Poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear factor B (NF-B) have both been demonstrated to play a pathophysiological role in a number of inflammatory disorders. We recently presented evidence that PARP-1 can act as a promoter-specific coactivator of NF-B in vivo independent of its enzymatic activity. PARP-1 directly interacts with p300 and both subunits of NF-B (p65 and p50) and synergistically coactivates NF-B-dependent transcription. Here we show that PARP-1 is acetylated in vivo at specific lysine residues by p300/CREB-binding protein upon stimulation. Furthermore, acetylation of PARP-1 at these residues is required for the interaction of PARP-1 with p50 and synergistic coactivation of NF-B by p300 and the Mediator complex in response to inflammatory stimuli. PARP-1 physically interacts with the Mediator. Interestingly, PARP-1 interacts in vivo with histone deacetylases (HDACs) 1-3 but not with HDACs 4 -6 and might be deacetylated in vivo by HDACs 1-3. Thus, acetylation of PARP-1 by p300/CREB-binding protein plays an important regulatory role in NF-B-dependent gene activation by enhancing its functional interaction with p300 and the Mediator complex.Nuclear factor B (NF-B) is a widely expressed transcription factor of particular importance to the regulation of cells of the immune system (1). NF-B encompasses a family of inducible transcription factors including RelA/p65, RelB, c-Rel, p50, and p52 (1). These proteins share a conserved 300-amino acid region within their amino termini, designated Rel-homology domain (RHD). This domain is responsible for dimerization, nuclear translocation, DNA binding, and interaction with heterologous transcription factors (1). NF-B is composed of homo-or heterodimers with a range of DNA binding and activation potentials. The most abundant and best-studied form of NF-B in cells is a heterodimer consisting of the two subunits, p50 (NF-B1) and p65 (RelA). NF-B plays a key role in the regulation of many genes involved in mammalian immune and inflammatory responses, apoptosis, cell proliferation, and differentiation (1, 2). NF-B has additionally been associated with neurodegenerative processes and cancer (3, 4). In unstimulated cells, NF-B is sequestered in the cytoplasm as an inactive transcription factor complex by its physical association with one of several inhibitors of NF-B (IBs) 2 (5). Treatment of cells with extracellular stimuli including cytokines, bacterial lipopolysaccharides (LPS), phorbol esters, or potent oxidants leads to rapid phosphorylation of IB␣, which results in ubiquitination of IB␣ and subsequent degradation by the 26 S proteasome (4, 5). Dissociation of NF-B unmasks the nuclear localization sequences of p65 and p50 subunits, which leads to nuclear translocation and binding of NF-B to specific B consensus sequences in the chromatin and activation of specific subsets of genes (3).NF-B-dependent gene expression requires growing families of transcriptional coactivators (6, 7). The two key coactivators of NF-B, histone acetyltransferases p300 and its homo...

Nuclear factor kappaB (NF-κB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65−/− cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor α was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFα identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-κB dependent gene expression.

Dendritic cells (DCs) direct CD4+ T cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a dramatically impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to activation of CD4+ T cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.