ABSTRACT
Beef is a major global source of protein, playing an essential role in the human diet. The worldwide production and consumption of beef continue to rise, reflecting a significant trend. However, despite the critical importance of beef cattle resources in agriculture, the diversity of cattle breeds faces severe challenges, with many breeds at risk of extinction. The initiation of the Beef Cattle Genome Project is crucial. By constructing a high-precision functional annotation map of their genome, it becomes possible to analyze the genetic mechanisms underlying important traits in beef cattle, laying a solid foundation for breeding more efficient and productive cattle breeds. This review details advances in genome sequencing and assembly technologies, iterative upgrades of the beef cattle reference genome, and its application in pan-genome research. Additionally, it summarizes relevant studies on the discovery of functional genes associated with key traits in beef cattle, such as growth, meat quality, reproduction, polled traits, disease resistance, and environmental adaptability. Finally, the review explores the potential of telomere-to-telomere (T2T) genome assembly, structural variations (SVs), and multi-omics techniques in future beef cattle genetic breeding. These advancements collectively offer promising avenues for enhancing beef cattle breeding and improving genetic traits.
Subject(s)
Genome , Animals , Cattle/genetics , Genomics/methods , Breeding/methods , Whole Genome Sequencing/methods , Red Meat , Quantitative Trait LociABSTRACT
The Chungtien schizothoracin (Ptychobarbus chungtienensis), an endangered fish species endemic to the Zhongdian Plateau, remains underexplored in terms of transcriptomic sequencing. This investigation used tissues from five distinct organs (heart, liver, spleen, kidney, and brain) of the Chungtien schizothoracin for PacBio Iso-seq and RNA-seq analyses, yielding a repertoire of 16,598 full-length transcripts spanning lengths from 363 bp to 7,157 bp. Gene family clustering and phylogenetic analysis encompassed a comprehensive set of 13 fish species, all of which were cyprinids, including the zebrafish and the examined species Ptychobarbus chungtienensis. Moreover, the identification of long non-coding RNAs (lncRNAs) and coding sequences was accomplished across all five tissues. Comprehensive analyses of gene expression profiles and differentially expressed genes among the above five tissues were performed. In summary, the obtained full-length transcripts and detailed gene expression profiles of the Chungtien schizothoracin tissues furnish crucial expression data and genetic sequences, laying the groundwork for future investigations and fostering a holistic comprehension of the adaptive mechanisms inherent in the Chungtien schizothoracin under various conditions.
Subject(s)
Cyprinidae , Phylogeny , Transcriptome , Animals , Cyprinidae/genetics , RNA-Seq , RNA, Long Noncoding/genetics , Endangered SpeciesABSTRACT
The development and characteristics of muscle fibers in broilers are critical determinants that influence their growth performance, as well as serve as essential prerequisites for the production of high-quality chicken meat. Guanidinoacetic acid (GAA) is a crucial endogenous substance in animal creatine synthesis, and its utilization as a feed additive has been demonstrated the capabilities to enhance animal performance, optimize muscle yield, and augment carcass quality. The objective of this study was to investigate the regulation and molecular mechanism underlying muscle development in broilers at different levels of GAA via multiple omics analysis. The 90 Cobb broilers, aged 1 day, were randomly allocated into three treatments consisting of five replicates of six chickens each. The control group was provided with a basal diet, while the Normal GAA and High GAA groups received a basal diet supplemented with 1.2 g/kg and 3.6 g/kg of GAA, respectively. After a feeding period of 42 days, the pectoralis muscles were collected for histomorphological observation, transcriptome and metabolomic analysis. The results demonstrated that the addition of 1.2 g/kg GAA in the diet led to an augmentation in muscle fiber diameter and up-regulation of IGF1, IHH, ASB2, and ANKRD2 gene expression. However, a high dose of 3.6 g/kg GAA in the diet potentially reversed the beneficial effects on chicken breast development by excessively activating the TGF-ß signaling pathway and reducing nucleotide metabolite content. These findings would provide a theoretical foundation for enhancing the performance and meat quality of broilers by incorporating GAA as a feed additive.
