ATP-dependent chromatin remodeling complex SWI/SNF in cardiogenesis and cardiac progenitor cell development

Abstract

The recent identification of cardiac progenitor cells (CPCs) provides a new paradigm for studying and treating heart disease. To realize the full potential of CPCs for therapeutic purposes, it is essential to understand the genetic and epigenetic mechanisms guiding CPC differentiation into cardiomyocytes, smooth muscle, or endothelial cells. ATP-dependent chromatin remodelers mediate one critical epigenetic mechanism. These large multiprotein complexes open up chromatin to modulate transcription factor access to DNA. SWI/SNF, one of the major types of chromatin remodelers, plays a key role in various aspects of development (de la Serna et al., 2006; Wu et al., 2009), including heart development and disease (Lickert et al., 2004; Wang et al., 2004; Huang et al., 2008; Stankunas et al., 2008; Hang et al., 2010). In this review, we describe the specific function of various SWI/SNF components in cardiogenesis and cardiac progenitor cell (CPC) self-renewal and differentiation. We envision that a detailed understanding of the SWI/SNF in heart development and CPC formation and differentiation will generate novel insights into epigenetic mechanisms that govern CPC differentiation and may have significant implications in understanding and treating heart disease.

Keywords

ATP-dependent chromatin remodeling / SWI/SNF / cardiogenesis and cardiac progenitor cell

The role of chromatin remodeling complexes in regulating chromatin structure during development

The differentiation of various types of cells with distinct function is one of the significant features in the development of mammals and other organisms. The differentiation processes depend on the coordinated regulation of different transcription factors. Moreover, chromatin remodeling complexes have been shown to contribute to an additional level of regulation in the developmental process. Genomic DNAs are packaged into chromatin in the forms of nucleosome. Alterations in the structure of chromatin at individual genes act as epigenetic modifications to facilitate the specific regulation of transcription factors during development. In mammals, two classes of chromatin modifying enzymes are found: ATP-dependent chromatin remodeling complexes and histone-modification enzymes. The histone-modification enzymes covalently modify histones with acetylation, methylation, and phosphorylation and so on. ATP-dependent chromatin remodeling complexes use the energy of ATP hydrolysis to reorganize the chromatin structure to regulate the DNA accessibility (Saha et al., 2006). Studies on these proteins have connected chromatin remodeling with cellular process during development. Here, we focus on the ATP-dependent chromatin remodeling complex and begin with the dynamic properties of nucleosomes on the chromatin.

Chromatin structure and dynamics

The nucleosome is the basic structure of eukaryotic chromatin, packing the genome into the nucleus. A nucleosome consists of approximately 147 base pairs of DNA, a histone protein core with H2A, H2B, H3, and H4 packaging the DNA into the nucleosome structure (Luger et€al., 1997). Nucleosomes serve as fundamental units of chromatin, and they play an essential role in the function of the genome (Francastel et al., 2000). The regulation of genome can be modulated by the changes of nucleosome positions and their covalent modification.

Modification of histones is correlated with the transcriptional status of genes (Fischle et al., 2003; Bernstein et al., 2005). Various histone modification sites are found, including acetylation, methylation, ubiquitination and phosphorylation (Berger, 2007). Developmental processes, especially the differentiation process of pluripotent or multipotent cells, accompany changes in histone modifications. For instance, an increase of repressed heterochromatin marks such as H3K9me3 and H3K27me2 are found during mouse ES cell differentiation (Martens et al., 2005). A decrease of global acetylated histones H3 and H4, which are normally found in decondensed chromatin, are also found during ES cell differentiation (Lee et al., 2004). These data indicate a transition of chromatin modifications from active to less active state during differentiation.

