We have previously demonstrated that the phosphorylation of p65 at serine 536 was differentially recruited to selective promoters following cell activation. We have recently demonstrated that the distance between the site of p65 binding and the transcription start site of a particular gene determines if p65 needs to be phosphorylated on serine 536. The phosphorylation of p65 was not involved in the formation of an enhanceosome, where the recruitment of histone modifying enzymes to proximal promoters was required. These findings suggested that the phosphorylation of p65 and the cis-acting elements of the promoter regulate the various NFkB responsive genes. We are currently investigating the role of various phosphorylation sites of p65 in controling the chromatin architecture surrounding p65 responsive genes. We are also examining how the various IkB members regulate the nuclear translocation of phosphorylated p65. In addition to serine 536, the phosphorylation of serine 529 of p65 has been shown to regulate transcriptional activity. We are currently investigating the relationship between the phosphorylation of serines 529 and 536 in regulating the chromatin architecture. We have shown previously that the phosphorylation of p65 at serine 536 resulted in an increase in Csf2 gene expression. Recently, it has been shown that the phosphorylation of p65 at serine 536 by IKKa was induced by lymphotoxin-beta receptor (LTbR) signaling in mouse fibroblast. We have observed that the treatment of 3T3 fibroblast cells with agonistic anti-LTbR antibody resulted in the phosphorylation of p65 at serine 536, but no expression of Csf2 gene. However, priming with anti-LTbR antibody resulted in a synergistic increase of TNF-mediated Csf2 expression. The synergistic enhancement required the activation of NIK and signaling through the alternative NFkB pathway. Furthermore, the nuclear translocation and recruitment of both p65 and RelB to the Csf2 promoter were observed during the LTbR priming of TNF-mediated Csf2 gene expression. We are currently examining the mechanism of priming.