ENO1 couples HDAC1 to regulate histone lactylation and gene transcription.

Histone deacetylases (HDACs) regulate transcription and catalyze deacetylation predominantly within canonical transcriptional complexes. Nevertheless, the mechanistic role of other HDAC-associated proteins in orchestrating this process remains incompletely understood. To systematically decode endogenous HDAC interactomes in living cells, we developed BimPL, a heterobifunctional molecule-enabled proximity labeling strategy. Leveraging BimPL and quantitative proteomics, we robustly captured established HDAC complexes and identified putative interactors, including glycolytic enzyme enolase-1 (ENO1). Importantly, we uncover that ENO1 translocates into the nucleus and interacts with HDAC1 at chromatin, which in turn blunts the activity of HDAC1 through locally generated phosphoenolpyruvate (PEP). Consequently, the ENO1-HDAC1 coupling promotes histone lysine lactylation (Kla), which drives transcriptional reprogramming of oncogenes in hepatic malignancies. Our study establishes BimPL as a versatile tool for mapping endogenous protein interactomes and reveals a metabolic enzyme-orchestrated HDAC regulatory mechanism for histone lactylation, highlighting ENO1's moonlighting function in epigenetic reprogramming.