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.

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.

Expression profiling of peripheral blood miRNA using RNAseq technology in dairy cows with Escherichia coli-induced mastitis.

E. coli is the main causative agent of mastitis in dairy cows, but the mechanism of molecular regulation underlying the occurrence and development of mastitis has not yet been fully elucidated. In this study, an E. coli-induced mastitis model was created and RNASeq technology was used to measure the miRNA expression profiles at different times post-infection (0, 1, 3, 5, 7 dpi), as well as to screen for differentially expressed miRNA. The results show detection of 2416 miRNAs, including 628 known miRNAs and 1788 newly discovered miRNAs. A total of 200 differentially expressed miRNAs were found at different time points. Bioinformatics analysis showed that these differentially expressed miRNAs may regulate the occurrence and development of mastitis in dairy cows through seven signal transduction pathways, namely cytokine-cytokine receptor interaction, MAPK signaling pathway, chemokine signaling pathway, leukocyte transendothelial migration, T cell receptor signaling pathway, Toll-like receptor signaling pathway, and cell adhesion molecules. In addition, bta-miR-200a, bta-miR-205, bta-miR-122, bta-miR-182 and the newly discovered conservative_15_7229 might be involved in immune process in late stage of E. coli-induced mastitis. The results of this study lay the foundation for molecular network analysis of mastitis and molecular breeding of dairy cows.

Comparison of microRNA Profiles between Bovine Mammary Glands Infected with Staphylococcus aureus and Escherichia coli.

MicroRNAs (miRNAs) play crucial roles in regulating innate and adaptive immunity in humans and animals. Infection with E. coli or S. aureus can cause inflammation of the mammary glands, which results in significant economic losses in dairy cattle. However, the regulatory mechanisms of miRNAs in response to E. coli or S. aureus infection in bovine mammary glands have not been thoroughly explored. To discover the differential expression of miRNA in bovine mammary gland challenged with E. coli or S. aureus, we performed miRNA sequencing on tissue samples. A total of 1838 miRNAs were identified, including 580 known-miRNAs (included in the miRbase database) and 1258 predicted novel miRNAs. The miRNA expression patterns indicated that, compared with control samples, 279 miRNAs and 305 miRNAs were differentially expressed miRNAs (DIE-miRNA) in S. aureus and E. coli infected tissues, respectively. Moreover, the results of comparison the DIE-miRNAs between the E. coli and S. aureus infected groups showed that 197 DIE-miRNAs are identical, 108 DIE-miRNAs are specific to the E. coli group, and 82 DIE-miRNAs are specific to the S. aureus group. Many DIE-miRNAs, such as bta-miR-144, bta-miR-451 and bta-miR-7863, might be the useful biomarkers of mastitis caused by E. coli and S. aureus. In addition, target genes of the DIE-miRNAs were predicted. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that these DIE-miRNAs are likely involved in many immune signaling pathways, including the Toll-like receptor signaling pathways, MAPK signaling pathway, cell adhesion molecules, TGF-ß signaling pathway, leukocyte trans endothelial migration, cytokine-cytokine receptor interaction, and chemokine signaling pathways. This study has provided supportive evidence that miRNAs may serve as diagnostic biomarkers of mastitis in dairy cows, and suggests potentially of effective strategies to combat mastitis.

Polymorphisms in adrenergic receptor genes in Qinchuan cattle show associations with selected carcass traits.

The beta-adrenergic receptors coded by the ADRB1, ADRB2 and ADRB3 genes play important roles in mediating metabolic effects, especially lipolysis, insulin resistance and energy balance. This study investigated the expression levels of these three genes in different tissues of Qinchuan cattle by real-time polymerase chain reaction (RT-PCR). Expressed levels of RNA from the ADRB2 gene were generally much higher than for ADRB1 and ADRB3. ADRB1 and ADRB2 expression levels were highest in subcutaneous fat and lower in muscle, whereas ADRB3 expression was higher in muscle tissue. Eight single nucleotide polymorphisms (SNPs) were discovered in 503 Qinchuan cattle by DNA sequencing, containing three missense mutations (g.1148G>C in ADRB1, g.1293C>T and g.1311T>C in ADRB2), four synonymous mutations (g.1054T>C, g.1122C>T and g.1143G>T in ADRB1 and g.506A>G in ADRB3), as well as one mutation in 3'untranslated region (3'UTR) (g.2799G>A in ADRB3). Interestingly, five of them were located in regions predicted to contain multiple repeats of CG nucleotides (CpG islands). Association analysis showed relationships between most of those SNPs or combined haplotypes and carcass traits of Qinchuan cattle. This study association analysis suggests that polymorphisms in these genes might be useful for selection in beef cattle breeding.

Characterization of the promoter region of the bovine SIX1 gene: Roles of MyoD, PAX7, CREB and MyoG.

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.