FOXO1 regulates bovine skeletal muscle cells differentiation by targeting MYH3.

The growth and development of bovine skeletal muscle and beef yield is closely intertwined. Our previous research found that forkhead box O1 (FOXO1) plays an important role in the regulation of beef muscle formation, but its specific mechanism is still unknown. In this study, we aimed to clarify the regulatory mechanism of FOXO1 in proliferation and differentiation of bovine skeletal muscle cells (BSMCs). The results showed that interfering with FOXO1 can promote proliferation and the cell G1/S phase of BSMCs by up-regulating the expression of PCNA, CDK1, CDK2, CCNA2, CCNB1, CCND1 and CCNE2. Besides, interfering with FOXO1 inhibited the apoptosis of BSMCs by up-regulating the expression of anti-apoptosis gene BCL2, while simultaneously down-regulating the expression of the pro-apoptosis genes BAD and BAX. Inversely, interfering with FOXO1 can promote the differentiation of BSMCs by up-regulating the expression of myogenic differentiation marker genes MYOD, MYOG, MYF5, MYF6 and MYHC. Furthermore, RNA-seq combined with western bolt, immunofluorescence and chromatin immunoprecipitation analysis showed that FOXO1 could regulate BSMCs differentiation process by influencing PI3K-Akt, Relaxin and TGF-beta signaling pathways, and target MYH3 for transcriptional inhibition. In conclusion, this study provides a basis for studying the role and molecular mechanism of FOXO1 in BSMCs.

FOXO1 regulates the formation of bovine fat by targeting CD36 and STEAP4.

Intramuscular fat content is closely related to the quality of beef, where the forkhead box protein O1 (FOXO1) is involved in adipocyte differentiation and lipid metabolism, but the specific mechanism of its involvement is still unclear. In this study, interfering with FOXO1 promoted the G1/S transformation of bovine adipocytes by enhancing the expression of proliferation marker genes PCNA, CDK1, CDK2, CCNA2, CCNB1, and CCNE2, thereby positively regulating the proliferation of bovine adipocytes. Additionally, interfering with FOXO1 negatively regulated the expression of adipogenic differentiation marker genes PPARG and CEBPA, as well as lipid anabolism marker genes ACC, FASN, SCD1, SREBP1, FABP4, ACSL1, LPL, and DGAT1, thus reducing triglyceride (TG) content and inhibiting the generation of lipid droplets in bovine adipocytes. A combination of transcriptomic and metabolomics analyses revealed that FOXO1 could regulate the lipogenesis of cattle by influencing the AMPK and PI3K/AKT pathways. Importantly, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis revealed that FOXO1 could regulate bovine lipogenesis by binding to the promoter regions of the CD36 and STEAP4 genes and affecting their transcriptional activities. These results provide a foundation for studying the role and molecular mechanism of FOXO1 in the bovine adipogenesis.

Interaction of C/EBPß with SMAD2 and SMAD4 genes induces the formation of lipid droplets in bovine myoblasts.

As a key component of Transforming growth factor-ß (TGF-ß) pathway, Smad2 has many crucial roles in a variety of cellular processes, but it cannot bind DNA without complex formation with Smad4. In the present study, the molecular mechanism in the progress of myogenesis underlying transcriptional regulation of SMAD2 and SMAD4 had been clarified. The result showed the inhibition between SMAD2 and SMAD4, which promotes and inhibits bovine myoblast differentiation, respectively. Further, the characterization of promoter region of SMAD2 and SMAD4 was analyzed, and identified C/EBPß directly bound to the core region of both SMAD2 and SMAD4 genes promoter and stimulated the transcriptional activity. However, C/EBPß has lower expression in myoblasts which plays vital function in the transcriptional networks controlling adipogenesis, while the overexpression of C/EBPß gene in myoblasts significantly increased SMAD2 and SMAD4 gene expression, induced the formation of lipid droplet in bovine myoblasts, and promoted the expression of adipogenesis-specific genes. Collectively, our results showed that C/EBPß may play an important role in the trans-differentiation and dynamic equilibrium of myoblasts into adipocyte cells via promoting an increase in SMAD2 and SMAD4 gene levels. These results will provide an important basis for further understanding of the TGFß pathway and C/EBPß gene during myogenic differentiation.

