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.

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Owen HC, Roberts SJ, Ahmed SF, Farquharson C. Dexamethasone-induced expression of the glucocorticoid response gene lipocalin 2 in chondrocytes. Am J Physiol Endocrinol Metab 294: E1023-E1034, 2008. First published April 1, 2008 doi:10.1152/ajpendo.00586.2007 are commonly used anti-inflammatory drugs, but long-term use can result in marked growth retardation in children due to their actions on growth plate chondrocytes. To gain an insight into the mechanisms involved in GC-induced growth retardation, we performed Affymetrix microarray analysis of the murine chondrogenic cell line ATDC5, incubated with 10 Ϫ6 M dexamethasone (Dex) for 24 h. Downregulated genes included secreted frizzled-related protein and IGF-I, and upregulated genes included serum/GC-regulated kinase, connective-tissue growth factor, and lipocalin 2. Lipocalin 2 expression increased 40-fold after 24-h Dex treatment. Expression increased further after 48-h (75-fold) and 96-h (84-fold) Dex treatment, and this response was Dex concentration dependent. Lipocalin 2 was immunolocalized to both proliferating and hypertrophic growth plate zones, and its expression was increased by Dex in primary chondrocytes at 6 h (3-fold, P Ͻ 0.05). The lipocalin 2 response was blocked by the GC-receptor antagonist RU-486 and was increased further by the protein synthesis blocker cycloheximide. Proliferation in lipocalin 2-overexpressing cells was less than in control cells (49%, P Ͻ 0.05), and overexpression caused an increase in collagen type X expression (4-fold, P Ͻ 0.05). The effects of lipocalin 2 overexpression on chondrocyte proliferation (64%, P Ͻ 0.05) and collagen type X expression (8-fold, P Ͻ 0.05) were further exacerbated with the addition of 10 Ϫ6

It has been shown that cell cycle genes play an important role in the coordination of chondrocyte proliferation and differentiation. The inhibitory effects of glucocorticoids (GCs) on chondrocyte proliferation are consistent with GCs disrupting cell cycle progression and promoting cell cycle exit. Cyclin-dependent kinase inhibitors (CDKIs) force cells to exit the cell cycle and differentiate, and studies have shown that expression of the CDKI p21(CIP1/WAF1) is increased in terminally differentiated cells. In this study, p21 mRNA and protein expression was increased during chondrocyte differentiation and after exposure to dexamethasone (Dex, 10(-6 )M) in murine chondrogenic ATDC5 cells. In 4-week-old mice lacking a functional p21 gene, Dex caused a reduction in body weight compared to saline control null mice, but this was consistent with the reduction in body weight observed in Dex-treated wild-type littermates. In addition, p21 ablation had no effect on the reduction in width of the growth plate or reduced mineral apposition rate in Dex-treated mice. However, an alteration in growth rate and epiphyseal structure is evident when comparing p21(-/-) and wild-type mice. These findings suggest that p21 does not directly contribute to GC-induced growth retardation in vivo but is involved in the maintenance of the growth plate.

Epigenetic regulation plays a key role in the link between inflammation and cancer. Here we examine Mbd2, which mediates epigenetic transcriptional silencing by binding to methylated DNA. In separate studies the Mbd2 −/− mouse has been shown (1) to be resistant to intestinal tumourigenesis and (2) to have an enhanced inflammatory/immune response, observations that are inconsistent with the links between inflammation and cancer. To clarify its role in tumourigenesis and inflammation, we used constitutive and conditional models of Mbd2 deletion to explore its epithelial and non‐epithelial roles in the intestine. Using a conditional model, we found that suppression of intestinal tumourigenesis is due primarily to the absence of Mbd2 within the epithelia. Next, we demonstrated, using the DSS colitis model, that non‐epithelial roles of Mbd2 are key in preventing the transition from acute to tumour‐promoting chronic inflammation. Combining models revealed that prior to inflammation the altered Mbd2 −/− immune response plays a role in intestinal tumour suppression. However, following inflammation the intestine converts from tumour suppressive to tumour promoting. To summarise, in the intestine the normal function of Mbd2 is exploited by cancer cells to enable tumourigenesis, while in the immune system it plays a key role in preventing tumour‐enabling inflammation. Which role is dominant depends on the inflammation status of the intestine. As environmental interactions within the intestine can alter DNA methylation patterns, we propose that Mbd2 plays a key role in determining whether these interactions are anti‐ or pro‐tumourigenic and this makes it a useful new epigenetic model for inflammation‐associated carcinogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

RelA (NF-kappaB) is a transcription factor inducible by distinct stimuli in many different cell types. To find new cell type specific cofactors of NF-kappaB dependent transcription, we isolated RelA transcription activation domain binding proteins from the nuclear extracts of three different cell types. Analysis by electrophoresis and liquid chromatography tandem mass spectrometry identified several novel putative molecular partners. Some were strongly enriched in the complex formed from the nuclear extracts of specific cell types.