Besides post-translational modifications of histone, the higher ordered organization of nucleosome also contributes to gene regulation during developmental process (Lodén and van Steensel, 2005; Jiang and Pugh, 2009). Changes in chromatin structure can affect the accessibility of regulatory proteins, mainly transcription factors, to their target sites, therefore regulating gene expression and function. Although DNA at the nucleosome surface can be accessed by transcriptional factors (Polach and Widom, 1996), reorganizing the nucleosome structure by chromatin remodeling complexes is required for the DNA located inside the nucleosome. The ATP-chromatin remodeling complexes reconstruct the nucleosome in many ways, including nucleosome sliding, nucleosome removal, as well as replacement of histone subunits (Fig. 1) (Lomvardas and Thanos, 2001; Mizuguchi et al., 2004; Konev et al., 2007). In mammals, nucleosome sliding, which is promoted by recruitment of chromatin remodeling complex SWI/SNF, is involved in the induction of interferon-β promoter. Loss of nucleosomes in the locus control region (LCR) allows the binding of hematopoietic activator NF-E2, while in other cell types, nucleosomes create a condensed chromatin around LCR region and repress the expression of genes such as globin (Oyake et al., 1996). As discussed above, nucleosomes establish different level of chromatin fluidity in different cell types. The architecture of nucleosomes provides essential components for specific gene expression profiles by packing genes to active and repressive chromatin structure. The core histones will be recruited or dissociated to genomic regions to silence or activate genes.

Classification of ATP-dependent chromatin remodeling complexes

ATP-dependent chromatin remodeling complexes have been shown involved in the remodeling or assembling of the chromatin structure (Kwon et al., 1994). ATP-dependent chromatin remodeling enzymes are specialized as multi-protein machinery with a core DNA-dependent ATPase. Based on the DNA binding domain of ATPase, the ATP-dependent chromatin modifiers can be divided into 4 groups: SWI/SNF family (also called BAF in mammals) (Fig. 2), imitation SWI (ISWI) family, chromodomain and helicase-like domain (CHD) family and INO80 family. The SWI/SNF contains brahma (Brm) or brahma-like 1 (Brg1), which consists of a bromo domain binding to the acetylated histones (Hassan et al., 2002). ISWI enzymes SNF2H and SNF2L have a histone binding domain called SANT domain (Boyer et al., 2004). The CHD family ATPase contains chromodomains which interact with methylated histone (Bannister etal., 2001; Lachner et al., 2001). The INO80 family contains INO80 and Snf2-related CBP activator protein (SRCAP) and regulates transcription by mediating nucleosome mobilization (Morrison and Shen, 2009). It is interesting that the regulation of genome requires so many ATP-dependent chromatin remodeling complexes. ATP-dependent chromatin remodeling enzymes exert their transcriptional regulation in different manner. BAF complexes can act as transcriptional activators or repressors, they can even act as both activators and repressors during development (Chi et al., 2003). The tissue-specific BAF can interact with different transcription factors during heart and neural development (Lickert et al., 2004; Wu et al., 2007). The understanding of ATP-dependent chromatin remodeling enzymes is still limited. New functions of the ATP-dependent chromatin remodeling enzymes besides transcription regulation have been found. For instance, INO80 has been shown to be involved in telomere regulation, checkpoint control and DNA replication during cell division (Morrison and Shen, 2009). Studies in mutation of chromatin remodeling complexes subunits also suggest that the various assemblies of ATP-dependent chromatin remodeling complexes could function in a non-redundant way in regulating chromatin.

Composition and specificity of SWI/SNF complexes

Biochemical studies have identified the combinatorial assembly of BAF complexes (Wang et al., 1996a, b; Zhao et al., 1998). A total of 11 subunits have been identified in the complex and form 288 predicted assemblies. The BAF complexes contain either Brg1 or Brm, the core ATPase. Two distinct subfamilies have been identified: BAF250a (Arid1a) and BAF250b (Arid1b) associated BAF complexes; BAF180 associated PBAF complex with BAF200 (Arid2) instead of BAF250 (Nie et al., 2000; Xue et al., 2000; Yan et al., 2005). Beside the different subunits, two subfamilies share ten subunits including Brg1, BAF170, BAF155, BAF60, BAF57, BAF53, BAF47, BAF45, and actin. These subunits have various functions during development (Table 1). In cultured cells, using specific antibodies for protein assays, these subunits are found to interaction with other subunits (Zhao et al., 1998). Furthermore, the subunits resist dissociation and they are not exchangeable (Zhao et al., 1998), suggesting stable and specific composition of BAF complex would be critical for diverse functions of the complexes. For example, during the development of neuron, the shift of BAF53a and BAF45a to BAF53b and BAF45b in BAF complex is critical for neural progenitor cells to exit cell cycle and differentiate into postmitotic neurons (Lessard et al., 2007). Thus, the combinatorial assemblies of BAF complexes contribute to the functional specificity and diversity in different systems.

