Abstract
miRNAs reportedly participate in various biological processes, such as skeletal muscle proliferation and differentiation. However, the regulation of differentially expressed (DE) miRNAs and their function in myogenesis remain unclear. Herein, miRNA expression profiles and regulation during C2C12 differentiation were analyzed in relation to chromatin states by RNA-seq, ATAC-seq, and ChIP-seq. We identified 19 known and nine novel differentially expressed miRNAs at days 0, 1, 2, and 4. The expression of the differentially expressed miRNAs was related to the chromatin states of the 113 surrounding open chromatin regions defined by ATAC-seq peaks. Of these open chromatin regions, 44.25% were colocalized with MyoD/MyoG binding sites. The remainder of the above open chromatin regions were enriched with motifs of the myoblast-expressed AP-1 family, Ctcf, and Bach2 transcription factors (TFs). Additionally, the target genes of the above differentially expressed miRNAs were enriched primarily in muscle growth and development pathways, especially the Hippo signaling pathway. Moreover, via combining a loss-of-function assay with Q-PCR, western blotting, and immunofluorescence, we confirmed that the Hippo signaling pathway was responsible for C2C12 myoblast differentiation. Thus, our results showed that these differentially expressed miRNAs were regulated by chromatin states and affected muscle differentiation through the Hippo signaling pathway. Our findings provide new insights into the function of these differentially expressed miRNAs and the regulation of their expression during myoblast differentiation.
Highlights
Skeletal muscles account for ~40% of the body weight of humans[1] and play important roles in motor movements and energy metabolism[2]
By using Integrative Genomics Viewer to visualize the binding profiles from ATAC-seq and ChIP-seq data surrounding these pri-miRNA loci, we found that the changes in the ATAC-seq signals around the differentially expressed miRNAs were consistent with the differences in the miRNA expression levels between day 0 and day 2.5 during C2C12 myoblast differentiation (Fig. 2a)
We further examined the expression patterns of these pathways by using the same time course rRNA-depleted RNA sequencing (RNA-seq) data of C2C12 myoblast differentiation and found that the levels of target genes involved in the Hippo and MAPK signaling pathways changed during C2C12 myoblast differentiation (Figs. 3b and S4)
Summary
Skeletal muscles account for ~40% of the body weight of humans[1] and play important roles in motor movements and energy metabolism[2]. As important regulators of gene expression, TFs regulate target genes, including miRNA genes, mainly through binding to their promoter regions surrounding transcription start sites or enhancer elements[23,24]. Previous chromatin immunoprecipitation followed by sequencing (ChIP-seq) analyses showed that MyoD and MyoG can bind to upstream regions of miR-1, miR-133, and miR-206 primary miRNAs (pri-miRNAs) and are likely to regulate the enhanced expression of these miRNAs during myogenesis[26]. MyoD, MEF2, and SRF can bind their respective sites in the upstream enhancer regions of mir-1-1 and mir-1-2 to directly regulate miR-1 expression[27], and the SRF expression was repressed by miR-133 in turn[9]. MyoD can cooperate with MEF2 to activate the transcription of the pri-miRNAs encoding miR-1-2 and miR133a-1 via an intragenic enhancer between the mir-1-2 and mir-133a-1 coding regions in skeletal muscle[28]. The function of chromatin states and their associated TFs in the regulation of miRNA expression was still a missing part and is the time course study of miRNA expression in myoblast differentiation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