ABSTRACT
Multi-omics-integrated analysis, known as panomics, represents an advanced methodology that harnesses various high-throughput technologies encompassing genomics, epigenomics, transcriptomics, proteomics, and metabolomics. Sheep, playing a pivotal role in agricultural sectors due to their substantial economic importance, have witnessed remarkable advancements in genetic breeding through the amalgamation of multiomics analyses, particularly with the evolution of high-throughput technologies. This integrative approach has established a robust theoretical foundation, enabling a deeper understanding of sheep genetics and fostering improvements in breeding strategies. The comprehensive insights obtained through this approach shed light on diverse facets of sheep development, including growth, reproduction, disease resistance, and the quality of livestock products. This review primarily focuses on the application of principal omics analysis technologies in sheep, emphasizing correlation studies between multiomics data and specific traits such as meat quality, wool characteristics, and reproductive features. Additionally, this paper anticipates forthcoming trends and potential developments in this field.
ABSTRACT
Preadipocyte differentiation represents a critical stage in adipogenesis, with mitochondria playing an undeniable pivotal role. Given the intricate interplay between transcription and metabolic signaling during adipogenesis, the regulation of sirtuin 5 (SIRT5) on mitochondrial function and lipid metabolism was revealed via multiple omics analysis. The findings suggest that SIRT5 plays a crucial role in promoting mitochondrial biosynthesis and maintaining mitochondrial function during preadipocyte differentiation. Moreover, SIRT5 modulates the metabolic levels of numerous bioactive substances by extensively regulating genes expression associated with differentiation, energy metabolism, lipid synthesis, and mitochondrial function. Finally, SIRT5 was found to suppress triacylglycerols (TAG) accumulation while enhancing the proportion and diversity of unsaturated fatty acids, and providing conditions for the expansion and stability of membrane structure during mitochondrial biosynthesis through numerous gene regulations. Our findings provide a foundation for the identification of crucial functional genes, signaling pathways, and metabolic substances associated with adipose tissue differentiation and metabolism.
Subject(s)
Lipid Metabolism , Sirtuins , Cattle , Animals , Sirtuins/genetics , Sirtuins/metabolism , Adipogenesis , Mitochondria/genetics , Adipose Tissue/metabolismABSTRACT
As an exemplary model for examining molecular mechanisms responsible for extreme phenotypic variations, plumage color has garnered significant interest. The Cygnus genus features two species, Cygnus olor and Cygnus atratus, that exhibit striking disparities in plumage color. However, the molecular foundation for this differentiation has remained elusive. Herein, we present two high-quality genomes for C. olor and C. atratus, procured using the Illumina and Nanopore technologies. The assembled genome of C. olor was 1.12 Gb in size with a contig N50 of 26.82 Mb, while its counterpart was 1.13 Gb in size with a contig N50 of 21.91 Mb. A comparative analysis unveiled three genes (TYR, SLC45A2, and SLC7A11) with structural variants in the melanogenic pathway. Notably, we also identified a novel gene, PWWP domain containing 2A (PWWP2A), that is related to plumage color, for the first time. Using targeted gene modification analysis, we demonstrated the potential genetic effect of the PWWP2A variant on pigment gene expression and melanin production. Finally, our findings offer insight into the intricate pattern of pigmentation and the role of polygenes in birds. Furthermore, these two high-quality genome references provide a comprehensive resource and perspective for comparative functional and genetic studies of evolution within the Cygnus genus.
Subject(s)
Birds , Genome , Animals , GenomicsABSTRACT
Mongolian cattle (MG, Bos taurus) and Minnan cattle (MN, Bos indicus) are two different breeds of Chinese indigenous cattle, representing North type and South type, respectively. However, their value and potential have not yet been discovered at the genomic level. In this study, 26 individuals of MN and MG were sequenced for the first time at an average of 13.9- and 12.8-fold, respectively. Large numbers of different variations were identified. In addition, the analyses of phylogenetic and population structure showed that these two cattle breeds are distinct from each other, and results of linkage disequilibrium analysis revealed that these two cattle breeds have undergone various degrees of intense natural or artificial selection. Subsequently, 496 and 306 potential selected genes (PSRs) were obtained in MN and MG, containing 1,096 and 529 potential selected genes (PSGs), respectively. These PSGs, together with the analyzed copy number variation (CNV)-related genes, showed potential relations with their phenotypic characteristics, including environmental adaptability (e.g., DVL2, HSPA4, CDHR4), feed efficiency (e.g., R3HDM1, PLAG1, XKR4), and meat/milk production (e.g., PDHB, LEMD3, APOF). The results of this study help to gain new insights into the genetic characteristics of two distinct cattle breeds and will contribute to future cattle breeding.
