Multiple PcG and TrxG genes are normally expressed in the mouse heart. Due to the essential function of PcG/TrxG genes, constitutive knockouts of key PcG or TrxG genes often result in lethality during early embryogenesis before cardiac phenotypes can be analyzed (Bultman et al.,2000; O'Carroll et al.,2001; Voncken et al.,2003). Aided by conditional knockout models, studies in the past decade have uncovered crucial roles for two PcG complexes and one TrxG complex during cardiac development and/or in the adult heart (Fig. 4; see Supp. Table S1, which is available online). The major findings of these studies are reviewed below.
Figure 4. Multiple steps of cardiac development require PcG/TrxG function. Precardiac mesoderm give rise to cardiac progenitors in the first heart field (FHF) and second heart field (SHF, also known as anterior heart field or AHF). The TrxG protein Baf60c likely regulates the induction of cardiac fate. Cells in the FHF form the linear heart tube, which gives rise to the bulk of the left ventricle (LV) and also serves as a scaffold for subsequent heart growth. As the heart tube loops, cells in the SHF migrate to join the linear heart tube and give rise to the outflow tract (O), right ventricle (RV), and atria (A). Both Baf60c and the PcG protein Phc1 have been shown to regulate this early phase of cardiac development. The formation of the chambered heart from the looped heart involves a number of morphogenic processes such as trabeculation, proliferation, and septation. Multiple PcG and TrxG proteins, including Brg1, Baf60c, Ezh2, Eed, and Jmj, have been shown to regulate these processes. The dashed line between Jmj and Ezh2/Eed represents possible functional interaction. In addition to the FHF and SHF, cells from cardiac neural crest and proepicardium also contribute to the heart (not diagrammed).
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Polycomb Repressor Complex 2 (PRC2) Core Components: Ezh2 and Eed
Ezh2 is a SET-domain histone methyltransferase and the core subunit of PRC2 (Czermin et al.,2002; Müller et al.,2002). Homozygous Ezh2 knockout embryos die before completion of gastrulation (O'Carroll et al.,2001), suggesting that Ezh2 is essential for early embryonic development. Ezh2 is highly expressed in the developing heart but down-regulated in the adult heart, while its homolog Ezh1 shows the reverse pattern (Sdek et al.,2011). Two recent studies that inactivated Ezh2 in specific cell populations in the heart showed that Ezh2 plays important roles both during heart development and in the adult heart (He et al.,2012a; Delgado-Olguin et al.,2012).
Inactivation of Ezh2 in ventricular cardiomyocytes using Nkx2.5::Cre (Ezh2NK) resulted in perinatal lethality and an array of cardiac abnormalities including hypoplasia in the compact myocardium, excessive trabeculation, septal defects, and dilation in the right atrium (He et al.,2012a). Perinatal lethality and thinning of the myocardium were also observed when Eed was deleted in differentiated cardiomyocytes by TnT::Cre, which is active slightly later than Nkx2.5::Cre (He et al.,2012a). However, EedTnT embryos did not exhibit septal defects or atrial dilation. Taken together, these results suggest that PRC2 activity is required for multiple aspects of heart morphogenesis at multiple time points. What is the molecular basis for the morphological defects in Ezh2NK and EedTnT hearts? He et al. (2012a) have identified more than 50 genes that are directly repressed by PRC2 in the developing heart. The list includes multiple transcription factors with known roles in various steps of heart morphogenesis, such as Isl1, Tbx2, Tbx3, Hand1, Irx5, and Six1 (Cai et al.,2003; Costantini et al.,2005; Guo et al.,2011; McFadden et al.,2005; Mesbah et al.,2008; Risebro et al.,2006; Ribeiro et al.,2007; Riley et al.,1998; Singh et al.,2011). This suggests that PRC2 is critically involved in the developmental coordination of cardiac gene expression programs. In addition, PRC2 directly represses the cyclin-dependent kinase inhibitors Ink4a/b, which may explain the hypoplasia phenotype in Ezh2NK embryos (He et al.,2012a) (Fig. 5A).
