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J. Biol. Chem., Vol. 278, Issue 33, 31233-31239, August 15, 2003
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-Myosin Heavy Chain Promoter*
**
From the
Department of Molecular, Cellular and
Developmental Biology, University of Colorado, Boulder, Colorado 80309,
||Department of Cardiology, School of Medicine,
University of Colorado Health Sciences Center, Denver, Colorado 80262, and
¶Department of Medicine, Denver Health Medical
Center, Denver, Colorado 80204
Received for publication, February 24, 2003 , and in revised form, May 8, 2003.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-myosin heavy chain
(MyHC) gene, which, with 
MyHC are the molecular motors
of the heart. MyHC mRNA and protein levels are down-regulated in
hypertrophy and heart failure, and this is thought to be detrimental for
cardiac contractility. We show that YY1 specifically interacts with the
MyHC promoter and that overexpression of YY1 in cardiac cells
represses the activity of the MyHC promoter. We also show that
the 170–200-amino acid region of YY1, important for its interaction with
histone acetyl transferases and histone deacetylases, is important for its
repressive activity and that YY1 deleted in this region is an activator of the
MyHC promoter. Moreover, we show that YY1 levels and DNA
binding activity are increased in failing human left ventricles and in a mouse
model of hypertrophic cardiomyopathy, where MyHC levels are decreased.
These results suggest that YY1 is a negative regulator of MyHC
gene expression. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
, differ in the rate of ATP hydrolysis, with MyHC having an
2 fold greater rate than MyHC
(1). The contractile velocity
of cardiac muscle correlates with MyHC isoform composition, suggesting a
causal relationship between MyHC isoform composition and cardiac contractility
(1). Cardiac myosin composition
varies among species, with small rodents expressing predominantly
MyHC and large mammals expressing predominantly
MyHC. Independent of species, antithetical transitions in
cardiac MyHC isoform expression have been observed in response to various
pathological and physiologic stimuli. Many of these stimuli result in cardiac
hypertrophy accompanied by changes in gene expression and increases in cardiac
myocyte size. In rodent models, pathological stimuli result in down-regulation
of MyHC and up-regulation of MyHC
(2). In the normal human heart,
20–30% of total MyHC mRNA consists of MyHC mRNA, whereas in
the failing heart, MyHC expression represents less than 2% of total
MyHC
(3–5).
At the protein level, MyHC in normal hearts constitutes 11% of
total MyHCs, but it is undetectable in the failing heart
(6). Moreover, in humans,
increases in MyHC isoform expression are closely associated with
improvements in left ventricular function and chamber remodeling, in contrast
to the expression of several of the other genes known to regulate contractile
function or pathological hypertrophy
(7). Therefore, rodent and
human models of hypertrophy and failure have directionally similar MyHC
isoform shifts.
In contrast to pathological stimuli, the hypertrophy induced by exercise
(physiological hypertrophy) is accompanied by an induction of MyHC and
repression of MyHC in rats
(8) and in C57/B16 mice
(9). Furthermore, this
hypertrophy is beneficial and is associated with cardiovascular conditioning
(9,
10). Recent evidence suggests
that even a small difference in MyHC isoform composition in the heart can have
important consequences for contractility. Transgenic mice expressing 12% of
their cardiac myosin as MyHC have decreased contractility and
myofibrillar ATPase (11). In
contrast, isolated cardiac myocytes that express only 12% of their MyHCs as
have 52% greater power output than those expressing only
(12). Thus, the decrease in
MyHC in human heart failure may play an important role in the reduction
of contractility observed in the failing heart and is likely to be
attributable, at least in part, to transcriptional regulation.
Through transfection, gene injection, and transgenic experiments, it has
been possible to identify a number of regulatory sites in the rodent
MyHC promoter region
(13,
14, reviewed in Ref.
15). Although most of these
elements act positively, there is one report characterizing an element that
acts as a repressor of the rat MyHC promoter. This element,
located in the first intron of the gene, is an ets binding site
(16) but is not present in
either the proximal or distal promoter elements of the human
MyHC
promoter.2 Relatively
little is known about the regulatory elements of the human
MyHC gene. However, the proximal promoter regions
(–340/+20 bp) of the human and rat MyHC genes show 80%
sequence identity, including most of the potential DNA-binding sites
(Fig. 1). Previous studies have
shown that the –340/+20 bp promoter region of the rat
MyHC gene is sufficient to direct cardiac-specific expression
in cells (17). This fragment
contains binding sites for GATA4, MEF2C, SRF, TEF, and thyroid hormone
(15).
