Arginine (315) is required for the PLIN2-CGI-58 interface and plays a functional role in regulating nascent LDs formation in bovine adipocytes.

Perilipin-2 (PLIN2) can anchor to lipid droplets (LDs) and play a crucial role in regulating nascent LDs formation. Bimolecular fluorescence complementation (BiFC) and flow cytometry were examined to verify the PLIN2-CGI-58 interaction efficiency in bovine adipocytes. GST-Pulldown assay was used to detect the key site arginine315 function in PLIN2-CGI-58 interaction. Experiments were also examined to research these mutations function of PLIN2 in LDs formation during adipocytes differentiation, LDs were measured after staining by BODIPY, lipogenesis-related genes were also detected. Results showed that Leucine (L371A, L311A) and glycine (G369A, G376A) mutations reduced interaction efficiencies. Serine (S367A) mutations enhanced the interaction efficiency. Arginine (R315A) mutations resulted in loss of fluorescence in the cytoplasm and disrupted the interaction with CGI-58, as verified by pulldown assay. R315W mutations resulted in a significant increase in the number of LDs compared with wild-type (WT) PLIN2 or the R315A mutations. Lipogenesis-related genes were either up- or downregulated when mutated PLIN2 interacted with CGI-58. Arginine315 in PLIN2 is required for the PLIN2-CGI-58 interface and could regulate nascent LD formation and lipogenesis. This study is the first to study amino acids on the PLIN2 interface during interaction with CGI-58 in bovine and highlight the role played by PLIN2 in the regulation of bovine adipocyte lipogenesis.

Genome-wide analysis reveals genomic diversity and signatures of selection in Qinchuan beef cattle.

BACKGROUND: Indigenous Chinese cattle have abundant genetic diversity and a long history of artificial selection, giving local breeds advantages in adaptability, forage tolerance and resistance. The detection of selective sweeps and comparative genome analysis of selected breeds and ancestral populations provide a basis for understanding differences among breeds and for the identification and utilization of candidate genes. We investigated genetic diversity, population structure, and signatures of selection using genome-wide sequencing data for a new breed of Qinchuan cattle (QNC, n = 21), ancestral Qinchuan cattle (QCC, n = 20), and Zaosheng cattle (ZSC, n = 19). RESULTS: A population structure analysis showed that the ancestry components of QNC and ZSC were similar. In addition, the QNC and ZSC groups showed higher proportions of European taurine ancestry than that of QCC, and this may explain the larger body size of QNC, approaching that of European cattle under long-term domestication and selection. A neighbor-joining tree revealed that QCC individuals were closely related, whereas QNC formed a distinct group. To search for signatures of selection in the QNC genome, we evaluated nucleotide diversity (θπ), the fixation index (FST) and Tajima's D. Overlapping selective sweeps were enriched for one KEGG pathway, the apelin signaling pathway, and included five candidate genes (MEF2A, SMAD2, CAMK4, RPS6, and PIK3CG). We performed a comprehensive review of genomic variants in QNC, QCC, and ZSC using whole-genome sequencing data. QCC was rich in novel genetic diversity, while diversity in QNC and ZSC cattle was reduced due to strong artificial selection, with divergence from the original cattle. CONCLUSIONS: We identified candidate genes associated with production traits. These results support the success of selective breeding and can guide further breeding and resource conservation of Qinchuan cattle.

Integrated multi-omics analysis reveals variation in intramuscular fat among muscle locations of Qinchuan cattle.

BACKGROUND: Intramuscular fat (IMF) is closely related to the tenderness, marbling, juiciness, and flavor of meat. We used a combined transcriptome and metabolome analysis to investigate the molecular mechanisms underlying phenotypic variation among Qinchuan cattle. RESULTS: The IMF content was relatively high in the meat of Qinchuan cattle bulls and differed among muscle locations, namely the high rib (15.86%), ribeye (14%), striploin (10.44%), and tenderloin (8.67%). CCDC80 and the HOX gene cluster may regulate intramuscular adipose tissue deposition. Moreover, erucic acid (EA) was found to be the main metabolite in Qinchuan beef cattle, with a high concentration in IMF. The deposition of IMF could be regulated by the metabolic pathway for unsaturated fatty acids involving EA and the ACOX3, HACD2, and SCD5 genes. In addition, differentially expressed genes and metabolites were enriched in three major KEGG pathways: purine metabolism, pyrimidine metabolism, and the metabolism of glycine, serine, and threonine. CONCLUSIONS: We identified a significant metabolite, EA, with variation in IMF. Its closely related genes, ACOX3, HACD2, and SCD5, co-regulate the metabolism of unsaturated fatty acids, ultimately affecting the accumulation of intramuscular adipose tissue in Qinchuan cattle. Consequently, Qinchuan cattle are an elite cultivar for high-quality beef production and have great potential for breeding.

