Differential Regulation of Male-Hormones-Related Enhancers Revealed by Chromatin Accessibility and Transcriptional Profiles in Pig Liver.

Surgical castration can effectively avoid boar taint and improve pork quality by removing the synthesis of androstenone in the testis, thereby reducing its deposition in adipose tissue. The expression of genes involved in testis-derived hormone metabolism was altered following surgical castration, but the upstream regulatory factors and underlying mechanism remain unclear. In this study, we systematically profiled chromatin accessibility and transcriptional dynamics in liver tissue of castrated and intact full-sibling Yorkshire pigs. First, we identified 897 differentially expressed genes and 6864 differential accessible regions (DARs) using RNA- and ATAC-seq. By integrating the RNA- and ATAC-seq results, 227 genes were identified, and a significant positive correlation was revealed between differential gene expression and the ATAC-seq signal. We constructed a transcription factor regulatory network after motif analysis of DARs and identified a candidate transcription factor (TF) SP1 that targeted the HSD3B1 gene, which was responsible for the metabolism of androstenone. Subsequently, we annotated DARs by incorporating H3K27ac ChIP-seq data, marking 2234 typical enhancers and 245 super enhancers involved in the regulation of all testis-derived hormones. Among these, four typical enhancers associated with HSD3B1 were identified. Furthermore, an in-depth investigation was conducted on the androstenone-related enhancers, and an androstenone-related mutation was identified in a newfound candidatetypical enhancer (andEN) with dual-luciferase assays. These findings provide further insights into how enhancers function as links between phenotypic and non-coding area variations. The discovery of upstream TF and enhancers of HSD3B1 contributes to understanding the regulatory networks of androstenone metabolism and provides an important foundation for improving pork quality.

Novel Haplotype in the HHEX Gene Promoter Associated with Body Length in Pigs.

The screening of important candidate genes and the identification of genetic markers are important for molecular selection in the pig industry. The hematopoietically expressed homeobox (HHEX) gene plays an important role in embryonic development and organogenesis; however, the genetic variation and expression pattern of the porcine HHEX gene remains to be clarified. In this study, semiquantitative RT-PCR and immunohistochemistry results showed the specific expression of the HHEX gene in porcine cartilage tissues. A novel haplotype consisting of two SNPs rs80901185 (T > C) and rs80934526 (A > G) was detected in the promoter region of the HHEX gene. The expression of the HHEX gene was significantly higher in Yorkshire pigs (TA haplotype) than in Wuzhishan pigs (CG haplotype), and a population analysis showed that this haplotype was significantly associated with body length. An analysis subsequently revealed that the -586 to -1 bp region of the HHEX gene promoter showed the highest activity. Furthermore, we found that the activity of the TA haplotype was significantly higher than that of the CG haplotype by changing the potential binding of transcription factors YY1 and HDAC2. In summary, we conclude that the porcine HHEX gene may contribute to the breeding of pigs for body length traits.

Integrated analysis of the whole transcriptome of skeletal muscle reveals the ceRNA regulatory network related to the formation of muscle fibers in Tan sheep.

Meat quality is highly influenced by the kind of muscle fiber, and it can be significantly improved by increasing the percentage of slow-twitch fibers. It is still not known which genes control the formation of muscle fibers or how those genes control the process of forming in sheep until now. In this study, we used high-throughput RNA sequencing to assess the expression profiles of coding and noncoding RNAs in muscle tissue of Tan sheep and Dorper sheep. To investigate the molecular processes involved in the formation of muscle fibers, we collected two different muscle tissues, longissimus dorsi and biceps femoris, from Tan sheep and Dorper sheep. The longissimus dorsi of Tan sheep and Dorper sheep displayed significantly differential expression levels for 214 lncRNAs, 25 mRNAs, 4 miRNAs, and 91 circRNAs. Similarly, 172 lncRNAs, 35 mRNAs, 12 miRNAs, and 95 circRNAs were differentially expressed in the biceps femoris of Tan sheep and Dorper sheep according to the expression profiling. GO and KEGG annotation revealed that these differentially expressed genes and noncoding RNAs were related to pathways of the formation of muscle fiber, such as the Ca2+, FoxO, and AMPK signaling pathways. Several key genes are involved in the formation of muscle fibers, including ACACB, ATP6V0A1, ASAH1, EFHB, MYL3, C1QTNF7, SFSWAP, and FBXL5. RT-qPCR verified that the expression patterns of randomly selected differentially expressed transcripts were highly consistent with those obtained by RNA sequencing. A total of 10 lncRNAs, 12 miRNAs, 20 circRNAs, and 19 genes formed lncRNA/circRNA-miRNA-gene networks, indicating that the formation of muscle fiber in Tan sheep is controlled by intricate regulatory networks of coding and noncoding genes. Our findings suggested that specific ceRNA subnetworks, such as circ_0017336-miR-23a-FBXL5, may be critical in the regulation of the development of muscle fibers, offering a valuable resource for future study of the development of muscle fibers in this animal species. The findings increase our understanding of the variety in how muscle fibers originate in various domestic animals and lay the groundwork for future research into new systems that regulate the development of muscle.

