Deciphering Hierarchical Transcriptional Control of Fatty Acid Metabolism in Megalobrama Amblycephala: Insights from Promoter Architecture, PPAR Synergy, and Network Biology.

This study provides a comprehensive, multi-level analysis of the transcriptional regulatory architecture controlling key enzymes in the fatty acid elongation and desaturation pathways of Megalobrama amblycephala, an economically significant freshwater aquaculture species. The research integrates in silicopromoter analysis, functional validation through dual-luciferase reporter assays, and systems-level transcriptional regulatory network (TRN) modeling to elucidate the complex mechanisms governing lipid metabolism. Our investigation focused on the promoter regions of five crucial genes—ELOVL5, ELVL2, FADS2, ELOVL4A, and ELOVL4B—which encode enzymes responsible for determining fatty acid chain length and saturation levels, critically influencing cellular membrane composition, energy storage, and signaling molecule precursors . Computational dissection of the approximately 2.0–2.5 kb promoter regions revealed distinct and complex cis-regulatory landscapes for each gene, with unique enrichments for specific transcription factor binding motifs. The ELOVL5promoter showed significant enrichment for Sp1-family factors (particularly Sp1.5), while the ELOVL2promoter exhibited a predominance of GATA-1.1 binding sites. Analysis of the ELOVL4paralogs revealed divergent evolutionary trajectories: ELOVL4Adisplayed the highest overall cis-element density with clusters of sites for Sp1.4, EGR1, and GATA-1.1, whereas ELOVL4Bwas overwhelmingly dominated by Elk-1.1 sites. The FADS2promoter was characterized by a prevalence of CACCC-box binding factors. This bioinformatic mapping provided strong evidence for complex, gene-specific regulatory logic and identified potential peroxisome proliferator-activated receptor response elements (PPREs) within these promoter contexts . Functional validation using dual-luciferase reporter assays in HEK293T cells demonstrated that all five promoters are direct transcriptional targets of both PPARA and PPARG, with individual overexpression of either transcription factor inducing statistically significant transactivation (p < 0.0001). Notably, we identified a gene-specific hierarchy of co-regulation: PPARA and PPARG acted synergistically on the ELOVL5, ELOVL2, and FADS2promoters, with combined overexpression resulting in luciferase activity significantly exceeding the sum of individual effects (p < 0.001). In contrast, their regulation of the ELOVL4paralogs was merely additive or non-synergistic, suggesting distinct regulatory paradigms for different branches of the fatty acid metabolic network. This differential regulation may reflect promoter-intrinsic properties that determine the cooperative potential of PPARA and PPARG, possibly through mechanisms involving complex stabilization or recruitment of shared co-activators . At a systems level, we reconstructed a genome-scale transcriptional regulatory network for M. amblycephalaby integrating data from five functionally characterized sub-networks. The consolidated TRN comprised approximately 5,000 nodes (genes/TFs) and 45,000 edges (regulatory relationships), exhibiting scale-free topology characteristic of robust biological systems. Network analysis identified Ppara, SREBP, and Mstn1as central hubs with the highest betweenness centrality and degree scores. The Mstn1hub functioned as a pivotal integrator, connecting immune-response modules with growth and metabolic regulators, suggesting a role in coupling immune signals with metabolic homeostasis—a feature more elaborated in M. amblycephalathan in grass carp based on comparative analysis. This network topology reveals a sophisticated hierarchical architecture where core metabolic processes are wired with immune and stress response pathways, providing a framework for understanding system-level metabolic control . This integrated approach elucidates a sophisticated, hierarchical model of transcriptional control for lipid metabolism in teleosts, revealing how gene-specific regulatory logic at the promoter level integrates into a broader network architecture. The findings not only advance fundamental knowledge of metabolic regulation in fish but also offer practical insights for aquaculture nutrition, providing a rational basis for developing targeted dietary strategies to optimize fatty acid composition and improve metabolic health in this economically vital species.