Competition Between Negative Acting YY1 versus Positive Acting Serum Response Factor and Tinman Homologue Nkx-2.5 Regulates Cardiac {alpha}-Actin Promoter Activity

Ching-Yi Chen and Robert J. Schwartz

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcription of sarcomeric {alpha}-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 {alpha}-actin promoter. In addition, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac {alpha}-actin gene in 10T1/2 fibroblasts, by a mechanism that involved coassociation of SRF and Nkx-2.5 on intact SREs of the {alpha}-actin promoter. Here, we show that the second SRE of the avian cardiac {alpha}-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 {alpha}-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 {alpha}-actin gene expression during cardiogenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cardiac and skeletal {alpha}-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 {alpha}-actins are expressed predominantly in the respective sarcomeric tissues of the mature animal (4), the two genes are regulated independently: cardiac {alpha}-actin is the major striated actin isoform in embryonic skeletal muscle, as well as in the heart, and is gradually replaced by skeletal {alpha}-actin as skeletal muscle matures (5). In warm-blooded vertebrates, cardiac {alpha}-actin transcripts are first detected in the presumptive ventricular myocardium of the heart primordia (6). As cardiac morphogenesis proceeds, cardiac {alpha}-actin mRNA accumulates throughout the myocardium of the tubular heart. The spread of cardiac {alpha}-actin mRNA and the appearance of skeletal {alpha}-actin transcripts to other regions of the myocardium parallel the regional progression of myofibril formation in the developing chick heart (7). Thus, expression of {alpha}-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 {alpha}-actin genes are largely unknown.

Studies of muscle {alpha}-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 {alpha}-actin gene, the capacity for selective expression of the chicken skeletal {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-actin promoter are also located predominantly within 300-bp upstream sequences (11, 12, 13). Sequence comparison of the muscle-specific {alpha}-actin promoters has revealed several regions of sequence conservation present in the chicken, mouse, rat, and human cardiac and skeletal {alpha}-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 {alpha}-actin genes (8, 14). Mutagenesis studies of vertebrate cardiac and skeletal {alpha}-actin promoters demonstrate that SREs play a positive role in myogenic induction (8, 10, 15, 16).

Nuclear proteins have been shown to interact with {alpha}-actin CArG sequences (17, 18, 19). A CArG box-binding factor reported to regulate the transcription of {alpha}-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 {alpha}-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), {delta} (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 {alpha}-actin promoters (28, 31). We proposed that the skeletal {alpha}-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 {alpha}-actin promoter in differentiation-blocked myoblasts (3)1, and like the closely related cardiac {alpha}-actin promoter, SRF- binding activity was also required for the cardiac {alpha}-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 {alpha}-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 {alpha}-actin promoter and increased transcription of the endogenous cardiac {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-actin SREs, which is thought to result in transcriptional activition of the cardiac {alpha}-actin promoter during cardiogenesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Coexpression of Nkx 2.5 and SRF Activates the Avian Cardiac {alpha}-Actin Promoter in 10T1/2 Fibroblasts
SRF alone can induce modest activation of the skeletal {alpha}-actin promoter in differentiation-blocked myoblasts (31) and, like the closely related cardiac {alpha}-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 {alpha}-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. 1Go). Expression of either Nkx-2.5 or SRF stimulated the cardiac {alpha}-actin promoter activity about 3- to 5-fold, which is in the range of SRF-stimulated {alpha}-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. 1AGo) and as shown previously (35, 36). Nkx-2.5 and SRF also weakly activated the {alpha}-skeletal actin promoter (Fig. 1DGo). Nkx-2.5 and SRF did not cotransactivate a minimal c-fos SRE construct as well as a solitary skeletal {alpha}-actin proximal SRE1 construct (Fig. 1Go, 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. 1Go, B and C). These results suggest that Nkx 2.5 and SRF cotransactivation was restricted to the cardiac {alpha}-actin promoter.