The Novel-miR-659/SPP1 Interaction Regulates Fat Deposition in Castrated Male Pigs.

Castration is usually used to remove boar taint in commercial pork production, but the adipose accumulation was increased excessively, which affected the meat quality of pigs. Based on our previous study, secreted phosphoprotein 1 (SPP1) was significantly differentially expressed between castrated and intact male pigs. However, the role of SPP1 in regulating adipose growth and fat storage caused by castration is unknown. In this study, SPP1 was identified to inhibit adipogenesis by the expression of adipogenic markers PPARγ and FABP4 as well as Oil red staining assay during differentiation of porcine bone marrow mesenchymal stem cells (pBMSCs). Subsequently, testosterone was used to treat pBMSCs to simulate the androgen status of intact pigs. Compared with the control groups without testosterone, the SPP1 expression in the testosterone group was markedly increased in the late stage of pBMSCs differentiation. Furthermore, novel-miR-659 was predicted by TargetScan and miRDB to target SPP1 and verified through a dual-luciferase reporter assay. Oil Red O staining assay indicated that novel-miR-659 overexpression significantly promoted adipogenesis, whereas novel-miR-659 inhibition suppressed adipogenesis. The expressions of adipogenic markers PPARγ and FABP4 showed the same tendency. Taken together, our study found that the targeted interaction between novel-miR-659 and SPP1 is involved in regulation of fat deposition in castrated male pigs.

A native, highly active Tc1/mariner transposon from zebrafish (ZB) offers an efficient genetic manipulation tool for vertebrates.

New genetic tools and strategies are currently under development to facilitate functional genomics analyses. Here, we describe an active member of the Tc1/mariner transposon superfamily, named ZB, which invaded the zebrafish genome very recently. ZB exhibits high activity in vertebrate cells, in the range of those of the widely used transposons piggyBac (PB), Sleeping Beauty (SB) and Tol2. ZB has a similar structural organization and target site sequence preference to SB, but a different integration profile with respect to genome-wide preference among mammalian functional annotation features. Namely, ZB displays a preference for integration into transcriptional regulatory regions of genes. Accordingly, we demonstrate the utility of ZB for enhancer trapping in zebrafish embryos and in the mouse germline. These results indicate that ZB may be a powerful tool for genetic manipulation in vertebrate model species.

Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish.

Enhancers are key transcriptional drivers of gene expression. The identification of enhancers in the genome is central for understanding gene-expression programs. Although transposon-mediated enhancer trapping (ET) is a powerful approach to the identification of enhancers in zebrafish, its efficiency varies considerably. To improve the ET efficiency, we constructed Tol2-mediated ET vectors with a reporter gene (mCherry) expression box driven by four minimal promoters (Gata, Myc, Krt4 and Oct4), respectively. The ET efficiency and expression background were compared among the four promoters by zebrafish embryo injection at the one-cell stage. The results showed that the Gata minimal promoter yielded the lowest basic expression and the second-highest trapping efficiency (44.6% at 12 hpf (hour post-fertilization) and 23.1% at 72 hpf, n = 305 and n = 307). The Krt4 promoter had the highest trapping efficiency (64% at 12 hpf and 67.1% at 72 hpf, n = 302 and n = 301) and the strongest basic expression. To detect enhancer activity, chicken 5'HS4 double insulators were cloned into the two ET vectors with the Gata or Krt4 minimal promoter, flanking the mCherry expression box. The resulting detection vectors were injected into zebrafish embryos. mCherry expression driven by the Gata promoter (about 5%, n = 301) was decreased significantly compared with that observed for embryos injected with the ET vectors (23% at 72 hpf, n = 308). These results suggest that the insulators block the genome-position effects and that this vector is fit for enhancer-activity evaluation. To assess the compatibility between the enhancers and the minimal promoters, four enhancers (CNS1, Z48, Hand2 and Hs769) were cloned upstream of the Gata or Beta-globin minimal promoter in the enhancer-activity-detection vectors. The resulting recombinant vectors were assayed by zebrafish embryo injection. We found that Z48 and CNS1 responded to the Gata minimal promoter, and that Hand2 only responded to the Beta-globin minimal promoter. In contrast, Hs769 did not respond to either the Gata or Beta-globin minimal promoters. These results suggest the existence of compatibility between enhancers and minimal promoters. This study represents a systematic approach to the discovery of optional ET and enhancer-detection vectors. We are eager to provide a superior tool for understanding functional genomics.