ABSTRACT
This study investigated the effect of ELOVL6 (elongation of very long chain fatty acids protein 6) and its underlying mechanism on lipid metabolism in bovine adipocytes. The ELOVL6 gene was overexpressed in bovine adipocytes by adenoviruses, and RNA sequencing was performed. Overexpression of ELOVL6 showed reduced proportions of C14:0 (Myristic) and C16:0 (palmitate) fatty acids and increased proportions of C18.0 (stearate) and C20:4n6 (arachidonic) fatty acids in adipocytes. In addition, a total of 2170 differentially expressed genes (DEGs) were found, containing 1802 up-regulated and 368 down-regulated genes. KEGG pathway analysis revealed that the down-regulated genes were linked with the regulation of lipolysis and the Wnt signaling pathway. The up-regulated genes were mainly involved in the FoxO signaling pathway; the PI3K-Akt signaling pathway; and the cAMP signaling pathway. In conclusion, our results suggest that ELOVL6 could affect the fatty acid composition in bovine adipocytes. We identified numerous related DEGs and pathways, which may provide a basis for studying the function and molecular mechanism of the ELOVL6 gene in regulating lipid metabolism.
Subject(s)
Adipocytes/metabolism , Cattle/metabolism , Fatty Acid Elongases/metabolism , Lipid Metabolism , Adipocytes/chemistry , Animals , Cattle/genetics , Cells, Cultured , Fatty Acid Elongases/chemistry , Fatty Acid Elongases/genetics , Fatty Acids/analysis , Gene Expression , High-Throughput Nucleotide Sequencing , Lipid Metabolism/genetics , Lipolysis/genetics , Phylogeny , Sequence Alignment , Sequence Analysis, ProteinABSTRACT
Preadipocyte differentiation and lipid synthesis are critical steps for intramuscular fat (IMF) deposition and lipid metabolism homeostasis. IMF content of beef not only determines the ratio of muscle to adipose, but also determines the beef quality, flavor, and sensory characteristics. Maintaining lipid metabolism homeostasis is the key means of preventing and treating diabetes, obesity, and other metabolic diseases. SIRT6, which is an ADP-ribosyltransferase and NAD+-dependent deacetylase of acetyl and long-chain fatty acyl groups, playing central roles in lipid and glucose metabolism, is closely related to the occurrence of diabetes and obesity caused by overnutrition and aging. This study was based on bovine preadipocyte differentiation and an obese mice model, and comprehensively used transcriptome sequencing (RNA-seq) and morphological identification methods to explore the effects of inhibition of SIRT6 on differentiation and lipid synthesis, and related molecular mechanisms. Additionally, the feedback synergistic regulation of SIRT5 and SIRT6 on differentiation and lipid deposition was analyzed. The results showed that in the differentiation process of bovine preadipocytes, inhibition of SIRT5 significantly promoted SIRT6 expression. In addition, SIRT6 inhibited bovine preadipocyte differentiation and lipid synthesis, cooperating with SIRT5 to decrease lipid deposition, and repressed cell cycle arrest of preadipocytes. Moreover, in vivo verification experiments also obtained consistent results. Furthermore, SIRT6 inhibited preadipocyte differentiation and lipid deposition by activating the adenosine monophosphate activated protein kinase alpha (AMPKα) pathway. The above results provided a novel approach for understanding the functions of SIRT6 in regulating bovine adipocyte differentiation and lipid metabolism, as well as a new target for the treatment of diabetes and obesity in a clinical setting.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Adipogenesis , Lipid Metabolism , Signal Transduction , Sirtuins/metabolism , Adipocytes/cytology , Animals , Cattle , Cell Differentiation , Cells, CulturedABSTRACT
SIRT5 (sirtuin 5) is located in the mitochondria and plays an important role in biological processes such as maintaining the balance of lipid metabolism and promoting fatty acid oxidation mobilization. In this study, the bovine preadipocyte differentiation and obese mouse models were constructed; combined with transcriptome sequencing (RNA-seq) and morphological identification, the regulatory and molecular mechanisms underlying the effects of SIRT5 on bovine preadipocyte differentiation and lipid metabolism were studied. The results reveal that during the differentiation of preadipocytes, SIRT5 inhibited the expression of key genes that promote lipid formation and differentiation in fatty acid biosynthesis and PPAR pathways. SIRT5 significantly activated the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway and repressed the mitogen-activated protein kinase (MAPK) pathway. Eventually, SIRT5 significantly inhibited the differentiation of bovine preadipocytes and simultaneously inhibited lipid synthesis and lipid deposition in adipocytes. The verification experiments performed using obese mice also yielded consistent results in vivo.