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Yin Yang 1 (YY1) is a transcription factor that has been shown to regulate a variety of promoters (reviewed in Ref. 18). In cardiac myocytes, it has been shown to act largely as a repressor of transcription, with one report showing that it can act as an activator (19–21). YY1 has been shown to interact with histone deacetylases (HDACs) 1, 2, and 3 in HeLa cells, consistent with its role as a negative regulator of transcription (22, 23). Other studies have shown that YY1 interacts with the histone acetyltransferase (HATs) pCAF and p300, possibly explaining its mechanism in transcription activation (24, 25). Recently, YY1 has been shown to have a polycomb-like function in Drosophila, acting as a repressor of developmentally regulated genes (26). The activity of YY1 itself is regulated through acetylation by p300 and PCAF and deacetylation by HDACs (27). Acetylation of YY1 augments its repressor activity, possibly by facilitating a tighter interaction with HDACs (27).
To investigate the molecular mechanisms whereby MyHC is decreased in
heart failure, we have analyzed the activity of the human MyHC
promoter and proximal regulatory region with respect to the effect of YY1. We
show that YY1 binds to the MyHC promoter region and is a
negative regulator of transcription in neonatal cardiac myocytes (neonatal rat
ventricular myocytes, NRVM) but is a positive regulator of the same promoter
in non-cardiac cells. Additionally, we show that YY1 protein abundance is
significantly increased in pathological models where MyHC is decreased,
failing human hearts and in a transgenic mouse model of hypertrophic
cardiomyopathy. Conversely, YY1 levels are decreased in exercise-induced
hypertrophy in which expression of MyHC is induced. Our data
suggest that YY1 is an important contributor to the repression of
MyHC expression seen in cardiac hypertrophy and failure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MyHC antibody was a gift from
Dr. Kathy Schreiber, Myogen.
Plasmid Construct—The –454/+32 bp fragment of the
MyHC promoter was cloned into the pGL3 basic vector (Promega).
Mutations were created in the YY1 binding sites by generating oligonucleotides
(see "EMSA") containing the mutation of interest and amplifying
the fragments containing the mutation by PCR. The YY1 expression construct was
a gift from Dr. Michael Atchison (University of Pennsylvania). The YY1
170-200del construct was a gift of Dr. Edward Seto (University of South
Florida). The DNA constructs were purified using the Qiagen method.
Cell Culture and Transfection—Neonatal rat ventricular myocytes (NRVMs) were prepared according to the method described in Waspe et al. (28). Briefly, 150,000 cells/well were plated in 12-well tissue culture plates coated with gelatin. Eighteen h later, the medium was changed to minimum Eagle's medium supplemented with Hank's salt and L-glutamine. 20 mM Hepes pH 7.5, penicillin, vitamin B12, bovine serum albumin, insulin, and transferrin were added to the medium. Transfections were carried out by the FuGENE 6 (Roche Applied Science) method according to the manufacturer's recommendations; 0.75 µl of FuGENE/0.25 µg of plasmid DNA was transfected in each well. In the cotransfection experiments, the total amount of DNA was kept constant by the addition of a plasmid containing the cytomegalovirus (CMV) promoter not driving the expression of any gene. H9C2 cells were maintained according to American Type Culture Collection recommendation. Transfection in H9C2 cells was done by the FuGENE 6 method; 1.8 µl of FuGENE/0.6 µg of DNA was transfected in each well on a 24-well plate. Retinoic acid (RA) differentiation of H9C2 cells was done by treating the cells with 10 mM RA every day for 5–7 days in medium with 1% fetal bovine serum.
