Genetic and Functional Dissection of the NFKB2 Gene: Implications for Milk Fatty Acid Biosynthesis in Dairy Cattle

Abstract

Milk fat, an essential component of bovine milk, significantly impacts human health and dairy industry profitability. Our previous research identified that the NFKB2 gene was a key candidate for milk fatty acids (FAs) in dairy cattle. This study aimed to investigate the role of NFKB2 in regulating milk FAs in Holstein cattle through post-GWAS analysis. Knockdown of NFKB2 using siRNA in bovine mammary epithelial cells (BMECs) significantly reduced triglyceride concentrations, lipid droplet secretion, and the expressions of 17 key genes involved in lipid metabolism pathways, including PI3K-AKT, NF-κB, MAPK, adipocytokine, PPAR, and AMPK signaling pathways. These findings highlighted NFKB2 as a critical regulator of milk FA composition through its interactions with key lipid biosynthesis pathways. Further, this study constructed a new Chinese Holstein population including 1065 cows and performed genotyping and milk FAs measurements in these individuals. By SNP identification and association analysis, SNP g.22889812C/T in the 5' flanking region of NFKB2 was significantly associated with multiple milk FAs, including higher C14:1, C17:0, C18:0, and C14 index levels in allele C group, and higher C6:0, C8:0, C14:0, C16:1, and C16 index levels in allele T group. Using computational predictions, luciferase assays, EMSA and super-shift EMSA, we revealed that SNP g.22889812C/T downregulated NFKB2 transcription activity with allele T selectively binding with transcription factor (TF) ZNF282. This study not only advanced the understanding of genetic mechanisms underlying milk fat production but also identified NFKB2 and its causal mutation g.22889812C/T as promising molecular markers for selective breeding to optimize milk fat traits.

Keywords: EMSA; dairy cattle; fatty acids; knocked‐down of NFKB2; transcriptional activity.

FIGURE 1

Identification transcription factor (TF). (A) Transient reporter gene expression assays were conducted using constructs featuring the g.22889812C/T (rs109915272) variant in the NFKB2 gene, with the SNP in the sequences highlighted in red. (B) Luciferase activity analysis was performed on the recombinant plasmids incorporating the g.22889812C/T (rs109915272) variant in HEK‐293T cells. The reported values for each construct represent the mean of three independent experiments, each conducted in triplicate. ** denotes p < 0.01. (C) Electrophoretic mobility shift assay (EMSA) was conducted using C and T oligonucleotides corresponding to the g.22889812C/T (rs109915272) site, with CK representing the blank control. The 150‐fold excess of an unlabeled consensus oligonucleotide probe was utilized, indicated as 150×. (D) Super‐shift analysis of g.22889812C/T (rs109915272) was performed to validate the specific TF predicted to be ZNF282. CK represented nuclear extracts without oligonucleotide probes. Lanes 3 and 4 illustrated the DNA‐binding capacities of the alleles in the absence of antibody. Lanes 5 and 6 showed the DNA‐binding abilities of the alleles when bound to the ZNF282 antibody, while lanes 7 and 8 depicted the binding capabilities with PRDM4 antibody. Lanes 9 and 10 demonstrated the DNA‐binding abilities when using IgG antibody. Lanes 11 and 12 corresponded to the use of 150× unlabeled consensus oligonucleotide probes. P1 denoted the probe containing the allele C of g.22889812C/T (rs109915272), and P2 signified the probe with the allele T.

FIGURE 2

Effect of NFKB2 knocked‐down on lipid secretion in BMECs. (A) Relative expression levels of NFKB2 normalized to GAPDH before and after knockdown. (B) Relative expression levels of NFKB2 normalized to MARVELD1 before and after knockdown. (C) Lipid secretion profile in BMECs prior to NFKB2 knockdown. (D and E) Lipid secretion profiles in BMECs following NFKB2 knockdown. (F) Changes in intracellular triglyceride concentrations in BMECs after NFKB2 knockdown. * and ** denote p < 0.05 and p < 0.01, respectively.

FIGURE 3

Differentially expressed genes following NFKB2 knockdown in BMECs were found in PI3K‐AKT, NF‐κB, MAPK, adipocytokine, PPAR, and AMPK signaling pathways. Downregulated genes after NFKB2 knockdown were indicated by green frames, and the upregulated genes were highlighted in brownish red.