Genome-wide identification of enhancers, transcription factors, and mechanisms that control skeletal muscle differentiation in cattle

Abstract

Skeletal muscle development and growth involve significant changes in gene expression. The overall objective of this dissertation project was to identify transcription factors, enhancers, and mechanisms that control gene expression during skeletal muscle development and growth on a genome-wide scale. Three independent studies were conducted in this project. The objective of the first study was to identify potentially novel mechanisms that mediate myoblast differentiation, a process whereby the mononuclear muscle precursor cells myoblasts express skeletal muscle-specific genes and fuse with each other to form multinucleated myotubes. Comparing gene expression profiles in C2C12 cells, a widely used model of myoblasts, before and 6 days after induced myogenic differentiation by RNA sequencing (RNA-seq) revealed 11,046 differentially expressed genes, of which 5,615 and 5,431 were upregulated and downregulated, respectively. Functional enrichment analyses revealed that the upregulated genes were associated with biological processes or cellular components such as skeletal muscle contraction, autophagy, and sarcomere. In contrast, the downregulated genes were associated with biological processes or cellular components such as ribonucleoprotein complex biogenesis, mRNA processing, and ribosome. Western blot analyses showed an increased conversion of LC3-I to LC3-II protein during myoblast differentiation, further demonstrating the upregulation of autophagy during myoblast differentiation. Blocking the autophagic flux in C2C12 cells with chloroquine inhibited the expression of skeletal muscle-specific genes and the formation of myotubes, confirming a positive role of autophagy in myoblast differentiation and fusion. The aim of the second study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells, which are the myogenic precursor cells in adult skeletal muscle, into myotubes. In this study chromatin immunoprecipitation followed by sequencing (ChIP-seq) was used to identify active enhancers, i.e., genomic regions marked with histone modification H3K27ac (acetylation of lysine 27 of H3 histone protein). 19,027 and 47,669 H3K27ac-marked enhancers were identified from undifferentiated and differentiating bovine satellite cells, respectively. Of these enhancers, 5,882 and 35,723were specific to undifferentiated and differentiating bovine satellite cells, respectively while 13,199 were shared by both undifferentiated and differentiating bovine satellite cells. Many of the H3K27ac-marked enhancers specific to differentiating bovine satellite cells were associated with muscle structure and development genes and were enriched with binding sites for MyoD, AP-1, AP-4, KLF, TEAD, and MEF2 transcription factors. Through siRNA-mediated knockdown, AP-4 was found to be essential for differentiation of bovine satellite cells into myotubes. The objective of the third study was to identify enhancers and transcription factors that control differential gene expression in skeletal muscle between neonatal and adult cattle. First, RNA-seq was performed to compare gene expression profiles in skeletal muscle between neonatal calves and adult steers. This analysis identified 924 genes downregulated and 1,021 upregulated from calf to steer muscle. Among genes downregulated in steer muscle were myosin heavy chain3 (MYH3) and MYH8, and among genes upregulated in steer muscle were MYH7 and myoglobin. Surprisingly, many so-called adult muscle genes, such as MYH1 and MYH2, were not differentially expressed between calf and steer muscle. Gene ontology analyses showed that many genes downregulated in steer muscle are involved in protein synthesis and glycolysis and that many genes upregulated in steer muscle function in blood vessel development and immune cell activation. Next, ChIP-seq was performed to identify genomic regions marked with H3K27ac, i.e., active enhancers, in the skeletal muscle of neonatal calves and adult steers. This experiment led to the finding of 20,163 enhancers specifically active in the calf muscle, 14,909 enhancers specifically active in the steer muscle, and 27,002 enhancers active in both the calf and steer muscle. Motif enrichment analyses revealed the enrichment of binding sites for the KLF family and TEAD family transcription factors in enhancers active specifically in the calf muscle, the enrichment of binding sites for the FOXO family and the SMAD family transcription factors in enhancers specifically active in the steer muscle, and the enrichment of binding sites for the MRF family and MEF2 family transcription factors in enhancers active in both the calf and steer muscle. . These results shed light on the differences in gene expression and biology between newborn calf and adult steer skeletal muscle. These results also shed light on the enhancers and transcription factors that control these differences.