Many efforts have been made to reveal the mechanisms underlying skeletal muscle development because of its importance in animals. However, knowledge on chromatin accessibility, a prerequisite for gene expression, remains limited. Here, dynamic changes in chromatin accessibility were analyzed in the skeletal muscles of Min pigs at the ages of 30, 90, and 210 d using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). A total of 16,301 differentially accessible regions (DARs) associated with 7455 genes were identified among three developmental stages. Seven out of eight DARs selected for a functional analysis were found to regulate reporter gene expression significantly (p < 0.05), indicating that DARs are active in gene expression. A total of 2219 differentially expressed genes (DEGs) were identified with RNA sequencing (RNA-seq). Through integrated analyses of ATAC-seq and RNA-seq data, 54 DEG_DAR_genes and 61 transcription factors (TFs) were characterized as critical for muscle development. Among them, Kruppel-like factor 5 (KLF5), targeted to 36 DEG_DAR_genes, was the most important TF. The effects of KLF5 on DEG_DAR_gene expression were then analyzed with molecular biology techniques. KLF5 was found to regulate SLPI (secretory leukocyte proteinase inhibitor) expression by directly binding to the promoter; KLF5 was also involved in APOA1 (apolipoprotein A-I) expression through affecting the regulatory role of DAR located in the intron. These results indicate that the TFs identified were functional. Altogether, the chromatin accessibility region, TFs, and genes important for muscle development in Min pigs were identified. The results provide novel data for further revealing the mechanisms underlying the epigenetic regulation of muscle development.

The cAMP response element-binding protein (CREB), a basic leucine zipper transcription factor, is involved in the activation of numerous genes in a variety of cell types. The CREB gene is rich in alternative splicing (AS) events. However, studies on the AS of CREB genes in pigs are limited, and few reports have compared the roles of isoforms in activating gene expression. Here, five AS transcripts, V1–5, were characterized by RT-PCR and two, V3 and V5, were new identifications. Both V1 and V2 have all the functional domains of the CREB protein, with similar tissue expression profiles and mRNA stability, suggesting that they have similar roles. The transcriptional transactivation activities of four isoforms encoding complete polypeptides were analyzed on the expression of the B-cell CLL/lymphoma 2-like protein 2 and the poly (A)-binding protein, nuclear 1 genes with a dual-luciferase reporter system, and differential activities were observed. Both V1 and V2 have promoting effects, but their roles are gene-specific. V3 has no effect on the promoter of the two genes, while V4 functions as a repressor. The mechanisms underlying the differential roles of V1 and V2 were analyzed with RNA-seq, and the genes specifically regulated by V1 and V2 were identified. These results will contribute to further revealing the role of CREB and to analyzing the significance of AS in genes.