Preparation of Protein Extracts—Protein extracts from human normal and failing (idiopathic cardiomyopathy) left ventricles and transgenic hypertrophic cardiomyopathy (HCM) mice and littermate controls, 4 months of age, were prepared according to Molkentin and Marklan (29) with minor modifications. 0.5 g of tissue was homogenized with a Teflon homogenizer attached to a drill (SKIL-PA6-GF30) at 50% of maximum power. The resulting preparation was sonicated in a cell disruptor (Ultrasonics, W185F) at 50% of maximum power for 15 s. After cell lysis, the proteins were precipitated by the slow addition of the same volume of 4 M NH4SO4. These results were placed in a 50% final concentration of NH4SO4, allowing a greater protein recovery when compared with the 30% final concentration of NH4SO4 as described in the original method. Nuclear extracts from NRVMs were prepared according to Gupta et al. (30). HeLa cell nuclear extracts were prepared by the method of Dignam et al. (31).
EMSAs—EMSAs were carried out as described (32). Thirty-bp, double-stranded oligonucleotides were labeled by Klenow fill-in using [32P]dCTP: Wild type YY1-1, 5'-CAGGAGGAGGAAAGCCCATGGT-3'; Mut-YY1-1, 5'-CAGGAGGAGGAAAGCCCGCGGT-3'; Wild type YY1-2, 5'-GGTGACCCTCACCCATGTTTTCAGTTCACCC-3'; Mut-YY1-2, 5'-GGTGACCCTCACCCAACTTTTCAGTTCACCC-3'; Wild type YY1-3, 5'-GAGGTAAGGGCCATGGCAGGGTGGGAGAGG-3'; and Mut-YY1-3, 5'-GAGGTAAGGGCCCTGGCCTGGTGGGAGAGG-3' (mutations are in italic and bold). The reaction was performed using 100,000 cpm of wild type or mutant probe in a 30-µl binding reaction containing 10 mM Hepes, pH 7.9, 100 mM KCl, 4% glycerol, 1 mM EDTA, 0.1% Nonidet P-40, 1 µg of poly(dI-dC) (Amersham Biosciences), and 10 µg of extract. The resulting complex was resolved in a non-denaturing 4% acrylamide gel in 0.5x Tris-borate-EDTA.
Western Blots—Forty µg of protein extracts were resolved in a 7.5% acrylamide gel. The resulting gel was transferred to a polyvinylidene difluoride membrane for 1 h in 10% methanol at 400 mA. The membrane was blocked for 1 h in 1x PBS containing 10% low fat dry milk and 0.1% Tween. YY1 antibody was diluted 1:200 in 1x PBS containing 3% bovine serum albumin, 2% normal goat serum, and 0.1% Tween and incubated with the blot for 1 h at room temperature. The mouse secondary antibody conjugated to alkaline phosphatase was diluted 1:2500 in 1x PBS containing 5% low fat dry milk and 0.1% Tween and incubated with the blot for 1 h at room temperature. We used alkaline phosphatase-conjugated secondary antibodies, the Vistra (Amersham Biosciences) enhanced chemiluminescence reagent, an ABI STORM PhosphorImager, and ImageQuant software to visualize and quantify bound antibody.
Coomassie Staining of Polyvinylidene Difluoride Membranes—After Western blot experiments, the membrane was stripped of the antibodies and stained with Coomassie for 1 h. The membrane was than destained for at least 12 h. The proteins on the membrane were quantified on the LiCor scanner.
Exercised-induced Hypertrophy—HCM and wild-type mice were exercised in a voluntary wheel running model for 2 weeks (10).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MyHC Promoter Proximal Region—YY1 has a loose consensus
binding site, with the 5'CCAT3' core sequence being essential for
binding (33). The
–452-bp fragment of the MyHC promoter was scanned for
potential YY1 binding sites using the Vector NTI Suite v. 6.0 (InforMax).
Three regions containing the 5'CCAT3' core were found
(–425/–413 (YY1-1), –370/–360 (YY1-2), and
–325/–310 (YY1-3)). To test whether these regions were capable of
interacting with the YY1 transcription factor, 30-bp probes containing the
putative YY1 binding sites were created and used in EMSAs with nuclear
extracts prepared from NRVMs (see "Materials and Methods" for
probe description). Fig.
2A shows the result of EMSA experiments using the three
potential YY1 binding regions in NRVMs. A prominent complex was observed for
all three probes (Fig.
