Environmental contaminants affect a wide variety of biological events in many species. Dioxins are typical environmental contaminants that exert adverse oestrogen-related effects. Although their anti-oestrogenic actions are well described, dioxins can also induce endometriosis and oestrogen-dependent tumours, implying possible oestrogenic effects. However, the molecular mechanism underlying oestrogen-related actions of dioxins remains largely unknown. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix-loop-helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Here we show that the agonist-activated AhR/Arnt heterodimer directly associates with oestrogen receptors ER-alpha and ER-beta. This association results in the recruitment of unliganded ER and the co-activator p300 to oestrogen-responsive gene promoters, leading to activation of transcription and oestrogenic effects. The function of liganded ER is attenuated. Oestrogenic actions of AhR agonists were detected in wild-type ovariectomized mouse uteri, but were absent in AhR-/- or ER-alpha-/- ovariectomized mice. Our findings suggest a novel mechanism by which ER-mediated oestrogen signalling is modulated by a co-regulatory-like function of activated AhR/Arnt, giving rise to adverse oestrogen-related actions of dioxin-type environmental contaminants.

Fat-soluble ligands, including sex steroid hormones and environmental toxins, activate ligand-dependent DNA-sequence-specific transcriptional factors that transduce signals through target-gene-selective transcriptional regulation. However, the mechanisms of cellular perception of fat-soluble ligand signals through other target-selective systems remain unclear. The ubiquitin-proteasome system regulates selective protein degradation, in which the E3 ubiquitin ligases determine target specificity. Here we characterize a fat-soluble ligand-dependent ubiquitin ligase complex in human cell lines, in which dioxin receptor (AhR) is integrated as a component of a novel cullin 4B ubiquitin ligase complex, CUL4B(AhR). Complex assembly and ubiquitin ligase activity of CUL4B(AhR) in vitro and in vivo are dependent on the AhR ligand. In the CUL4B(AhR) complex, ligand-activated AhR acts as a substrate-specific adaptor component that targets sex steroid receptors for degradation. Thus, our findings uncover a function for AhR as an atypical component of the ubiquitin ligase complex and demonstrate a non-genomic signalling pathway in which fat-soluble ligands regulate target-protein-selective degradation through a ubiquitin ligase complex.

Histone modifications induced by activated signalling cascades are crucial to cell-lineage decisions. Osteoblast and adipocyte differentiation from common mesenchymal stem cells is under transcriptional control by numerous factors. Although PPAR-gamma (peroxisome proliferator activated receptor-gamma) has been established as a prime inducer of adipogenesis, cellular signalling factors that determine cell lineage in bone marrow remain generally unknown. Here, we show that the non-canonical Wnt pathway through CaMKII-TAK1-TAB2-NLK transcriptionally represses PPAR-gamma transactivation and induces Runx2 expression, promoting osteoblastogenesis in preference to adipogenesis in bone marrow mesenchymal progenitors. Wnt-5a activates NLK (Nemo-like kinase), which in turn phosphorylates a histone methyltransferase, SETDB1 (SET domain bifurcated 1), leading to the formation of a co-repressor complex that inactivates PPAR-gamma function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-gamma function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.

Chromatin reorganization is governed by multiple post-translational modifications of chromosomal proteins and DNA. These histone modifications are reversible, dynamic events that can regulate DNA-driven cellular processes. However, the molecular mechanisms that coordinate histone modification patterns remain largely unknown. In metazoans, reversible protein modification by O-linked N-acetylglucosamine (GlcNAc) is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). However, the significance of GlcNAcylation in chromatin reorganization remains elusive. Here we report that histone H2B is GlcNAcylated at residue S112 by OGT in vitro and in living cells. Histone GlcNAcylation fluctuated in response to extracellular glucose through the hexosamine biosynthesis pathway (HBP). H2B S112 GlcNAcylation promotes K120 monoubiquitination, in which the GlcNAc moiety can serve as an anchor for a histone H2B ubiquitin ligase. H2B S112 GlcNAc was localized to euchromatic areas on fly polytene chromosomes. In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over chromosomes including transcribed gene loci, with some sites co-localizing with H2B K120 monoubiquitination. These findings suggest that H2B S112 GlcNAcylation is a histone modification that facilitates H2BK120 monoubiquitination, presumably for transcriptional activation.

Pluripotent mesenchymal stem cells in bone marrow differentiate into adipocytes, osteoblasts and other cells. Balanced cytodifferentiation of stem cells is essential for the formation and maintenance of bone marrow; however, the mechanisms that control this balance remain largely unknown. Whereas cytokines such as interleukin-1 (IL-1) and tumour-necrosis factor-alpha (TNF-alpha) inhibit adipogenesis, the ligand-induced transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma), is a key inducer of adipogenesis. Therefore, regulatory coupling between cytokine- and PPAR-gamma-mediated signals might occur during adipogenesis. Here we show that the ligand-induced transactivation function of PPAR-gamma is suppressed by IL-1 and TNF-alpha, and that this suppression is mediated through NF-kappaB activated by the TAK1/TAB1/NF-kappaB-inducing kinase (NIK) cascade, a downstream cascade associated with IL-1 and TNF-alpha signalling. Unlike suppression of the PPAR-gamma transactivation function by mitogen-activated protein kinase-induced growth factor signalling through phosphorylation of the A/B domain, NF-kappaB blocks PPAR-gamma binding to DNA by forming a complex with PPAR-gamma and its AF-1-specific co-activator PGC-2. Our results suggest that expression of IL-1 and TNF-alpha in bone marrow may alter the fate of pluripotent mesenchymal stem cells, directing cellular differentiation towards osteoblasts rather than adipocytes by suppressing PPAR-gamma function through NF-kappaB activated by the TAK1/TAB1/NIK cascade.

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Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of ␤-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent ␤-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc Min/؉ mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.cecal cancer ͉ ubiquitin ligase ͉ ␤-catenin ͉ tumor chemoprevention

Polyubiquitin chains of different topologies regulate diverse cellular processes. K48- and K63-linked chains, the two most abundant chain types, regulate proteolytic and signaling pathways, respectively. Although recent studies reported important roles for heterogeneous chains, the functions of branched ubiquitin chains remain unclear. Here, we show that the ubiquitin chain branched at K48 and K63 regulates nuclear factor κB (NF-κB) signaling. A mass-spectrometry-based quantification strategy revealed that K48-K63 branched ubiquitin linkages are abundant in cells. In response to interleukin-1β, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains formed by TRAF6, yielding K48-K63 branched chains. The K48-K63 branched linkage permits recognition by TAB2 but protects K63 linkages from CYLD-mediated deubiquitylation, thereby amplifying NF-κB signals. These results reveal a previously unappreciated cooperation between K48 and K63 linkages that generates a unique coding signal: ubiquitin chain branching differentially controls readout of the ubiquitin code by specific reader and eraser proteins to activate NF-κB signaling.