Subject(s)
Adipocytes/metabolism , Cell Differentiation , Lipid Metabolism , MAP Kinase Signaling System , Protein Kinases/metabolism , Sirtuins/metabolism , AMP-Activated Protein Kinase Kinases , Adipocytes/cytology , Animals , Cattle , Cells, Cultured , HEK293 Cells , Humans , Male , MiceABSTRACT
The elongation of very long chain fatty acids protein 6 (ELOVL6) gene encodes a key enzyme that plays a role in lipogenesis through the catalytic elongation of both saturated and monounsaturated fatty acids. Previous studies have described the high expression of bovine ELOVL6 in adipose tissues. However, transcriptional regulation and the functional role of ELOVL6 in lipid metabolism and adipocyte proliferation remain unexplored. Here, a 1.5 kb fragment of the 5'-untranslated region promoter region of ELOVL6 was amplified from the genomic DNA of Qinchuan cattle and sequenced. The core promoter region was identified through unidirectional 5'-end deletion of the promoter plasmid vector. In silico analysis predicted important transcription factors that were then validated through site-directed mutation and small interfering RNA interference with an electrophoretic mobility shift assay. We found that the binding of KLF6 and PU.1 transcription factors occurred in the region -168/+69. Both perform a vital regulatory function in the transcription of bovine ELOVL6. Overexpression of ELOVL6 significantly upregulated the expression of peroxisome proliferator activated receptor γ (PPARγ), but inhibited the expression of fatty acid-binding protein 4 (FABP4), while silencing of ELOVL6 negatively regulated the messenger RNA expression level of PPARγ, FABP4, ACSL, and FATP1. In addition, ELOVL6 promotes adipocyte proliferation by regulating the cell-cycle genes' expression. Taken together, these findings provide useful information about the transcriptional regulation and functional mechanisms of bovine ELOVL6 in lipid metabolism and adipocyte proliferation in Qinchuan cattle.
Subject(s)
Adipocytes/cytology , Adipocytes/metabolism , Fatty Acid Elongases/genetics , Gene Expression Regulation , Lipid Metabolism/genetics , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Cattle , Cell Proliferation/genetics , Fatty Acid Elongases/metabolism , Kruppel-Like Factor 6/metabolism , Promoter Regions, Genetic , Protein Binding/genetics , Proto-Oncogene Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/metabolism , Sequence Deletion , Subcellular Fractions/metabolism , Trans-Activators/metabolismABSTRACT
Sirtuin 4 (SIRT4) belongs to the mitochondrial sirtuin protein family, a class of NAD+-dependent protein deacylases that remove post-translational acyl modifications from cellular substrates during the regulation of various biological pathways. SIRT4 has been shown to regulate lipid homeostasis. However, the mechanism by which the bovine SIRT4 gene is transcriptionally regulated remains unknown. To explore the molecular mechanism of SIRT4 expression, we obtained a 400-kb fragment of the 5'-regulatory region of bovine SIRT4 by molecular cloning, which contained a CpG island. Electrophoretic mobility shift assays and luciferase reporter gene assays identified the nuclear respiratory factor 1 (NRF1) and myb proto-oncogene protein (CMYB) binding sites as transcriptional repression and activation sites in the SIRT4 promoter region, respectively. We further verified that NRF1 and CMYB bind to the SIRT4 promoter using chromatin immunoprecipitation assays. In addition, from DNA methylation and reporter gene assays, results revealed that SIRT4 promoter activity was enhanced by demethylation. Further, NRF1-mediated transcriptional inhibition and CMYB-mediated transcriptional activation of SIRT4 expression were strengthened by demethylation during bovine adipocyte differentiation. Taken together, our results shed light on the mechanism underlying the promoter methylation and transcriptional regulation of SIRT4 expression in bovine adipocytes.