View larger version (31K):
[in this window]
[in a new window]
 
Figure 1. Nkx-2.5 and SRF Activated the Avian {alpha}-Cardiac Actin Promoter in 10T1/2 Fibroblast Cells

A variety of promoter reporter genes were cotransfected in combinations with Nkx-2.5 or SRF expression vectors in 10T1/2 cells as shown in the panels above and as described in Materials and Methods. A, Cotransfection assays with the avian cardiac {alpha}-actin promoter construct ({alpha}-CA-LUC); B, SV40 promoter (SV-LUC); C, TK minimal promoter (TK-LUC); D, the avian skeletal {alpha}-actin promoter ({alpha}-SK-LUC); E, the c-fos SRE linked to the TK minimal promoter (c-fos SRE-TK-LUC); and F, the avian skeletal actin SRE1 linked to TK minimal promoter (MRE-TK-LUC). Results were averages of two independent duplicate transfection experiments normalized to ß-gal activity, an internal standard. The relative value of each promoter obtained with an empty expression vector was set at 1.

 
Nkx-2.5 and SRF Displaces YY1 Binding to CaSRE2
How might Nkx-2.5 and SRF collaborate to stimulate cardiac {alpha}-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. 2AGo), 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. 2Go 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. 2BGo), suggesting their mutually exclusive binding to the CaSRE2 site.



View larger version (59K):
[in this window]
[in a new window]
 
Figure 2. Mutually Exclusive DNA-Binding Activities of YY1 vs. Nkx-2.5 or SRF to the CaSRE2 Site

A, Displacement of YY1 binding by Nkx-2.5 at CaSRE2. A labeled CaSRE2 oligonucleotide was incubated with YY1-transfected 10T1/2 nuclear extracts (5 µg) in the presence of increasing amounts of purified MBP-Nkx-2.5 protein (50, 100, 200, or 400 ng; lanes 2–5) or MBP-Nkx-2.5 pm (lanes 6–9). YY1 DNA-binding activity in transfected 10T1/2 cells was shown in lane 1. B, Displacement of YY1 binding by SRF at CaSRE2. The same nuclear extracts used in panel A was incubated with CaSRE2 probe in the presence of increasing amounts of bacterial purified SRF (50, 100, 200, or 400 ng, lanes 2–5). SRF-binding activity alone was also shown (lanes 6–9). YY1* represented truncated YY1.

 
The Cardiac {alpha}-Actin Promoter Sequence Between -150 and -100 Contains a Strong Negative Element
Since YY1 was shown to repress the skeletal {alpha}-actin promoter (44), we wanted to determine whether YY1 could also repress the cardiac {alpha}-actin promoter. We asked whether YY1-binding sites in the cardiac {alpha}-actin promoter acted as negative regulatory elements. Several cardiac {alpha}-actin promoter deletion mutants (Fig. 3AGo) linked to the luciferase reporter gene were constructed and were transfected into primary chicken cardiomyocytes to assay their promoter activities (Fig. 3BGo). 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. 1Go and in Chen et al. (35) that coexpression of Nkx-2.5 and SRF was able to transactivate the cardiac {alpha}-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. 3CGo, 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.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 3. Serial Deletion Mutagenesis Reveals a Potent Negative Element in the Cardiac {alpha}-Actin Promoter

A, Schematic diagram of the cardiac {alpha}-actin promoter deletion constructs. SREs were indicated by closed boxes. B, Transfection analysis of various repoter constructs in 12-day-old primary chicken cardiomyocytes. The luciferase activity of Del-58 in these cells was set at 1. C, Cotransfection analysis of reporter constructs with Nkx-2.5 and SRF expression vectors in 10T1/2 cells. The promoter activity of Del-58 in these cells cotransfected with an empty vector was set at 1.

