Integration of multi-omics data reveals cis-regulatory variants that are associated with phenotypic differentiation of eastern from western pigs.

BACKGROUND: The genetic mechanisms that underlie phenotypic differentiation in breeding animals have important implications in evolutionary biology and agriculture. However, the contribution of cis-regulatory variants to pig phenotypes is poorly understood. Therefore, our aim was to elucidate the molecular mechanisms by which non-coding variants cause phenotypic differences in pigs by combining evolutionary biology analyses and functional genomics. RESULTS: We obtained a high-resolution phased chromosome-scale reference genome with a contig N50 of 18.03 Mb for the Luchuan pig breed (a representative eastern breed) and profiled potential selective sweeps in eastern and western pigs by resequencing the genomes of 234 pigs. Multi-tissue transcriptome and chromatin accessibility analyses of these regions suggest that tissue-specific selection pressure is mediated by promoters and distal cis-regulatory elements. Promoter variants that are associated with increased expression of the lysozyme (LYZ) gene in the small intestine might enhance the immunity of the gastrointestinal tract and roughage tolerance in pigs. In skeletal muscle, an enhancer-modulating single-nucleotide polymorphism that is associated with up-regulation of the expression of the troponin C1, slow skeletal and cardiac type (TNNC1) gene might increase the proportion of slow muscle fibers and affect meat quality. CONCLUSIONS: Our work sheds light on the molecular mechanisms by which non-coding variants shape phenotypic differences in pigs and provides valuable resources and novel perspectives to dissect the role of gene regulatory evolution in animal domestication and breeding.

MiR-743a-5p regulates differentiation of myoblast by targeting Mob1b in skeletal muscle development and regeneration.

The microRNAs (miRNAs) play an important role in regulating myogenesis by targeting mRNA. However, the understanding of miRNAs in skeletal muscle development and diseases is unclear. In this study, we firstly performed the transcriptome profiling in differentiating C2C12 myoblast cells. Totally, we identified 187 miRNAs and 4260 mRNAs significantly differentially expressed that were involved in myoblast differentiation. We carried out validation of microarray data based on 5 mRNAs and 5 miRNAs differentially expressed and got a consistent result. Then we constructed and validated the significantly up- and down-regulated mRNA-miRNA interaction networks. Four interaction pairs (miR-145a-5p-Fscn1, miR-200c-5p-Tmigd1, miR-27a-5p-Sln and miR-743a-5p-Mob1b) with targeted relationships in differentiated myoblast cells were demonstrated. They are all closely related to myoblast development. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated cell cycle signals important for exploring skeletal muscle development and disease. Functionally, we discovered that miR-743a targeting gene Mps One Binder Kinase Activator-Like 1B (Mob1b) gene in differentiated C2C12. The up-regulated miR-743a can promote the differentiation of C2C12 myoblast. While the down-regulated Mob1b plays a negative role in differentiation. In addition, the expression profile of miR-743a and Mob1b are consistent with skeletal muscle recovery after Cardiotoxin (CTX) injury. Our study revealed that miR-743a-5p regulates myoblast differentiation by targeting Mob1b involved in skeletal muscle development and regeneration. Our findings made a further exploration for mechanisms in myogenesis and might provide potential possible miRNA-based target therapies for skeletal muscle regeneration and disease in the near future.

Landscape of tissue-specific RNA Editome provides insight into co-regulated and altered gene expression in pigs (Sus-scrofa).

