Yu-Jia Sun

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.

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.

Haplotypes and effects on growth traits of bovine Wnt7a gene in Chinese Qinchuan cattle.

Wnt7a is a member of the WNT gene family, which encodes secreted signaling proteins and responds to many biological processes. Specifically Wnt7a influences satellite stem cells and regulates the regenerative potential of the muscle. However, similar researches about the bovine Wnt7a gene are lacking. Therefore, in this study, polymorphisms of the bovine Wnt7a gene were detected in 488 individuals from Chinese Qinchuan cattle by DNA pooling, forced PCR-RFLP, and DNA sequencing methods. 3 novel SNPs were identified, two SNPs (g.T4926C and g.A21943G) were in the intron and the last one (g.C63777T) was in the exon. Five haplotypes involved in these three variant sites in the Wnt7a gene were identified and their effects on growth traits were analyzed. The results revealed that haplotype 1 had the highest haplotype frequencies and was highly significantly associated with body height (P<0.01), body weight (P<0.05), chest width (P<0.05) and height at hip cross (P<0.01) respectively.

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.

Relationship of polymorphisms within ZBED6 gene and growth traits in beef cattle.

ZBED6 is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle. The aim of this study was to examine the association of the ZBED6 polymorphism with growth traits in beef cattle breed. Three single nucleotide polymorphisms (SNPs) were identified in the bovine ZBED6 by sequencing pooled DNA samples (Pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (Forced PCR-RFLP) methods. Overall, we reported one mutation (SNP1) in the promoter region and two missense mutations (SNP2 and 3) in the coding region (single exon) within the bovine ZBED6 gene, and the haplotype variability and extent of linkage disequilibrium (LD) in 1522 individuals representing four main cattle breeds from China (Nanyang, NY; Qinchuan, QC; Jiaxian, JX; and Chinese Holstein, CH). We also investigated haplotype frequencies and linkage disequilibrium coefficients for three SNPs in all study populations. LD and haplotype structure of ZBED6 were different between breeds. The result of haplotype analysis of three SNPs showed that eight different haplotypes were identified in all breeds. The wild-type haplotype (Hap 1: GCA) and mutant-type haplotype (Hap 8: AGG) shared by all four populations accounted for 15.1, 29.8, 21.7, 57.5% and 9.5, 8.6, 16.7, 0% of all haplotypes were observed in NY, QC, JX and CH, respectively. The statistical analyses indicated that three SNPs were significantly associated with growth traits in NY cattle population (P<0.05 or P<0.01) at five different ages. The results of this study suggest that the ZBED6 gene possibly is a strong candidate gene that affects growth traits in beef cattle breeding program.

Comparative analysis of the IGF2 and ZBED6 gene variants and haplotypes reveals significant effect of growth traits in cattle

Muscle growth is a complex phenomenon regulated by many factors, whereby net growth results from the combined action of synthesis and turnover. 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; Zinc finger, BED-type containing 6 (ZBED6) is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle. In this study, a total of seven single nucleotide polymorphisms (SNPs) were identified, four SNPs in intron 8 of IGF2 and one promoter SNP and two missense mutations in the coding region of ZBED6, two of which were in complete linkage disequilibrium (LD) in the bovine IGF2. The 58 haplotypes were inferred in 1522 individuals representing four purebred cattle breeds from China. The seven SNPs, 79 and 66 combined diplotypes were revealed for association with body mass in Nanyang and Jiaxian cattle populations at five different ages (P < 0.05 or 0.01). The mutant-type variants and haplotype 58 (likely in LD with the beneficial quantitative trait nucleotide allele) was superior for body mass; the heterozygote diplotype of the most common haplotypes 58 was associated with higher body mass compared to either heterozygote or homozygote. The statistical analyses indicated that the mutant-type variants and haplotypes are significantly associated with body mass in study cattle populations at different ages. These data demonstrate that variants and haplotypes are associated with growth traits, and these results may provide important biological insights into the phenotypic differentiation that is associated with adaptation and specialization of cattle breeds.