenopus, chicken, and human CaSRE2 contain two
half-YY1-binding motifs at both ends of the SRE. YY1 was shown to bind
to the skeletal actin SRE1 (SkSRE1) and the c-fos SRE (19, 28, 31, 40). In both cases, YY1 competes with SRF for binding to these
SREs. Several lines of evidence suggest that YY1 is a regulator of
these SRE elements. Overexpression of YY1 represses basal and regulated
expression from the c-fos SRE and the skeletal actin SRE1.
This YY1-mediated trans-repression can be reversed by
overexpression of SRF (28). Thus, a subset of SREs serve as binding
sites for YY1. This functional antagonism between SRF and YY1 may
result from the competition for binding to the DNA- regulatory element.
However, binding of YY1 to the c-fos SRE was recently shown
to enhance the binding of SRF and stimulate SRF-responsive promoter
(41). Although the discrepancy between these data remain elusive, we
observed that Nkx-2.5 competed for YY1 binding to CaSRE2. Competitive
binding between Nkx-2.5 and YY1 to CaSRE2 depended upon Nkx-2.5
DNA-binding activity, inasmuch as a single point mutation of the third
helix of Nkx-2.5 abolished DNA binding and its competition with YY1.
Competitive binding over CaSRE2 is probably due to the overlapping DNA
contact sites of Nkx-2.5 and YY1, as was described for SRF and YY1
binding to the c-fos and skeletal actin SREs (19, 28).
Recently, Natesan and Gilman (41) suggested that YY1 and Phox-1
made significant major-groove contacts in the AT core of the
c-fos SRE, whereas SRF might instead contact the DNA solely
in the minor groove. Indeed, DNA contacts of SRF to the minor groove in
the AT core of the c-fos SRE was demonstrated by x-ray
crystallographic study of SRF core-SRE complex (42). These observations
may explain the mutually exclusive binding of Nkx-2.5 and YY1 to
CaSRE2, which may result from their DNA contacts made in the minor
groove, and also allow for the mutually inclusive binding of SRF and
Nkx-2.5 to CaSRE2.
|
-actin genes. YY1 protein
contents are enriched in replicating myoblasts and nonmuscle cells (19, 31), where expression of this -actin gene is inhibited or silent. By
contrast, its protein levels are gradually reduced in normal myogenesis
as myoblasts progress through fusion and become differentiated
myotubes, which is correlated with the increased transcription of the
skeletal -actin promoter (39). Moreover, stimulation of the skeletal
-actin promoter activity was observed by selectively weakening
YY1-SRE1 interaction without disrupting SRF-SRE1-binding activity (19).
Furthermore, transfected SRF activated the skeletal -actin promoter
in BrdUrd-treated myoblasts, a condition that reduced endogenous SRF
content while increasing YY1 binding activity (31).
As shown here, YY1 also repressed the avian cardiac -actin gene. YY1
was found to bind to the chicken CaSRE2 and CaSRE3 (36), and
elimination of the SRE2-binding site increased transcription activity.
Our deletion analysis of the chicken cardiac -actin promoter in
primary cardiomyocytes suggested a negative transcriptional regulator
binding to the promoter sequence between -150 and -100, which is
likely due to binding of SRE2 site by YY1. Binding of YY1 to this SRE
inhibited the promoter activity of a construct Del-150 in transfected
cardiomyocytes, even though it contained the SRE1 site, suggesting that
repression of this truncated promoter by YY1 was dominant over binding
of SRF to CaSRE, which could not be rescued by SRF and Nkx-2.5. The
inhibition by YY1 could be due to its association with other proteins
that could make it a more dominant repressor, e.g. YY1
interacts with E1A, p300, Sp1, and TBP. Also, YY1 might exert its
repression by its bending of DNA, as demonstrated in the
c-fos promoter (40). Possibly, YY1 may bind to and induce a
phased DNA bend at the CaSRE2 site in this promoter, which may preclude
SRF from binding to CaSRE1 and eventually repress promoter activity.