RNA editing generates genetic diversity in mammals by altering amino acid sequences, miRNA targeting site sequences, influencing the stability of targeted RNAs, and causing changes in gene expression. However, the extent to which RNA editing affect gene expression via modifying miRNA binding site remains unexplored. Here, we first profiled the dynamic A-to-I RNA editome across tissues of Duroc and Luchuan pigs. The RNA editing events at the miRNA binding sites were generated. The biological function of the differentially edited gene in skeletal muscle was further characterized in pig muscle-derived satellite cells. RNA editome analysis revealed a total of 171,909 A-to-I RNA editing sites (RESs), and examination of its features showed that these A-to-I editing sites were mainly located in SINE retrotransposons PRE-1/Pre0_SS element. Analysis of differentially edited sites (DESs) revealed a total of 4,552 DESs across tissues between Duroc and Luchuan pigs, and functional category enrichment analysis of differentially edited gene (DEG) sets highlighted a significant association and enrichment of tissue-developmental pathways including TGF-beta, PI3K-Akt, AMPK, and Wnt signaling pathways. Moreover, we found that RNA editing events at the miRNA binding sites in the 3'-UTR of HSPA12B mRNA could prevent the miRNA-mediated mRNA downregulation of HSPA12B in the muscle-derived satellite (MDS) cell, consistent with the results obtained from the Luchuan skeletal muscle. This study represents the most systematic attempt to characterize the significance of RNA editing in regulating gene expression, particularly in skeletal muscle, constituting a new layer of regulation to understand the genetic mechanisms behind phenotype variance in animals.Abbreviations: A-to-I: Adenosine-to-inosine; ADAR: Adenosine deaminase acting on RNA; RES: RNA editing site; DEG: Differentially edited gene; DES: Differentially edited site; FDR: False discovery rate; GO: Gene Ontology; KEGG: Kyoto Encyclopaedia of Genes and Genomes; MDS cell: musclederived satellite cell; RPKM: Reads per kilobase of exon model in a gene per million mapped reads; UTR: Untranslated coding regions.

A comprehensive epigenome atlas reveals DNA methylation regulating skeletal muscle development.

DNA methylation is important for the epigenetic regulation of gene expression and plays a critical role in mammalian development. However, the dynamic regulation of genome-wide DNA methylation in skeletal muscle development remains largely unknown. Here, we generated the first single-base resolution DNA methylome and transcriptome maps of porcine skeletal muscle across 27 developmental stages. The overall methylation level decreased from the embryo to the adult, which was highly correlated with the downregulated expression of DNMT1 and an increase in partially methylated domains. Notably, we identified over 40 000 developmentally differentially methylated CpGs (dDMCs) that reconstitute the developmental trajectory of skeletal muscle and associate with muscle developmental genes and transcription factors (TFs). The dDMCs were significantly under-represented in promoter regulatory regions but strongly enriched as enhancer histone markers and in chromatin-accessible regions. Integrative analysis revealed the negative regulation of both promoter and gene body methylation in genes associated with muscle contraction and insulin signaling during skeletal muscle development. Mechanistically, DNA methylation affected the expression of muscle-related genes by modulating the accessibly of upstream myogenesis TF binding, indicating the involvement of the DNA methylation/SP1/IGF2BP3 axis in skeletal myogenesis. Our results highlight the function and regulation of dynamic DNA methylation in skeletal muscle development.

Comprehensive Profiles of mRNAs and miRNAs Reveal Molecular Characteristics of Multiple Organ Physiologies and Development in Pigs.

The pig (Sus scrofa) is not only an important livestock animal but also widely used as a biomedical model. However, the understanding of the molecular characteristics of organs and of the developmental skeletal muscle of the pig is severely limited. Here, we performed a comprehensive transcriptome profiling of mRNAs and miRNAs across nine tissues and three skeletal muscle developmental stages in the Guizhou miniature pig. The reproductive organs (ovary and testis) had greater transcriptome complexity and activity than other tissues, and the highest transcriptome similarity was between skeletal muscle and heart (R = 0.79). We identified 1,819 mRNAs and 96 miRNAs to be tissue-specific in nine organs. Testis had the largest number of tissue-specific mRNAs (992) and miRNAs (40). Only 15 genes and two miRNAs were specifically expressed in skeletal muscle and fat, respectively. During postnatal skeletal muscle development, the mRNAs associated with focal adhesion, Notch signaling, protein digestion, and absorption pathways were up-regulated from D0 to D30 and then down-regulated from D30 and D240, while genes with opposing expression patterns were significantly enriched in the oxidative phosphorylation and proteasome pathways. The miRNAs mainly regulated genes associated with insulin, Wnt, fatty acid biosynthesis, Notch, MAPK, TGF-beta, insulin secretion, ECM-receptor interaction, focal adhesion, and calcium signaling pathways. We also identified 37 new miRNA-mRNA interaction pairs involved in skeletal muscle development. Overall, our data not only provide a rich resource for understanding pig organ physiology and development but also aid the study of the molecular functions of mRNA and miRNA in mammals.

ISEcp1-mediated transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid in Escherichia coli.