Selection signatures of Qinchuan cattle based on whole-genome sequences.

Qinchuan cattle has gradually improved in body shape and growth rate in the long-term breeding process from the draft cattle to beef cattle. As the head of the five local yellow cattle in China, the Qinchuan cattle has been designated as a specialized beef cattle breed. We investigated the selection signatures using whole genome sequencing data in Qinchuan cattle. Based on Fst, we detected hundreds of candidate genes under selection across Qinchuan, Red Angus, and Japanese Black cattle. Through protein-protein interaction analysis and functional annotation of candidate genes, the results revealed that KMT2E, LTBP1 and NIPBL were related to brain size, body characteristics, and limb development, respectively, suggesting that these potential genes may affect the growth and development traits in Qinchuan cattle. ARIH2, DACT1 and DNM2, et al. are related to meat quality. Meanwhile, TBXA2R can be used as a gene associated with reproductive function, and USH2A affect coat color. This provided a glimpse into the formation of breeds and molecular genetic breeding. Our findings will promote genome-assisted breeding to improve animal production and health.

The role of BBS2 in regulating adipogenesis and the association of its sequence variants with meat quality in Qinchuan cattle.

The BBS2 gene plays a vital role in human obesity and fat deposition. However, little is known about it in beef cattle. Therefore, this study investigates the function of BBS2 in the fat deposition of beef cattle and screens the effective SNPs marker for meat quality traits in cattle breeding. The expression of BBS2 is negatively correlated with marbling ratios of beef cattle. Moreover, the knockdown of BBS2 promoted adipogenesis and lipid accumulation of bovine preadipocytes by stimulating PPARγ, FABP4, and FASN expression (P < 0.01). Four novel SNPs in the exons of BBS2 in Chinese Qinchuan cattle were identified and of which the g.24226239C > T (Q527), g.24223562G > A (V441I), and g.24227851A > G (Q627R) were significantly associated with the meat quality of Qinchuan cattle (P < 0.01, P < 0.05). The findings suggested that BBS2 could be used as a candidate gene for meat quality improvement in Qinchuan cattle. Furthermore, these genotypes can be exploited as molecular markers in future beef breeding projects.

A genome-wide landscape of mRNAs, lncRNAs, circRNAs and miRNAs during intramuscular adipogenesis in cattle.

BACKGROUND: Intramuscular preadipocyte differentiation plays a critical role in bovine intramuscular fat (IMF) deposition. However, the roles of different RNAs, including mRNAs, circRNAs, lncRNAs and miRNAs, in regulating the adipogenic differentiation of intramuscular preadipocytes remain largely unclear. RESULTS: In the present study, a whole transcriptome sequencing and analysis, including the analysis of mRNAs, circRNAs, lncRNAs and miRNAs, during different differentiation stages (0, 3, 6, and 9 d) of intramuscular preadipocytes from Qinchuan cattle was performed. All samples were prepared with 3 biological replicates. Here, a total of 27,153 mRNAs, 14,070 circRNAs, 7035 lncRNAs, and 427 miRNAs were annotated. Among them, we identified 4848 differentially expressed mRNAs (DEMs), 181 DE circRNAs (DECs), 501 DE lncRNAs (DELs) and 77 DE miRNAs (DEmiRs) between 0 d and other differentiation days (3, 6, and 9 d). GO and KEGG functional enrichment analyses showed that these differentially expressed genes were mainly enriched in cell differentiation, fat metabolism and adipogenesis-related pathways. Furthermore, weighted gene coexpression network analysis (WGCNA) and co-expression network analysis screened out multiple important mRNAs, circRNAs and lncRNAs related to intramuscular adipogenesis. Based on the competing endogenous RNA (ceRNA) regulatory mechanism, we finally identified 24 potential ceRNA networks and 31 potential key genes, including FOXO1/miR-330/circRNA2018/MSTRG.20301, GPAM/miR-27b/ciRNA489 and SESN3/miR-433/circRNA2627MSTRG.20342. CONCLUSIONS: This study provides new insights into the differential expression patterns of different transcript types (i.e., mRNAs, circRNAs, lncRNAs and miRNAs) in intramuscular preadipocyte differentiation. Our findings provide data support for studying the molecular mechanism of key mRNAs and noncoding RNAs in IMF deposition, and provide new candidate markers for the molecular breeding of beef cattle.

