Junn Yanagisawa

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.

Purification and Identification of p68 RNA Helicase Acting as a Transcriptional Coactivator Specific for the Activation Function 1 of Human Estrogen Receptor α

ABSTRACT The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERα (hERα) is enhanced through phosphorylation of the Ser118 residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERα. Phosphorylation of hERα Ser118 potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERα in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERβ, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERα AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERα A/B domain was essential for the full activity of hERα AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERα AF-1 and strongly suggest that the interaction between p68 and the hERα A/B domain is regulated by MAPK-induced phosphorylation of Ser118.

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.

Inactivating Mutations in the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene in Patients with Pseudovitamin D–Deficiency Rickets

BACKGROUND Pseudovitamin D-deficiency rickets is characterized by the early onset of rickets with hypocalcemia and is thought to be caused by a deficit in renal 25-hydroxyvitamin D3 1alpha-hydroxylase, the key enzyme for the synthesis of 1alpha,25-dihydroxyvitamin D3. METHODS We cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase complementary DNA (cDNA) using a mouse 1alpha-hydroxylase cDNA fragment as a probe. Its genomic structure was determined, and its chromosomal location was mapped by fluorescence in situ hybridization. We then identified mutations in the 1alpha-hydroxylase gene in four unrelated patients with pseudovitamin D-deficiency rickets by DNA-sequence analysis. Both the normal and the mutant 1alpha-hydroxylase proteins were expressed in COS-1 cells and were assayed for 1alpha-hydroxylase activity. RESULTS The gene for 25-hydroxyvitamin D3 1alpha-hydroxylase was mapped to chromosome 12q13.3, which had previously been reported to be the locus for pseudovitamin D-deficiency rickets by linkage analysis. Four different homozygous missense mutations were detected in this gene in the four patients with pseudovitamin D-deficiency rickets. The unaffected parents and one sibling tested were heterozygous for the mutations. Functional analysis of the mutant 1alpha-hydroxylase protein revealed that all four mutations abolished 1alpha-hydroxylase activity. CONCLUSIONS Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene are a cause of pseudovitamin D-deficiency rickets.

Retracted: A subfamily of RNA‐binding DEAD‐box proteins acts as an estrogen receptor α coactivator through the N‐terminal activation domain (AF‐1) with an RNA coactivator, SRA

One class of the nuclear receptor AF‐2 coactivator complexes contains the SRC‐1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA‐binding DEAD‐box proteins (p72/p68) as a human estrogen receptor α (hERα) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC‐1/TIF2 family protein and the hERα A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC‐1) and SRA were co‐immunoprecipitated with estrogen‐bound hERα in MCF7 cells and in partially purified complexes associated with hERα from HeLa nuclear extracts. Estrogen induced co‐localization of p72 with hERα and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen‐induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen‐induced transactivation of hERα. These findings indicate that p72/p68 acts as an ER subtype‐selective coactivator through ERα AF‐1 by associating with the coactivator complex to bind its AF‐2 through direct binding with SRA and the SRC‐1/TIF2 family proteins.

Epigenetic control of rDNA loci in response to intracellular energy status

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

The chromatin-remodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams syndrome

S phase progression. WINAC mediates the recruitment Hirochika Kitagawa,1,2 Ryoji Fujiki,1 Kimihiro Yoshimura,1 Yoshihiro Mezaki,1 Yoshikatsu Uematsu,1 Daisuke Matsui,1 Satoko Ogawa,1 Kiyoe Unno,1,3 Mataichi Okubo,3 Akifumi Tokita,3 Takeya Nakagawa,4 Takashi Ito,4 Yukio Ishimi,5 of unliganded VDR to VDR target sites in promoters, Hiromichi Nagasawa,6 Toshio Matsumoto,2 while subsequent binding of coregulators requires liJunn Yanagisawa,1,7 and Shigeaki Kato1,7,* gand binding. This recruitment order exemplifies that Institute of Molecular and Cellular Biosciences an interaction of a sequence-specific regulator with a University of Tokyo chromatin-remodeling complex can organize nucleo1-1-1 Yayoi somal arrays at specific local sites in order to make Bunkyo-ku promoters accessible for coregulators. Furthermore, Tokyo 113-0032 overexpression of WSTF could restore the impaired Japan recruitment of VDR to vitamin D regulated promoters 2 First Department of Internal Medicine in fibroblasts from Williams syndrome patients. This University of Tokushima School of Medicine suggests that WINAC dysfunction contributes to 3-18-15 Kuramoto-cho Williams syndrome, which could therefore be considTokushima 770-8503 ered, at least in part, a chromatin-remodeling factor Japan disease. 3 Department of Pediatrics

Selective Interaction of Vitamin D Receptor with Transcriptional Coactivators by a Vitamin D Analog

ABSTRACT The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1α,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

Nuclear Receptor Function Requires a TFTC-Type Histone Acetyl Transferase Complex.

Nuclear receptors (NRs) regulate transcription in a ligand-dependent way through two types of coactivator complexes: the p160/CBP histone acetyl transferase (HAT) complex and the DRIP/TRAP/SMCC complex without HAT activity. Here we identified a large human (h) coactivator complex necessary for the estrogen receptor alpha (ERalpha) transactivation. This complex contains the GCN5 HAT, the c-Myc interacting protein TRRAP/PAF400, TAF(II)30, and other subunits. Similarly to known TFTC (TBP-free TAF(II)-containing)-type HAT complexes (hTFTC, hPCAF, and hSTAGA), TRRP directly interacted with liganded ER alpha, or other NRs. ER alpha transactivation was enhanced by the purified complex in vitro. Antisense TRRAP RNA inhibited estrogen-dependent cell growth of breast cancer cells. Thus, the isolated TFTC-type HAT complex acts as a third class of coactivator complex for NR function.

Transrepression by a liganded nuclear receptor via a bHLH activator through co-regulator switching

Vitamin D receptor (VDR) is essential for ligand-induced gene repression of 25(OH)D3 1α-hydroxylase (1α(OH)ase) in mammalian kidney, while this gene expression is activated by protein kinase A (PKA) signaling downstream of the parathyroid hormone action. The mapped negative vitamin D response element (1αnVDRE) in the human 1α(OH)ase gene promoter (around 530 bp) was distinct from those of the reported DR3-like nVDREs, composed of two E-box-like motifs. Unlike the reported nVDREs, no direct binding of VDR/RXR heterodimer to 1αnVDRE was detected. A bHLH-type factor, designated VDIR, was identified as a direct sequence-specific activator of 1αnVDRE. The transactivation function of VDIR was further potentiated by activated-PKA signaling through phosphorylation of serine residues in the transactivation domains, with the recruitment of a p300 histone acetyltransferase co-activator. The ligand-dependent association of VDR/RXR heterodimer with VDIR bound to 1αnVDRE caused the dissociation of p300 co-activators from VDIR, and the association of HDAC co-repressor complex components resulting in ligand-induced transrepression. Thus, the present study deciphers a novel mechanism of ligand-induced transrepression by nuclear receptor via co-regulator switching.