2A, lanes 2, 5, and 8). To confirm that
this complex contains YY1, an anti-YY1 antibody added to the reaction
prevented the formation of the complex
(Fig. 2A, lanes 3,
6, and 9). The YY1 antibody was used against GATA4 and Sp1
binding complexes, and no alteration to their binding ability was observed,
consistent with the specificity of the antibody (data not shown). To
characterize further the interaction of YY1 and the MyHC
promoter, mutations in the YY1-1, YY1-2, and YY1-3 binding sites that should
disrupt YY1 binding were designed (see "Materials and Methods").
The mutant oligonucleotides were labeled and used as probes against NRVM
nuclear extracts. The mutants failed to interact with any proteins in the
extract (Fig. 2B,
lanes 2, 4, and 6). Specificity of this binding was tested
using the wild-type and the mutant double-stranded oligonucleotides as cold
competitors. As seen in Fig.
2C (lanes 3, 4, and 5), increasing
amounts of the YY1-3 wild-type site as a cold competitor decreased YY1 binding
in a concentration-dependent manner. Addition of the mutant cold competitor
did not affect YY1 binding, suggesting that the binding is specific
(Fig. 2D, lanes
3 and 4).
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The YY1 Transcription Factor Is a Repressor of MyHC
Promoter Activity—YY1 has been shown to act as a positive or
negative regulator of transcription
(18). To test the effect of
YY1 on the activity of the MyHC promoter, cotransfection
experiments were performed in NRVMs. As shown in
Fig. 3A, –452 bp
of the MyHC promoter driving the expression of the luciferase gene was
cotransfected with increasing amounts of a YY1 expression construct.
Dose-dependent repression of promoter activity was observed with 0.04 to 0.2
µg of CMV-YY1 DNA transfected into the cells
(Fig. 3A). Because YY1
can act as a positive or negative regulator of transcription depending on the
promoter and/or cell context, we tested whether its repressive effect on
MyHC promoter activity was specific for NRVMs. H9C2 is a rat
atrial embryonic cell line that has characteristics of both skeletal and
cardiac muscle (34).
Differentiation of H9C2 cells with RA has been shown to activate a more
cardiac phenotype (34).
Undifferentiated H9C2 cells were cotransfected with the MyHC
promoter and CMV-YY1. As shown in Fig.
3B, YY1 significantly increases the activity of the
MyHC promoter in H9C2 cells. Interestingly, upon
differentiation of H9C2 cells by RA, YY1 becomes a repressor of the
MyHC promoter. Cotransfection of a YY1 expression construct,
and the MyHC promoter-luciferase construct into HeLa cells
also resulted in an increase in the activity of the promoter (data not shown).
These results suggest that repression of MyHC promoter
activity by YY1 occurs in a cell type-dependent manner.
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EMSA experiments showed that the MyHC promoter has three
sites that can interact with YY1. To test whether abolishing the interaction
of the YY1 transcription factor with the MyHC promoter would
result in an up-regulation of this promoter in cells, point mutations in all
three YY1 binding sites and all possible combinations were generated in the
context of the MyHC promoter. The resulting constructs were
transfected into NRVMs. As shown in Fig.
4A, each of the mutant constructs showed 2–4-fold
higher activity when compared with the wild-type promoter construct.
Combinations of mutations in the different sites did not result in an increase
in the activity of the promoter when compared with mutations in single sites,
suggesting that a single YY1 site is sufficient for complete repression by YY1
(see "Discussion").
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To show further that the effect of YY1 on the MyHC promoter
is attributable to a direct interaction of YY1 with the promoter,
cotransfection experiments using the YY1 cDNA and the wild-type
MyHC promoter, as well as promoter constructs containing each
of the sites mutated individually, or a combination of the three sites
mutated, were done in NRVMs. As shown in
Fig. 4B, the wild-type
and singly mutant constructs were repressed by YY1, but the construct
containing all three sites mutated did not respond to YY1. These results
suggest that YY1 repression of the MyHC promoter is not the
result of a non-specific squelching effect attributable to the presence of an
exogenous DNA into the transfection experiments. To the contrary, an
MyHC promoter construct that cannot bind YY1 has increased
activity in NRVMs.