MiR-145 reduces the activity of PI3K/Akt and MAPK signaling pathways and inhibits adipogenesis in bovine preadipocytes.

Adipose tissue is the largest metabolic organ because of adipogenesis controlled by numerous miRNAs. MiR-145 is classified into the same cluster with famous miR-143. However, few studies have investigated the role of miR-145 in adipogenesis. In the current study, we observed that the expression of miR-145 was downregulated during bovine adipogenesis in vivo and in vitro. The results of RNA-Seq analysis showed that miR-145 mainly disturb the PI3K/Akt and MAPK signaling pathways in bovine preadipocytes. MiR-145 inhibited bovine preadipocyte differentiation and downregulated phosphorylation level of Akt and ERK1/2 proteins. Furthermore, insulin, as a powerful inducer initiating adipogenesis and an activator of the PI3K/Akt and MAPK signaling pathways, was able to rescue the downregulation of Akt and ERK1/2 phosphorylation levels caused by miR-145. Taken together, our findings suggest that miR-145 is a potent inhibitor of adipogenesis that may function by reducing the activity of PI3K/Akt and MAPK signaling pathways.

SIRT5 inhibits bovine preadipocyte differentiation and lipid deposition by activating AMPK and repressing MAPK signal pathways.

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.

Effect of ELOVL6 on the lipid metabolism of bovine adipocytes.

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.

Expression of the bovine KLF6 gene polymorphisms and their association with carcass and body measures in Qinchuan cattle (Bos Taurus).

Kruppel-like factor 6 (KLF6) genes plays a significant role in the regulation of cell differentiation, proliferation and muscle development. The aim of this study is to investigate the genetic variation and the haplotype combination of the KLF6 gene in Qinchuan cattle and verify its contribution to bovine carcass traits and body measurements. The data were analyzed by real-time quantitative PCR (qPCR) to detect the expression profile of the KLF6 gene in the various tissues of Qinchuan cattle. PCR amplicons sequencing explored three novel SNPs at loci 3332C > G; 3413C > T and 3521G > A in the 2nd exon region of the KLF6 gene. The expression of KLF6 in the liver, kidney and lung was greater than that of other tissues. Allelic and genotypic frequencies of these SNPs were found to be in Hardy Weinberg equilibrium (P < 0.05). In SNP1, genotype CC, in SNP2, genotype CT and in SNP3 genotype GG were associated (P < 0.05) with larger body and carcass measurements. Association analysis results indicated that individuals with the Hap1/4 diplotype had a longer body and rump, were taller at the withers, and were wider at the hip than the other combinations. In terms of ultrasound carcass measures, Hap1/4 was associated with a larger muscle area and more intramuscular fat than other combinations. The bioinformatics study of the KLF6 protein showed a high degree of conservation in different mammalian species. The above results suggest that the KLF6 gene can used as potential candidate markers gene for the beef breed improvement through marker assisted selection of Qinchuan cattle.

Copy number variation detection in Chinese indigenous cattle by whole genome sequencing.

Copy number variation (CNV) refers to a kind of structural variation, having functional and evolutionary effects on phenotypes. Thus far, further elucidation of the CNVs in different Chinese indigenous cattle breeds by whole genome sequencing have yet not been done. In this study, a comprehensive genomic analysis was performed on 75 cattle individuals including six Chinese indigenous cattle breeds and two non-native specialized beef cattle breeds. Based on the 11,486 CNVRs discovered, population analysis was performed, showed that all the cattle breeds clustered in to three clades, consistent with their lineages Bos taurus, Bos taurus × Bos indicus and Bos indicus. Importantly, a set of CNVRs related genes were found to be associated with the traits of interest, which include meat production or quality (CAST, ACTC1, etc.), adaption (BLA-DQB, EGLN2, etc.) and coat color (KIT, MITF, etc.). These results provide valuable full genome variation resources for Chinese bovine genome research and would be helpful for cattle breeding and selection programs in the future.

Bta-miR-149-5p inhibits proliferation and differentiation of bovine adipocytes through targeting CRTCs at both transcriptional and posttranscriptional levels.