Perhaps, the presence of multiple SREs in the -actin gene promoters,
in which SRF binding over these actin SREs is marked by cooperative
binding events (19), may be essential for SRF to prevent YY1 from
binding to the -actin promoters. This evidence is further provided
by the deleted promoter constructs as described in Fig. 3B, in which we
observed a full promoter activity of the wild type promoter and a
reduced promoter activity of Del-200 with removal of the most distal
SRE4 site. Finally, YY1 might repress -actin promoter activity by
indirect mechanisms involving activation of repressors of myogenesis,
such as c-Myc (29).
As observed for the skeletal -actin promoter, transfected SRF and
Nkx-2.5 resulted in overcoming the inhibitory effect of YY1 on the
cardiac -actin promoter, as assayed by cotransfection experiments in
10T1/2 cells. This functional antagonism might be the consequence of
displacement of YY1 from the CaSRE2 (and perhaps CaSRE3) site by SRF
and Nkx-2.5, as a Nkx-2.5 mutant (Nkx-2.5 pm) that was unable to bind
to DNA (37) and failed to reverse YY1-directed repression (Fig. 4).
Transfection assays in 10T1/2 cells and in primary cardiomyocytes
indicated that mutation of the CaSRE2 site, in which the YY1-SRE2
interaction was eliminated (Fig. 5), resulted in increased promoter
activity (Fig. 6), which further suggests that YY1 binding to CaSRE2 is
necessary for repression of the cardiac -actin promoter. In
addition, we showed that disruption of SRF-SRE2 binding did not impair
the cardiac -actin promoter activity, thus suggesting that SRF
binding to the SRE2 was not a critical event for activation of the
promoter by SRF. Because this SRE2-mutated promoter provided functional
cooperation between Nkx-2.5 and SRF in a cotransfection assay (Fig. 6A), and the mutated SRE2 site continued to bind Nkx-2.5 (Fig. 5), it
was concluded that Nkx-2.5 was a primary binder on the cardiac
-actin SRE2. Alternatively, the increase in promoter activity with
SRE2 mutation might be caused merely by elimination of YY1 binding to
the mutated promoter.
Therefore, our results suggest that one way of stimulating the cardiac
-actin promoter by Nkx-2.5 and SRF is in part preventing binding of
YY1 from the promoter and suggested that removal of a specific
repressor such as YY1, which can be achieved by the developmental
down-regulation of its protein contents and/or by increasing SRF or
Nkx-2.5 contents, might be a first step in activation of the cardiac
-actin gene. Because of the homology of SREs in all vertebrate
cardiac -actin genes, the mutually exclusive and inclusive binding
of these nuclear factors to the CaSRE2 site is likely conserved. Thus,
displacement of YY1 from CaSRE2 by simultaneous increase in Nkx-2.5 and
SRF cellular contents is likely a general mechanism that stimulates
transcription of the cardiac -actin genes in vertebrate cardiac
myocytes. In addition, results in this study may also provide insights
into how the cell type- and tissue-specific expression of the cardiac
-actin genes can be tightly controlled by positive- and
negative-acting factors and thereby foster our understanding of the
molecular basis for actin isoform switch during development.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-actin promoter
construct and its deletion derivatives, Del-200, Del-100, and Del-58,
were described previously (35). Del-150 was constructed by digesting
SRE3m-LUC, which was obtained by inserting a HindIII
fragment isolated from SRE3m-CAT (13) into the HindIII site
of pGL2-basic luciferase vector (Promega, Madison, WI), with
BglII, followed by religation with T4 DNA ligase. The
cardiac actin SRE2-mutated promoter, SRE2m-LUC, was constructed by
inserting a HindIII fragment from SRE2m-CAT (13) into the
HindIII site of pGL2. Ca SRE1-TATA-LUC was constructed by
digesting -CA-LUC with BglII and NcoI to
remove the promoter sequences from -310 to -100. The skeletal
-actin reporter, -SK-LUC, was previously constructed by
subcloning nucleotides -394 to +24 of the chicken skeletal -actin
gene into pGL2-basic (39). The c-fos SRE and the proximal
chicken skeletal actin SRE1 (MRE) were both cloned upstream into the
herpes simplex virus minimal promoter (nucleotide -105 to +51). The
herpes simplex virus thymidine kinase promoter (nucleotide -105 to
+51) and the SV40 early promoter and enhancer were each cloned into
pGL2-basic. Bacterial expression vectors, pMAL-c2-Nkx-2.5 and
pMAL-c2-Nkx-2.5HD, were described previously (37). A bacterial
expression vector expressing histidine-tagged human SRF was kindly
provided by Dr. R. Prywes (Department of Biological Sciences, Columbia
University, New York, NY).
