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.

Systematic Discovery of a New Catalogue of Tyrosine-Type Integrases in Bacterial Genomic Islands.

Site-specific recombinases (integrases) can mediate the horizontal transfer of genomic islands. The ability to integrate large DNA sequences into target sites is very important for genetic engineering in prokaryotic and eukaryotic cells. Here, we characterized an unprecedented catalogue of 530 tyrosine-type integrases by examining genes potentially encoding tyrosine integrases in bacterial genomic islands. The phylogeny of putative tyrosine integrases revealed that these integrases form an evolutionary clade that is distinct from those already known and are affiliated with novel integrase groups. We systematically searched for candidate integrase genes, and their integration activities were validated in a bacterial model. We verified the integration functions of six representative novel integrases by using a two-plasmid integration system consisting of a donor plasmid carrying the integrase gene and attP site and a recipient plasmid harboring an attB site in recA-deficient Escherichia coli. Further quantitative reverse transcription-PCR (qRT-PCR) assays validated that the six selected integrases can be expressed with their native promoters in E. coli. The attP region reductions showed that the extent of attP sites of integrases is approximately 200 bp for integration capacity. In addition, mutational analysis showed that the conserved tyrosine at the C terminus is essential for catalysis, confirming that these candidate proteins belong to the tyrosine-type recombinase superfamily, i.e., tyrosine integrases. This study revealed that the novel integrases from bacterial genomic islands have site-specific recombination functions, which is of physiological significance for their genomic islands in bacterial chromosomes. More importantly, our discovery expands the toolbox for genetic engineering, especially for efficient integration activity. IMPORTANCE Site-specific recombinases or integrases have high specificity for DNA large fragment integration, which is urgently needed for gene editing. However, known integrases are not sufficient for meeting multiple integrations. In this work, we discovered an array of integrases through bioinformatics analysis in bacterial genomes. Phylogeny and functional assays revealed that these new integrases belong to tyrosine-type integrases and have the ability to conduct site-specific recombination. Moreover, attP region extent and catalysis site analysis were characterized. Our study provides the methodology for discovery of novel integrases and increases the capacity of weapon pool for genetic engineering in bacteria.

miR-302b promotes bovine preadipocyte differentiation and inhibits proliferation by targeting CDK2.

MicroRNAs have been recently reported to act as key regulators of adipogenesis, a multifactorial complex process. One miRNA, miR-302b, is an important regulator of cell proliferation and differentiation and controls cancer development, but we speculate that miR-302b may also regulate bovine adipogenesis. Herein we have evaluated the role of this miRNA in bovine adipocyte differentiation using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), Oil Red O staining, a dual-luciferase reporter. CDK2 was identified as the target gene of miR-302b, and miR-302b agomir promoted mRNA and protein expression levels of adipocyte-specific genes. In addition, a CCK-8 kit was used to show that miR-302b agomir, but not the negative control, inhibits preadipocyte proliferation. In conclusion, miR-302b promotes bovine preadipocyte differentiation and inhibits proliferation by targeting CDK2.

Research progress of intramuscular fat formation based on co-culture.

Intramuscular fat (IMF) is closely related to the meat quality of livestock and poultry. As a new cell culture technique in vitro, cell co-culture has been gradually applied to the related research of IMF formation because it can simulate the changes of microenvironment in vivo during the process of IMF cell formation. In the co-culture model, in addition to studying the effects of skeletal muscle cells on the proliferation and differentiation of IMF, we can also consider the role of many secretion factors in the formation of IMF, thus making the cell research in vitro closer to the real level in vivo. This paper reviewed the generation and origin of IMF, summarized the existing co-culture methods and systems, and discussed the advantages and disadvantages of each method as well as the challenges faced in the establishment of the system, with emphasis on the current status of research on the formation of IMF for human and animal based on co-culture technology.

Efficient Suppression of Natural Plasmid-Borne Gene Expression in Carbapenem-Resistant Klebsiella pneumoniae Using a Compact CRISPR Interference System.