YY1 Repression of the MyHC Promoter Is Suppressed upon
Deletion of the 170–200-Amino Acid Region—The region of YY1
encompassing 170–200 amino acids has been shown to be important for its
interactions with HDACs and HATs
(27). A YY1 construct deleted
for this region was used in cotransfection experiments with the
MyHC promoter. As shown in
Fig. 5, this construct not only
fails to repress the activity of the MyHC promoter but
increases the activity of the promoter 2–3-fold. These results suggest
that the 170–200-amino acid region of the YY1 transcription factor is of
fundamental importance to its function as a repressor of MyHC
promoter activity in NRVMs. Because this is the region that has been shown to
be important in the interaction with HDACs and HATs in HeLa cells, it is
possible that the function of YY1 may be regulated by these proteins in
NRVMs.
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YY1 Levels Are Increased in Failing Human Left Ventricle and in Mouse
HCM—Considering that MyHC expression (mRNA and protein) is
decreased significantly in myocardial pathological hypertrophy and failure, we
next tested the hypothesis that the amount of YY1 would be increased in these
settings. Western blots were performed using protein extracts from non-failing
and failing human left ventricle as well as from cardiac tissue from wild-type
mice and a transgenic mouse model of HCM
(35). YY1 levels are increased
2-fold in failing human left ventricle
(Fig. 6A) and in the
hearts from the HCM transgenic mouse model
(Fig. 7A). Six
different non-failing and failing left ventricle samples were used, and the
experiment was repeated twice. The failing left ventricle samples came from
patients 39, 66, 54, 49, 55, and 54 years of age, and the non-failing left
ventricle samples came from patients 50, 19, 20, 75, 56, and 55 years of age.
Four different heart samples were used for the wild-type and HCM samples.
Because hypertrophy increases the general transcription/translation activity
of cardiac myocytes, the loading control for the Western blots was done by
measuring total protein in each lane through Coomassie staining of the
polyvinylidene difluoride membranes (see "Materials and Methods").
To test whether YY1 binding activity correlated with the increase in protein
levels observed in the failing hearts, the YY1-2 probe from the
MyHC gene was used as a probe in EMSAs against normal and
failing extracts. As shown in Fig.
6B, binding of YY1 was increased 3-fold in the failing
heart samples. There is, therefore, an antithetic relationship between the
amount and binding activity of the YY1 transcription factor and MyHC
mRNA and protein. These results are consistent with the hypothesis that YY1
up-regulation in failing hearts could be contributing to the down-regulation
of human MyHC gene expression in hypertrophy and heart
failure.
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YY1 Is Decreased in Exercise-induced Hypertrophy—As
described earlier, exercise induces hypertrophy and has been shown to be
associated with an increase in MyHC gene expression
(8,
34). Exercise has also been
shown to counteract the deleterious effects of hypertrophy
(36). Considering that YY1
decreases the activity of the MyHC promoter in NRVMs and its
levels are increased in human failing hearts, we hypothesized that the
elevated YY1 levels in HCM mice would be decreased by exercise. Sedentary HCM
mouse hearts have also been shown to have elevated MyHC and decreased
MyHC (37). To test this
hypothesis, male HCM mice, 4 months of age, were exercised on a voluntary cage
wheel for 2 weeks. Wild-type mice ran an average of 5.7 km/24 h, and the HCM
mice ran an average of 5.4 km/24 h. Protein extracts were prepared, and YY1
levels were assessed by Western blot. YY1 levels were indistinguishable in
sedentary and exercised wild-type mice, correlating with MyHC levels
(wild-type young adult mouse hearts express 100% of their MyHC as ;
therefore, there can theoretically be no increase). As hypothesized, YY1
levels in exercised HCM mice returned to wild-type levels
(Fig. 7A). To
determine whether MyHC protein levels were up-regulated in exercised
HCM mice, a Western blot using an antibody specific for the isoform
was performed. As expected, MyHC levels in exercised HCM mice were
up-regulated when compared with sedentary HCM mice
(Fig. 7B). These
results suggest a strong link between YY1 and MyHC levels. Hearts that
show an up-regulation of YY1 protein levels also show a decrease in
MyHC protein levels and vice versa, suggesting that YY1 plays an
important role in the regulation of MyHC gene expression in
physiological and pathological states.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MyHC is decreased in pathological hypertrophy and
heart failure. The transcription factors that have been shown to be important
for the regulation of the rat MyHC promoter, i.e.