Subject(s)
Adipocytes/metabolism , DNA Methylation/genetics , Mitochondrial Proteins/metabolism , Nuclear Respiratory Factor 1/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Animals , Cattle , Cell Culture Techniques , Chromatin Immunoprecipitation/methods , Cloning, Organism/methods , Electrophoretic Mobility Shift Assay/methods , Mutagenesis, Site-Directed/methods , Promoter Regions, Genetic , Real-Time Polymerase Chain ReactionABSTRACT
Sirtuin 5 (SIRT5) belongs to the mitochondrial sirtuin family, which constitutes a highly conserved family of nicotinamide adenine dinucleotide NAD+-dependent deacetylases and ADP-ribosyltransferases that play important regulatory roles in stress resistance and metabolic homeostasis. SIRT5 was shown to have deacetylase, desuccinylase, and demalonylase activities. However, the mechanisms regulating SIRT5 transcription remain unclear. To explore the molecular regulation of bovine SIRT5 expression, we obtained a 500-base pair fragment of the 5'-regulatory region of bovine SIRT5 by molecular cloning, which contained a region with 3 CpG islands. Electrophoretic mobility shift assays and luciferase reporter assays revealed the E2F transcription factor 4 (E2F4) and Kruppel-like factor 6 (KLF6) binding sites as transcriptional activators or repressors in the promoter region of SIRT5. We further verified that E2F4 and KLF6 bind to the SIRT5 promoter by chromatin immunoprecipitation assays. Additionally, methylation and luciferase report assays showed that SIRT5 promoter activity was enhanced by demethylation, and transcriptional activation by E2F4 and transcriptional inhibition by KLF6 of SIRT5 expression was strengthened by demethylation during adipocytes differentiation. This study focused on the mechanism underlying the methylation and transcriptional regulation of SIRT5 expression in bovine adipocytes.
Subject(s)
E2F4 Transcription Factor/metabolism , Kruppel-Like Factor 6/metabolism , Sirtuins/genetics , Adipocytes/metabolism , Animals , Binding Sites/genetics , Cattle , CpG Islands/genetics , DNA Methylation/genetics , E2F4 Transcription Factor/physiology , Electrophoretic Mobility Shift Assay , Gene Expression Regulation/genetics , Kruppel-Like Factor 6/physiology , Kruppel-Like Transcription Factors/genetics , Promoter Regions, Genetic/genetics , Sirtuins/metabolism , Transcription Factors/genetics , Transcriptional ActivationABSTRACT
Sirtuins, NAD+-dependent deacylases and ADP-ribosyltransferases, are critical regulators of metabolism involved in many biological processes, and are involved in mediating adaptive responses to the cellular environment. SIRT4 is a mitochondrial sirtuin and has been shown to play a critical role in maintaining insulin secretion and glucose homeostasis. As a regulator of lipid homeostasis, SIRT4 can repress fatty acid oxidation and promote lipid anabolism in nutrient-replete conditions. Using real-time quantitative PCR (qPCR) to explore the molecular mechanisms of transcriptional regulation of bovine SIRT4 during adipocyte differentiation, we found that bovine SIRT4 is expressed at high levels in bovine subcutaneous adipose tissue. SIRT4 knockdown led to decreased expression of adipogenic differentiation marker genes during adipocyte differentiation. The core promoter of bovine SIRT4 was identified in the -402/-60 bp region of the cloned 2-kb fragment containing the 5'-regulatory region. Binding sites were identified in this region for E2F transcription factor-1 (E2F1), CCAAT/enhancer-binding protein ß (CEBPß), homeobox A5 (HOXA5), interferon regulatory factor 4 (IRF4), paired box 4 (PAX4), and cAMP responsive element-binding protein 1 (CREB1) by using Electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. We also found that E2F1, CEBPß, and HOXA5 transcriptionally activate SIRT4 expression, whereas, IRF4, PAX4, and CREB1 transcriptionally repress SIRT4 expression. We further verified that SIRT4 knockdown could affect the ability of these transcription factors (TFs) to regulate the differentiation of bovine adipocytes. In conclusion, our results shed light on the mechanisms underlying the transcriptional regulation of SIRT4 expression in bovine adipocytes.