There is an urgent need for efficient tools for genetic manipulation to assess plasmid function in clinical drug-resistant bacterial strains. To address this need, we developed an all-in-one CRISPR interference (CRISPRi) system that easily inhibited the gene expression of a natural multidrug-resistant plasmid in an sequence type 23 (ST23) Klebsiella pneumoniae isolate. We established an integrative CRISPRi system plasmid, pdCas9gRNA, harboring a dcas9 gene and a single guide RNA (sgRNA) unit under the control of anhydrotetracycline-induced and J23119 promoters, respectively, using a one-step cloning method. This system can repress the single resistance gene blaNDM-1, with a >1,000-fold reduction in the meropenem MIC, or simultaneously silence the resistance genes blaNDM-1 and blaSHV-12, with a 16-fold and 8-fold respective reduction in the meropenem and aztreonam MIC on a large natural multidrug-resistant pNK01067-NDM-1 plasmid in an ST23 K. pneumoniae isolate. Furthermore, an sgRNA targeting the blaNDM-1 promoter region can silence the entire blaNDM-1-bleMBL-trpF operon, confirming the existence of the operon. We also used this tool to knock down the multicopy resistance gene blaKPC-2 in pathogenic Escherichia coli, increasing the susceptibility to meropenem. In a word, the all-in-one CRISPRi system can be used for efficient interrogation of indigenous plasmid-borne gene functions, providing a rapid, easy genetic manipulation tool for clinical K. pneumoniae isolates.

Sphingomonas baiyangensis sp. nov., isolated from water in Baiyang Lake.

A novel Gram-stain-negative, short-rod-shaped, orange-pigmented bacterial strain, designated L-1-4 w-11T, was isolated from Baiyang Lake in China. The strain grew at 15-35 °C (optimum 30 °C) and pH 7-8 (optimum pH 7) in TSA medium. The predominant polar lipids of strain L-1-4 w-11T were sphingoglycolipid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid and three unidentified lipids; the major cellular fatty acids were C17 : 1ω6c and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c); and the major respiratory quinone was ubiquinone 10 (Q-10). Strain L-1-4 w-11T showed the highest 16S rRNA gene sequence similarity to Sphingomonas japonica JCM 15438T (98.3 %) and S. spermidinifaciens GDMCC 1.657T (98.0 %). The draft genome size of strain L-1-4 w-11T was 3.3 Mbp, and the G+C content was 67.8 mol%. Digital DNA-DNA hydridization and average nucleotide identity values between the genome sequences of strain L-1-4 w-11T and S. spermidinifaciens GDMCC 1.657T (76.9 and 21.0 %), S. japonica JCM 15438T (76.0 and 19.9 %) and S. paucimobilis CGMCC 1.12825T (72.8 and 19.6 %) were far below the thresholds for prokaryotic conspecific assignment. With the evidence from the phylogenetic, chemotaxonomic and genotypic analyses, we propose that strain L-1-4 w-11T represents a novel Sphingomonas species with the name S. baiyangensis sp. nov. The type strain is L-1-4 w-11T (=CGMCC 1.13572T=JCM 33962T).

Distinct dynamics of Vibrio parahaemolyticus populations in two farming models.

AIMS: Despite the recent prosperity of shrimp cultivation in China, very little is known about how different shrimp farming models influence the dynamics of Vibrio parahaemolyticus populations and the antibiotic resistance of this bacterium. METHODS AND RESULTS: To this end, we conducted continuous surveillance of V. parahaemolyticus on four farms over 3 years: two traditional shrimp farms with daily water exchange and two farms operated in the recirculating aquaculture systems (RAS). No antibiotics were used in these farms to exclude the potential impacts of antibiotics on the emergence of antibacterial resistance. Multilocus sequence typing was utilized to characterize the dynamics of V. parahaemolyticus populations. Whole-genome sequencing (WGS) was conducted to determine the representative sequence types (STs) at each farm. Results revealed that the population structure of V. parahaemolyticus remained stable over time in both RAS farms, with only nine and four STs observed at each. In contrast, annual replacement of V. parahaemolyticus populations was observed in traditional farms with 26 and 28 STs identified in rearing water. WGS of 50 isolates divided them into five clusters, of which ST917a isolates harboured a genomic island that disrupted the gene recA. Pair-wised genomic comparison of isolates from the same STs showed that they were genetically related but belonged to different clones associated with geographical distribution. CONCLUSIONS: These results suggested that RAS presented a specific ecological niche by minimizing the water exchanges with the external environment. In contrast, traditional farming might pose a food safety issue by introducing new V. parahaemolyticus populations with antibiotic resistance genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results expose the potential food safety issue associated with conventional agriculture and should encourage the development of preventive strategies to reduce the emergence of resistant V. parahaemolyticus populations.