Interaction of MyoD and MyoG with Myoz2 gene in bovine myoblast differentiation.

This study aims to explore the functional role of Myoz2 in myoblast differentiation, and elucidate the potential factors interact with Myoz2 in promoter transcriptional regulation. The temporal-spatial expression results showed that the bovine Myoz2 gene was highest expressed in longissimus dorsi, and in individual growth stages and myoblast differentiation stages. Knockdown of Myoz2 inhibited the differentiation of myoblast, and negative effect of MyoD, MyoG, MyH and MEF2A expression on mRNA levels. Subsequently, the promoter region of bovine Myoz2 gene with 1.7 Kb sequence was extracted, and then it was set as eight series of deleted fragments, which were ligated into pGL3-basic to detect core promoter regions of Myoz2 gene in myoblasts and myotubes. Transcription factors MyoD and MyoG were identified as important cis-acting elements in the core promoter region (-159/+1). Also, it was highly conserved in different species based on dual-luciferase analysis and multiple sequence alignment analysis, respectively. Furthermore, a chromatin immunoprecipitation (ChIP) analysis combined with site-directed mutation and siRNA interference and overexpression confirmed that the combination of MyoD and MyoG occurred in region -159/+1, and played an important role in the regulation of bovine Myoz2 gene. These findings explored the regulatory network mechanism of Myoz2 gene during the development of bovine skeletal muscle.

Roles of MEF2A and MyoG in the transcriptional regulation of bovine LATS2 gene.

As an important downstream effector gene in the hippo signaling pathway, large tumor suppressor gene 2 (LATS2) is involved in cell proliferation and differentiation, organ size and tissue regeneration, and plays an important role in regulating the growth and development of animal muscles. The purpose of this study is to explore the temporal expression of bovine LATS2 gene, and determine the key transcription factors for regulating bovine LATS2 gene. The result showed that bovine LATS2 gene was highly expressed in liver and longissimus dorsi, and was up-regulated in infancy muscle. In addition, it was highly expressed on the 2th day during the differentiation stage of myoblast. The upstream 1.7 Kb sequence of the 5 'translation region of bovine LATS2 gene was cloned, and 7 different deletion fragments were amplified by the upstream primers. These fragments were constructed into double luciferase reporter vectors and transfected into myoblasts and myotubes cells, respectively to detect the core promoter regions. In addition, the key transcription factors of the core promoter sequence of the bovine LATS2 gene were analyzed and predicted by online software. Combining with site-directed mutations, siRNA interference and chromatin immunoprecipitation technology, it was identified that MEF2A and MyoG combined in core promoter region (-248/-56) to regulate the transcription activity of bovine LATS2 gene. The results have laid a theoretical foundation for exploring the molecular regulation mechanism of LATS2 gene in the process of muscle growth.

Tissue Expression Analysis, Cloning, and Characterization of the 5'-Regulatory Region of the Bovine LATS1 Gene.

As a member of the large tumor suppressor (LATS) gene family, LATS1 plays an important role in regulating muscle growth and development. In this study, we determined the distinct exhibit patterns of tissue expression of bovine LATS1. Further, we determined the functional proximal minimal promoter of bovine LATS1 and identified the key transcription factors in the core promoter region to elucidate its molecular regulation mechanism. The results showed that bovine LATS1 was highly expressed in the longissimus thoracis and upregulation in infancy muscle. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with site-directed mutation and small interfering RNA (siRNA) interference demonstrated that myogenic differentiation 1 (Myod1) and myocyte enhancer factor 2A (MEF2A) binding in the core promoter region (-298/-123 bp) play important roles in the transcriptional regulation of the bovine LATS1 promoter. Taken together, these interactions provide insight into the regulatory mechanisms of LATS1 transcription in mediating skeletal muscle growth in cattle.

Genetic diversity of Gymnocypris chilianensis (Cypriniformes, Cyprinidae) unveiled by the mitochondrial DNA D-loop region.