Subject(s)
Adipocytes/cytology , Adipogenesis , Promoter Regions, Genetic , Sirtuins/genetics , Transcription Factors/metabolism , Transcriptional Activation , Adipocytes/metabolism , Animals , Base Sequence , Cattle , Cells, Cultured , Gene Expression , Sirtuins/analysisABSTRACT
Sirtuin 5 (SIRT5) belongs to the mitochondrial sirtuin family, which constitutes a highly conserved family of nicotinamide adenine dinucleotide NAD+-dependent deacetylases and ADP-ribosyltransferases that play an important regulatory role in stress resistance and metabolic homeostasis. SIRT5, a member of the mitochondrial sirtuins, has been confirmed to exhibit deacetylase, desuccinylase, and demalonylase enzymatic activities. First, we showed that SIRT5 was expressed at the highest level in the bovine testis, followed by longissimus thoracis and subcutaneous adipose tissue, using real-time quantitative PCR and mRNA levels of SIRT5 during adipocyte differentiation, which increased before the first day and then decreased rapidly. To explore the molecular regulation of bovine SIRT5 expression, we cloned a 2-kb fragment of the 5'-regulatory region and the functional proximal minimal promoter of bovine SIRT5. Electrophoretic mobility shift assays and luciferase reporter assays identified Kruppel-like factor 2 (KLF2), CCAAT enhancer binding protein beta (CEBPß), peroxisome proliferator-activated receptor alpha (PPARα), myogenic differentiation 1 (MYOD), and nuclear respiratory factor 1 (NRF1) binding sites as transcriptional activators or repressors in the core promoter region of SIRT5. In brief, our study focused on the mechanism underlying the transcriptional regulation of SIRT5 expression in bovine adipocytes.
Subject(s)
Adipocytes/metabolism , CCAAT-Enhancer-Binding Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , MyoD Protein/metabolism , Nuclear Respiratory Factor 1/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Sirtuins/genetics , 3T3 Cells , Animals , Cattle , Cell Differentiation , Male , Mice , Protein Binding , Sirtuins/metabolism , Testis/metabolismABSTRACT
Sirtuin6 (SIRT6) is an ADP-ribosyltransferase and NAD+-dependent deacylase of acetyl groups and long-chain fatty acyl groups, and has been shown as a regulator of insulin secretion, glucose metabolism, lipid metabolism, and cancer. In this study, we determined that the bovine SIRT6 showed higher levels of mRNA expression in the testis, longissimus thoracis, and subcutaneous fat tissue. To elucidate the molecular regulation mechanism of bovine SIRT6 expression, we obtained a 2-kb fragment containing the 5'-regulatory region, and the functional proximal minimal promoter of bovine SIRT6 was identified in the -472/-73 bp region. The CCAAT enhancer binding protein beta (CEBPß), paired box 6 (PAX6), Kruppel-like factor 2 (KLF2), myb proto-oncogene protein (CMYB), nuclear respiratory factor 1 (NRF1), and E2F transcription factor 1 (E2F1) binding sites, as transcriptional activators or repressors in the core promoter region of SIRT6, were determined by electrophoretic mobility shift assay (EMSA) experiments and luciferase reporter assays. In addition, the results from methylation assay and luciferase report assay showed that the bovine SIRT6 promoter activity was coordinately regulated by methylation and NRF1 or E2F1 during bovine adipocyte differentiation. Taken together, this study illuminated the underlying mechanism of methylation and transcription regulation of SIRT6 expression in bovine adipocytes.