Figure 5. Roles of Ezh2, Brg1, and Jmj in cardiomyocyte proliferation and trabeulation. A: Pathways by which Ezh2, Brg1, and Jmj regulate fetal cardiomyocyte proliferation. Cyclins (such as cyclin D) activates cyclin-dependent kinases (such as Cdc4), which phosphorylate Rb and relieve Rb repression of a number of genes essential for cell cycle progression. Ezh2 promotes fetal cardiomyocyte proliferation by direct repression of the cyclin-dependent kinase inhibitor Ink4a/b. Brg1 also promotes fetal cardiomyocyte proliferation, and it does so by activating Bmp10, which in turn represses another cyclin-dependent kinase inhibitor p57kip2. Jmj inhibits fetal cardiomyocyte proliferation by repressing cyclin D and by acting as a co-repressor for Rb. It is unclear whether Jmj and Ezh2 functionally interact with each other in the repression of cyclin D and Ink4a/b, and if they do, whether Jmj promotes or inhibits PRC2 activity in these contexts (hence dashed line between Jmj and Ezh2). Over-proliferation of fetal cardiomyocytes may result in delayed differentiation. B: Regulation of trabeculation by Ezh2, Brg1, and Jmj. The diagram shows a trabecula. Formation of these finger-like trabeculae is induced by signaling between the endocardium and the myocardium. Proteoglycans in the cardiac jelly modulates the trabeculation process by modulating the function of signaling molecules (x). Ezh2 expression in the myocardium is required for trabeculation, possibly by repressing an as-yet unidentified downstream effector (y). Brg1 expression in the endocardium promotes termination of trabeculation by activating the secreted proteinase ADAMTS1, which mediates the degradation of extracellular proteoglycans. Jmj expression in the endocardium negatively regulates trabeculation by repressing Notch.
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A study by Delgado-Olguin et al. used a MEF2c-ANF::Cre to inactivate Ezh2 from E7.5 on in cardiac progenitors of the second heart field (SHF, also known as the anterior heart field or AHF) and their derivatives. Interestingly, Ezh2SHF mice did not exhibit overt defects in cardiac morphogenesis; instead, such animals survived to adulthood but developed cardiac hypertrophy and fibrosis in the SHF-derived right ventricle (Delgado-Olguin et al.,2012) (Fig. 6). Six1 was identified as the main effector of Ezh2 function in cardiac hypertrophy. Removing one copy of Six1 rescued the hypertrophy and fibrosis phenotypes caused by SHF-specific deletion of Ezh2 (Delgado-Olguin et al.,2012). Six1 is normally expressed in progenitors in the SHF at E7.5–E8.5, but is down-regulated quickly upon differentiation (Guo et al.,2011). In Ezh2SHF hearts, Six1 expression persists throughout cardiogenesis and in the adult myocardium. An interesting question is whether Ezh2 (and PRC2 activity) plays an initiating or maintenance role in the developmental silencing of Six1 and other cardiac targets. In Drosophila, the developmental silencing of Hox genes can be divided into initiating and maintenance phases, which require distinct regulatory elements, and PcG activity is specifically required during the maintenance phase (Ringrose and Paro,2007). Thus we might expect that the activity of mammalian PRC2 is continuously required, throughout adulthood, to keep Six1 in a silent state. In other words, Six1 repression may have been initiated in Ezh2SHF hearts but was not maintained. Alternatively, mammalian PRC2 may be needed transiently for initiating Six1 silencing but becomes dispensable afterwards. Differentiating between these two scenarios will impact on the design of therapies that target the PRC2 pathway. It is worth noting that the adult heart predominantly expresses Ezh1 instead of Ezh2 (Sdek et al.,2011). However, Ezh1 function was not sufficient to repress Six1 in adult Ezh2SHF hearts (Delgado-Olguin et al.,2012). This may be due to functional divergence between Ezh1 and Ezh2. Although Ezh1 has been shown to methylate H3K27 and complement Ezh2 in ES cells and skin tissue (Ezhkova et al.,2011; Shen et al.,2008), two studies have highlighted important differences between the functions of the two homologues, including a transcriptional activating role for Ezh1 (Margueron et al.,2008; Mousavi et al., 2012).
Figure 6. Opposing roles of Ezh2 and Brg1 in the regulation of hypertrophic response. The adult heart responds to stress, such as pressure overload or β-adrenergic stimulation, by hypertrophic growth of cardiomyocytes. This results in thickened myocardial walls and smaller ventricular chamber(s). The PcG protein Ezh2 represses cardiac hypertrophy through a Six1-dependent pathway. The TrxG protein Brg1 is required for development of the hypertrophic response.
