Cedeno-Cedeno, Y.; Taylor, S. J.; Gamble, M. J.; Steidl, U.; Coleman, R. A.

Gene expression relies on transcriptional bursts driven by dynamic chromatin-modifying enzymes that depend on metabolites like NAD+. Depletion of NAD+ contributes to cancer, metabolic disorders, and aging, emphasizing the significance of tight regulation of NAD+ production in the cell. NMNAT1, a nuclear NAD+ -synthetase enzyme, supports chromatin-modifying enzymes such as PARP1 and SIRT1; however, its direct role in transcriptional regulation remains unclear. Using integrated multi-omics, we present the first high-resolution, genome-wide study of NMNAT1s regulatory functions. We demonstrate that NMNAT1 binds to the promoters and enhancers of actively transcribed genes involved in DNA replication, cell cycle progression, and chromatin regulation. RNA-seq and CUT&Tag analyses indicate reduced RNA Polymerase II occupancy at downregulated genes in NMNAT1 knockout cells, implicating NMNAT1 in transcriptional activation through Pol II engagement. These findings position NMNAT1 as a key node linking localized NAD+ production to gene-specific transcription, offering new insights into metabolic regulation of gene expression.