Overview of heart morphogenesis

The heart is the first functional organ formed during embryogenesis in vertebrates. The heart is first formed as a linear heart tube consists of the myocardium layer and endocardial layer. Then heart looping and chamber maturation takes place to form a four-chamber heart in mammals. Sophisticated genetic studies in mouse and chick reveal a comprehensive regulatory network including transcription factors, their downstream targets and upstream regulators that direct cardiac development. Beside the well documented genetic regulation of cardiac development, epigenetic regulation such as chromatin remodeling and histone modification are found to play essential roles during cardiac development.

Mammalian hearts are composed of various cell types, including cardiomycytes, smooth muscle cells and endothelial cells. The origin of these cells can be divided into the first heart field, secondary heart field, proepicardium and cardiac neural crest cells. The formation of cardiac crescent marks the initiation of cardiogenesis. The primary heart field (FHF) is derived from lateral plate mesoderm. The FHF forms cardiac crescent and gives rise first to the linear heart tube and later to the left ventricle. The second heart field, which has been identified later, originates in the pharyngeal mesoderm next to the cardiac crescent. The second heart field lineage contributes to the outflow tract, right ventricle and most of the atria (Kelly and Buckingham, 2002; Buckingham et al., 2005).

Regulation of FHF and SHF progenitor cells

The regulation of heart development involves comprehensive signals and transcriptional networks. Regulations of FHF and SHF seem to have both common cardiogenic signals and distinct regulatory genes. At cardiac crescent stage, Nkx2.5 (NK2 transcription factor related, locus5) and GATA4 (GATA binding protein 4) are activated by receiving positive signals such as BMPs and FGFs at the FHF lineage, whereas the SHF is closer to the negative WNTs (wingless-related MMTV integration sites) signals (Brand, 2003). BMPs and FGFs are required to activate Nkx2.5 in the SHF (Cai et al., 2003a, b; Kelly et al., 2001). These evidence indicate that both FHF and SHF share similar patterning signals. The cardiac transcription factors can directly interact with each other and coorporately activate target genes. Foxh1 (forkhead box H1) is expressed in the SHF progenitor cells. The Foxh1 mutant embryos have cardiac defects similar to those found in Isl1 and Mef2c mutants, including right ventricle and outflow tract defects (von Both et al., 2004). Moreover, Isl1 and Foxh1 can cooperate with Gata factors and Nkx2.5 to regulate the cardiac enhancer of Mef2c, therefore activating the expression of Mef2c during cardiac development (Arceci etal., 1993; Dodou et al., 2004). Furthermore, Mef2c directly regulates Smyd1 (SET-domain protein, Bop). Smyd1 is shown to regulate Hand2 (heart and neural crest derivatives expressed transcript 2) in SHF lineage, suggesting a regulatory cascade of Smyd1 and Hand2 by Isl1/GATA and Foxh1/Nkx2.5 (Gottlieb et al., 2002; Phan et al., 2005). On the other hand, in vitro, Foxa2, Foxc1 and Foxc2 can activate Tbx1 enhancer which direct specific Tbx1 expression in SHF, suggesting a possible Tbx1 regulation during SHF development (Maeda et al., 2006). Tbx1 regulates the production of growth factors such as Fgf8, which regulates the differentiation of cardiac neural crest derived cells and expression of Isl1 in outflow tract (Abu-Issa et al., 2002; Frank et al., 2002; Hu et al., 2004; Park et al., 2006). Thus, the cardiac transcription factors form an interactive network to strictly regulate cardiac development.

