An RNA-sequencing analysis to determine potential upstream transcriptional regulators of essential amino acid deficiency responses in bovine mammary epithelial cells.

Essential amino acids (EAA) may alter bovine mammary epithelial cell (BMEC) activities through regulation of gene transcription. For this study, we used pathway analysis of RNA-sequencing data to identify upstream transcription factors (TF) that potentially mediate the effects of single EAA deficiency on BMEC. Differentiated BMEC were cultured in 1 of 4 treatment media representing normal physiological concentrations of all amino acids (CTL), or ¼ the normal concentration of histidine (LH), lysine (LK), or methionine (LM). After 48 h, rates of DNA synthesis were reduced 30 to 40% by each of the deficiencies. Protein synthesis rate was 30 to 50% lower in LK and LM but was unaffected by LH. Up-regulated upstream TF included those related to nutrient stress, interferon signaling, and suppression of cell population growth. Down-regulated TF were primarily related to cell cycle progression. An array of classic ATF4 targets increased in response to EAA deficiencies including amino acid transporters, branched-chain aminotransferase, aminoacyl-tRNA synthetases, sestrin2 (a leucine sensor), and activators of apoptosis. Interferon signaling emerged as a novel pathway activated by EAA deficiencies, possibly contributing to reduced cell proliferation and protein synthesis. In contrast, the MAPK signaling pathway was unaffected. Expression of hypoxia-inducible factor 1α (HIF1A) increased, consistent with an anti-proliferative role. Downregulation of Forkhead box protein M1 (FOXM1) likely contributed to slower cell proliferation. These findings reveal a complex network of transcriptional regulators and signaling pathways influenced by EAA availability and shed light on the nutritional regulation of milk protein production by dairy cows.