A comprehensive epigenome atlas reveals DNA methylation regulating skeletal muscle development

Abstract

DNA methylation is important for the epigenetic regulation of gene expression and plays a critical role in mammalian development. However, the dynamic regulation of genome-wide DNA methylation in skeletal muscle development remains largely unknown. Here, we generated the first single-base resolution DNA methylome and transcriptome maps of porcine skeletal muscle across 27 developmental stages. The overall methylation level decreased from the embryo to the adult, which was highly correlated with the downregulated expression of DNMT1 and an increase in partially methylated domains. Notably, we identified over 40 000 developmentally differentially methylated CpGs (dDMCs) that reconstitute the developmental trajectory of skeletal muscle and associate with muscle developmental genes and transcription factors (TFs). The dDMCs were significantly under-represented in promoter regulatory regions but strongly enriched as enhancer histone markers and in chromatin-accessible regions. Integrative analysis revealed the negative regulation of both promoter and gene body methylation in genes associated with muscle contraction and insulin signaling during skeletal muscle development. Mechanistically, DNA methylation affected the expression of muscle-related genes by modulating the accessibly of upstream myogenesis TF binding, indicating the involvement of the DNA methylation/SP1/IGF2BP3 axis in skeletal myogenesis. Our results highlight the function and regulation of dynamic DNA methylation in skeletal muscle development.

Sp1-Mediated circRNA circHipk2 Regulates Myogenesis by Targeting Ribosomal Protein Rpl7

“…Sp1 is a well-known activator of MyoD and a suppressor of CDKN1A . It plays an important role in muscle cell proliferation and differentiation [ 27 , 33 , 34 ]. We cloned three continuous regions containing these binding sites (B1–B3) and constructed a series of luciferase reporter vectors ( Figure 5 b).…”

“…Sp1 is a zinc finger transcription factor that binds GC-rich DNA motifs to regulate thousands of genes, and is involved in many critical cellular functions, such as cell growth, differentiation, and apoptosis [ 60 , 61 ]. Recent studies suggested that Sp1 could function as a DNA methylation-related modulator and an activator of myogenic differentiation, and plays a critical role in skeletal muscle development [ 27 , 33 ]. In this study, we verified that Sp1 directly binds to the promoter of circHipk2 and promoted the expression of both Hipk2 and circHipk2 in C2C12 myoblasts.…”

“…Analysis of gene expression was performed with SYBR Green Master Mix (ABI) by quantitative real-time PCR (RT-qPCR). RT-qPCR data were analyzed using the ΔΔCt method as in our previous report [ 27 ]. The primer sequences used in the present study are listed in Table S1 .…”

“…A ChIP assay was performed using a ChIP kit (EMD Millipore Corporation, Billerica, MA, USA) following the manufacturer’s instructions as previously described [ 27 ]. Briefly, the crosslinking reaction was terminated by glycine in Sp1 overexpression C2C12 cells treated with 1% formaldehyde.…”

“…The results revealed that circHipk2 inhibited myoblast proliferation and promoted differentiation by targeting ribosomal protein L7 (Rpl7), a ribosomal protein that is a component of the 60S subunit. Our recent study suggested that overexpression of transcription factor Sp1 promoted differentiation and repressed proliferation in C2C12 myoblasts [ 27 ]. In this study, we verified that Sp1 could directly bind to the promoter of circHipk2 and thus affect its transcription activity.…”