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In addition to methylating H3K27, Ezh2 was also found to methylate the cardiac transcription factor GATA4 and inhibit GATA4 activity both in vivo and in vitro (He et al.,2012b) (Fig. 7). This makes GATA4 the first known non-histone substrate of PRC2 and reveals a novel mechanism by which PRC2 may regulate transcription. Future experiments are needed to elucidate the relative contribution by H3K27 methylation versus GATA4 methylation toward PRC2-mediated gene silencing of GATA4 target genes. Moreover, it is possible that GATA4 is not an isolated example and PRC2 has other non-histone substrates that remain to be identified.
Figure 7. Known interactions between PcG/TrxG proteins and cardiac transcription factors. The TrxG complex BAF physically interacts with cardiac transcription factors GATA4, Nkx2.5, and Tbx5 and potentiates their activity on target promoters. A genetic interaction between Brg1 and the transcription factor Tbx20 has been shown, but it is unclear whether Tbx20 physically interacts with Brg1, Baf60c, or other subunit(s) of BAF. GATA4 also physically interacts with the PcG protein Ezh2, which methylates GATA4 and inhibits its activity. Jmj interacts with both GATA4 and Nkx2.5 and inhibits their activities through an as-yet unknown mechanism.
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PRC2 Accessory Component: Jumonji/Jarid2
Jumonji (Jmj, also known as Jarid2) is the founding member of the Jumonji family that consists of 27 proteins in humans. Many of the Jumonji family proteins are predicted to be histone lysine demethylases (KDM) (reviewed in Cloos et al.,2008). However, Jmj itself is predicted to be enzymatically inactive because it carries several amino acid substitutions in the cofactor Fe(II) binding region that is essential for KDM activity (Klose et al., 2006). Jmj is associated with PRC2 in ES cells and several other cell types and tissue (Landeira et al.,2010; Li et al.,2010a; Pasini et al.,2010; Peng et al.,2009; Shen et al.,2009). Consistent with this association, Jmj and PRC2 co-localize at many chromatin regions and each promotes the efficient recruitment of the other to target chromatin. However, it is uncertain whether Jmj enhances or inhibits the HMTase activity of PRC2, and it is possible that Jmj can do both depending on its protein level relative to PRC2. Finally, Landeira et al. (2010) showed that Jmj is required for maintaining PRC2 target promoters in a “primed” state, bound by RNA polymerase II phosphorylated at Ser 5 (the “poised” RNA pol II) and ready to be activated when differentiation cues are received.
Jmj plays critical roles in cardiac development. Jmj expression in the ventricles initiates in the trabecular layer at E10.5 and expands into the compact layer by E12.5; it is broadly expressed in both layers by E18.5 and in postnatal stages (Toyoda et al.,2003). The effect of Jmj mutation is highly influenced by genetic background. Jmj−/− in a C3H/He background died by around E11.5 with hyper-proliferation in the trabecular layer, which could have prevented blood circulation and caused lethality (Takeuchi et al.,1999). On the other hand, Jmj−/− embryos in a C57BL/6 background survived to perinatal stages and exhibited double-outlet right ventricle (DORV), ventricular septal defects (VSD), and severe noncompaction of the ventricular wall (Lee et al.,2000).