Mutations in cardiac transcription factors have dramatic consequences of cardiac development and leads to congenital heart disease.

Regulation of chamber formation

Ventricular chamber formation is an important event in cardiac development and for normal cardiac function (Moorman and Christoffels, 2003). The chambers are specified as the heart undergoes looping at E9.5. The endocardial cells migrate through the cardiac jelly and trigger myocardial cells to form projections (primitive trabeculae) toward the ventricular lumen. The primitive trabeculae undergo proliferation and differentiation and eventually form mature trabeculae, the highly organized cardiomyocytes. The trabeculae will then collapse to form compact myocardium (Moorman and Lamers, 1999). Tbx5 regulates chamber formation. Tbx5 is detected in trabeculae of right ventricle. Ablation of Tbx5 abolishes the expression of Nppa and Gja5 (Connexin 40) (Bruneau et al., 1999). In vitro assay shows that Tbx5 synergistically cooperates with Nkx2.5 and Gata4 to activates Nppa and Gja5 (Hiroi et al., 2001). Ectopic expression of Tbx5 inhibits ventricle formation and trabeculation is defective (Liberatore et al., 2000). Tbx20 is another T box member that promotes chamber formation. Tbx20 is expressed at the outer curvature of looping heart (Kraus et al., 2001). Tbx20 knockout mutants do not express Nppa and Cited1 (Stennard et al., 2005), suggesting that differentiation of cardiomyocytes is affected. Tbx2 is upregulated in Tbx20 mutant, indicating that Tbx20 represses the expression of Tbx2 in normal development (Singh et al., 2005; Stennard etal., 2005). Tbx2 marks the myocardium of outflow tract, atrio-ventricular canal and inflow tract (Christoffels et al., 2004). Tbx2 knockout mutants display ectopic expression of Nppa, Cx40 and Cited1 (Harrelson et al., 2004). Tbx2 represses the target genes in non-chamber myocardium by interacting with Nkx2.5 and inhibiting the formation of Tbx5 and Nkx2.5 complex which activates chamber formation genes (Habets et al., 2002). Taken together, the T box members function in the differentiation of chamber and non-chamber myocardium.

Although the regulation of early specification of chamber is clear, little is known about the late development of chamber trabeculation. In Nrg1, ErbB2, and ErbB4 knockout mutants, the growth of ventricular trabeculae is abolished (Meyer and Birchmeier, 1995; Gassmann et al., 1995; Lee et al., 1995). Nrg1 have been further shown to promote the migration and differentiation of early cardiomyocytes to trabeculae myocardium by activating focal adhesion kinase which increases cell motility and migration (Kuramochi et al., 2006). Bone morphogenetic proteins (Bmp) are involved in different stage of heart development, including induction of cardiogenic mesoderm, valves development as well as chamber formation (Schneider et al., 2003). Genetic analysis shows that Bmp10 is required for the proliferation and differentiation of trabecular myocardium by regulating cell cycle genes and cardiogenic factors such as Nkx2.5 and Mef2c (Chen et al., 2004). Notch signaling has been reported as a common regulator of Bmp10 signals and Nrg1 signaling during cardiac trabeculation (Grego-Bessa et al., 2007). Notch1 and Rpbjk mutant have trabeculation defects due to abnormal proliferation of cardiomyocytes and differentiation of trabecular myocardium (Grego-Bessa et al., 2007). These evidence suggest that Notch signal regulates the proliferation and differentiation of trabecular myocytes via mediating Bmp10 and EphB2/Nrg1.

Cardiac development regulated by ATP-dependent chromatin remodeling

In addition to transcriptional regulation, epigenetic events also take an essential role during cardiogenesis (Bruneau, 2010). Among the many epigenetic regulations, The ATP-dependent chromatin remodeling complexes utilize the energy of ATP hydrolysis to change the chromatin accessibility and are essential for many aspect of organ development. The association of ATP-dependent chromatin remodeling complexes with cardiac transcription factors provides an additional level of specificity and fine-tunes the regulation of heart development.

