Smad4 and p65 integration at Samd9l regulatory elements shapes BMP protection against inflammatory cell death

Abstract Inflammation-induced osteoblast death disrupts bone homeostasis. However, how pro-survival bone morphogenetic protein (BMP) signaling intersects with the tumor necrosis factor (TNF)–NF-κB pathways remains unclear. Here we show that BMP4 protection of mouse osteoblasts from TNF-α–triggered inflammatory cell death requires Smad4 and NF-κB p65. ChIP-seq and ATAC-seq under TNF-α and BMP4 co-stimulation reveal broad overlap of Smad4 and p65 binding. Peak-shape–based quality scoring ranks two Samd9l -proximal accessible elements as the top candidates among ~20,000 peaks. These elements contact the Samd9l promoter, act as TNF-α–responsive enhancers and are required for Samd9l induction. One element mediates BMP4–Smad4-dependent attenuation of TNF-α–driven output. Samd9l depletion reduces TNF-α–induced cell-death-associated responses, and enhances ectopic bone formation in vivo. The corresponding mouse elements map to a regulatory region near the human SAMD9L locus. Together, these data delineate an enhancer-resolution BMP4–Smad4 brake on inflammatory NF-κB output that limits Samd9l -dependent osteoblast death, providing a mechanistic framework for supporting osteoblast survival during inflammation.