 
SRF and Nkx-2.5 Overcome YY1 Repression on the {alpha}-Cardiac Actin Promoter
To investigate the functional roles of these three protein factors regulating the cardiac {alpha}-actin gene, the wild type cardiac {alpha}-actin promoter construct was transfected into 10T1/2 cells together with various combinations of protein expression vectors. As shown in Fig. 4Go, YY1 did not repress the basal activity of cardiac {alpha}-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. 4Go). Repression of the SRF-stimulated cardiac {alpha}-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 {alpha}-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 {alpha}-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. 3Go, 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 {alpha}-actin promoters.



View larger version (42K):
[in this window]
[in a new window]
 
Figure 4. Overcoming YY1 Repression of Cardiac {alpha}-Actin Promoter Activity by Nkx-2.5 and SRF

10T1/2 cells were cotransfected with the wild type cardiac {alpha}-actin promoter construct in the presence of various combinations of effectors: pEMSV-YY1 (1.5 µg; lanes 2, 4, 6, 7, and 8), pEMSV-Nkx-2.5 (3 µg; lanes 3, 4, 8, and 9), pCGN-SRF (75 ng; lanes 5, 6, 7, 8, and 9), or pEMSV-Nkx-2.5 pm (3 µg; lane 7). The luciferase activity obtained with cotransfection of the promoter construct and pCGN plus pEMSV empty vectors was set at 1. Results represented the averages of two separate transfection assays.

 
Elimination of DNA-Binding Activities of SRF and YY1, but Not Nkx-2.5, at the CaSRE2 Site Stimulated the Cardiac {alpha}-Actin Promoter Activity
To test the idea that YY1 acts as a repressor in restricting cardiac {alpha}-actin promoter activity, we wanted to eliminate YY1-binding activity to CaSRE2, by generating a cardiac {alpha}-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. 5AGo). 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. 5BGo). 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. 6Go). 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. 6BGo]). The promoter activities of these two reporter genes were also examined in primary chicken cardiomyocytes. As shown in Fig. 6CGo, 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 {alpha}-actin promoter, and elimination of YY1 binding enhanced transcription of the cardiac {alpha}-actin promoter in transfected cells.



View larger version (68K):
[in this window]
[in a new window]
 
Figure 5. Electrophoretic Mobility Shift Assays with Wild Type and Mutated CaSRE2 Oligonucleotides

A, Binding of SRF, YY1, and Nkx-2.5HD to wild type or mutated CaSRE2 oligonucleotide. Gel mobility shift assays with a wild type SRE 2 or mutated SRE2 (SRE2m) oligonucleotide. Two increasing inputs of proteins were shown. MBP-Nkx-2.5HD (75 or 150 ng) was purified bacterial protein. SRF- and YY1-binding activities were 10T1/2 nuclear extracts (5 or 10 µg) transfected with either an SRF or YY1 expression vector. The strong bands in lanes 7 and 8 were due to an unknown protein binding to the SRE2m oligonucleotide. B, EMSA competition assays. Nkx-2.5-transfected C2C12 nuclear extracts (10 µg) were incubated with CaSRE2 probe and challenged with cold SRE2 or SRE2m oligonucleotides at competitor-probe molar ratios of 50 or 100. The incomplete competition was due to the nonspecific binding of protein in C2C12 extracts, which happened to comigrate with the Nkx-2.5-binding complex.

 


View larger version (37K):
[in this window]
[in a new window]
 
Figure 6. Stimulation of Cardiac {alpha}-Actin Promoter Activity by Weakening YY1 Binding to CaSRE2 Site

A, Schematic diagram of the cardiac {alpha}-actin promoter constructs. A site-directed mutagenesis of SRE2 is indicated by a striped box. B, A cotransfection assay of these reporters in 10T1/2 cells with Nkx-2.5 and SRF expression vectors. The luciferase activity obtained with the wild type promoter in the presence of empty vectors was set at 1. C, Promoter activities of these constructs in transfected primary chicken cardiomyocytes. The promoter activity obtained with Del-58 in Fig. 3BGo was set at 1.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Quitschke et al. (11) identified a negative factor enriched in nonmouscle cells, which bound to the avian cardiac actin SRE2. We demonstrated here that this negative factor was most likely YY1. Sequence comparison of the vertebrate cardiac {alpha}-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. 7Go). 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.



