Impact of lysine, methionine, and histidine deficiencies on gene expression and upstream transcriptional regulators in bovine mammary epithelial cells: an RNA-sequencing analysis

Essential amino acids (EAA) may alter bovine mammary epithelial cell functions by impacting mRNA expression of genes through their upstream transcription factors. This study investigates the transcriptional responses in bovine mammary epithelial cells under EAA deprivation (lysine, methionine, and histidine) using RNA-sequencing and pathway analysis. Differential expression genes with absolute log2 fold change = 1 and a false discovery rate of < 0.05 were considered differentially expressed. QIAGEN Ingenuity Pathway Analysis identified kinetochore metaphase signaling pathway as the most significant across all treatments, suggesting a strong link to cell proliferation and apoptosis during EAA deficiency. Activation of activating transcription factor 4 (ATF4) regulates genes involved in amino acid transport, stress response, and apoptosis. Interferon signaling emerged as a novel pathway activated by EAA deficiency, contributing to reduced cell proliferation and protein synthesis. In contrast, the RAS/RAF/MEK/ERK signaling pathway was unaffected. Hypoxia-inducible factor 1a (HIF1A) increased expression, suggesting an anti-proliferative role. Downregulation of Forkhead box protein M1 (FOXM1), a key cell cycle progression factor, likely contributed to slower cell proliferation. Additionally, tumor suppressor p53 (TP53) and ER stress-related transcription factor DNA damage inducible transcript 3 (DDIT3) were identified as mediators of apoptosis. These findings reveal a complex network of transcriptional regulators and signaling pathways influenced by EAA availability, providing new insights into the regulation of milk protein synthesis and potential strategies for optimizing milk production in dairy cows. Overall design: To identify potentially novel transcription factors that mediate the effects of essential amino acid deficiency on mammary cell activity, we used a genome-wide approach based on RNA-sequencing of gene expression in bovine mammary epithelial cell exposed to lysine, methionine, or histidine deprivation for 60 h.