Fumiaki Ohtake

Modulation of oestrogen receptor signalling by association with the activated dioxin receptor

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-α and ER-β. 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-α-/- 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.

A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-gamma transactivation.

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-γ (peroxisome proliferator activated receptor-γ) 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-γ 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-γ function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-γ function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.

Dioxin receptor is a ligand-dependent E3 ubiquitin ligase

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, CUL4BAhR. Complex assembly and ubiquitin ligase activity of CUL4BAhR in vitro and in vivo are dependent on the AhR ligand. In the CUL4BAhR 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.

Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB1/NIK cascade.

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-α (TNF-α) inhibit adipogenesis, the ligand-induced transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), is a key inducer of adipogenesis. Therefore, regulatory coupling between cytokine- and PPAR-γ-mediated signals might occur during adipogenesis. Here we show that the ligand-induced transactivation function of PPAR-γ is suppressed by IL-1 and TNF-α, and that this suppression is mediated through NF-κB activated by the TAK1/TAB1/NF-κB-inducing kinase (NIK) cascade, a downstream cascade associated with IL-1 and TNF-α signalling. Unlike suppression of the PPAR-γ transactivation function by mitogen-activated protein kinase-induced growth factor signalling through phosphorylation of the A/B domain, NF-κB blocks PPAR-γ binding to DNA by forming a complex with PPAR-γ and its AF-1-specific co-activator PGC-2. Our results suggest that expression of IL-1 and TNF-α in bone marrow may alter the fate of pluripotent mesenchymal stem cells, directing cellular differentiation towards osteoblasts rather than adipocytes by suppressing PPAR-γ function through NF-κB activated by the TAK1/TAB1/NIK cascade.

DNA demethylation in hormone-induced transcriptional derepression

Epigenetic modifications at the histone level affect gene regulation in response to extracellular signals. However, regulated epigenetic modifications at the DNA level, especially active DNA demethylation, in gene activation are not well understood. Here we report that DNA methylation/demethylation is hormonally switched to control transcription of the cytochrome p450 27B1 (CYP27B1) gene. Reflecting vitamin-D-mediated transrepression of the CYP27B1 gene by the negative vitamin D response element (nVDRE), methylation of CpG sites (5mCpG) is induced by vitamin D in this gene promoter. Conversely, treatment with parathyroid hormone, a hormone known to activate the CYP27B1 gene, induces active demethylation of the 5mCpG sites in this promoter. Biochemical purification of a complex associated with the nVDRE-binding protein (VDIR, also known as TCF3) identified two DNA methyltransferases, DNMT1 and DNMT3B, for methylation of CpG sites, as well as a DNA glycosylase, MBD4 (ref. 10). Protein-kinase-C-phosphorylated MBD4 by parathyroid hormone stimulation promotes incision of methylated DNA through glycosylase activity, and a base-excision repair process seems to complete DNA demethylation in the MBD4-bound promoter. Such parathyroid-hormone-induced DNA demethylation and subsequent transcriptional derepression are impaired in Mbd4-/- mice. Thus, the present findings suggest that methylation switching at the DNA level contributes to the hormonal control of transcription.

GlcNAcylation of histone H2B facilitates its monoubiquitination

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.

Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands

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 β-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 β-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 ApcMin/+ mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

PKA-dependent regulation of the histone lysine demethylase complex PHF2–ARID5B

Reversible histone methylation and demethylation are highly regulated processes that are crucial for chromatin reorganization and regulation of gene transcription in response to extracellular conditions. However, the mechanisms that regulate histone-modifying enzymes are largely unknown. Here, we characterized a protein kinase A (PKA)-dependent histone lysine demethylase complex, PHF2–ARID5B. PHF2, a jmjC demethylase, is enzymatically inactive by itself, but becomes an active H3K9Me2 demethylase through PKA-mediated phosphorylation. We found that phosphorylated PHF2 then associates with ARID5B, a DNA-binding protein, and induce demethylation of methylated ARID5B. This modification leads to targeting of the PHF2–ARID5B complex to its target promoters, where it removes the repressive H3K9Me2 mark. These findings suggest that the PHF2–ARID5B complex is a signal-sensing modulator of histone methylation and gene transcription, in which phosphorylation of PHF2 enables subsequent formation of a competent and specific histone demethylase complex.

AhR acts as an E3 ubiquitin ligase to modulate steroid receptor functions.

The arylhydrocarbon receptor (AhR) mediates the adverse effects of dioxins, including modulation of sex steroid hormone signaling. The role of AhR as a transcription factor is well described. AhR regulates the expression of target genes such as CYP1A1; however, the mechanisms of AhR function through other target-selective systems remain elusive. Accumulating evidence suggests that AhR modulates the functions of other transcription factors. The ligand-activated AhR directly associates with estrogen or androgen receptors (ERalpha or AR) and modulates their function both positively and negatively. This may, in part explain the sex steroid hormone-related adverse effects of dioxins. AhR has recently been shown to promote the proteolysis of ERalpha/AR through assembling a ubiquitin ligase complex, CUL4B(AhR). In the CUL4B(AhR) complex, AhR acts as a substrate-recognition subunit to recruit ERalpha/AR. This action defines a novel role for AhR as a ligand-dependent E3 ubiquitin ligase. We propose that target-specific regulation of protein destruction, as well as gene expression, is modulated by environmental toxins through the E3 ubiquitin ligase activity of AhR.

Intrinsic AhR function underlies cross-talk of dioxins with sex hormone signalings.

The arylhydrocarbon receptor (AhR) mediates sex steroid hormone-related actions in both normal physiology and in dioxin toxicity. In addition to regulation of direct target genes, the ligand-activated AhR associates with estrogen or androgen receptors (ERalpha or AR) to regulate transcription as a functional unit. Given that endogenous and exogenous AhR-ligands are structurally diverse, it is unclear whether cross-talk regulation of ERalpha/AR by the activated AhR is an intrinsic function of the AhR or the result of ligand-type-selective differences. To ensure uniform activity of the AhR irrespective of ligand-type-specific differences, we employed CA-AhR, which lacks the ligand-binding domain and has a constitutive activity. We found that CA-AhR, in the absence of a ligand, acted as a transcriptional co-regulator for the unliganded ERalpha/AR as well as for mutants of ERalpha/AR lacking a ligand-binding domain. CA-AhR was recruited to estrogen-/androgen-responsive promoters with endogenous ERalpha/AR. Moreover, CA-AhR had an E3 ubiquitin ligase activity and promoted proteasomal degradation of ERalpha/AR. Thus, these findings indicate that the cross-talk function of the AhR with sex hormone receptors is an intrinsic function of the AhR.