Mingxun Li

Novel 9-bp indel in visfatin gene and its associations with chicken growth

1. The effects of polymorphisms of the visfatin gene on growth performance, carcase traits, meat quality and serum variables were investigated in an F2 resource population of Gushi chickens crossed with Anka broilers. 2. A 9-bp (‘TAACCTGTG’) insertion/deletion in intron10 of the visfatin gene was found and a total of 964 individuals were genotyped in the resource population. Genotypes (II, ID and DD) were identified based on the 9-bp insertion (allele I) or deletion (allele D). The insertion/deletion polymorphism was used for analysing associations of the gene with growth traits, carcase traits and meat quality traits in 414 F2 chickens. 3. The DD genotype was not detected in those 66 F1 chickens and the I allelic frequency (0·724–0·879) was obviously higher than the D allelic frequency (0·121–0·276) in the birds of three generations. 4. The 9-bp insertion/deletion was associated with the traits of 8-week shank length, 12-week shank length, 4-week pectoral angle and pancreas weight. The relationships with other traits: body weight, carcase traits, meat quality traits and serum variables, were not significant. 5. It was concluded that allele D (9-bp deletion) of the visfatin gene had a negative effect on skeletal growth.

Long non-coding RNA ADNCR suppresses adipogenic differentiation by targeting miR-204

Adipogenesis is a complex and precisely orchestrated process mediated by a network of adipogenic regulatory factors. Several studies have highlighted the relevance of lncRNAs in adipocyte differentiation, but the precise molecular mechanism has largely remained elusive. In the present study, we performed Ribo-Zero RNA-Seq to investigate both the poly(A)+and poly(A)-lncRNAs of in vitro cultured bovine preadipocytes and differentiated adipocytes. A stringent set of 2882 lncRNAs was finally identified. A comparison of the lncRNAs expression profiles revealed that 16 lncRNAs are differentially expressed during adipocyte differentiation. We focused on the most downregulated lncRNA, which we named adipocyte differentiation-associated long noncoding RNA (ADNCR). Mechanistically, ADNCR inhibited adipocyte differentiation by functioning as a competing endogenous RNA (ceRNA) for miR-204, thereby augmenting the expression of the miR-204 target gene, SIRT1, which is known to inhibit adipocyte differentiation and adipogenic gene expression by docking with NCoR and SMART to repress PPARγ activity. Our data not only provide a valuable genomic resource for the identification of lncRNAs with functional roles in adipocyte differentiation but also reveal new insights into understanding the mechanisms of adipogenic differentiation.

A novel single-nucleotide polymorphism of the visfatin gene and its associations with performance traits in the chicken

Visfatin is a peptide that is predominantly expressed in visceral adipose tissue and is hypothesized to be related to obesity and insulin resistance. In this study, a novel silent single-nucleotide polymorphism (SNP) was found in exon 7 of the chickenvisfatin gene (also known asPBEF1) by single-stranded conformation polymorphism (SSCP) and DNA sequencing. In total, 836 chickens forming an F2 resource population of Gushi chicken crossed with Anka broiler were genotyped by XbaI forced RFLP, and the associations of this polymorphism with chicken growth, carcass characteristics, and meat quality were analyzed. Significant associations were found between the polymorphism and 4-week body weight (BW4), 6-week body weight (BW6), 4-week body slanting length (BSL4), fat bandwidth (FBW), breast muscle water loss rate (BWLR) and breast muscle fiber density (BFD) (P < 0.05), as well as 4-week breastbone length (BBL4) (P < 0.01). These observations suggested that the polymorphism in exon7 of thevisfatin gene had significant effects on the early growth traits of chicken.

The developmental transcriptome sequencing of bovine skeletal muscle reveals a long noncoding RNA, lncMD, promotes muscle differentiation by sponging miR-125b.