BAF60c

BAF60c is expressed in the developing heart and skeletal muscle (Lickert et al., 2004). RNAi knockdown of BAF60c in mice results in embryonic lethality by E11 and cardiac abnormailites including ventricular hypoplasia and shortened outflow tract (Lickert et al., 2004). Genes such as Hand2, Bmp10 and Irx3 are dysregulated in the knockdown embryos. BAF60c is important to establish interactions between BAF complex and transcription factors such as Gata4, Nkx2.5, and Tbx5 (Takeuchi and Bruneau, 2009), which could potentiate activaton the downstream genes. In skeletal muscle develo-pment, BAF60c is required for the activation of MyoD (Simone et al., 2004).

BAF60c either adds an additional spatial and quantitative regulation of genes in developmental process or maintains a specific chromatin state for target genes to stabilizing a gene expression program. The function of BAF60c or BAF complex would therefore like catalyst. This concept is tested by gain of function study in mouse embryos (Takeuchi and Bruneau, 2009). Ectopic transfection of BAF60c with Gata4, Nkx2.5 and Tbx5 can efficiently induce cardiac genes expression and beating cardiac cells, whereas the induction of cardiac cells using Gata4, Nkx2.5 and Tbx5 or their combination without BAF60c is inefficient. Replacement of BAF60a or BAF60b to BAF60c also makes the formation of cardiac cells inefficient. Interestingly, a recent study of BAF60c in zebrafish identified its role in guiding cardiac prognitor cell migration to the developing heart field (Lou etal., 2011). Therefore, these results suggest that BAF60c acts as both a catalyst and a determining factor in directing the cardiac program and crosstalk between BAF complex and transcription factors are critical for specific gene activation during development.

It is surprising that Gata4 and other transcription factors could not efficiently activate downstream genes. During liver development, Gata4 can access the target genes without the chromatin remodeling factors (Cirillo et al., 2002). However, Gata4 is not able to bind to the target genes in the absence of BAF60c and Brg1 (Lickert et al., 2004). This suggests that in cardiac developmental process, interaction of BAF60c and transcription factors are essential for the recruitment of transcription factors to downstream targets. The mechanism of activation by BAF complex and transcription factors is still unclear. Gata4 may act as a potential pioneer factor that binds to target loci and recruits BAF60c and BAF complex for additional function. Alternatively, BAF complex may also change the chromatin structure on target genes by unknown mechanism, thus Gata4 and other transcription factors could easily activate the target genes.

Brg1

Brg1 is essential for the developmental function of BAF complex (Bultman et al., 2000). Deletion of Brg1 eliminates BAF complex function (Bultman et al., 2000). Genetic studies show that Brg1 regulates cardiac development.

Brg1 regulates the cardiac trabeculation by specifically regulating ADAMTS1 (Stankunas et al., 2008). ADAMTS1 is a extracellular matrix (ECM) proteinase which is required for cardiac jelly development. ADAMTS1 has been shown to be repressed by endocardial Brg1 at E9.5 to E11.5 and allow cardiac jelly development (Stankunas et al., 2008). From E12.5 to E14.5, ADAMTS1 is then derepressed to degrade the ECM and terminate cardiac trabeculation. Mice lacking Brg1 prematurely activate ADAMTS1 and lead to trabeculation defect by early degradation of cardiac jelly.

Brg1 also functions in cardiac progenitor cells to regulate cardiac development. Brg1 has been shown to regulate the cardiomyocytes proliferation by maintaining the expression of Bmp10 and p57^kip2. Bmp10 regulates cardiac cells proliferation (Chen et al., 2004). p57^kip2 is an important negative cell cycle regulator (Chen et al., 2004). Mice lacking Brg1 in cardiac progenitor cells die at E10.5 due to myocardial trabeculation defects and interventricular septal defects. These defects are due to lack of Brg1 which leads to Bmp10 downregulation and subsequently upregulation of p57^kip2.

