Zhang Dongjie, Zhang Qian, Wu Xiaoxu, Wang Liang, Zhang Xiaohan, Liu Di, Yang Xiuqin
Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China.
College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China.
Cells. 2024 Dec 20;13(24):2118. doi: 10.3390/cells13242118.
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 ( < 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 (secretory leukocyte proteinase inhibitor) expression by directly binding to the promoter; KLF5 was also involved in (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.
由于骨骼肌发育在动物中具有重要性，人们已做出许多努力来揭示其潜在机制。然而，关于染色质可及性（基因表达的一个先决条件）的认识仍然有限。在此，利用转座酶可及染色质高通量测序技术（ATAC-seq）分析了30日龄、90日龄和210日龄的民猪骨骼肌中染色质可及性的动态变化。在三个发育阶段共鉴定出16301个与7455个基因相关的差异可及区域（DAR）。在选择用于功能分析的8个DAR中，有7个被发现能显著调节报告基因的表达（P<0.05），这表明DAR在基因表达中具有活性。通过RNA测序（RNA-seq）共鉴定出2219个差异表达基因（DEG）。通过对ATAC-seq和RNA-seq数据的综合分析，54个DEG_DAR_基因和61个转录因子（TF）被确定为对肌肉发育至关重要。其中，靶向36个DEG_DAR_基因的Kruppel样因子5（KLF5）是最重要的TF。然后用分子生物学技术分析了KLF5对DEG_DAR_基因表达的影响。发现KLF5通过直接结合启动子来调节分泌型白细胞蛋白酶抑制剂（SLPI）的表达；KLF5还通过影响位于内含子中的DAR的调控作用参与载脂蛋白A-I（ApoA-I）的表达。这些结果表明所鉴定的TF具有功能。总之，确定了民猪肌肉发育中重要的染色质可及性区域、TF和基因。这些结果为进一步揭示肌肉发育表观遗传调控的机制提供了新的数据。
