Acetylation of p65 at lysine 314 is important for late NF-k(kappa)B-dependent gene expression

We provided earlier evidence that acetylation of p65 at lysines 310, 314 and 315 is important for the expression of a defined subset of genes; acetylation at these residues regulates both positively and negatively gene expression, in a gene-specific manner. These earlier studies provided a first glance of the functional relevance of p65 acetylation, since gene expression was measured only after 45 minutes of TNFα stimulation. In order to know if the requirement for site-specific acetylation is maintained for the same genes after longer exposure to TNFα, and to identify possible new genes regulated through p65 acetylation, we decided to extent our analysis to 3 hours of stimulation. For this, we used p65(-/-) MEFs complemented with non-acetylatable mutants, where the target lysines for acetylation were mutated to arginines. p65(-/-) cells complemented either with wild type p65, an empty plasmid as control (pTV), the acetylation-deficient double mutant K314/315R or the triple mutant KTR (K310/314/315R), were stimulated by TNFα for 3 hours and total RNA was isolated in three independent replicates from these cells. RNA was amplified, labeled and hybridized to the Agilent Whole Mouse Genome Array. After statistical analysis of the expression profiles, differentially expressed genes were identified.