GATA4, MEF2C, NFAT3, and SRF among others, are positive regulators of
transcription (15). There is
only one report of an ets binding region in the rat MyHC
promoter that has been shown to act as a repressor
(16), but this region is not
present in the human MyHC proximal promoter region (data not
shown). Here we show that YY1 binds to the MyHC promoter in a
sequence-specific manner and represses its transcription in cardiac cells. We
also show that YY1 is increased in the failing human heart and in a transgenic
mouse model of HCM but decreased in exercised-induced hypertrophy.
Cotransfection experiments in NRVMs suggest that YY1 is a strong repressor
of human MyHC promoter activity. Squelching of transcription
in the presence of an excess of a transcription factor is a phenomenon that
has been extensively described
(38). In our studies, we have
three experiments that show that the repression mediated by YY1 is not
attributable to a non-specific squelching effect. First, point mutations in
the YY1 binding sites of the human MyHC promoter prevented the
binding of YY1 to this promoter. Mutation of these sites also resulted in its
up-regulation, suggesting again that the effects of YY1 on the
MyHC promoter are as a repressor. Second, cotransfection of
the MyHC promoter and the YY1 cDNA in undifferentiated H9C2 cells and
in HeLa cells resulted in an up-regulation of activity of this promoter, and
upon differentiation of H9C2 cells into a phenotype that is more cardiac-like,
YY1 became a repressor of the MyHC promoter, demonstrating
that YY1 can have a dual activity even on the same promoter, depending on the
cell type context or differentiation state. The cause for this dual activity
of YY1 in different cell types is not known, but it is likely attributable to
the factors that interact with YY1 and possibly change its function.
Transfection experiments using the constructs containing single sites
mutated as well as combinations of these sites mutated suggest that the
repressive effect of YY1 on the MyHC promoter is not additive
but equivalent for all three sites. Combinations of mutations in the three
different sites did not result in a further increase in promoter activity when
compared with constructs containing single mutations. At this point, we do not
know the reason for this finding, but it could be attributable to the fact
that repression by YY1 requires all three sites and that disruption of a
single site abolishes YY1 repression activity.
Why YY1 represses transcription of the MyHC promoter in
cell types in which this promoter is normally active is not yet known. One
hypothesis is that YY1 is a general repressor of transcription in cardiac
cells. However, YY1 can activate transcription of brain naturietic peptide in
cardiac cells (21). Another
hypothesis is that there is an equilibrium between YY1 (as a repressor) and
activators of the MyHC promoter. In a normal physiological
state, YY1 repression of the promoter is counteracted by positive regulators,
but in a pathological state, when MyHC activity is repressed,
YY1 is capable of overcoming the activators and effectively represses the
promoter.
The mechanism by which YY1 represses or activates transcription is not
clear. Various groups have attempted to characterize functional domains of YY1
that are responsible for its opposing effects. The reports in the literature
are controversial, and some regions that are characterized as activators by
one group are characterized as repressors by other groups
(18). Recently, YY1 has been
shown to be itself acetylated and deacetylated by HATs and HDACs,
respectively. These studies were done in HeLa cells and two HATs, pCAF and
p300, were found to acetylate the 170–200-amino acid region of YY1,
whereas the C-terminal zinc finger was acetylated only by pCAF. Acetylation of
this central region increases the repressive activity of YY1 and results in a
more efficient interaction with HDACs
(27). Acetylation of the
C-terminal DNA-binding region most likely decreases the repression activity of
YY1 by decreasing its affinity for DNA
(27). Our results suggest that
the 170–200-amino acid region plays a fundamental role in the repression
of MyHC promoter activity mediated by YY1 in NRVMs. However,
preliminary studies from our laboratory suggest that in NRVMs neither pCAF nor
p300 is capable of reversing the repressor activity of YY1 (data not shown).
YY1 has been shown to be deacetylated by class I HDACs (1, 2, and 3) in HeLa
cells. In addition to Class I HDACs, cardiac cells also express the class II
HDACs (4, 5, and 6). It is unknown whether the class II HDACs play a role in
the repression of YY1 in cardiac cells. In these cells, the best characterized
example of HDAC/HAT regulation is the MEF2C-HDAC-calcium calmodulin-dependent
kinase pathway (39), and it is
possible that YY1 regulation might follow a similar pathway in this context.
