Identification of a functional single nucleotide polymorphism in the FASN promoter associated with milk fat traits in dairy cattle

In the past decades, multiple genome-wide association studies have been performed to identify loci affecting milk production traits, including some using imputed whole-genome sequence data (i.e. Daetwyler et al., 2014; Fang & Pausch, 2019; Sanchez et al., 2017). Several genome-wide association studies have linked a polymorphism (rs136067046, BTA19: g.50775172 C>G on ARS-UCD1.2) located in the upstream region of FASN (encoding fatty acid synthase) to milk fat traits in dairy cattle (Cai et al., 2020; Sanchez et al., 2019; Tribout et al., 2020). For example, Cai and collaborators showed that rs136067046 is associated to milk fat traits in Nordic Holstein cows. FASN is an enzyme that plays a critical role in de novo fatty acid synthesis in milk. Interestingly, rs136067046 is located within an ATAC peak (chr.19: 50 773 523–50 795 886) found in the mammary gland (Yuan et al., 2023). It is thus possible that rs136067046 is a functional variant that directly impacts these traits.

The ability of this SNP to alter transcription factor binding sites was then predicted with a custom script, as previously described (Ramírez-Ayala et al., 2021). This analysis suggests that rs136067046 modifies the binding sites of nine transcription factors (Table 1). Five of those transcription factors are expressed (transcript per million reads ≥0.5) in mammary gland tissue (Fang et al., 2020). Interestingly, three of those five transcription factors are from the Krüppel-like factor (KLF) family, a conserved class of transcription factors. It has been shown that KLF4 promotes milk fat synthesis in bovine mammary epithelial cells by targeting the FASN promoter region (Wu et al., 2024). The authors have shown, using yeast one-hybrid assay, that KLF4 interacts directly with a part of the FASN promoter region, encompassing the location of rs136067046. It has also been shown that KLF5 controls the expression of FASN through an interaction with SREBBP-1 (Lee et al., 2009). In addition, inactivation of KLF6 in bovine mammary epithelial cells increases FASN expression (Iqbal et al., 2022). These studies point to the important role played by these KLF transcription factors in the regulation of FASN. All these findings suggest that, consequently, rs136067046 is potentially a regulatory variant that might alter the expression of FASN.

To investigate the potential regulatory function of the rs136067046 variant, we constructed and then functionally tested two allele-specific recombinant promoter vectors using the dual-luciferase reporter system and the bovine mammary epithelial MAC-T cell line (Huynh et al., 1991). Details on the plasmid constructions and luciferase assays are provided in Supplementary Material S1. The construct with the G allele showed a significant 2.41 ± 0.12 (mean value ± standard deviation) fold increase (Student's t-test, p = 0.0008) of luciferase activity compared to the construct carrying the C allele (Figure 1). These results show that rs136067046 is a regulatory variant and that the G allele might lead to an increase in FASN mRNA synthesis by increasing its transcription rate.

In conclusion, we have demonstrated that the rs136067046 variant has a regulatory effect on FASN promoter activity. Further work is needed to demonstrate that it is a causal polymorphism and directly responsible for its association with milk fat traits.

D.R. planned and coordinated the study, analysed the results and wrote the manuscript. M.F. and M.P. prepared the allele-specific plasmids. L.B.B. and M.P. performed the luciferase assays. M.B.B. and M.C. did the bioinformatics analyses. A.B., H.T., N.D. and V.B. contributed to data analyses. All authors reviewed the manuscript.

The work was part of the BovReg project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 815668. M.B.B. was the recipient of a postdoctoral fellowship from the Animal Genetics division of INRAE.

The authors declare no competing interests.