Roles of MEF2A and HOXA5 in the transcriptional regulation of the bovine FoxO1 gene.

The Forkhead box factor 1 (FoxO1) gene plays a vital role in the growth and development of skeletal muscle. In the present study, expression analysis of the bovine FoxO1 gene exhibited the highest expression in longissimus dorsi muscle followed by its expression in adipose tissue. Moreover, high mRNA expression of FoxO1 gene was found in differentiated bovine myoblasts and adipocytes at day 6 of induced differentiation (p < 0.05). The regulatory pattern of the bovine FoxO1 gene was investigated through screening and dual-luciferase activity of the 1.7 kb 5'UTR (untranslated region) within pGL3-basic vector and a core promoter region was explored at (-285/-27) upstream of the transcription start site. The transcription factors (TFs) MEF2A and HOXA5 within the core promoter region (-285/-27) were found as the regulatory cis-acting element. The siRNA interference of the TFs, chromatin immunoprecipitation (ChIP) assay, and site-directed mutation validated that MEF2A and HOXA5 binding occurs in the region -285/-27 bp and performs an essential role in the transcriptional regulation of bovine FoxO1 gene. These findings explored the regulatory network mechanism of the FoxO1 gene in skeletal muscle development and adipogenesis for the bovine breed improvement program.

Genome-wide identification of cyclin-dependent kinase (CDK) genes affecting adipocyte differentiation in cattle.

BACKGROUND: Cyclin-dependent kinases (CDKs) are protein kinases regulating important cellular processes such as cell cycle and transcription. Many CDK genes also play a critical role during adipogenic differentiation, but the role of CDK gene family in regulating bovine adipocyte differentiation has not been studied. Therefore, the present study aims to characterize the CDK gene family in bovine and study their expression pattern during adipocyte differentiation. RESULTS: We performed a genome-wide analysis and identified a number of CDK genes in several bovine species. The CDK genes were classified into 8 subfamilies through phylogenetic analysis. We found that 25 bovine CDK genes were distributed in 16 different chromosomes. Collinearity analysis revealed that the CDK gene family in Bos taurus is homologous with Bos indicus, Hybrid-Bos taurus, Hybrid Bos indicus, Bos grunniens and Bubalus bubalis. Several CDK genes had higher expression levels in preadipocytes than in differentiated adipocytes, as shown by RNA-seq analysis and qPCR, suggesting a role in the growth of emerging lipid droplets. CONCLUSION: In this research, 185 CDK genes were identified and grouped into eight distinct clades in Bovidae, showing extensively homology. Global expression analysis of different bovine tissues and specific expression analysis during adipocytes differentiation revealed CDK4, CDK7, CDK8, CDK9 and CDK14 may be involved in bovine adipocyte differentiation. The results provide a basis for further study to determine the roles of CDK gene family in regulating adipocyte differentiation, which is beneficial for beef quality improvement.

ncRNAs regulate bovine adipose tissue deposition.

Lipid metabolism, which encompasses synthesis and degradation of lipids, is critical for a wide range of cellular functions, including structural and morphological properties of organelles, energy storage, signalling, and the stability and function of membrane proteins. Adipose tissue is a dynamic tissue type that performs a lot of significant physiological functions, including secretion, and is involved in maintaining homeostasis and in regulatory roles of other tissues based on paracrine or endocrine. More recently, several classes of non-coding RNAs (ncRNAs), such as long non-coding RNA (lncRNA), microRNA (miRNA) and circular RNA (circRNA), have been discovered in adipocytes, and they act as critical regulators of gene expression in adipogenesis and regulate adipogenesis through multiple pathways. In the present paper, we discussed several classes of non-coding RNAs and summarized the latest research on the regulatory role of ncRNAs in bovine adipogenesis. We gave examples for known modes of action to look forward to providing reference information future scientific research in cattle breeding.

MiR-125b regulates inflammation in bovine mammary epithelial cells by targeting the NKIRAS2 gene.