Nuclear Extract Preparation
Nuclear extracts of transfected cells were prepared by using a
mini-extract procedure (38). The protein concentration was determined
by the method of Bradford using a Bio-Rad kit (Richmond, CA).
Purification of Bacterially Expressed SRF and Nkx-2.5
Bacterially expressed MBP-Nkx-2.5 and MBP-Nkx-2.5HD proteins
were purified by an amylose column (New England Biolabs, Inc., Beverly,
MA) as previously described (37). Histidine-tagged SRF was purified
over a nickel column according to the manufacturer’s procedures
(Quiagen, Chatsworth, CA).
EMSAs
EMSAs were performed with 20-µl reaction mixtures at room
temperature as previously described (35), in which 0.5 µg poly
(dG-dC) was used as a nonspecific competitor. For EMSA competition and
antibody interference assays, proteins were incubated with cold
competitors or antiserum for 5 min before addition of the probe. The
oligonucleotide sequences (one strand) were as follows: CaSRE1,
5'-CGCCCGGCCAAATAAGGAGAAGG-3'; CaSRE2,
5'-CGACCTG-CCATTCATGGCCGCG-3'; CaSRE3, 5'-CGACCTGCCTTAGATGGCC
CGC-3'; CaSRE4, 5'-CGAGGCCCCTATTTGGCCATG-3'; CaSRE2m, 5'-CGACCTGC
CATAGATCTCCGCG-3'; SkSRE1, 5'-CGGACACCAAATATGGCGACG-3'; Nkx-2.1,
5'-TCGGGATCGCCCAGTCAAGTGC-3'.
Transfection Assays
Growing C3H10T1/2 mouse fibroblasts cultured in DMEM containing
10% newborn calf serum were transfected with a total of 8 µg plasmid
mixed with 15 µl LipofectaMINE (GIBCO, BRL, Gaithersburg, MD) for 20
min in 1.5 ml serum-free mediun. Cells were then exposed to the
DNA-lipid mixture for 5 h in serum-free medium, after which an
equal volume of medium supplemented with 20% newborn calf serum was
added to the cells. Primary chicken cardiomyocytes were plated at a
density of 1 x 106 cells per 35-mm dish and
transfected with 1 µg of each reporter construct and 2 µg
pSV40-§gal by LipofectaMINE (6 µl) as previously described (35, 36).
Cells were harvested at an appropriate time, and luciferase activity
was determined using the Luminometer Monolight 2010 (Analytical
Luminescence Laboratories, San Diego, CA) as previously described (35, 36). ß-Galactosidase activity was determined using
o-nitrophenyl-ß-D-galactopyranoside (ONPG;
Sigma Chemical Co.) as a substrate, and activity was measured as
absorbance at 410 nm. Experimental data were presented as the average
of three independent duplicate transfection assays normalized by
ß-gal activity.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This study was supported by NIH Grants R01 HL-50422 and P01 HL-49953.
Received for publication February 25, 1997. Revision received March 21, 1997. Accepted for publication March 24, 1997.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-actin genes during avian cardiogenesis: vascular smooth muscle
-actin transcripts mark the onset of cardiomyocyte differentiation.