View larger version (33K):
[in this window]
[in a new window]
 
Figure 7. DNA Sequence Comparision of Vertebrate Cardiac {alpha}-Actin SRE2 and SRE3 and the YY1 Consensus Sequence

DNA sequence comparison of vertebrate cardiac {alpha}-actin SRE2 and SRE3 (11, 17) and the YY1 consensus sequence (31). Boldface letters indicate conserved nucleotide nucleotides. The chicken SRE2 is conserved in the reverse orientation in relation to the human, mouse, and Xenopus SRE2. The chicken, human, and Xenopus SRE2 contains the half-YY1-binding site (ATGGN) in both orientations.

 
Several lines of evidence suggest that YY1 is most likely a transcriptional repressor of the striated {alpha}-actin genes. YY1 protein contents are enriched in replicating myoblasts and nonmuscle cells (19, 31), where expression of this {alpha}-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 {alpha}-actin promoter (39). Moreover, stimulation of the skeletal {alpha}-actin promoter activity was observed by selectively weakening YY1-SRE1 interaction without disrupting SRF-SRE1-binding activity (19). Furthermore, transfected SRF activated the skeletal {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-actin promoters. This evidence is further provided by the deleted promoter constructs as described in Fig. 3BGo, 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 {alpha}-actin promoter activity by indirect mechanisms involving activation of repressors of myogenesis, such as c-Myc (29).

As observed for the skeletal {alpha}-actin promoter, transfected SRF and Nkx-2.5 resulted in overcoming the inhibitory effect of YY1 on the cardiac {alpha}-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. 4Go). 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. 5Go), resulted in increased promoter activity (Fig. 6Go), which further suggests that YY1 binding to CaSRE2 is necessary for repression of the cardiac {alpha}-actin promoter. In addition, we showed that disruption of SRF-SRE2 binding did not impair the cardiac {alpha}-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. 6AGo), and the mutated SRE2 site continued to bind Nkx-2.5 (Fig. 5Go), it was concluded that Nkx-2.5 was a primary binder on the cardiac {alpha}-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 {alpha}-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 {alpha}-actin gene. Because of the homology of SREs in all vertebrate cardiac {alpha}-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 {alpha}-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 {alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid DNA
The SRF expression vector, pCGN-SRF, and Nkx-2.5 expression vectors, pEMSV-Nkx-2.5 and pEMSV-Nkx-2.5 pm, were described previously (35, 36). The YY1 expression vector driven by the EMSV promoter was described previously (39). The wild type cardiac {alpha}-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 {alpha}-CA-LUC with BglII and NcoI to remove the promoter sequences from -310 to -100. The skeletal {alpha}-actin reporter, {alpha}-SK-LUC, was previously constructed by subcloning nucleotides -394 to +24 of the chicken skeletal {alpha}-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
 
This article is dedicated in honor of our esteemed colleague, mentor, and friend, Dr. Bert O’ Malley, on the occasion of his 60th birthday.