In order to analyze the genetic diversity and genetic differentiation of Gymnocypris chilianensis, D-loop region of the mitochondrial DNA was sequenced in 50 individuals of G. chilianensis obtained from 2 geographic locations (Heihe River and Shule River) and 25 individuals of G. przewalskii (Qinghai Lake). Twenty-five homologous sequences of another G. eckloni (Yellow River) downloaded from GenBank were analyzed together. The sequences were analyzed by using the MEGA (version 7.0) and DnaSP (version 6.0) software. The results revealed that 82 haplotypes were detected among 100 individuals. The haplotype diversity (Hd) and nucleotide diversity (Pi) of G. chilianensis of the Shule River were 0.963 ± 0.029 and 0.00414 ± 0.00069, which were lower than those of 3 other populations. The genetic distance of G. chilianensis in both Heihe River and Shule River was 0.0013. The genetic distances between the 2 G. chilianensis populations and the G. eckloni were 0.0148 and 0.0141, respectively. Population differentiation values (Fst) and gene flow (Nm) showed that 4 population had occurred obvious genetic differentiation (Fst: 0.20811 ∼ 0.98863. p < 0.01; Nm < 1). Compared with G. przewalskii and G. eckloni, the differentiation degree was more significant between Heihe River G. chilianensis and Shule River G. chilianensis (Fst = 0.98863, p < 0.01; Nm = 0.00287). Maximum Likelihood (ML) phylogenetic tree showed that G. chilianensis had further genetic distance with G. eckloni and G. przewalskii. In conclution, G. chilianensis (HH&SL) had lower genetic diversity and further genetic distance than G. przewalskii (QH) and G. eckloni (YL). We suggest strengthen the protection of genetic resources of G. chilianensis.

Characterization of the promoter region of the bovine IRX3 gene: roles of SREBF2 and PPARG.

As a member of the Iroquois homeobox gene family, the IRX3 gene plays an important role in regulating the growth, development and fat deposition of chordates. In the present study, we found, using real-time PCR, that the bovine IRX3 gene was highly expressed in lung, kidney, heart, subcutaneous fat and longissimus dorsi muscle. We cloned the full-length sequence of the bovine IRX3 gene promoter and constructed eight series of 5' deletion promoter plasmid luciferase reporter assays and then transfected them to 3T3-L1 and C2C12 cell lines to detect its core promoter regions. The results showed that the core promoter of bovine IRX3 was located within a -292/-42 bp region relative to the transcriptional start site. Furthermore, sequence analysis identified eight CpG islands in the promoter region. A chromatin immunoprecipitation assay in combination with site-directed mutation and siRNA interference demonstrated that SREBF2 and PPARG binding occurs in region -292/-42 and is essential in bovine IRX3 transcription. These results lay an important theoretical foundation for exploring the molecular regulation mechanism of the IRX3 gene in bovine fat deposition.

Polymorphism in promoter of SIX4 gene shows association with its transcription and body measurement traits in Qinchuan cattle.

The sine oculis homeobox homolog 4 (SIX4) gene belongs to the SIX gene family, which plays a critical role in muscle regeneration and early stages of ontogeny. This study aimed to detect promoter variations of bovine SIX4 genes in Qinchuan cattle, and to evaluate the effect of transcription regulations and body measurement traits. Quantitative real-time PCR (qPCR) results showed that the mRNA expression levels of SIX4 gene were found significantly highest in longissimus thoracis tissue and individual before attaining the stage of physiological maturity. Using sequencing technology on a total of 428 Qinchuan cattle, seven single nucleotide polymorphisms (SNPs) were identified in the promoter region of SIX4, and seven haplotypes representing 18 potential transcription factor compositions of polymorphic potential cis-acting elements. Association analysis indicated that the H3-H3 diplotype performed greater withers height, chest depth, chest circumference, back fat thickness and ultrasound loin muscle area (P < 0.05) than H5-H6, which were consistent with the promoter activity of Hap3 haplotype was higher than the Hap5 and Hap6 haplotype in vitro. These potential transcription factor information and combined genotypes H3-H3 of the SIX4 gene can be used as a molecular marker for selection of economic traits in Qinchuan cattle.