Temporal organization of leptin signaling reveals distinct regulatory roles for STAT3 and ETV6.

Leptin regulates energy homeostasis through activation of its long-form receptor (LepRb), primarily via JAK2-dependent phosphorylation of STAT3. However, the temporal organization of leptin-induced transcriptional responses and how parallel signaling pathways contribute to these dynamics remain incompletely understood. Here, we established a LepRb-expressing hypothalamic cellular model to investigate the temporal architecture of leptin-induced transcription factors and pathways identified by transcriptome and proteome profiling at baseline (0 min), early (30 min), and late (180 min) time points. Overall changes in acute leptin stimulation were primarily reflected at the level of transcriptional and regulatory reprogramming. Proteomic remodeling mirrored the transcriptome but appeared smaller, likely due to assay insensitivity and slower dynamics of protein regulation, making the transcriptome the more informative kinetic readout. Structured transcriptional dynamics included the rapid activation of stress- and inflammatory-associated genes followed by sustained cytokine and interferon signaling modules. Transcription factor regulon and pathway activity inference identified coordinated activation of STAT, NF-κB, and MAPK-associated regulatory programs underlying these expression trajectories. CRISPR-Cas9-based deletion of Stat3 markedly altered basal transcriptional states and attenuated leptin-induced gene expression while revealing activation of alternative signaling pathways. In contrast, CRISPR-Cas9-based deletion of the ETS transcriptional repressor Etv6 strongly affected baseline transcription but had minimal impact on leptin-induced regulatory dynamics. Together, these findings reveal a temporally organized regulatory architecture in which STAT3 governs stimulus-responsive transcriptional dynamics, whereas ETV6 primarily modulates baseline transcriptional states.