Biochemical mechanisms by which NF-κB transcriptional activity is modulated by nuclear protein Daxx and SPOP were proposed. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-κB in HeLa cells. Overexpression of Daxx repressed the expression of NF-κB-regulated genes such as IκBα and IL8. Coimmunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-κB stimulated by TNFα. The sensitization of TNFα-induced apoptosis by Daxx involves the downregulation of NF-κB dependent transcription of antiapoptotic c-FLIP in HeLa cells undergoing TNFα-induced apoptosis. Daxx-mediated repression of NF-κB transactivation is suggested to correlate with the inhibition of p65 acetylation by Daxx. Based on the finding that Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, the physical interaction between Daxx and p65 may provide a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-κB transcriptional activity. Also observed was the inhibition of NF-κB transcriptional capacity by SPOP which interfere with the DNA binding ability of NF-κB to its recognition in the nuclei of cotransfected HeLa cells.