J Cell Biol 107:2575–2586-actin gene. Mol Cell Biol 6:2462–2475-actin promoter is tissue
specific in transgenic mice. Mol Cell Biol 9:3785–3792-actin gene requires MyoD1, CArG-box
binding factor, and SP1. Genes Dev 4:1811–1822-actin
promoter. Mol Cell Biol 11:2439–2450-actin promoter is regulated through a pair of complex elements
composed of E boxes and serum response elements that binds both
positive- and negative-acting factors. J Biol Chem 269:12731–12740-actin gene: evolution
of functional regulatory domains. Genomics 3:323–336[CrossRef][Medline]
-actin gene. Mol Cell Biol 10:528–538-actin gene transcription: the cooperative formation of
serum response factor-binding complexes over positive
cis-acting promoter serum response elements displaces a
negative-acting nuclear factor enriched in replicating myoblasts and
nonmyogenic cells. Mol Cell Biol 11:5090–5100-actin transcription in embryonic myoblasts. Proc Natl Acad Sci USA 89:9814–9818-actin
promoter depends upon serum response factor, tinman
homologue, Nkx-2.5, and intact serum response elements. Dev Genet 19:119–130[CrossRef][Medline]
-actin gene
transcription. Mol Cell Biol 16:6372–6384[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  K. Kawai-Kowase and G. K. Owens Multiple repressor pathways contribute to phenotypic switching of vascular smooth muscle cells Am J Physiol Cell Physiol, January 1, 2007; 292(1): C59 - C69. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Sucharov, S. Langer, M. Bristow, and L. Leinwand Shuttling of HDAC5 in H9C2 cells regulates YY1 function through CaMKIV/PKD and PP2A Am J Physiol Cell Physiol, November 1, 2006; 291(5): C1029 - C1037. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Favot, S. M. Hall, S. G. Haworth, and P. R. Kemp Cytoplasmic YY1 Is Associated with Increased Smooth Muscle-Specific Gene Expression: Implications for Neonatal Pulmonary Hypertension Am. J. Pathol., December 1, 2005; 167(6): 1497 - 1509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Rene, M. Taulan, F. Iral, J. Doudement, A. L'Honore, C. Gerbon, J. Demaille, M. Claustres, and M.-C. Romey Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway Nucleic Acids Res., September 16, 2005; 33(16): 5271 - 5290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. X. Zhang, E. Garcia-Gras, D. R. Wycuff, S. J. Marriot, N. Kadeer, W. Yu, E. N. Olson, D. J. Garry, M. S. Parmacek, and R. J. Schwartz Identification of Direct Serum-response Factor Gene Targets during Me2SO-induced P19 Cardiac Cell Differentiation J. Biol. Chem., May 13, 2005; 280(19): 19115 - 19126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-H. Lee, S. Evans, T. Y. Ruan, and A. B. Lassar SMAD-mediated modulation of YY1 activity regulates the BMP response and cardiac-specific expression of a GATA4/5/6-dependent chick Nkx2.5 enhancer Development, October 1, 2004; 131(19): 4709 - 4723. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Himeda, J. A. Ranish, J. C. Angello, P. Maire, R. Aebersold, and S. D. Hauschka Quantitative Proteomic Identification of Six4 as the Trex-Binding Factor in the Muscle Creatine Kinase Enhancer Mol. Cell. Biol., March 1, 2004; 24(5): 2132 - 2143. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Reinhold, D. G. McEwen, and M. C. Naski Fibroblast Growth Factor Receptor 3 Gene: Regulation by Serum Response Factor Mol. Endocrinol., January 1, 2004; 18(1): 241 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-S. Yeh, Y.-M. Lin, R.-H. Hsieh, and M.