The remarkable similarity of phenotypes between zebrafish having reduced levels of Brg1, mutated Dicer enzyme, or reduced levels of myogenic miRNAs suggested that Brg1, Dicer, and myogenic miRNAs function in the same pathway in vivo to regulate proper skeletal muscle development and structure. In zebrafish muscle, miR-1 and miR-133 account for more than 50% of the total miRNA-mediated gene regulation (31
). These two microRNAs target mRNAs encoding proteins associated with actin function, and the knockdown of these miRNAs or of the Dicer enzyme results in altered actin organization in muscle sarcomeres (31
). A notable feature of the altered actin organization in zebrafish lacking miR-1 and miR-133 or in zebrafish lacking Dicer is the near absence of striation and an inability to distinguish I bands. Strikingly, when the actin organization in Brg1-MO-injected fish with stunted tails was examined, a similarly altered organization, including the lack of distinct bands in the sarcomeres, was observed. These data indicate that in an in vivo setting, deficiency in Brg1 equates with the deficiency in Dicer or deficiency in myogenic microRNAs. The simplest explanation is that Brg1, Dicer, and myogenic microRNAs function in the same pathway to regulate actin organization in skeletal muscle tissue. Although there are multiple means by which these proteins might functionally relate to each other, a likely explanation given the known properties of these molecules is that the Brg1 chromatin remodeling enzyme is required for the expression of Dicer or the microRNAs. Direct analysis revealed that Dicer levels were unaffected in the Brg1-MO-injected zebrafish, whereas myogenic microRNA expression was compromised (Fig. ). Thus, we identified myogenic microRNAs as target genes for Brg1 in the zebrafish.
We note that numerous zebrafish genes involved in retinal formation and function have been identified as Brg1 targets (27
), and that in mammalian systems, Brg1 and SWI/SNF enzymes have been identified as regulators of genes involved in nearly every tissue differentiation process examined (13
), including skeletal muscle (11
). Thus, this work extends the role of Brg1 beyond the regulation of mRNAs to include the regulation of microRNA expression. Because Brg1 is not solely required for miRNA production, we did not attempt to rescue the Brg1 deficiency by the introduction of miR-1 and miR-133. In addition to the likelihood that there are other myogenic microRNAs that are deficient due to the reduction in Brg1 levels, myogenic mRNA expression also would be compromised, rendering the attempt at rescuing the phenotype of Brg1 unproductive. We note, however, that other workers have rescued retinal and neurogenesis defects due to Brg1 morpholino injection via the introduction of either a Brg1 cDNA or a genomic P1-derived artificial chromosome that includes the Brg1 locus (17
) or via the introduction of Brg1 mRNA (14
). The morpholino used in our experiments was the same one used by Gregg et al. (17
), suggesting that off-target effects are not the cause of the observed phenotypes.
miR-1 and mir-133, in addition to being expressed in skeletal muscle, are expressed in cardiac muscle (6
). Brg1 and two other subunits of the mammalian SWI/SNF enzyme complex, Baf180 and Baf60, have been shown to contribute to cardiac development and function in mouse models (20
). This raises the question of whether the Brg1-MO-injected fish also showed heart defects. Although we did not perform any heart analysis in these animals, we observed that beating hearts were present in all animals showing skeletal muscle defects (data not shown). However, we did note pericardial swelling in animals that survived to 48 hpf and later time points (data not shown). Pericardial swelling was a reported characteristic of the yng
mutant fish line (29
), which was later shown to be mutated in the Brg1 gene (17
). Thus, the nonskeletal muscle phenotypes observed in our Brg1-MO-injected zebrafish are consistent with previous reports.
We performed subsequent studies to address the requirement for and the mechanism controlling the function of Brg1 in myogenic microRNA regulation via the direct analysis of skeletal muscle tissue and by the manipulation of Brg1 levels in primary cells cultured ex vivo and in a tissue culture model for myogenesis. Although we cannot definitively state that Brg1 is directly acting at microRNA regulatory sequences in zebrafish skeletal muscle tissue, the data obtained from these multiple experimental systems all are consistent with the conclusion that Brg1 promotes chromatin remodeling and gene expression at myogenic miRNA regulatory sequences. Our analysis of MyoD binding to regions upstream of individual myogenic microRNA coding sequences in both primary tissue and tissue culture cells revealed MyoD interactions with at least one consensus E box located within 3 kb of each of the mature microRNA start sites. However, additional consensus E boxes further upstream did not show interactions with MyoD. Interestingly, the expression of dominant-negative Brg1 did not inhibit MyoD binding to these sites, indicating that MyoD is able to access these sequences in the absence of functional chromatin remodeling by Brg1-based SWI/SNF complexes. This is similar to observations made regarding MyoD binding to myogenic genes encoding mRNAs that are expressed at later times of differentiation (34
) but distinct from events at the myogenin promoter, where the homeodomain factor Pbx-1 initiates the cascade of transcription factor binding and chromatin remodeling (3
). Whether MyoD possesses an intrinsic ability to interact with its binding sites upstream of myogenic microRNAs or whether different chromatin modifying or remodeling events are required remains unknown.
ChIP experiments showed that the Brg1 ATPase of SWI/SNF enzymes was present at precisely the same sequence regions upstream of myogenic microRNAs as those that were bound by MyoD. Again, there was exact agreement between Brg1 binding in muscle tissue and binding in tissue culture cells. The data suggest that the Brg1 remodeling enzyme is targeted to the sequences upstream of the myogenic microRNAs by MyoD. This regulatory event is consistent with prior data showing that MyoD coimmunoprecipitates with Brg1 from cell extracts and targets Brg1 to some myogenic protein coding genes (12
). The functionality of Brg1 was demonstrated by the exact correlation, again both in muscle tissue and in tissue culture cells, between the presence of MyoD and wild-type Brg1 and increased nuclease accessibility at sites of MyoD and Brg1 binding, while sites not bound by MyoD and Brg1 showed no change in chromatin accessibility.
Collectively, these data indicate that Brg1 is required for skeletal muscle organization and that a previously unappreciated function of the Brg1-based SWI/SNF chromatin remodeling enzyme during myogenesis is to promote the expression of myogenic miRNAs that are important contributors to vertebrate myogenic development and function. This work establishes the concept that the tissue-specific induction of microRNA expression, like mRNA expression, is regulated by Brg1-based ATP-dependent chromatin remodeling enzymes.