    FOOTNOTES
 
Address requests for reprints to: Robert J. Schwartz, Ph.D., Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Vandekerckhove J, Weber K 1978 At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino- terminal tryptic peptide. J Mol Biol 126:782–802
  2. Schwartz RJ, Rothblum KN 1981 Gene switching in myogenesis: differential expression of the actin multigene family. Biochemistry 20:4122–4129[Medline]
  3. Chang KS, Zimmer Jr WE, Bergsma DJ, Dodgson JB, Schwartz RJ 1984 Isolation and characterization of six different chicken actin genes. Mol Cell Biol 4:2498–2508[Medline]
  4. Hayward LJ, Schwartz RJ 1986 Sequential expression of chicken actin genes during myogenesis. J Cell Biol 102:1485–1493[Abstract]
  5. Minty A, Alonso S, Caravatti M, Buckingham M 1982 A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA. Cell 30:185–192[Medline]
  6. Sasson D, Lyons G, Wright WE, Lin V, Lassar A, Weintraub H, Buckingham M 1989 Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 341:303–307[CrossRef][Medline]
  7. Ruzicka DL, Schwartz RJ 1988 Sequential activation of {alpha}-actin genes during avian cardiogenesis: vascular smooth muscle {alpha}-actin transcripts mark the onset of cardiomyocyte differentiation. J Cell Biol 107:2575–2586[Abstract]
  8. Bergsma DJ, Grichnik JM, Gosset LMA, Schwartz RJ 1986 Delimitization and characterization of cis-acting DNA sequences required for the regulated expression and transcriptional control of the chicken skeletal {alpha}-actin gene. Mol Cell Biol 6:2462–2475[Medline]
  9. Petropoulos CJ, Rosenberg MP, Jenkins NA, Copeland NG, Hughes SH 1989 The chicken skeletal muscle {alpha}-actin promoter is tissue specific in transgenic mice. Mol Cell Biol 9:3785–3792[Medline]
  10. Sartorelli V, Webster KA, Kedes L 1990 Muscle-specific expression of the cardiac {alpha}-actin gene requires MyoD1, CArG-box binding factor, and SP1. Genes Dev 4:1811–1822[Abstract]
  11. Quitschke WW, DePonti-Zilli L, Lin ZY, Paterson B 1989 Identification of two nuclear factor-binding domains on the chicken cardiac actin promoter: implications for regulation of the gene. Mol Cell Biol 9:3218–3230[Medline]
  12. French BA, Chow KL, Olson EN, Schwartz RJ 1991 Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac {alpha}-actin promoter. Mol Cell Biol 11:2439–2450[Medline]
  13. Moss JB, McQuinn TC, Schwartz RJ 1994 The avian cardiac {alpha}-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[Abstract/Free Full Text]
  14. Taylor A, Erba HP, Muscat GEO, Kedes L 1988 Nucleotide sequence and expression of the human skeletal {alpha}-actin gene: evolution of functional regulatory domains. Genomics 3:323–336[Medline]
  15. Walsh K, Schimmel P 1988 DNA-bindinbg site for two skeletal actin promoter factors is important for expression in muscle cells. Mol Cell Biol 8:1800–1810[Medline]
  16. Chow KL, Schwartz RJ 1990 A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal {alpha}-actin gene. Mol Cell Biol 10:528–538[Medline]
  17. Gustafon TA, Miwa T, Boxer L, Kedes L 1988 Interaction of nuclear proteins with muscle specific regulatory sequences of the human cardiac actin promoter. Mol Cell Biol 7:4100–4119
  18. Walsh K, Schimmel P 1987 Two nuclear factors compete for the skeletal muscle actin promoter. J Biol Chem 262:9429–9432[Abstract/Free Full Text]
  19. Lee TC, Chow KL, Fang P, Schwartz RJ 1991 Activation of skeletal {alpha}-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[Medline]
  20. Treisman R 1986 Interaction of a protein-binding site that mediates transcription response of the c-fos gene to serum factors. Cell 46:567–574[Medline]
  21. Boxer LM, Prywes R, Roeder RG, Kedes L 1989 Identification and characterization of a factor that binds to two human sarcomeric actin promoters. Mol Cell Biol 9:515–522[Medline]