Brg1 also functions in the heart after birth. In mice, α-MHC is expressed in the adult hearts and β-MHC is expressed embryonic hearts (Hang et al., 2010). During embryonic development, Brg1 interacts with HDACs and PARPs to repress α-MHC and maintain β-MHC expression. Mice lacking Brg1 result in prematuration of α-MHC from β-MHC in embryonic hearts. Brg1 is turned off at adult myocardial cells (Hang et al., 2010). However, under cardiac stress, Brg1 is reactivated to induce the adult α-MHC into fetal β-MHC in cooperating with HDAC/PARP. Brg1 is upregulated in hypertrophic cardiomyopathy patients. These suggest that Brg1 have a role in both embryonic and adult hearts.

The function of Brg1 has been shown to be dosage-dependent (Han et al., 2011; Takeuchi et al., 2011). Brg1 heterozygous mice cause congenital heart defects, including ventricular septal defects and cardiac dilatation. Double heterozygous Brg1 with Tbx5, Nkx2.5 or Tbx20 cause more severe cardiac defects. These data suggest that Brg1 interacts with other transcription factors during cardiac development in a dosage-dependent manner.

BAF180

BAF180, a polybromo subunit which recognizes acetylated histone tails, is involved in cardiac development. BAF180 knockout mice have trophoblast placental defects and hypoplastic ventricle defects. It has been shown that BAF180 may synergistic interact with RA pathway to regulate cardiac development. BAF180 regulates retinoic acid target genes such as RARβ2 and CRABPII (Wang et al., 2004). In addition, BAF180 is required for the coronary development through regulating the proper epithelial mesenchymal transformation (Huang et al., 2008).

BAF250a

BAF250a is a critical regulatory subunit in SWI/SNF. Our studies show that BAF250a is essential for the proper differentiation of ES cells into beating cardiomyocytes and that BAF250a is involved in normal cardiogenesis (Gao et al., 2008). To further investigate the function of BAF250a-containing BAF complex during mammalian cardiac develo-pment, we applied a conditional gene targeting approach. The BAF250a gene was knocked out in different cardiac cell lineages using Nkx2.5-cre, Mef2c-cre and Mlc2v-cre mice (Lei et al. in preparation). Nkx2.5-cre is activated in both first heart field (FHF) and second heart field (SHF); Mef2c-cre in SHF and Mlc2v-cre in differentiated myocardium. Phenotypic analysis showed that the lack of BAF250a in both FHF and SHF resulted in the formation of a common ventricle, trabeculation defects and retarded heart development. Mice lacking BAF250a in SHF showed trabeculation defects in the right ventricle, ventricular spetal defect, persistent truncus arteriosus and reduced myocardial proliferation, while BAF250a deletion in ventricular cardiomyocytes did not lead to any observable abnormalities. These results suggest that BAF250a is required in cardiac progenitor cells stage but not in the differentiated myocardium stage.

To examine the effect of BAF250a in regulating the differentiation potential of SHF cardiac progenitor cells (CPCs), in vitro differentiation of ESC-derived CPCs was performed. Immunostaining using troponin T (cTnT) antibody on the CPCs differentiated cells showed that the formation of cardiomyocytes were significantly reduced in the absence of BAF250a. Moreover, the myocardial transcripts were downregulated during differentiation of BAF250a null cells. Noticeably, the mRNA level of endothelial marker Flk1 and smooth muscle cell marker SM-MHC in BAF250a null cells was increased, suggesting that function of BAF250a in directing CPCs differentiation was specific for myocardial lineage.

