An increasing number of miRNAs, both muscle-specific and ubiquitously or widely expressed, have been shown to affect muscle cell proliferation, development and differentiation (45–48
). In addition, there is a growing appreciation that specific miRNAs are involved in the onset of disease states affecting all three types of muscle (8
). miRNAs, therefore, play an important and essential role in the regulation of muscle formation and function, and defining the principles controlling the expression of these miRNAs is critical to better understanding muscle biology and muscle disease.
We have focused on the transcriptional regulation of expression of several miRNAs that are induced during muscle differentiation and are largely muscle specific in their expression. The miRNAs are critical regulators of myoblast proliferation and differentiation (5
). Previous studies have implicated MyoD, myogenin, Mef2 and the respective binding sites for these factors in myogenic miRNA regulatory sequences in the induction of miRNA expression (5
). Thus, the data support the idea that myogenic, DNA-binding transcription factors that control myogenic mRNA expression also control myogenic miRNA expression.
Considerable effort has been made to understand how epigenetic regulators influence myogenic mRNA expression. These studies have revealed roles for numerous histone-modifying enzymes, specific histone modifications and ATP-dependent chromatin remodeling enzymes (49–51
). Whether such chromatin modifiers also contribute myogenic miRNA expression has only recently begun to be investigated. Recently, we used morpholinos to reduce Brg1 levels in developing zebrafish and observed that ~40% had altered tail development with morphologically altered somite structure. In addition, these embryos had altered sarcomeric actin organization in the tissue. Analysis of myogenic miRNA expression in these altered tissues showed that there was a significant decrease in expression relative to the controls (13
). This phenotype was remarkably similar to that observed by others when the microRNA processing enzyme, Dicer, was mutated or when the levels of myogenic miRNAs miR-1 and miR-133 were reduced by morpholino injection (52
). Together, the two studies suggest that Brg1 and miRNAs are part of the same regulatory pathway. Subsequent studies in Brg1-deficient myoblasts, in primary skeletal muscle tissue and in a cell culture model for skeletal muscle differentiation indicated that Brg1 is not only required for skeletal muscle mRNA expression and differentiation, as previously reported (37
), but also directly required for myogenic miRNA expression (13
). To date, no other chromatin modifiers or remodeling enzymes have been shown to contribute to myogenic miRNA regulation.
Given the recently demonstrated roles for the arginine methyltransferases Prmt5 and Carm1/Prmt4 in myogenic mRNA expression, we asked whether these enzymes are involved in myogenic miRNA regulation. We previously showed that Prmt5 is directly required for expression of the myogenin gene (21
), but despite binding to regulatory sequences controlling myogenic genes expressed at late times of myogenesis in cultured cells, it was not required for the expression of these genes. Carm1/Prmt4, in contrast, bound to late gene regulatory sequences and was required for expression during differentiation in culture (20
). Our studies of Prmt5 and Carm1/Prmt4 function during myogenic miRNA expression revealed that the requirements for these arginine methyltransferases were similar to their functions in regulating myogenic late gene mRNA expression. Thus, there is conservation between Prmt5 and Carm1/Prmt4 function in the regulation of myogenic miRNAs and in the regulation of a subset of myogenic mRNAs. It is important to note, however, that despite the similarity to the regulation of myogenic late gene mRNAs, this does not indicate that the myogenic miRNAs should be considered ‘late’ expressing genes. In vivo
analysis of the expression of transgene constructs controlled by regulatory sequences of several myogenic miRNAs indicates that they can be detected in somites at E9.5 and at later stages of development (6
The ChIP experiments performed in the Carm1/Prmt4-deficient cells revealed several mechanistic insights into how Carm1/Prmt4 functions at myogenic miRNA regulatory sequences. First, incorporation of the Carm1/Prmt4 substrate, dimethylated H3R17, at these sequences is absolutely dependent upon the presence of Carm1/Prmt4 ( and ). Second, the subset of E box containing sequences that showed Carm1/Prmt4 binding correlated exactly with those sequences previously found to bind both MyoD and the Brg1 SWI/SNF ATPase (13
). This suggests that factors regulating myogenic miRNA expression are functioning at the same cis
-acting sequences. Third, cells lacking Carm1/Prmt4 not only were deficient for the H3R17 modification, but also failed to target the Brg1 ATPase of SWI/SNF chromatin remodeling enzymes to the regulatory sequences ( and ). We have previously demonstrated that chromatin remodeling at these miRNA sequences and microRNA expression are entirely dependent upon Brg1 (13
). The absence of Brg1 indicates that these sequences cannot undergo chromatin remodeling and are therefore not accessible for active gene expression. The mechanism(s) by which Carm1/Prmt4 directly or indirectly facilitates Brg1 binding remains to be determined, but we can speculate that Carm1/Prmt4 mediated histone modifications likely contribute to targeting the ATP-dependent chromatin remodeler.