  22. Walsh K 1989 Cross-binding of factors to functionally different promoter elements in the c-fos and skeletal actin genes. Mol Cell Biol 9:2192–2201
  23. Taylor M, Treisman R, Garrett N, Mohun T 1989 Muscle-specific (CArG) and serum-responsive (SRE) promoter elements are functionally interchangeable in Xenopus embryos and mouse fibroblasts. Development 106:67–78[Abstract]
  24. Shi Y, Seto E, Chang LS, Shenk T 1991 Transcriptional repression by YY1, a human GLI-Kruppel-related protein, and releif of repression by adenovirus E1A protein. Cell 67:377–388[Medline]
  25. Park K, Atchison ML 1991 Isolation of a candidate repressor/activator, NF-E1 YY-1, delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site. Proc Natl Acad Sci USA 88:9804–9808[Abstract]
  26. Hariharan N, Kelly DE, Perry RP 1991 Delta, a transcription factor which binds to downstream elements in several polymerase II promoters, is a functionally versatile zinc finger protein. Proc Natl Acad Sci USA 88:9799–9870[Abstract]
  27. Flanagan JR, Becker KG, Ennist DL, Gleason SL, Driggers PH, Levi BZ, Apella E, Ozato K 1992 Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus. Mol Cell Biol 12:38–44[Abstract]
  28. Gualberto A, LePage D, Pons G, Mader SL, Park P, Atchison ML, Walsh K 1992 Isolation of a candidate repressor/activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site. Mol Cell Biol 12:4209–4214[Abstract]
  29. Riggs KJ, Saleque S, Wong KK, Merrell KT, Lee JS, Shi Y, Calame K 1993 Yin-Yang -1 activates the c-myc promoter. Mol Cell Biol 13:7487–7495[Abstract]
  30. Seto E, Shi Y, Shenk T 1991 YY1 is an initiator sequence-binding protein that directs and activates transcription in vitro. Nature 354:241–245[CrossRef][Medline]
  31. Lee TC, Shi Y, Schwartz RJ 1992 Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal {alpha}-actin transcription in embryonic myoblasts. Proc Natl Acad Sci USA 89:9814–9818[Abstract]
  32. Lints TJ, Parsons LM, Hartley L, Lyons I, Harvey RP 1993 Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119:419–431[Abstract/Free Full Text]
  33. Komuro I, Izumo S 1993 Csx: a murine homeobox-containing gene specifically expressed in the developing heart. Proc Natl Acad Sci USA 90:8145–8149[Abstract/Free Full Text]
  34. Bodmer R 1993 A new homeobox-containing gene, msh-2, is transiently expressed early during mesoderm formation of Drosophila. Development 118:719–729[Abstract/Free Full Text]
  35. Chen CY, Croissant J, Majesky M, Topouzis S, McQuinn T, Frankovsky M, Schwartz RJ 1996 Activation of the cardiac {alpha}-actin promoter depends upon serum response factor, tinman homologue, Nkx-2.5, and intact serum response elements. Dev Genet 19:119–130[CrossRef][Medline]
  36. Chen CY, Schwartz RJ 1996 Recruitment of the tinman homologue, Nkx-2. 5 by serum response factor activates cardiac {alpha}-actin gene transcription. Mol Cell Biol 16:6372–6384[Abstract]
  37. Chen CY, Schwartz RJ 1995 Identification of novel DNA binding targets and regulatory domains of a murine tinman homeodomain factor, Nkx-2.5. J Biol Chem 270:15628–15633[Abstract/Free Full Text]
  38. Bohinski RJ, Di Lauro R, Whitsett JA 1994 Cis-acting elements controlling lung cell-specific expression of human pulmonary surfactant protein. Mol Cell Biol 14 5671–5681
  39. Lee TC, Zhang Y, Schwartz RJ 1994 Bifunctional transcriptional properties of YY1 in regulating muscle actin and c-myc gene expression during myogenesis. Oncogene 9:1047–1052[Medline]
  40. Natesan S, Gilman MZ 1993 DNA bending and orientation-dependent function of YY1 in the c-fos promoter. Genes Dev 7:2497–2509[Abstract]
  41. Natesan S Gilman MZ 1995 YY1 facilitates the association of serum response factor with the c-fos serum response element. Mol Cell Biol 15:5975–5982[Abstract]
  42. Pellegrini L Tan S Richmond TJ 1995 Structure of serum response factor core bound to DNA. Nature 376:490–498[CrossRef][Medline]