While the molecular mechanism of Jmj function during cardiac development is far from well understood, a number of studies have shed light on it in various ways. First of all, Jmj interacts with two important cardiac transcription factors, Nkx2.5 and GATA4, and inhibits the activation of Nkx2.5 and GATA4 target promoters (Kim et al.,2004). This may explain why Jmj−/− hearts failed to down-regulate atrial natriuretic factor (ANF, encoded by the Nppa gene), which is a known target of Nkx2.5 and GATA4 (Lee et al.,2000). Secondly, Jmj regulates cardiomyocyte cell cycle in two ways (Fig. 5A): on the one hand, it represses cyclin D1 expression (and hence inhibits Rb phosphorylation); on the other hand, it interacts with Rb and functions as an Rb co-repressor (Toyoda et al.,2003; Jung et al.,2005). Both will result in repression of E2F target genes, which are required for cell cycle progression. Jmj is able to bind to the cyclin D1 promoter in transfected cells and recruit H3K9 methylation activity (Shirato et al.,2009), thus directly repressing cyclin D1. Thirdly, in the endocardium, Jmj is a negative regulator of Notch signaling, which serves as a mitogenic signal to the myocardium and regulates the trabeculation process (Fig. 5) (for a review of Notch signaling in trabeculation, see High and Epstein,2008). Jmj is associated with a conserved region in the Notch1 locus, and Notch1 protein level is significantly elevated in Jmj−/− hearts (Mysliwiec et al.,2011). Remarkably, an endothelial-specific knockout of Jmj, which deleted Jmj in the endocardium and other cells of the endothelial lineage, recapitulated most of the cardiac phenotypes of Jmj−/− mice (Mysliwiec et al.,2011). This suggests that Notch and possibly other signals originating in the endocardium mediate a significant portion of Jmj function, in a cell non-autonomous manner. Finally, Jmj is required for the expression of differentiation markers, such as Myh6 (encoding α-MHC), α-cardiac actin, and actinin, in fetal cardiomyocytes (Takeuchi et al.,1999; Nakajima et al.,2011). However, this may be an indirect effect stemming from Jmj function in regulating proliferation versus differentiation. Increased expression of cyclin D in Jmj−/− hearts causes hyper-proliferation and prevents the expression of GATA4, which is a transcriptional activator of these differentiation genes (Nakajima et al.,2011).
Polycomb Repressor Complex 1 (PRC1) Core Component: Phc1
Phc1 (also known as Rae28) is a mammalian homolog of the Drosophila PcG protein Polyhomeotic (Ph). Phc1 and Ph are components of the mammalian and Drosophila PRC1 complex, respectively. Phc1 mutant embryos are defective in an early and important step of cardiac morphogenesis—cardiac looping—which takes place between E8.5 and E9.5 (Shirai et al.,2002). While the mutant heart is able to form chambers, it displays VSD and other cardiac abnormalities (Takihara et al.,1997; Shirai et al.,2002). These defects appear to stem from a loss of expression of the cardiac transcription factor Nkx2.5 (Shirai et al.,2002). Phc1 is not required for the initiation of Nkx2.5 expression, but is required for its continued expression. Given that PcG proteins generally function to silence genes, regulation of Nkx2.5 by Phc1 may be indirect, but the exact mechanism remains to be determined.
Interestingly, transgenic studies showed that while ubiquitous expression of a Phc1 transgene could restore Nkx2.5 expression and rescue cardiac morphogenesis defects in Phc1−/− embryos, cardiomyocyte-specific expression could not (Shirai et al.,2002; Koga et al.,2002). This suggests that normal cardiac morphogenesis requires the function of Phc1 in a cell population other than cardiomyocytes. Cardiomyocyte-specific over-expression of Phc1 neither rescued congenital heart defects in Phc1−/− embryos nor disrupted cardiac morphogenesis in embryos of wild-type background. However, continued expression of Phc1 in adult cardiomyocytes is deleterious and leads to disorganization of sarcomeres, cardiomyocyte apoptosis, chamber dilation, and heart failure (Koga et al.,2002). Because PcG proteins function in multi-subunit complexes, the activity of a complex, such as PRC1, is likely influenced by both subunit composition and stoichiometry. Thus, it is difficult to predict whether constitutive expression of Phc1 in adult cardiomyocytes would boost or interfere with PRC1 activity. Nonetheless, we can conclude from the Phc1 transgenic studies that the fine regulation of PcG activity in adult stages is essential for the maintenance of cardiomyocyte function. It awaits future studies to decipher the molecular function(s) of PRC1 in the adult heart.
BAF Complex Core Component: Brg1/Smarca4
The Drosophila TrxG protein Brm is a core component of the BRM complex, which mediates ATP-dependent chromatin remodeling (Papoulas et al.,1998). In humans, the BRG1-associated-factor (BAF) complex shares multiple conserved subunits with the Drosophila BRM complex (Wang et al.,1996). The ATPase core of hBAF can be either hBRG1 (also known as SMARCA4) or hBRM (SMARCA2), both of which are homologous to Drosophila Brm.