BACKGROUND: CMY-2 is the most prevalent pAmpC ß-lactamase, but the chromosomal blaCMY-2 gene transfer via horizontal transmission has been seldom reported. This study aimed to describe an ISEcp1-mediated transposition of a chromosomal blaCMY-2 gene from Escherichia coli into a small endogenous ColE1-like plasmid, resulting in elevated resistance to extended-spectrum cephalosporins. METHODS: Three ESCs-resistant ST641 E. coli strains EC6413, EC4103 and EC5106 harbored the blaCMY-2 gene. S1-PFGE, I-ceu I-PFGE, Southern blotting and electroporation experiments were performed to investigate the location and transferability of blaCMY-2. The genetic context and gene expression of blaCMY-2 in the original isolates and the corresponding electroporants were explored by PCR mapping, primer walking strategy and RT-qPCR. RESULTS: The blaCMY-2-containing region (ISEcp1-blaCMY-2-∆blc-∆yggR-∆tnp1-orf7-orf8-orf9-∆tnp2-∆hsdR) was transposed into endogenous ColE1-like plasmid pSC137 in the process of electroporation at very low frequencies (10-8-10-9). The transpositions resulted in novel larger blaCMY-2-harboring ColE1-like plasmids with size of 14,845 bp, enabling increase in MICs of 2 to 8-fold for cefotaxime, ceftiofur, and ceftazidime in recipient strains over their respective original counterparts. Transcriptional level analysis revealed that the increased blaCMY-2 expression was correlated with elevated MIC values of cephalosporins. The blaCMY-2 transposition unit was identical to that in a clinical isolate E. coli TN44889 from France isolated in 2004. CONCLUSIONS: Our results firstly demonstrated that ISEcp1 mediated a transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid by electroporation. Amplification of the blaCMY-2 gene facilitates the strain adaptation to a changed environment with an elevated antibiotic pressure.

Prevalence of cfr in Enterococcus faecalis strains isolated from swine farms in China: Predominated cfr-carrying pCPPF5-like plasmids conferring "non-linezolid resistance" phenotype.

The cfr gene associated with linezolid resistance has attracted wide attention. However, little is known about its prevalence and mode of transmission in Enterococcus faecalis. In this study, we investigate the prevalence and genetic environment of the cfr gene in 91 E. faecalis isolates collected from swine faecal swabs in 30 farms in Guangdong Province, China in 2012. A relatively high prevalence of cfr was identified in E. faecalis isolates (11/91, 12.1%) by PCR. All the cfr-positive E. faecalis strains had a multidrug-resistance phenotype including erythrocin, tetracycline, gentamicin, kanamicin and ciprofloxacin, except vancomycin and linezolid. Molecular typing indicated that ST475 and ST16 were the most common types in cfr-positive E. faecalis strains. In addition, we demonstrated that all the cfr genes were located on plasmids by S1-PFGE and Southern blotting. A 12 kb cfr-positive plasmid (pE30) was identified in most (9/11) E. faecalis strains, but it couldn't mediate resistance to linezolid in the transconjugant. Sequence analysis showed that the pE30 was a pCPPF5-like plasmid and the region surrounding the cfr gene was the same as a cfr-carrying ISEnfa5-composite element in Streptococcus plasmid pStrcfr with 4 bp direct repeat (GTAT) on both sides. In conclusion, the cfr gene which had no linezolid resistance phenotype was present in multidrug-resistance E. faecalis strains, and the clonal spread of ST475 and ST16 strains and the horizontal transfer of the pCPPF5-like plasmids have contributed to the dissemination of cfr.

Multilevel selection of bcrABDR-mediated bacitracin resistance in Enterococcus faecalis from chicken farms.

In this study we isolated 109 Enterococcus faecalis from chicken faecal samples in 6 provinces of China to investigate the prevalence and transmission mechanism of the bacitracin resistance locus bcrABDR in E. faecalis. Thirty-seven bcrABDR-positive E. faecalis were detected with 26 different PFGE clusters. The MLST of 14 positive strains belonged to ST16 and we also detected three new sequence types. S1-PFGE analysis indicated that the locus was located on plasmids presenting different sizes, with the most prevalent size being ~50 kb (13/37). Sequence analysis revealed that 17 out of the 37 strains harbored a 5400-bp central region, in which locus bcrABDR was bracketed by two ISEnfa1 of the same orientation. Two types of bcrABDR alleles, differing in around 10% of their sequence were found. In silico analysis showed that bcrABDR is present in a variety of bacteria including the chicken commensal Enterococcus cecorum. Our results indicate that the use of bacitracin at farms might trigger the emergence and spread of the bacitracin resistance determinant bcrABDR among human bacterial pathogens. The finding of bcrABDR in the chicken commensal E. cecorum indicates that farm animals microbiota can be an important reservoir of resistance genes with relevance for human health.

