The quality of pork is significantly impacted by intramuscular fat (IMF). However, the regulatory mechanism of IMF depositions remains unclear. We performed whole-transcriptome sequencing of the longissimus dorsi muscle (IMF) from the high (5.1 ± 0.08) and low (2.9 ± 0.51) IMF groups (%) to elucidate potential mechanisms. In summary, 285 differentially expressed genes (DEGs), 14 differentially expressed miRNAs (DEMIs), 83 differentially expressed lncRNAs (DELs), and 79 differentially expressed circRNAs (DECs) were identified. DEGs were widely associated with IMF deposition and liposome differentiation. Furthermore, competing endogenous RNA (ceRNA) regulatory networks were constructed through co-differential expression analyses, which included circRNA-miRNA-mRNA (containing 6 DEMIs, 6 DEGs, 47 DECs) and lncRNA-miRNA-mRNA (containing 6 DEMIs, 6 DEGs, 36 DELs) regulatory networks. The circRNAs sus-TRPM7_0005, sus-MTUS1_0004, the lncRNAs SMSTRG.4269.1, and MSTRG.7983.2 regulate the expression of six lipid metabolism-related target genes, including PLCB1, BAD, and GADD45G, through the binding sites of 2-4068, miR-7134-3p, and miR-190a. For instance, MSTRG.4269.1 regulates its targets PLCB1 and BAD via miRNA 2_4068. Meanwhile, sus-TRPM7_0005 controls its target LRP5 through ssc-miR-7134-3P. These findings indicate molecular regulatory networks that could potentially be applied for the marker-assisted selection of IMF to enhance pork quality.

Meat production traits in pigs are critical economic characteristics, primarily influenced by the formation and development of skeletal muscle. Skeletal muscle development is regulated by a complex transcriptional network, which partly relies on chromatin accessibility for initiation. Ningxiang pigs, a renowned Chinese indigenous breed, are highly valued for their tender meat. However, studies focusing on skeletal muscle development in Ningxiang pigs, particularly from the perspective of chromatin accessibility, have not yet been reported. Based on this, the present study selected several key time points in the skeletal muscle development of Ningxiang pigs to perform Transposase-Accessible Chromatin Sequencing (ATAC-seq) and RNA sequencing (RNA-seq). This was carried out to identify key open chromatin regions and genes during different growth stages, which could influence skeletal muscle development in Ningxiang pigs. We collected longissimus dorsi muscle samples at postnatal days 14 (D14), 28 (D28), 85 (D85), 165 (D165), and 250 (D250). For each age, three individuals were collected for ATAC-seq and RNA-seq. After initial differential analysis among different ages, we identified 6412 differentially accessible chromatin peaks and 1464 differentially expressed genes. To clarify the key candidate transcription factors affecting the development of skeletal muscle in Ningxiang pigs, motif analysis of differential peaks revealed potential cis-regulatory elements with binding sites for transcription factors, including Fosl2 and JunB. Correlation analysis identified 56 overlapping genes and a significant positive correlation (r = 0.73, p = 1 × 10⁻¹⁴) between gene expression and chromatin accessibility. Key candidate genes such as HOXA10, closely related to skeletal muscle development, were specifically examined. These results enhance our understanding of the genetic and epigenetic regulatory mechanisms of porcine skeletal muscle development, providing a robust foundation for future molecular studies.

In order to investigate the potential mechanisms affecting the intramuscular fat content, high-throughput RNA sequencing was employed to assess the expression profiles of circRNAs in the longissimus dorsi tissues of Duroc pigs with high and low IMF content. A total of 230 circRNAs showed significantly differential expression levels between the HIMF and LIMF groups. GO annotation revealed that the host genes of these circRNAs were associated with responses to lipids, such as lipid tube assembly, lipid metabolic process, etc. Moreover, the KEGG enrichment analysis indicated that the host genes of these circRNAs were enriched in the AMP-activated protein kinase signaling pathway, fatty acid metabolism, etc. Several key circRNAs involved in lipid metabolism were screened, including novel_circ_0001751, novel_circ_0018596, novel_circ_0000494, etc. In addition, five circRNA-miRNA interaction networks were constructed. Notably, this study demonstrates for the first time the role of novel_circ_0018596 and novel_circ_0000494 in controlling IMF deposition in Duroc pigs.

Pioneer transcription factors (TFs) can directly establish higher-order chromatin interactions to instruct gene transcription. Here, we present a protocol for capturing TF-mediated 3D chromatin interactions using affinity tag-based bridge linker (BL)-Hi-chromatin immunoprecipitation (HiChIP). We describe steps for constructing FLAG-tagged TF, performing BL-HiChIP, and preparing the library. We then detail procedures for sequencing, data analysis, and quality control. This protocol has potential applications in 3D chromatin analysis centered on any specific TF in any type of cells without the need of optimal antibodies.For complete details on the use and execution of this protocol, please refer to Ren et al. (2022).1

Recent research has identified ADAR1 as a participant in the regulation of lipid accumulation in mice. However, there are no reports on the roles of ADAR1 in proliferation, apoptosis and differentiation of porcine preadipocytes. In this study, we investigated the role of ADAR1 in differentiation, proliferation and apoptosis of porcine preadipocytes using CCK-8, EdU staining, cell cycle detection, RT-qPCR, Western blot, a triglyceride assay and Oil Red O staining. The over-expression of ADAR1 significantly promoted proliferation but inhibited the differentiation and apoptosis of porcine preadipocytes. The inhibition of ADAR1 had the opposite effect on the proliferation, differentiation and apoptosis of porcine preadipocytes with over-expressed ADAR1. Then, the regulation mechanisms of ADAR1 on preadipocyte proliferation were identified using RNA-seq, and 197 DEGs in response to ADAR1 knockdown were identified. The MAPK signaling pathway is significantly enriched, indicating its importance in mediating fat accumulation regulated by ADAR1. The study’s findings will aid in uncovering the mechanisms that regulate fat accumulation through ADAR1.

Background: A recent study reported that a deficiency of Col5a3 reduces dermal fat. However, the regulatory mechanism of the Col5a3 gene on adipose deposition remains unclear. Methods: In this study, we assessed the effects of Col5a3 interference on the proliferation and differentiation of 3T3-L1 preadipocytes through CCK-8, EdU staining, cell cycle detection, RT-qPCR, Western blot, a triglyceride assay, and Oil Red O staining. RNA-seq was then performed on differentiated adipocytes to identify key differentially expressed genes (DEGs) and signaling pathways. Results: Col5a3 interference significantly promoted the proliferation of 3T3-L1 cells but inhibited their differentiation. RNA-seq analysis identified 368 DEGs, with the most significant enrichment observed in the oxidative phosphorylation pathway. Conclusions: This study demonstrates the regulatory role of Col5a3 in the proliferation and differentiation of preadipocytes, identifying various genes regulated by Col5a3 in adipogenesis. We speculate that Col5a3 may influence adipogenesis through the oxidative phosphorylation pathway in 3T3-L1 cells. The findings help gain a better understanding of the molecular mechanisms underlying fat deposition and obesity-related metabolic diseases.