MicroRNAs are small, single stranded, and noncoding RNAs that have been proven to be potent regulators of adipogenesis. However, the role of bta-miR-149-5p in regulating bovine adipogenesis is still unclear. Expression profiling in different stages of adipogenesis revealed that bta-miR-149-5p was enriched in the proliferation stage, and also on Day 9 of differentiation in bovine adipocytes. Our gain of function study showed that bta-miR-149-5p can negatively regulate both bovine adipocyte proliferation and differentiation. Overexpression of bta-miR-149-5p suppressed the expression of proliferation marker genes at both the messenger RNA (mRNA) and protein levels, markedly decreased the percentage of S-phase cells, decreased the number of EdU-stained cells, and substantially reduced adipocyte proliferation vitality in the cell count assay. Collectively, these findings elucidated that bta-miR-149-5p inhibits adipocyte proliferation. Furthermore, overexpression of bta-miR-149-5p also suppressed the expression of adipogenic genes at both the mRNA and protein levels, inhibited lipid accumulation, and reduced the secretion of adiponectin in bovine adipocytes. Furthermore, a luciferase activity assay explored how bta-miR-149-5p targeted CRTCs (CRTC1 and CRTC2) directly. This targeting was further validated by the mRNA and protein level expression of CRTC1 and CRTC2, which were down regulated by bta-miR-149-5p overexpression. Moreover, bta-miR-149-5p indirectly targeted CRTC1 and CRTC2 through regulating their key transcription factors. Overexpression of bta-miR-149-5p suppressed the expression of SMAD3, while enriched the expression of NRF1, which are the key transcription factors and proven regulators of CRTC1. Overexpression of bta-miR-149-5p also repressed the expression of C/EBPγ, XBP1, INSM1, and ZNF263, which are the key regulators of CRTCs, at both the mRNA and protein levels. These findings suggest that bta-miR-149-5p is a negative regulator of CRTC1 and CRTC2 both at transcriptional and posttranscriptional level. Taken together, these findings suggest that bta-miR-149-5p can regulate adipogenesis, which implies that bta-miR-149-5p could be a target for increasing intramuscular fat in beef cattle.

SIRT6 cooperates with SIRT5 to regulate bovine preadipocyte differentiation and lipid metabolism via the AMPKα signaling pathway.

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.

The role of BAMBI in regulating adipogenesis and myogenesis and the association between its polymorphisms and growth traits in cattle.

Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein that affects the growth, development and muscle regeneration of the body by regulating the TGF-ß, BMP and Wnt signaling pathways. Studies have found that BAMBI has important regulatory functions in skeletal muscle and preadipocytes in vivo and in vitro. However, research on this protein in cattle is lacking. In this study, to determine the role of BAMBI in the growth and development of cattle, we first found that the expression of BAMBI in adipose tissue and longissimus muscle of newborn and adult Qinchuan beef cattle was significantly different. Then we showed that BAMBI knockdown promoted the differentiation of bovine preadipocytes and suppressed myoblast myogenesis, as indicated by the increased lipid droplets and the decreased myotubes, as well as the corresponding significant changes in the expression of PPARγ, C/EBPα, C/EBPß, FABP4, MyoD, MyoG and Myf6. Finally, to further verify the effect of BAMBI on the growth performance of cattle, we identified seven novel SNPs in the BAMBI genomic region, which were significantly correlated with one or more growth traits (p < 0.05). Furthermore, individuals with haplotype H1H4 (TC-GA-CT-CA-AT-AT-AG) had a higher body and carcass quality than those with other haplotypes (p < 0.05). In brief, BAMBI may be a functional gene for the differentiation of bovine preadipocytes and myoblasts, and variations in the BAMBI genomic region, especially the combined haplotype H1H4, may benefit marker-assisted selection in cattle.

Genome-wide analysis reveals the effects of artificial selection on production and meat quality traits in Qinchuan cattle.

A new strain of Qinchuan cattle (QNS) has been obtained after more than forty years of selective breeding, and it shows good performance and production traits. To characterize the genetic changes that have resulted from breeding, we sequenced 10 QNS and 10 of the original breed Qinchuan cattle (QCC) for the first time, with average of 12.5-fold depth. A total of 31,242,284 and 29,612,517 SNPs were identified in the QCC and QNS genomes, 47.81% and 44.36% of which were found to be novel, respectively. Furthermore, population structure analysis revealed the selection that these cattle had experienced. Then, 332 and 571 potential selected genes were obtained, associated with enhanced immunity and acclimatization in QCC (CD5, SMARCA2, CATHL2, etc.) and production or meat quality traits in QNS (PLCD3, MB, PPARGC1A, etc.). These results revealed the efforts of selective breeding for Chinese Qinchuan cattle, and will be helpful for future cattle breeding.

