The transcription factor NFB plays a critical role in normal and pathophysiological immune responses. Therefore, NFB and the signaling pathways that regulate its activation have become a major focus of drug development programs. Withania somnifera (WS) is a medicinal plant that is widely used in Palestine for the treatment of various inflammatory disorders. In this study we show that the leave extract of WS, as well as its major constituent withaferin A (WA), potently inhibits NFB activation by preventing the tumor necrosis factor-induced activation of IB kinase ␤ via a thioalkylation-sensitive redox mechanism, whereas other WS-derived steroidal lactones, such as withanolide A and 12-deoxywithastramonolide, are far less effective. To our knowledge, this is the first communication of IB kinase ␤ inhibition by a plant-derived inhibitor, coinciding with MEK1/ ERK-dependent Ser-181 hyperphosphorylation. This prevents IB phosphorylation and degradation, which subsequently blocks NFB translocation, NFB/DNA binding, and gene transcription. Taken together, our results indicate that pure WA or WA-enriched WS extracts can be considered as a novel class of NFB inhibitors, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

Methylation of cytosines in cytosine-guanine (CpG) dinucleotides is one of the most important epigenetic alterations in animals. The presence of methylcytosine in the promoter of specific genes has profound consequences on local chromatin structure and on the regulation of gene expression. Changes in DNA methylation play a central role in carcinogenesis. Hypermethylation and consecutive transcriptional silencing of tumor-suppressor genes has been documented in numerous cancers. The identification of target genes silenced by this modification has a great impact on diagnosis, classification, definition of risk groups and prognosis of cancer patients. Here we outline genome-wide techniques aiming at the identification of relevant methylated promoters. Methods and applications allowing clinicians to monitor the methylation of target genes will be also reviewed.

IKK␣ regulates many chromatin events in the nuclear phase of the NF-B program, including phosphorylation of histone H3 and removal of co-repressors from NF-B-dependent promoters. However, all of the nuclear functions of IKK␣ are not understood. In this study, using mouse embryonic fibroblasts IKK␣ knock-out and reexpressing IKK␣ after retroviral transduction, we demonstrate that IKK␣ contributes to NF-B/p65 DNA binding activity on an exogenous B element and on some, but not all, endogenous NF-B-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKK␣ is crucial for p65 binding on B sites of icam-1 and mcp-1 promoters but not on ib␣ promoter. The mutation of IKK␣ putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKK␣ activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the ib␣ promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKK␣ in a promoter-specific manner. Indeed, whereas the absence of IKK␣ induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the ib␣ promoter, where a better p65 binding occurs. We conclude that nuclear IKK␣ is required for p65 DNA binding in a gene-specific manner.

No abstract

NF-B is