Our data demonstrate that the ability of myocardin to induce expression of most smooth muscle-specific genes is regulated by the activity of the SWI/SNF ATP-dependent chromatin remodeling complex. We suggest a model in which myocardin associates with the SWI/SNF complex through direct binding to the Brg1 or Brm ATPase subunit. This association is required in order for myocardin to increase SRF binding to the promoters of smooth muscle-specific genes within intact chromatin, thereby leading to activation of these genes during differentiation of vascular smooth muscle cells.
Although Brg1/Brm containing SWI/SNF complexes are required for myocardin to induce expression of many smooth muscle-specific genes the induction of SM α-actin by myocardin was largely independent of SWI/SNF (– and supplementary figure 1
). In addition, in SW13 cells that lack Brg1 and Brm, myocardin was still able to induce SM α-actin expression (). This correlates with the relatively high basal levels of SM α-actin expression in many of these cells in the absence of added myocardin. Thus it is likely that the SM α-actin promoter is already in an active transcriptionally favorable conformation in the absence of myocardin. This may suggest that the SWI/SNF complex is dispensable for myocardin-induced activation of genes, such as SM α-actin, in cells in which these genes are already transcriptionally active. In contrast, SWI/SNF activity is required for myocardin to induce expression of genes that are otherwise transcriptionally silent in a given cell type. Similar to SM α-actin expression of SM22α in primary smooth muscle cells was found to be largely independent of Brg1. This may also reflect the contribution of myocardin independent pathways in driving chromatin remodeling and transcription of the SM22α locus in smooth muscle cells. For example, both SM α-actin and SM22α can be induced by TGFβ in 10T1/2 cells by a myocardin independent pathway 18, 19
. This model is, however, likely to be an over-simplification, as in primary cultures of mouse aortic smooth muscle cells we observed that the myocardin-induced increase in expression of smooth muscle-specific genes was at least partially attenuated by DN-Brg1 (). As all of the genes examined were expressed prior to myocardin over-expression, this may suggest that even if a gene is transcriptionally active, SWI/SNF induced changes in chromatin can further augment myocardin’s myogenic activity.
Domain mapping experiments suggest that Brg1 and Brm interact with the region of myocardin that spans the SRF interaction domain (basic and poly Q region). Despite the overlapping binding sites, co-immunoprecipitation studies show that myocardin is present in a complex that includes both Brg1 and SRF within intact cells. The Brg1 binding site in myocardin is present in both the long, cardiac-selective isoform (1
–935) and the shorter, smooth muscle-selective isoform (80–935). These results suggest that both cardiac- and smooth muscle-selective myocardin isoforms will interact with and be regulated by SWI/SNF. In support of this proposal, the myogenic activity of both the cardiac myocardin (Supplemental figure I
), and the smooth muscle myocardin (–) was attenuated by dominant negative Brg1. In addition, the ability of myocardin to induce expression of cardiac-specific genes in 10T1/2 cells was also attenuated by DN-Brg1. These observations suggest that the myogenic activity of myocardin both in the heart and in vascular SMCs is regulated by SWI/SNF.
Results from experiments in which we reconstituted Brg1 or Brm expression in SW13 cells suggest that either wild-type Brg1 or Brm containing SWI/SNF complexes are equally effective at supporting myocardin’s myogenic activity (). In addition, knockdown of either Brg1 or Brm in aortic SMCs attenuated expression of smooth muscle-specific genes () and both Brg1 and Brm can directly bind to myocardin in vitro
(). These data suggest that Brg1 or Brm containing SWI/SNF complexes may both play important roles in smooth muscle cells. In contrast to myocardin, the LIM domain protein CRP2 has recently been shown to interact specifically with Brg1, but not Brm, in order to induce expression of smooth muscle-specific genes in cardiomyocytes20
. Despite the ability of either Brg1 or Brm to support myocardin’s myogenic activity in SW13 cells, knocking-down both Brg1 and Brm in smooth muscle cells did not result in any further attenuation of smooth muscle-specific genes as compared to knockout of either protein alone (). The lack of an additive effect of the double knockdown, may suggest that Brg1- and Brm-containing SWI/SNF complexes act together in smooth muscle cells to regulate myocardin. This must however, be interpreted with caution as knockdown of Brg1 also attenuated expression of Brm. Further studies analyzing tissue specific single or double knockouts of Brg1 and Brm, in vivo
, will be required to clarify the role of individual SWI/SNF complexes in regulating smooth muscle differentiation.
In addition to ATP-dependent chromatin remodeling complexes, enzymes that covalently modify histones are important to mediate myocardin activation of smooth muscle-specific genes21, 22
. For example, myocardin has been shown to bind to p300 and to promote acetylation of histones associated with the promoters of smooth muscle-specific genes6, 21
. As the bromodomains of Brg1 and Brm are known to bind to acetylated histones we initially speculated that myocardin-recruited HAT activity may help recruit SWI/SNF to promote transcriptional activation of genes in smooth muscle cells. However, data showing that the bromodomains of Brg1 and Brm are not important for supporting myocardin’s myogenic function argue against this proposal (). Similarly, it is unlikely that direct DNA binding by the AT-hook domain of Brg1 or Brm is required to recruit myocardin to chromatin, as the C-terminal truncation of Brm that we analyzed also lacks this domain23
yet was still able to support myocardin’s myogenic activity ().
In skeletal muscle, weak binding of MyoD to the myogenin promoter via MyoD interactions with Pbx, facilitates SWI-SNF recruitment through direct binding of MyoD and Brg1 24, 25
. Chromatin remodeling by SWI/SNF then facilitates tight binding of MyoD to the E box within the myogenin promoter, facilitating promoter activation and skeletal muscle cell differentiation. By analogy we propose a model in which in undifferentiated SMC or in nonmuscle cells, SRF has a low binding affinity for CArG box elements in the promoters of smooth muscle-specific genes within intact chromatin. Little transcription activity of smooth muscle-specific genes such as telokin and SM-MHC, thus occurs in these cells. To induce smooth muscle differentiation, myocardin complexed with p300 and SWI/SNF, interacts with SRF weakly bound to the promoters of smooth muscle-specific genes. SWI/SNF binding to the promoter regions then leads to ATP-dependent chromatin remodeling and rearrangement of the nucleosomes that facilitates tight binding of SRF. This may also allow binding of additional activators to the adjacent DNA segments. These activators, together with the SRF/myocardin/p300 complex can then further modify chromatin to facilitate recruitment of general transcriptional factors, including RNA polymerase II, resulting in transcriptional activation of smooth muscle-specific genes.