Recently, a research paper titled "Epigenomics Analysis of miRNA cis-regulatory Elements in Pig Muscle and Fat Tissues" was published by the team of Professor Zhao Shuhong and Professor Xu Xuewen from the College of Animal Science and Technology at Huazhong Agricultural University in the Genomics (JCR Q2, Impact Factor 5.73). This paper has analyzed and integrated epigenomic data from published studies on pig muscle and fat tissues, and identified functional cis-regulatory elements for miRNAs, which has provides a theoretical basis for understanding the molecular mechanisms of miRNA regulation in pig muscle and fat. Yingzi Genes provided resource support for the epigenomic analysis in this study. 

miRNAs play a crucial role in the growth and development as well as energy metabolism in pig skeletal muscle and fat tissues by regulating downstream target genes; meanwhile, the expression of miRNAs is also regulated by upstream promoters and enhancers. Despite the continuous improvement of the Encyclopedia of DNA Elements (ENCODE) project, which has identified a large number of cis-regulatory elements for protein-coding and long-chain non-coding genes in pigs, there have been few reports on the identification of cis-regulatory elements for pig miRNAs. Therefore, this study integrated and analyzed miRNAome data from two-week-old pigs' skeletal muscle and fat tissues, along with published ChIP-seq, ATAC-seq, RNA-seq, and Hi-C data, to systematically annotate the miRNA cis-regulatory elements in pig skeletal muscle and fat tissues.

Materials: Two longissimus muscles and two subcutaneous fatty tissues on the back from two-week-old pigs

Sequencing: Small RNA

Analysis: Conjoint analysis of miRNA and published epigenomic data (ChIP-seq, ATAC-seq, RNA-seq, Hi-C)

3.1 Small RNA Sequencing Analysi

Through the small RNA sequencing analysis of samples from two longissimus muscles and two subcutaneous fatty tissues on the back from two-week-old pigs, a total of 419 miRNAs were identified, including 359 known and 60 novel miRNAs (Figure 1b). Pearson's Correlation Coefficient (PCC) analysis demonstrated the reliability of the data (Figure 1c).

Figure 1. Identification of miRNAs in Muscle and Fat Tissues of Landrace pigs

3.2 Identification of miRNA Cis-Regulatory Elements in Pig Muscle and Fat Tissues

Analysis of H3K4me3, H3K27ac histone ChIP-seq, and ATAC-seq revealed that these epigenetic modification signals were significantly enriched around the majority of pre-miRNAs, and the signals were highly consistent (Figure 2a, b). Further analysis found that among the expressed pre-miRNAs in muscle and fat, 84.21% and 85.51%, respectively, were likely associated with a promoter, with 69.64% and 72.46% of the promoters identified as positive, and the remainder referred to as candidate promoters (Figure 2c). Furthermore, over 70% and 75% of the promoters overlapped with CpG islands and were identified as active promoters, indicating the accuracy of miRNA promoter identification (Figure 2d). The authors further identified enhancers, and the results showed that in muscle and fat, 65.18% and 65.25%, respectively of promoter-related pre-miRNAs were associated with at least one enhancer (Figure 2e), with 76.03% and 78.57% of these pre-miRNAs being regulated by multiple enhancers (Figure 2f).

Figure 2. Identification of miRNA Cis-Regulatory Elements

3.3 Analysis of miRNA Cis-Regulatory Elements in the Pig Genome

To research the relationship between miRNA transcriptional regulation and the 3D structure of the genome, the authors first utilized Hi-C data to distinguish miRNAs located in Compartment A and B, and the results showed that miRNA expression levels in Compartment A were significantly higher than those in Compartment B (Figure 3a, b). Subsequently, an analysis of H3K27ac ChIP-seq data was performed to assess the relationship between miRNAs and epigenetic modifications, and it was found that miRNAs with active promoters were significantly more abundant than those without active promoters (Figure 3c, d). Moreover, miRNAs associated with super-enhancers exhibited significantly higher expression levels compared with those associated with regular enhancers (Figure 3e, f). The authors then compared miRNA cis-regulatory elements between the pig and human genomes, and the results revealed that over 90% of miRNA promoters and approximately 85% of miRNA enhancers maintained sequence consistency with the human genome (Figure 3g); Approximately 55% of promoters and 21% of miRNA enhancers were conserved in both sequence and function (Figure 3g).