It is also possible that the regulation of YY1 in cardiac cells is not
mediated by acetylation/deacetylation.
The increase in YY1 protein levels in human failing heart extracts and in
the HCM mouse model, where MyHC levels are very low, is consistent with
the hypothesis that it is a repressor of MyHC gene expression
in vivo. Our data are in agreement with a previous report that YY1
levels are increased in failing human heart and in animal models submitted to
left coronary occlusion (40).
In both cases, a limitation of the technique is that YY1 levels are not
measured in individual myocytes. The decrease in YY1 levels in exercised HCM
mice is of particular interest. In physiological hypertrophy in wild-type
mice, there is a 10% increase in heart weight, no change in MyHC
expression, and YY1 levels do not change. However, in HCM, where there is
pathological hypertrophy and a decrease in MyHC, exercise reverses the
induction of YY1 levels and increases MyHC levels. Together, these
results suggest that MyHC levels are tightly linked to YY1.
In conclusion, our results show that YY1 and MyHC
are regulated in a antithetical manner and suggest that transcriptional
activity of the MyHC promoter is dependent on low levels of
YY1 and vice versa. Our results also show for the first time a transcription
factor that represses the activity of the human MyHC promoter.
Although further investigation is required, YY1 could be part of a pathway
that functions to repress the MyHC gene expression in cardiac
hypertrophy and failure, contributing to the pathology of human heart
failure.
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FOOTNOTES |
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Present address: Dept. of Cardiology, School of Medicine, University of
Colorado Health Sciences Center, Denver, CO 80262.
** To whom correspondence should be addressed: Dept. of Molecular, Cellular and Developmental Biology, CB 347, University of Colorado, Boulder, CO 80309. Tel.: 303-492-7606; Fax: 303-492-8907; E-mail: Leslie.Leinwand{at}colorado.edu.
1 The abbreviations used are: MyHC, myosin heavy chain; YY1, Yin Yang 1;
HDAC, histone deacetylase; HAT, histone acetyltransferase; RA, retinoic acid;
NRVM, neonatal rat ventricular myocyte; EMSA, electrophoretic mobility shift
assay; CMV, cytomegalovirus; HCM, hypertrophic cardiomyopathy.
2 C. Sucharov, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 S. K. Ambler, Y. K. Hodges, G. M. Jones, C. S. Long, and L. D. Horwitz Prolonged administration of a dithiol antioxidant protects against ventricular remodeling due to ischemia-reperfusion in mice Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1303 - H1310. [Abstract] [Full Text] [PDF]
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 Q. Liu, K. A. Merkler, X. Zhang, and M. P. McLean Prostaglandin F2{alpha} Suppresses Rat Steroidogenic Acute Regulatory Protein Expression via Induction of Yin Yang 1 Protein and Recruitment of Histone Deacetylase 1 Protein Endocrinology, November 1, 2007; 148(11): 5209 - 5219. [Abstract] [Full Text] [PDF]
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 S. Srivastava, B. Chandrasekar, Y. Gu, J. Luo, T. Hamid, B. G. Hill, and S. D. Prabhu Downregulation of CuZn-superoxide dismutase contributes to {beta}-adrenergic receptor-mediated oxidative stress in the heart Cardiovasc Res, June 1, 2007; 74(3): 445 - 455. [Abstract] [Full Text] [PDF]
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 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]
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C. C. Sucharov, P. D. Mariner, K. R. Nunley, C. Long, L. Leinwand, and M. R. Bristow A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1299 - H1308. [Abstract] [Full Text] [PDF]
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J. B. Pillai, H. M. Russell, J. Raman, V. Jeevanandam, and M. P. Gupta Increased expression of poly(ADP-ribose) polymerase-1 contributes to caspase-independent myocyte cell death during heart failure Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H486 - H496. [Abstract] [Full Text] [PDF]
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 C. C. Sucharov, S. M. Helmke, S. J. Langer, M. B. Perryman, M. Bristow, and L. Leinwand The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses {alpha} Myosin Heavy-Chain Gene Expression Mol. Cell. Biol., October 1, 2004; 24(19): 8705 - 8715. [Abstract] [Full Text] [PDF]
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 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]
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