In the mouse, Brg1 is widely expressed in embryonic heart, and its expression in different regions appears to serve different roles. Brg1 expression in the endocardium is crucial for trabeculation in the ventricular myocardium (Stankunas et al.,2008) (Fig. 5B). During the process of trabeculation, signaling between the endocardium and the myocardium induces myocardial cells to form finger-like projections, or trabeculae. Trabeculation is a temporally regulated process that initiates at ∼E9.0, slows down around E12.5, and completes by E14.5. An appropriate degree of trabeculation is critical for normal contraction and hemodynamics of the embryonic heart, and is essential for the survival of the embryo. The extracellular matrix between endocardium and myocardium, known as cardiac jelly, plays important roles in trabeculation by affecting the diffusion and function of signaling molecules and by providing a microenvironment that supports the extensive cellular movements needed for trabeculae formation (for a comprehensive review of the role of ECM in cell signaling, see Kim et al.,2011). Brg1 is required for the repression of ADAMTS1, a secreted matrix metalloproteinase that degrades Versican and possibly other proteoglycans in the cardiac jelly, and thereby terminates the trabeculation process (Stankunas et al.,2008). When Brg1 is deleted in the endocardium, ADAMST1 becomes de-repressed prematurelly, resulting in early degradation of cardiac jelly and hypo-trabeculation. A small-molecule inhibitor of ADAMST1 can rescue the hypo-trabeculation phenotype in cultured Brg1 mutant embryos, suggesting that the main function of Brg1 in the trabeculation process is to regulate ADAMST1.
Brg1 is also expressed throughout the embryonic myocardium, and this expression is required for normal proliferation and differentiation of cardiomyocytes (Hang et al.,2010). Myocardium-specific deletion of Brg1 resulted in significantly reduced cardiomyocyte proliferation, reduced expression of Bmp10 (a cardiomyocyte growth factor), and increased expression of p57kip2 (a cyclin-dependent kinase inhibitor) (Fig. 5A). In addition, Brg1−/− cardiomyocytes exhibited premature formation of organized sarcomeres, elevated expression of the “adult” MHC isoform α-MHC, and reduced expression of β-MHC (encoded by the Myh7 gene), the “fetal” isoform that is primarily expressed by embryonic hearts.
In addition to being required for heart development, Brg1 also has roles in heart disease in the adult. With the exception of a small number of non-cardiomyocyte cells, the adult heart does not express Brg1. However, Brg1 can be reactivated by stress signals. The reactivation of Brg1 is essential for the development of hypertrophy in TAC-operated mice (Hang et al.,2010) (Fig. 6). The adult murine heart predominantly expresses α-MHC. One of the hallmark events during the hypertrophic process is α/β-MHC isoform switching: the re-activation of Myh7, and often a concurrent repression of Myh6. Consistent with its role in the embryonic heart, Brg1 is required for Myh7 activation and Myh6 repression. This may partially explain why Brg1 is required for hypertrophy, though additional mechanism(s) may also be at work.
While the recruitment of PcG proteins to target chromatin sites is thought to involve specialized regulatory elements (reviewed in Muller and Kassis, 2002; Ringrose and Paro,2007) and/or long non-coding RNA (Kotake et al.,2011; Pandey et al.,2008; Rinn et al.,2007; Plath et al.,2003; Zhao et al.,2008), a large number of studies suggest that transcription factors play important roles in the recruitment of BAF and other chromatin-remodeling complexes (reviewed in Peterson and Workman,2000; Sudarsanam and Winston,2000). Consistent with this view, Brg1 and the BAF complex functionally interact with multiple cardiac transcription factors (Lickert et al.,2004; Lou et al.,2011; Takeuchi and Bruneau,2009; Takeuchi et al.,2011) (Fig. 7). In the mouse, Brg1 genetically interacts with Tbx5, Nkx2.5, and Tbx20 (Takeuchi et al.,2011). Double heterozygotes of Brg1 and any of these transcription factors die before E14.5 and exhibit various cardiac morphogenic defects, demonstrating mutual genetic enhancement between mutations in Brg1 and in these transcription factors. Functional interaction has also been observed between BAF complex and GATA4 in both mouse and zebrafish (Lickert et al.,2004; Lou et al.,2011; Takeuchi and Bruneau,2009; Takeuchi et al.,2011).