Mastitis is a complex inflammatory disease caused by pathogenic infection of mammary tissue in dairy cows. The molecular mechanism behind its occurrence, development, and regulation consists of a multi-gene network including microRNA (miRNA). Until now, there is no report on the role of miR-125b in regulating mastitis in dairy cows. This study found that miR-125b expression is significantly decreased in lipopolysaccharide (LPS)-induced MAC-T bovine mammary epithelial cells. Also, its expression is negatively correlated with the expression of NF-κB inhibitor interacting Ras-like 2 (NKIRAS2) gene. MiR-125b target genes were identified using a double luciferase reporter gene assay, which showed that miR-125b can bind to the 3' untranslated region (3' UTR) of the NKIRAS2, but not the 3'UTR of the TNF-α induced protein 3 (TNFAIP3). In addition, miR-125b overexpression and silencing were used to investigate the role of miR-125b on inflammation in LPS-induced MAC-T. The results demonstrate that a reduction in miR-125b expression in LPS-induced MAC-T cells increases NKIRAS2 expression, which then reduces NF-κB activity, leading to low expression of the inflammatory factors IL-6 and TNF-α. Ultimately, this reduces the inflammatory response in MAC-T cells. These results indicate that miR-125b is a pro-inflammatory regulator and that its silencing can alleviate bovine mastitis. These findings lay a foundation for elucidating the molecular regulation mechanism of cow mastitis.

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.

Genome-wide association studies reveal novel loci associated with carcass and body measures in beef cattle.

Genomic selection has an essential role in the livestock economy by increasing selection productivity. Genomics provides a mechanism to increase the rate of genetic gain using marker-assisted selection. Various quantitative trait loci (QTL) associated with body, carcass and meat quality traits in beef cattle have been found. It is widely accepted that QTL traits in livestock species are regulated by several genes and factors from the environment. Genome-wide association studies (GWAS) are a powerful approach in identifying QTL and to establish genomic regions harboring the genes and polymorphisms associated with specific characteristics in beef cattle. Due to their impact on economic returns, growth, carcass and meat quality traits of cattle are frequently used as essential criteria in selection in breeding programs., GWAS has been used in beef cattle breeding and genetic program and some progress has been made. Furthermore, numerous genes and markers related to productivity traits in beef cattle have been found. This review summarizes the advances in the use of GWAS in beef cattle production and outlines the associations with growth, carcass, and meat quality.

Lateral force calibration for atomic force microscope cantilevers using a suspended nanowire.

Friction measurement via atomic force microscope (AFM) relies on accurate calibration for the torsional spring constant of the AFM cantilever and its lateral deflection sensitivity. Here we describe a method that employs a suspended nanowire (SNW) as a reference beam to quantify the torsional spring constant of AFM cantilevers. Based on the fact that a uniform SNW with cylindrical symmetry has an identical spring constant when bent in any direction perpendicular to its axis, the spring constant of the SNW in a normal direction is determined by an AFM cantilever with a known normal spring constant, and is subsequently used as a force transfer standard to calibrate the torsional spring constant of the AFM cantilever. The lateral deflection sensitivity can be accurately measured by pushing the AFM tip laterally on the groove edge. The calibration result is compared to the well-known diamagnetic lateral force calibrator method and shows an uncertainty of 15% or better. The presented method is applicable for the lateral force calibration of AFM cantilevers in a wide range of instruments including inverted configurations and in an ultrahigh vacuum.

Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis.

Type VI secretion system (T6SS) is a versatile protein export machinery widely distributed in Gram-negative bacteria. Known to translocate protein substrates to eukaryotic and prokaryotic target cells to cause cellular damage, the T6SS has been primarily recognized as a contact-dependent bacterial weapon for microbe-host and microbial interspecies competition. Here we report contact-independent functions of the T6SS for metal acquisition, bacteria competition, and resistance to oxidative stress. We demonstrate that the T6SS-4 in Burkholderia thailandensis is critical for survival under oxidative stress and is regulated by OxyR, a conserved oxidative stress regulator. The T6SS-4 is important for intracellular accumulation of manganese (Mn2+) under oxidative stress. Next, we identified a T6SS-4-dependent Mn2+-binding effector TseM, and its interacting partner MnoT, a Mn2+-specific TonB-dependent outer membrane transporter. Similar to the T6SS-4 genes, expression of mnoT is regulated by OxyR and is induced under oxidative stress and low Mn2+ conditions. Both TseM and MnoT are required for efficient uptake of Mn2+ across the outer membrane under Mn2+-limited and -oxidative stress conditions. The TseM-MnoT-mediated active Mn2+ transport system is also involved in contact-independent bacteria-bacteria competition and bacterial virulence. This finding provides a perspective for understanding the mechanisms of metal ion uptake and the roles of T6SS in bacteria-bacteria competition.