Pervasive transcription of the mammalian genome generates numerous long noncoding RNAs (lncRNAs), which are of crucial importance in diverse biological processes. Recent advances in high throughput sequencing technology have helped to accelerate the pace of lncRNA discovery. However, no study on the overall expression patterns of lncRNAs during muscle development has been conducted. We reported here the first analysis of lncRNA landscape in bovine embryonic, neonatal and adult skeletal muscle using Ribo-Zero RNA-Seq, a technology which can capture both poly(A)+ and poly(A)- transcripts. We finally defined 7692 high-confidence lncRNAs and uncovered 401 lncRNAs differentially expressed among three developmental stages, including lncMD, a novel muscle-specific lncRNA which is gradually up-regulated during myoblast differentiation. lncMD overexpression upregulated, whereas lncMD silencing decreased the expression of two well-established myogenic markers, myosin heavy chain (MHC) and myogenin (MyoG). In-depth analyses showed that lncMD acts as a molecular sponge for miR-125b and that insulin-like growth factor 2 (IGF2) is a direct target of miR-125b in cattle. Moreover, lncMD level was positively correlated with IGF2 mRNA level in bovine muscle tissues, a vital corollary to ceRNA function. Altogether, our research showed that lncMD acts as a ceRNA to sequester miR-125b, leading to heightened IGF2 expression and thus promotes muscle differentiation. Our findings also complement the reference genome annotation of cattle, which will likely be useful for further functional lncRNA cloning and more comprehensive studies on lncRNA regulation in muscle development.

Intragenic DNA methylation status down-regulates bovine IGF2 gene expression in different developmental stages

DNA methylation is a key epigenetic modification in mammals and has an essential and important role in muscle development. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to evaluate the expression of IGF2 and the methylation pattern on the differentially methylated region (DMR) of the last exon of IGF2 in six tissues with two different developmental stages. The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The quantitative real-time PCR (qPCR) analysis indicated that IGF2 has a broad tissue distribution and the adult bovine group showed significant lower mRNA expression levels than that in the fetal bovine group (P<0.05 or P<0.01). Moreover, the DNA methylation level analysis showed that the adult bovine group exhibited a significantly higher DNA methylation levels than that in the fetal bovine group (P<0.05 or P<0.01). These results indicate that IGF2 expression levels were negatively associated with the methylation status of the IGF2 DMR during the two developmental stages. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a marker-assisted selection for muscle developmental in beef cattle breeding program and as a model for studies in other species.

The developmental transcriptome landscape of bovine skeletal muscle defined by Ribo-Zero ribonucleic acid sequencing.

Ribonucleic acid sequencing (RNA-Seq) libraries are normally prepared with oligo(dT) selection of poly(A)+ mRNA, but it depends on intact total RNA samples. Recent studies have described Ribo-Zero technology, a novel method that can capture both poly(A)+ and poly(A)- transcripts from intact or fragmented RNA samples. We report here the first application of Ribo-Zero RNA-Seq for the analysis of the bovine embryonic, neonatal, and adult skeletal muscle whole transcriptome at an unprecedented depth. Overall, 19,893 genes were found to be expressed, with a high correlation of expression levels between the calf and the adult. Hundreds of genes were found to be highly expressed in the embryo and decreased at least 10-fold after birth, indicating their potential roles in embryonic muscle development. In addition, we present for the first time the analysis of global transcript isoform discovery in bovine skeletal muscle and identified 36,694 transcript isoforms. Transcriptomic data were also analyzed to unravel sequence variations; 185,036 putative SNP and 12,428 putative short insertions-deletions (InDel) were detected. Specifically, many stop-gain, stop-loss, and frameshift mutations were identified that probably change the relative protein production and sequentially affect the gene function. Notably, the numbers of stage-specific transcripts, alternative splicing events, SNP, and InDel were greater in the embryo than in the calf and the adult, suggesting that gene expression is most active in the embryo. The resulting view of the transcriptome at a single-base resolution greatly enhances the comprehensive transcript catalog and uncovers the global trends in gene expression during bovine skeletal muscle development.