Transcriptional regulation of bovine elongation of very long chain fatty acids protein 6 in lipid metabolism and adipocyte proliferation.

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.

Genetic Architecture and Selection of Chinese Cattle Revealed by Whole Genome Resequencing.

The bovine genetic resources in China are diverse, but their value and potential are yet to be discovered. To determine the genetic diversity and population structure of Chinese cattle, we analyzed the whole genomes of 46 cattle from six phenotypically and geographically representative Chinese cattle breeds, together with 18 Red Angus cattle genomes, 11 Japanese black cattle genomes and taurine and indicine genomes available from previous studies. Our results showed that Chinese cattle originated from hybridization between Bos taurus and Bos indicus. Moreover, we found that the level of genetic variation in Chinese cattle depends upon the degree of indicine content. We also discovered many potential selective sweep regions associated with domestication related to breed-specific characteristics, with selective sweep regions including genes associated with coat color (ERCC2, MC1R, ZBTB17, and MAP2K1), dairy traits (NCAPG, MAPK7, FST, ITFG1, SETMAR, PAG1, CSN3, and RPL37A), and meat production/quality traits (such as BBS2, R3HDM1, IGFBP2, IGFBP5, MYH9, MYH4, and MC5R). These findings substantially expand the catalogue of genetic variants in cattle and reveal new insights into the evolutionary history and domestication traits of Chinese cattle.

Performance Measurement and Comparative Transcriptome Analysis Revealed the Efforts on Hybrid Improvement of Qinchuan Cattle.

Crossbreeding can provide productive gains through heterosis, however, surveys about the effects of crossbreeding through global transcriptomic sequencing are few. This study revealed that Angus × Qinchuan cattle (AQF) have improved performance characteristics compared to Qinchuan cattle (QCF). We performed RNA-seq on the subcutaneous fat tissue of QCF and AQF. More than 42.2 million clean reads were obtained in each sample. We detected 40 and 21 breed-specific highly expressed genes (FPKM > 500) in QCF and AQF, respectively. Furthermore, a total of 353 differentially expressed genes (DEGs, |log2 ratio| ≥ 1 and Probability ≥ 0.8) were found between these two groups, of which 227 genes were upregulated in AQF and 126 genes were upregulated in QCF. Functional enrichment analyses showed that breed-specific highly expressed genes and DEGs were closely related to terms such as development in AQF, and adaption or immune in QCF. In addition, we also identified the novel transcript units, alternative splicing events, single-nucleotide polymorphisms and Indels. Our results revealed differences in inherent characteristics and genetic differences when comparing QCF with AQF.

MEF2A Regulates the MEG3-DIO3 miRNA Mega Cluster-Targeted PP2A Signaling in Bovine Skeletal Myoblast Differentiation.

Understanding the molecular mechanisms of skeletal myoblast differentiation is essential for studying muscle developmental biology. In our previous study, we reported that knockdown of myocyte enhancer factor 2A (MEF2A) inhibited myoblast differentiation. Here in this study, we further identified that MEF2A controlled this process through regulating the maternally expressed 3 (MEG3)-iodothyronine deiodinase 3 (DIO3) miRNA mega cluster and protein phosphatase 2A (PP2A) signaling. MEF2A was sufficient to induce MEG3 expression in bovine skeletal myoblasts. A subset of miRNAs in the MEG3-DIO3 miRNA cluster was predicted to target PP2A subunit genes. Consistent with these observations, MEF2A regulated PP2A signaling through its subunit gene protein phosphatase 2 regulatory subunit B, gamma (PPP2R2C) during bovine myoblast differentiation. MiR-758 and miR-543 in the MEG3-DIO3 miRNA cluster were down-regulated in MEF2A-depleted myocytes. Expression of miR-758 and miR-543 promoted myoblast differentiation and repressed PPP2R2C expression. Luciferase activity assay showed that PPP2R2C was post-transcriptionally targeted by miR-758 and miR-543. Taken together, these results reveal that the MEG3-DIO3 miRNAs function at downstream of MEF2A to modulate PP2A signaling in bovine myoblast differentiation.

Coordinate regulation by transcription factors and DNA methylation in the core promoter region of SIRT6 in bovine adipocytes.

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.