Figure 3. Regulatory Analysis and Validation of miRNA Cis-Regulatory Elements

3.4  Validation of miRNA Cis-Regulatory Elements in the Pig Genome

To validate the authenticity of identified miRNA cis-regulatory elements, the authors employed a dual fluorescence reporting system and CRISPR/Cas9 technology to confirm the activity and regulatory functions of miRNA enhancers and promoters (Figure 4).

Figure 4. Dual-Luciferase Reporter System Analysis of miRNA Cis-Regulatory Elements and Knockout of miRNA Enhancers 

3.5 Identification and Functional Analysis of Core miRNAs in Muscle and Fat Tissues

To identify potentially pivotal miRNAs in muscle and fat tissues, the authors separately selected the top 5% of miRNAs with the greatest changes in expression in muscle (red line) and fat (blue line). These miRNAs were designated as core miRNAs (Figure 5a, b). When these core miRNAs were compared with all differentially expressed miRNAs, the results indicated that the majority of core miRNAs exhibited higher changes (Figure 5b). Utilize TargetScan and miRDB to predict 27 known miRNAs, utilize IntaRNA and DIANA to predict 8 newly-discovered miRNAs, and select the overlapping results from these two programs as the final target genes (Figure 5c, d). Target gene prediction and GO enrichment analysis for these core miRNAs revealed that, in addition to common biological processes such as DNA transcription, cell migration, protein phosphorylation and cell cycle, the target genes of core miRNAs in muscle were involved in processes related to myoblast fate commitment, lipid metabolism, pri-miRNA transcription negative regulation, and energy metabolism (Figure 5g). On the other hand, the target genes of core miRNAs in fat were primarily enriched in processes related to glycolysis and steroid hormone metabolism (Figure 5g).

Figure 5. Selection and Target Gene Prediction of Core miRNAs in Pig Muscle and Fat

3.6 Histone Modification and Transcription Factor Binding in Core miRNA Cis-Regulatory Elements

The authors observed that, in the study of core miRNA cis-regulatory elements, changes in H3K27ac signals were consistent with differences in miRNA expression levels between muscle and fat tissues (Figure 6a). Among differentially expressed miRNAs between muscle and fat tissues, the fold change in H3K27ac signals for upregulated core miRNA cis-regulatory elements (promoters and enhancers) was significantly higher than that for non-differentially expressed miRNAs and downregulated differentially expressed miRNAs (Figure 6b). Furthermore, it was observed that the changes in the quantity of transcription factor bindings in differentially expressed miRNA cis-regulatory elements between muscle and fat tissues were consistent with changes in H3K27ac signals (Figure 6c). These results suggested that H3K27ac modification and transcription factors in miRNA cis-regulatory elements cooperated to regulate the expression of core miRNAs. Further analysis revealed that 19 and 21 significantly enriched transcription factors were detected in the core miRNA cis-regulatory elements of muscle tissue and fact tissues, respectively (Figure 6). 

Figure 6. Downstream Analysis of Histone Modification Changes in Core miRNA Cis-Regulatory Elements

This study utilized various omics research technologies, including miRNA, ChIP-seq, ATAC-seq, RNA-seq and Hi-C, to identify and describe miRNAs and their cis-regulatory elements in pig muscle and fat tissues. The study found that the number of miRNAs associated with super-enhancers, active promoters, and Compartment A was significantly higher than those associated with ordinary enhancers, H3K27ac-deficient promoters, and Compartment B, and the accuracy of these cis-regulatory elements was subsequently validated at the cellular level. The authors also identified core miRNAs associated with tissue-specific functions in muscle and fat, indicating that tissue-specific transcription factors are major determinants of the expression patterns of core miRNAs in muscle and fat. This research has contributed important information to the existing epigenetic data in pigs and provided essential resources for future in-depth studies of epigenetics in pigs.

Doctoral student Hu Mingyang, master student Kuang Renzhuo, and doctoral student Guo Yaping from the College of Animal Science and Technology at Huazhong Agricultural University are joint first authors of the paper. Associate researcher Zhao Yunxia and Professor Xu Xuewen from the College of Animal Science and Technology at Huazhong Agricultural University are joint corresponding authors of the paper. This research was supported by the National Natural Science Foundation of China, Natural Science Foundation of Hubei Province, Lingnan Modern Agriculture Laboratory Project, and National Key R&D Program.