The last significant conclusion from the ChIP experiments with Carm1/Prmt4 deficient cells is the novel observation that MyoD binding to these sequences was unaffected while myogenin binding was abolished ( and ). The data presented here and in our prior study (13
) indicate that MyoD binding to these sequences is independent of Brg1 function and the presence of Carm1/Prmt4. Thus, MyoD binding does not require these particular enzymes or the chromatin structural modifications that they mediate. Whether different chromatin-modifying or remodeling enzymes are required for MyoD binding at these sequences is not known. By contrast, myogenin binding is completely dependent on Carm1/Prmt4. Despite the sequence similarities between the MyoD and myogenin proteins, it has long been established that MyoD is intrinsically better at functioning in a chromatin environment than is myogenin (55
). How the structural differences between MyoD and myogenin relate to the Carm1/Prmt4 dependence of binding at miRNAs regulatory sequences is an obvious topic for further work. Nevertheless, the presence of MyoD at these sequences in both wild-type and Carm1/Prmt4-deficient cells suggests that MyoD binding is not sufficient for myogenic miRNA expression. Instead, the binding of myogenin correlates with transcriptional competence, suggesting that it is myogenin that is facilitating transcriptional activation. Previous cooperativity between Carm1/Prmt4 and both myogenin and Mef2 has been reported (20
), supporting the idea that Carm1/Prmt4 acts as a co-activator for myogenin and Mef2 to promote myogenic miRNA expression.
The functional interrelationships between two distinct types of Prmts and the Brg1 chromatin-remodeling enzyme during differentiation-mediated induction of myogenic miRNAs suggest that regulation of these myogenic miRNAs will be as complex as the regulation of myogenic mRNAs. It is therefore likely that histone acetyltransferases, lysine methyltransferases and other histone-modifying enzymes will also be involved in myogenic miRNA expression. Because histone modifications are dynamically regulated, there also will likely be roles for deacetylating and demethylating enzymes. How the activities of multiple chromatin modifiers are integrated with the functions of SWI/SNF enzymes and gene specific transcription factors to regulate myogenic miRNA expression will be an interesting topic for future studies.
Finally, the similarity in the regulation of the miR-1 and miR-133 myogenic microRNAs is interesting because miR-1 and miR-133 have opposing functions; miR-1 promotes differentiation, whereas miR-133 promotes myoblast proliferation (5
). We showed that differentiation-dependent kinetics of induction of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 are equivalent in MyoD-differentiated fibroblasts and that the four myogenic microRNAs are also induced with similar kinetics in myogenin/Mef2D1b-differentiated fibroblasts ( and ). This is consistent with the work of others who have seen differentiation-dependent increases in miR-1 and miR-133 expression in C2C12 cells (5
) and with data from transgenic animal studies indicating that the intragenic regions between the two miR-1 and miR-133 clusters both drive reporter gene expression in the heart and somites with similar kinetics (6
). The reason that the induction and regulation of two microRNAs that promote opposite developmental outcomes is similar is not known. However, others have speculated that differential regulation of the miRNAs at steps downstream of the transcription of these microRNAs might promote their differences in function, as might the obvious differences in the functions of the proteins encoded by their target mRNAs (16
). In addition, despite the similarities in regulation by chromatin-modifying and remodeling enzymes documented here and previously (13
), it remains possible that subtle, yet uncharacterized, differences in regulatory mechanisms or in the timing of microRNA expression could promote differences in miR-1 and miR-133 function.