Characterization of chromosomal qnrB and ampC alleles in Citrobacter freundii isolates from different origins.

The association of ESBLs (extended-spectrum beta-lactamases)/pAmpCs (plasmid-mediated AmpC ß-lactamases) with PMQR (plasmid mediated quinolone resistance) in gram-negative bacteria has been of great concern. The present study was performed to characterize the diversity, gene location, genetic context, and evolution of ampC and qnrB alleles in isolates of Citrobacter freundii. Fifteen isolates of C. freundii were identified from a total of 788 isolates of Enterobacteriaceae derived from humans, animals, animal food products, and the environment between 2010 and 2012. Co-existence of qnrB/ΔqnrB with ampC was detected in all C. freundii isolates. Both ampC and qnrB genes were found to be located on the chromosome, but were distantly separated on the chromosome. Seven and six novel alleles were discovered for the 10 ampC and qnrB variants detected in this study, respectively. Phylogenetic analysis showed that the new alleles differed a little from the variants of ampC/qnrB previously described in this genus. The genetic context surrounding ampC genes was AmpR-AmpC-Blc-SugE. However, five different genetic contexts surrounding qnrB/ΔqnrB genes were observed, but they occurred in all cases between the pspF and sapA genes. Additionally, cloning experiments showed that the regions containing different qnrB alleles, even with different genetic contexts, contributed to the reduction of quinolone susceptibility. Our results showed that the chromosomal ampC and qnrB alleles are closely related to C. freundii. However, unlike ampC, qnrB alleles seemed to be related to the genetic contexts surrounding them. The evolution of these two genes in C. freundii isolates might be through different pathways.

Dissemination of the chromosomally encoded CMY-2 cephalosporinase gene in Escherichia coli isolated from animals.

In this study, 619 individual Escherichia coli isolates from food-producing and companion animals were analysed to determine the prevalence of the cephalosporinase gene blaCMY-2. In total, 18 CMY-2-producers (2.9%) were detected and exhibited multidrug-resistant phenotypes. One of the CMY-2-producers was found to possess a novel blaCMY-2-like allele, blaCMY-130. The isolates belonged to distinct pulsotypes, suggesting that the blaCMY-2 gene was not disseminated by clonal expansion of blaCMY-2-positive strains. The blaCMY-2 genes were located on IncA/C-, IncHI2- or IncX-type plasmids in 9 (50%) of the 18 E. coli isolates. However, in the other nine isolates I-CeuI-PFGE and hybridisation analyses revealed that the blaCMY-2 gene was chromosomally located. A CMY gene-containing region composed of five open reading frames (ORFs) (ISEcp1-blaCMY-2-blc-sugE-ΔencR) was observed in plasmids from eight strains. A CMY gene-containing region composed of ten ORFs was observed in all of the nine chromosomally encoded blaCMY-2 genes, including a putative IS66-like element inserted in this conserved CMY genetic region in three strains. This conserved CMY genetic region was also found to be inserted into the oriVγ (putative gamma origin), part of the IncX plasmid backbone, by a complete transposition unit flanked by 5-bp DRs (direct repeat sequence) in pS62T. These results demonstrate the high prevalence of the chromosomally encoded blaCMY-2 gene in E. coli. This is the first study reporting a chromosomally encoded blaCMY-2 gene in E. coli. Chromosomally encoded blaCMY-2 might be a source of some plasmid-mediated blaCMY-2 genes and this probably facilitates the spread of cephalosporin-resistant strains.

Complete nucleotide sequence of cfr-carrying IncX4 plasmid pSD11 from Escherichia coli.

We report the complete nucleotide sequence of a plasmid carrying the multiresistance gene cfr. This plasmid was isolated from an Escherichia coli strain of swine origin in 2011. This 37,672-bp plasmid, pSD11, had an IncX4 backbone similar to those of the IncX4 plasmids obtained from the United States and Australia, in which the cfr gene was flanked by two copies of IS26 and a truncated Tn1331 was inserted.