Abstract
SETD3 is a member of SET-domain containing methyltransferase family, which plays critical roles in various biological events. It has been shown that SETD3 could regulate the transcription of myogenic regulatory genes in C2C12 differentiation and promote myoblast determination. However, how SETD3 is regulated during myoblast differentiation is still unknown. Here, we report that two important microRNAs (miRNAs) could repress SETD3 and negatively contribute to myoblast differentiation. Using microRNA (miRNA) prediction engines, we identify and characterize miR-15b and miR-322 as the primary miRNAs that repress the expression of SETD3 through directly targeting the 3’-untranslated region of SETD3 gene. Functionally, overexpression of miR-15b or miR-322 leads to the repression of endogenous SETD3 expression and the inhibition of myoblast differentiation, whereas inhibition of miR-15b or miR-322 derepresses endogenous SETD3 expression and facilitates myoblast differentiation. In addition, knockdown SETD3 in miR-15b or miR-322 repressed myoblasts is able to rescue the facilitated differentiation phenotype. More interestingly, we revealed that transcription factor E2F1 or FAM3B positively or negatively regulates miR-15b or miR-322 expression, respectively, during muscle cell differentiation, which in turn affects SETD3 expression. Therefore, our results establish two parallel cascade regulatory pathways, in which transcription factors regulate microRNAs fates, thereby controlling SETD3 expression and eventually determining skeletal muscle differentiation.
Highlights
Skeletal muscle differentiation is a complex process orchestrated by a family of myogenic regulatory factors (MRFs), including MyoD, myogenin, MRF4, and Myf[51,2]
To examine whether SETD3 is required for cell differentiation, C2C12 cells was induced by cultured in the differentiation medium (DM), and expression of SETD3 in both transcriptional levels and protein levels were examined
We found that the protein levels of SETD3 displayed an increase at the early stage of differentiation, but showed a reduction when MHC protein was significantly accumulated, which may suggest a complicated regulatory mechanism of SETD3 involved in muscle differentiation (Fig. S1c)
Summary
Skeletal muscle differentiation is a complex process orchestrated by a family of myogenic regulatory factors (MRFs), including MyoD, myogenin, MRF4, and Myf[51,2]. Expression of MyoD and Myf[5] in the initial stages of differentiation induces expression of myogenin and musclespecific transcription factors MEF2, whereas myogenin and MRF4 are expressed in the late stages of differentiation to activate the myogenic program by induction of muscle gene expression and silence of cell cycle-related gene expression[2,3,4]. MicroRNAs (miRNAs) modulate gene expression at the post-transcriptional level either by promoting mRNA degradation or inhibiting translation through complementary targeting 3’ untranslated regions (3’-UTRs) of specific mRNAs2,6. The muscle-specific miRNAs, miR-206, miR-1, and miR-133, are abundantly expressed during skeletal muscle differentiation, and promote muscle differentiation by inhibition specific transcription repressors[7,8,9,10]. Many non-muscle specific miRNAs regulate muscle differentiation by post-transcriptional mechanisms that affect the presence and functions of the myogenic factors, either positively or negatively
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