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory.

Characterization of the promoter region of bovine SIX4: Roles of E-box and MyoD in the regulation of basal transcription.

The sine oculis homeobox 4 (SIX4) gene belongs to the Six gene family and encodes an evolutionarily conserved transcription factor. Previous studies have demonstrated that SIX4 plays an essential role in proper muscle regeneration. However, the mechanisms regulating SIX4 transcription remain elusive. In the present study, we determined that bovine SIX4 was highly expressed in the longissimus thoracis and in undifferentiated Qinchuan cattle muscle cells (QCMCs) and that its protein localizes to both the cytoplasm and the nucleus. To elucidate the bovine the molecular mechanisms of SIX4 regulation, 1.3 kb of the 5'-regulatory region was obtained. MyoD and Ebox recognition sites were identified in the core promoter region at -522/-193 of the bovine SIX4 using a series of 5' deletion promoter plasmids in luciferase reporter assays. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with site-directed mutation and siRNA interference demonstrated that MyoD binding occurs at MyoD and Ebox recognition sites through direct and indirect mechanisms and play important roles in the transcriptional regulation of the bovine SIX4 promoter. Taken together, these interactions provide insight into the regulatory mechanisms of SIX4 transcription in mediating skeletal muscle growth in cattle.

Bta-miR-130a/b regulates preadipocyte differentiation by targeting PPARG and CYP2U1 in beef cattle.

Deposition of intramuscular fat (IMF) is one of the most important traits for the evaluation of beef carcass quality grade. MicroRNA (miRNA) is an endogenous non-coding RNA that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. Previously, we identified that bta-miR-130a regulates milk fat biosynthesis by targeting PPARG mRNA. However, the role of miR-130 in the regulation of bovine adipocyte differentiation remains unknown. In this study, we found that overexpression of bta-miR-130a/b led to significantly decreased cellular triacylglycerol (TAG) levels during adipogenesis process as well as reduced lipid droplet formation. In contrast, the inhibition of bta-miR-130a/b resulted in larger lipid droplets and TAG accumulation. In addition, overexpression of bta-miR-130a/b inhibited the expression of adipocyte differentiation-related genes, including PPARG, C/EBPα, C/EBPß, FABP4, LPIN1, and LPL. Western blot analysis verified qPCR results on the expression of PPARG and CYP2U1. A luciferase reporter assay further verified bta-miR-130a/b significantly affects PPARG and CYP2U1 expression by directly binding to their 3'-untranslated regions (UTR). We conducted in vitro rescue assay to confirm that bta-miR-130a/b affect bovine adipocyte differentiation by targeting PPARG and CYP2U1. This study shows that bta-miR-130a and bta-miR-130b play similar roles in the regulation of adipocyte differentiation in beef muscles by targeting the 3'UTR of PPARG and CYP2U1. Our result provides a reference for illustrating how noncoding RNAs affects beef quality traits in cattle.

Transcriptional Regulation by CpG Sites Methylation in the Core Promoter Region of the Bovine SIX1 Gene: Roles of Histone H4 and E2F2.

DNA methylation is a major epigenetic modification of the genome and has an essential role in muscle development. The SIX1 gene is thought to play a principal role in mediating skeletal muscle development. In the present study, we determined that bovine SIX1 expression levels were significantly higher in the fetal bovine group (FB) and in undifferentiated Qinchuan cattle muscle cells (QCMCs) than in the adult bovine group (AB) and in differentiated QCMCs. Moreover, a bisulfite sequencing polymerase chain reaction (BSP) analysis of DNA methylation levels showed that three CpG sites in the core promoter region (-216/-28) of the bovine SIX1 gene exhibited significantly higher DNA methylation levels in the AB and differentiated QCMCs groups. In addition, we found that DNA methylation of SIX1 core promoter in vitro obviously influences the promoter activities. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, in combination with site-directed mutation and siRNA interference, demonstrated that histone H4 and E2F2 bind to the -216/-28 region and play important roles in SIX1 methylation regulation during development. The results of this study provide the foundation for a better understanding of the regulation of bovine SIX1 expression via methylation and muscle developmental in beef cattle.