Tetra-primer ARMS-PCR is an efficient SNP genotyping method: An example from SIRT2

Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) offers fast detection and extreme simplicity at a negligible cost for SNP genotyping. SIRT2, the family member (sirtuins, SIRT1-7) with the greatest homology to the silent information regulator 2 (Sir2), is the most abundantly expressed sirtuins in adipocytes and has been implicated in promoting fatty acid oxidation (FAO) by deacetylating various substrates. In the current study, we have successfully genotyped a new identified bovine SIRT2 SNP g.4140A > G by T-ARMS-PCR method and validated the accuracy by PCR-RFLP assay using 1255 animals representing the five main Chinese breeds. The concordance between the two different methods was 98.8%. Individuals with discordant genotypes were retyped by direct DNA sequencing. 40% of the discrepancies could be attributed to incomplete digestion in the PCR-RFLP assay. 60% of discordant genotypes resulted from allele failure in the T-ARMS-PCR assay. Chi-square test shows that the frequencies of SNP g.4140A > G are in Hardy–Weinberg equilibrium in all the samples (P > 0.05), which suggested that the five populations are almost a dynamic equilibrium even in artificial selection. Association analysis showed that the g.4140A > G polymorphism is significantly related to 24 months-old body weight in Nanyang cattle. Our results provide direct evidence that T-ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping and g.4140A > G polymorphism in bovine SIRT2 is associated with growth efficiency traits. These findings may be used for marker-assisted selection and management in feedlot cattle.

Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

A novel c.-274C>G polymorphism in bovine SIRT1 gene contributes to diminished promoter activity and is associated with increased body size

SIRT1, a mammalian homologue for yeast silent information regulator 2 (SIR2), is a NAD(+) -dependent deacetylase that belongs to the class III histone deacetylases. It plays an important role in diverse cellular processes, including stress resistance, mitochondrial function, suppression of inflammation and DNA repair. In this study, we screened and identified a novel polymorphism (c.-274C>G) in the SIRT1 promoter region. In silico prediction reveals that this SNP is in the core of cell cycle-dependent element (CDE)-binding motif. Interestingly, the G allele abolished a CDE-binding site, which suggested its functional significance. In the luciferase assay system, we found that the G allele-containing construct displayed a strikingly lower promoter activity compared with the C allele, which may downregulate SIRT1 expression levels. Additionally, we observed a significant association between the c.-274C>G polymorphism and growth traits in Nanyang cattle, suggesting that anomalous transcription factor-based repression of SIRT1 may increase bovine fat mass and body size.

Tetra-primer ARMS-PCR identifies the novel genetic variations of bovine HNF-4α gene associating with growth traits.

Hepatocyte nuclear factor-4α (HNF-4α), a member of the hepatocyte nuclear factor family, plays an important role in regulating the expression of genes involved in the development, differentiation and normal function of liver and pancreatic β cells, as well as the maintenance of glucose homeostasis. Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a new method offering fast detection and extreme simplicity at a negligible cost for SNP genotyping. In this paper, we characterize the polymorphisms of the bovine HNF-4α gene in three Chinese indigenous cattle breeds (n=660). Six novel SNPs were identified including 1 mutation in the coding region and others in introns. The statistical analyses indicated that 4 SNPs (g.T53729C, g.A53861G, g.A65188C and g.T65444C) affected growth traits markedly (P<0.05) in Qinchuan cattle (2 years after birth). Besides, haplotypes involving these 4 SNP sites in the bovine HNF-4α gene were identified and their effects on growth traits were also analyzed. The results showed that haplotypes 2, 7, 9 and 11 were predominant and accounted for 73.2%, 59.6%, and 67.1% in Qinchuan, Nanyang and Jiaxian cattle breeds, respectively. Hap9 (TAAT) was extremely predominant in all test populations, which suggested that individuals with Hap9 were more adapted to the environment. Furthermore, 4 combined haplotypes were constructed to guarantee the reliability of analysis results in Qinchuan cattle. There were also significant differences in body length (P<0.05). These findings will benefit for the application of DNA marker related to the growth traits on marker-assisted selection (MAS), and improve the performance of beef cattle.