BAF Complex Core Component: Baf60c/Smarcd3
Baf60c (also known as Smarcd3) is another core component of the BAF complex. Its yeast homolog has been shown to be essential for the activity of the SWI/SNF complex, which is the yeast counterpart of BAF (Cairns et al.,1996). There are three Baf60 paralogues in the mammalian genome, Baf60a, b, and c. Among the three, Baf60c is the only one that is expressed in the developing heart (Lickert et al.,2004).
In contrast to the broad expression of Brg1, the expression pattern of Baf60c is highly tissue-specific. When Baf60c expression initiates at ∼E7.5, it is restricted to the cardiac crescent. It continues to be expressed at high levels in the heart tube and chambered heart. By E9.5, expression is also detected in the somites, dorsal neural tube, and limb bud. Using transgenic mice expressing siRNA against Baf60c, Lickert et al. showed that Baf60c was particularly important for development of the outflow tract (OFT), right ventricle (RV), and atrium, all of which are derivatives of the SHF (Lickert et al.,2004).
When Baf60c and GATA4 were co-transfected into wild-type E6.5–E8.75 mouse embryos, the early cardiac marker Actc1 was ectopically induced in normally non-cardiogenic mesoderm tissues (Takeuchi and Bruneau,2009). Addition of Tbx5 to the transfection mixture allowed further differentiation into beating cardiomyocytes. These results suggest that Baf60c may have a central function in the specification of cardiac fate in addition to its later role in cardiac morphogenesis. This function was not uncovered in the siBaf60c model, possibly because RNAi did not completely eliminate Baf60c expression (Lickert et al.,2004).
Although Brg1 and Baf60c are both core components of the BAF complex, Brg1- and Baf60c-deficient mice exhibited distinct, albeit overlapping, phenotypes. Both are required for trabeculation (Lickert et al.,2004; Stankunas et al.,2008). On the other hand, neither epicardium-specific nor myocardium-specific deletion of Brg1 exhibited gross defects in the SHF (Stankunas et al.,2008; Hang et al.,2010). The disparity in Brg1- and Baf60c-deficiency phenotypes may result from differences in the timing of Brg1 or Baf60c loss in the respective mouse models. A role for Brg1 in SHF development may have been missed in the conditional knockouts, in which deletion of Brg1 occurs after E9.5. Alternatively, it may be due to the distinct roles that the two proteins play within the BAF complex. In reporter assays, Baf60c potentiates the activity of Tbx5, Nkx2.5, and GATA4 by promoting the interaction between these cardiac transcription factors and Brg1 (Lickert et al.,2004) (Fig. 7). This suggests that Baf60c functions as a bridge between the BAF complex and select cardiac transcription factors. In addition to bringing chromatin remodeling activity contained in Brg1, Baf60c may allow its partner transcription factors to access other activities via the BAF complex, such as histone H2B ubiquitinase activity (Li et al.,2010b) or interaction with the basal transcription machinery (Cho et al.,1998; Lemieux and Gaudreau,2004; Neish et al.,1998; Wilson et al.,1996). Thus, different target genes may exhibit individual requirements for Brg1 and Baf60c, depending on whether their activation requires chromatin remodeling and/or other activities mediated by BAF.
Baf60c not only is important for the recruitment of Brg1 and other BAF-associated activities, but also is required for detectable binding of GATA4 to two of its target loci (Takeuchi and Bruneau,2009). A role for BAF in the recruitment of its interacting transcription factors has been previously observed. For example, SWI/SNF (the yeast counterpart of BAF) stimulates nucleosome binding by transcription factors Sp1, USF, and NF-κB in vitro (Utley et al.1997). In vivo, SWI/SNF is required for the efficient binding of GAL4 to low-affinity, nucleosomal sites, but not for GAL4 binding to high-affinity sites or nucleosome-free low-affinity sites (Burns and Peterson,1997; Kwon et al., 1994). It has been proposed that BAF may be recruited by a transcription factor binding to a high-affinity site, and in turn promotes the recruitment of other transcription factors that bind to weaker sites. Alternatively, BAF may interact with the latter transcription factor in solution before both are recruited to the target site (Peterson and Workman,2000; Sudarsanam and Winston,2000). Thus, Baf60c may be recruited by another factor and permits subsequent GATA4 binding, or it may be co-recruited with GATA4 and stabilizes GATA4-chromatin association that is otherwise weak or transient. Whether the recruitment of Baf60c/BAF is dependent on GATA4 or any of its interacting transcription factors needs to be directly tested by future experiments.