-J. Tseng Association of Transcription Factor YY1 with the High Molecular Weight Notch Complex Suppresses the Transactivation Activity of Notch J. Biol. Chem., October 24, 2003; 278(43): 41963 - 41969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Sucharov, P. Mariner, C. Long, M. Bristow, and L. Leinwand Yin Yang 1 Is Increased in Human Heart Failure and Represses the Activity of the Human {alpha}-Myosin Heavy Chain Promoter J. Biol. Chem., August 15, 2003; 278(33): 31233 - 31239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Konig, J. Burkman, J. Fitzgerald, M. Mitchell, L. Su, and H. Stedman Modular Organization of Phylogenetically Conserved Domains Controlling Developmental Regulation of the Human Skeletal Myosin Heavy Chain Gene Family J. Biol. Chem., July 26, 2002; 277(31): 27593 - 27605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Sepulveda, S. Vlahopoulos, D. Iyer, N. Belaguli, and R. J. Schwartz Combinatorial Expression of GATA4, Nkx2-5, and Serum Response Factor Directs Early Cardiac Gene Activity J. Biol. Chem., July 5, 2002; 277(28): 25775 - 25782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Mack, M. M. Thompson, S. Lawrenz-Smith, and G. K. Owens Smooth Muscle {alpha}-Actin CArG Elements Coordinate Formation of a Smooth Muscle Cell-Selective, Serum Response Factor-Containing Activation Complex Circ. Res., February 4, 2000; 86(2): 221 - 232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Ding, M. M. Witte, and R. E. Scott Transformation Blocks Differentiation-induced Inhibition of Serum Response Factor Interactions with Serum Response Elements Cancer Res., August 1, 1999; 59(15): 3795 - 3802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Spencer, M. L. Major, and R. P. Misra Basic Fibroblast Growth Factor Activates Serum Response Factor Gene Expression by Multiple Distinct Signaling Mechanisms Mol. Cell. Biol., June 1, 1999; 19(6): 3977 - 3988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ericsson, A. Usheva, and P. A. Edwards YY1 Is a Negative Regulator of Transcription of Three Sterol Regulatory Element-binding Protein-responsive Genes J. Biol. Chem., May 14, 1999; 274(20): 14508 - 14513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Qian, L. Kuo, Y.-T. Yu, and J. N. Rottman A Concise Promoter Region of the Heart Fatty Acid–Binding Protein Gene Dictates Tissue-Appropriate Expression Circ. Res., February 19, 1999; 84(3): 276 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Galvagni, E. Cartocci, and S. Oliviero The Dystrophin Promoter Is Negatively Regulated by YY1 in Undifferentiated Muscle Cells J. Biol. Chem., December 11, 1998; 273(50): 33708 - 33713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Sepulveda, N. Belaguli, V. Nigam, C.-Y. Chen, M. Nemer, and R. J. Schwartz GATA-4 and Nkx-2.5 Coactivate Nkx-2 DNA Binding Targets: Role for Regulating Early Cardiac Gene Expression Mol. Cell. Biol., June 1, 1998; 18(6): 3405 - 3415. [Abstract] [Full Text]
|
|
|
|
|
|
C. Garat, V. Van Putten, Z. A. Refaat, C. Dessev, S.-Y. Han, and R. A. Nemenoff Induction of Smooth Muscle alpha -Actin in Vascular Smooth Muscle Cells by Arginine Vasopressin Is Mediated by c-Jun Amino-terminal Kinases and p38 Mitogen-activated Protein Kinase J. Biol. Chem., July 14, 2000; 275(29): 22537 - 22543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Mack, A. V. Somlyo, M. Hautmann, A. P. Somlyo, and G. K. Owens Smooth Muscle Differentiation Marker Gene Expression Is Regulated by RhoA-mediated Actin Polymerization J. Biol. Chem., January 5, 2001; 276(1): 341 - 347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Carson, R. A. Fillmore, R. J. Schwartz, and W. E. Zimmer The Smooth Muscle gamma -Actin Gene Promoter Is a Molecular Target for the Mouse bagpipe Homologue, mNkx3-1, and Serum Response Factor J. Biol. Chem., December 8, 2000; 275(50): 39061 - 39072. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ficzycz, C. Eskiw, D. Meyer, K. E. Marley, M. Hurt, and N. Ovsenek Expression, Activity, and Subcellular Localization of the Yin Yang 1 Transcription Factor in Xenopus Oocytes and Embryos J. Biol. Chem., June 15, 2001; 276(25): 22819 - 22825. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Bhalla, L. Robitaille, and M. Nemer Cooperative Activation by GATA-4 and YY1 of the Cardiac B-type Natriuretic Peptide Promoter J. Biol. Chem., March 30, 2001; 276(14): 11439 - 11445. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