Subject(s)
Adipocytes/metabolism , DNA Methylation , Promoter Regions, Genetic/genetics , Sirtuins/genetics , Transcription Factors/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Animals , Cattle , Cell Differentiation , Gene Expression Regulation , Intracellular Space/metabolism , Mice , Phylogeny , Protein Transport , Sequence Analysis , Sirtuins/metabolismABSTRACT
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, 1.3â¯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.
Subject(s)
Cattle/genetics , E-Box Elements/genetics , Homeodomain Proteins/genetics , MyoD Protein/genetics , Promoter Regions, Genetic/genetics , Regulatory Elements, Transcriptional/genetics , Transcriptional Activation/genetics , Animals , Gene Expression Regulation/genetics , Trans-Activators/geneticsABSTRACT
Intramuscular fat (IMF) is known to enhance beef palatability and can be markedly increased by castration. However, there is little understanding of the molecular mechanism underlying the IMF deposition after castration of beef cattle. We hypothesize that genetic regulators function differently in IMF from bulls and steers. Therefore, after detecting serum testosterone and lipid parameter, as well as the contents of IMF at 6, 12, 18 and 24 months, we have investigated differentially expressed (DE) microRNAs (miRNAs) and mRNAs in IMF of bulls and steers at 24 months of age in Qinchuan cattle using next-generation sequencing, and then explored the possible biopathways regulating IMF deposition. Serum testosterone levels were significantly decreased in steers, whereas IMF content, serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TGs) were markedly increased in steers. Comparing the results of steers and bulls, 580 upregulated genes and 1,120 downregulated genes in IMF tissues were identified as DE genes correlated with IMF deposition. The upregulated genes were mainly associated with lipid metabolism, lipogenesis and fatty acid transportation signalling pathways, and the downregulated genes were correlated with immune response and intracellular signal transduction. Concurrently, the DE miRNAs-important players in adipose tissue accumulation induced by castration-were also examined in IMF tissues; 52 DE miRNAs were identified. The expression profiles of selected genes and miRNAs were also confirmed by quantitative real-time PCR (qRT-PCR) assays. Using integrated analysis, we constructed the microRNA-target regulatory network which was supported by target validation using the dual luciferase reporter system. Moreover, Ingenuity Pathway Analysis (IPA) software was used to construct a molecular interaction network that could be involved in regulating IMF after castration. The detected molecular network is closely associated with lipid metabolism and adipocyte differentiation, which is supported by functional identification results of bta-let-7i on bovine preadipocytes. These results provided valuable insights into the molecular mechanisms of the IMF phenotype differences between steers and bulls.
Subject(s)
Adipose Tissue/metabolism , Cattle/genetics , MicroRNAs/genetics , Muscle, Skeletal/metabolism , RNA, Messenger/genetics , Transcriptome , Animals , China , Cholesterol/blood , Male , Testosterone/blood , Triglycerides/bloodABSTRACT
The SIX1 gene belongs to the family of six homeodomain transcription factors (TFs), that regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and mediate skeletal muscle growth and regeneration. Previous studies have demonstrated that SIX1 is positively correlated with body measurement traits (BMTs). However, the transcriptional regulation of SIX1 remains unclear. In the present study, we determined that bovine SIX1 was highly expressed in the longissimus thoracis. To elucidate the molecular mechanisms involved in bovine SIX1 regulation, 2-kb of the 5' regulatory region were obtained. Sequence analysis identified neither a consensus TATA box nor a CCAAT box in the 5' flanking region of bovine SIX1. However, a CpG island was predicted in the region -235 to +658 relative to the transcriptional start site (TSS). An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with serial deletion constructs of the 5' flanking region, site-directed mutation and siRNA interference demonstrated that MyoD, PAX7 and CREB binding occur in region -689/-40 and play important roles in bovine SIX1 transcription. In addition, MyoG drives SIX1 transcription indirectly via the MEF3 motif. Taken together these interactions suggest a key functional role for SIX1 in mediating skeletal muscle growth in cattle.