To understand the molecular basis of BAF250a in regulating SHF cardiac development, expression of genes involved in cardiac development was examined by qRT-PCR. Deletion of BAF250a in SHF caused downregulation of myocardial proliferation related genes including Bmp10, p57^kip2, Ccnd2, CDK4 and Cdc20; trabeculation related genes ErbB2 and ErbB4 as well as cardiac transcription factors Nkx2.5 and Mef2c. To address whether BAF250a/BAF complex could directly regulate the differentially expressed genes, chromatin immune-precipitation (ChIP) was performed using BAF250a and Brg1 antibodies. It was found that BAF250a was recruited to the promoter regions of Nkx2.5, Mef2c and Bmp10. The binding of Brg1 to BAF250a targets was significantly reduced in the absence of BAF250a. Furthermore, the nucleosome densities of BAF250a target loci were increased in BAF250a knockout cells. These results suggest that BAF250a mediates chromatin remodeling at the specific genes to control proper cardiac gene expression program (Fig. 3).

WSTF

The BAF complex is indirectly correlated with the CHARGE syndrome (Coloboma, Heart defects, Atresia choanae, Retarded growth and development, Genital hypoplasia and Ear deafness) (Bajpai et al., 2010) and Williams syndrome (Kitagawa et al., 2003). Williams syndrome is a disease that characterized by growth defect, dysmorphic facial features, vitamin D metabolism defects, and cardiac defects. The cardiac defects include aortic stenosis, pulmonary arterial stenosis, and atrioventricular septal defect. There are 28 genes found which are lost by heterozygous deletion in Williams syndrome. Among these genes, the Williams syndrome transcription factor (WSTF) is correlated with the function of BAF complex. It accounts for some of the cardiac defects in Williams syndrome patients. Wstf knockout mice show hypotrabeculation, thin compact myocardium,atrial septal defect, and ventricular septal defects (Yoshimura et al., 2009). The pharyngeal arch arteries are hypoplastic, results in left carotid and subclavian arteries defects (Yoshimura et al., 2009). These cardiac defects are also found in some of the Wstf heterozygous mice, suggesting a haploinsufficiency requirement of Wstf and a possible role of Wstf in Williams syndrome. Wstf are involved in some chromatin-remodeling complexes: WSTF-including nucleosome assembly complex (WINAC) using Brg1 as core ATPase (Kitagawa et al., 2003) and WSTF-ISWI chromatin-remodeling complex (WICH) using Snf2h as core ATPase (Poot et al., 2004). Wstf recruits Brg1, BAF155, and other cardiac transcription factors such as Nkx2.5, Gata4 and Tbx5 to activate the expression of connexin40 in E9.5 hearts (Yoshimura et al., 2009). Further studies using ChIP and reporter assays suggest that Wstf regulates cardiac development via the interaction of Brg1 and WINAC complex. However, the WINAC complex could not explain all the phenotypes of Wstf knockout mice. Therefore, there is also a possible role of Snf2h/WICH during cardiac development. Further investigation of Williams syndrome could reveal the fully picture of chromatin remodeling in cardiac development.

Future Directions

Congenital heart diseases are the most common congenital diseases in human. As discussed in this review, cardiac development is strictly regulated at both genetic and epigenetic levels. The transcriptional networks that control cardiac development have been well-studied. However, investigations of the epigenetic regulation of cardiac development has just started in the last few years. In many aspects, the epigenetic regulation of cardiac development is still under debate. Do the epigenetic factors function as pioneer factors to direct cardiac transcription factors expression or are they recruited by cardiac factors to enforce the regulation of cardiac genes? Are the distinct compositions of chromatin remodeling complex required for tissue-specific function? How does chromatin remodeling regulate gene expression? All these questions are still currently under investigation. Future studies will be to elucidate the functions of SWI/SNF chromatin remodeling factors at different stages of cardiac development and establish a connection between chromatin regulation and cardiac gene expression. Such studies would include (1) characterization of the roles of SWI/SNF components in different cardiac lineages development, particularly in the SHF development using a variety of conditional knockout mice in different cardiac lineages; (2) identification of the roles of SWI/SNF in the differentiation potential of CPCs using and in vivo lineage tracing in vitro ESC differentiation systems; and (3) identification of SWI/SNF targets during cardiac development and elucidation of the chromatin structures of the target genes regulated by SWI/SNF.

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