Enhancer Trapping and Annotation in Zebrafish Mediated with Sleeping Beauty, piggyBac and Tol2 Transposons.

Although transposon-mediated enhancer trapping (ET) is successfully applied in diverse models, the efficiency of various transposon systems varies significantly, and little information is available regarding efficiency of enhancer trapping by various transposons in zebrafish. Most potential enhancers (Ens) still lack evidence of actual En activity. Here, we compared the differences in ET efficiency between sleeping beauty (SB), piggyBac (PB) and Tol2 transposons. Tol2 represented the highest germline transfer efficiencies at 55.56% (NF0 = 165), followed by SB (38.36%, NF0 = 151) and PB (32.65%, NF0 = 149). ET lines generated by the Tol2 transposon tended to produce offspring with a single expression pattern per line, while PB and SB tended to generate embryos with multiple expression patterns. In our tests, 10 putative Ens (En1⁻10) were identified by splinkerette PCR and comparative genomic analysis. Combining the GFP expression profiles and mRNA expression patterns revealed that En1 and En2 may be involved in regulation of the expression of dlx1a and dlx2a, while En6 may be involved in regulation of the expression of line TK4 transgene and rps26, and En7 may be involved in the regulation of the expression of wnt1 and wnt10b. Most identified Ens were found to be transcribed in zebrafish embryos, and their regulatory function may involve eRNAs.

[Enhancer trapping nearby rps26 gene in zebrafish mediated by the Tol2 transposon and it's annotation].

With the completion of large-scale genome sequencing of human beings and other organisms, understanding the expression of control elements on the genome has become an important research task in the post-genome era. The enhancer trapping technology is an effective method for identifying enhancer elements in the genome and understanding its mechanism for gene expression regulation. In this study, we selected the stable enhancer trapping line TK4 (head and trunk specific GFP expression), which is generated with the mediation of Tol2 transposon system, and analyzed the trapped enhancers with the techniques of Splinkerette PCR (sp-PCR), in situ hybridization and comparative genomics. We crossed F1 individuals of TK4 line with wild-type zebrafish, collected fertilized eggs, and then detected the expression pattern of green fluorescent protein reporter gene by fluorescence microscopy at six different developmental stages, 6 hpf (hour post fertilization), 24 hpf, 48 hpf, 3 dpf (day post fertilization), 4 dpf and 5 dpf . The zebrafish genome flank sequence near the insertion site of Tol2 transposon was cloned by sp-PCR, and the results revealed that the insertion located at the position 27749253 of chromosome 23, and the transgene inserted reversely inside the intron 1 of rps26 gene. Within the 100 kb region of the insertion site, totally, seven genes including arf3a, wnt10b, wnt1, rps26, IKZF4, dnajc22 and lmbr1l were identified. Comparative genomic analysis by VISTA program revealed that there were two potential enhancer elements in the downstream of rps26 gene, which were conserved non-coding sequence (CNS) 1 and CNS2. The results of in situ hybridization showed that two transcripts of rps26 gene were maternal expression, the expression of rps26-201 in zygote was earlier than that of rps26-001, and the GFP signal of TK4 line zebrafish was not detectable before 6hpf, the expression patterns of rps26 and GFP at the late stages display similarity, and also represent differences, which suggested that the expression of rps26 and GFP may be controlled by the same enhancer, and also by the different enhancer, and two potential enhancers (CNS1 and CNS2) may play a differential regulation roles on the spatial and temporal expression of nearby genes (including rps26). In this study, we successfully obtained two potential enhancers near rps26 gene for the first time, which laid a foundation for further study of the regulation mechanism between these two enhancers and nearby genes in the genome, and the combination technique used in this study also provides a reference for enhancer analysis.

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1.

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5'- and 3'-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.