Maiko Okada

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.

The Androgen Receptor in Health and Disease

Androgens play pivotal roles in the regulation of male development and physiological processes, particularly in the male reproductive system. Most biological effects of androgens are mediated by the action of nuclear androgen receptor (AR). AR acts as a master regulator of downstream androgen-dependent signaling pathway networks. This ligand-dependent transcriptional factor modulates gene expression through the recruitment of various coregulator complexes, the induction of chromatin reorganization, and epigenetic histone modifications at target genomic loci. Dysregulation of androgen/AR signaling perturbs normal reproductive development and accounts for a wide range of pathological conditions such as androgen-insensitive syndrome, prostate cancer, and spinal bulbar muscular atrophy. In this review we summarize recent advances in understanding of the epigenetic mechanisms of AR action as well as newly recognized aspects of AR-mediated androgen signaling in both men and women. In addition, we offer a perspective on the use of animal genetic model systems aimed at eventually developing novel therapeutic AR ligands.

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.

Retraction: ‘A cell cycle‐dependent co‐repressor mediates photoreceptor cell‐specific nuclear receptor function’

Photoreceptor cell‐specific nuclear receptor (PNR) (NR2E3) acts as a sequence‐specific repressor that controls neuronal differentiation in the developing retina. We identified a novel PNR co‐repressor, Ret‐CoR, that is expressed in the developing retina and brain. Biochemical purification of Ret‐CoR identified a multiprotein complex that included E2F/Myb‐associated proteins, histone deacetylases (HDACs) and NCoR/HDAC complex‐related components. Ret‐CoR appeared to function as a platform protein for the complex, and interacted with PNR via two CoRNR motifs. Purified Ret‐CoR complex exhibited HDAC activity, co‐repressed PNR transrepression function in vitro, and co‐repressed PNR function in PNR target gene promoters, presumably in the retinal progenitor cells. Notably, the appearance of Ret‐CoR protein was cell‐cycle‐stage‐dependent (from G1 to S). Therefore, Ret‐CoR appears to act as a component of an HDAC co‐repressor complex that supports PNR repression function in the developing retina, and may represent a co‐regulator class that supports transcriptional regulator function via cell‐cycle‐dependent expression.

Noncanonical Wnt signaling mediates androgen-dependent tumor growth in a mouse model of prostate cancer

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2–mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.

Switching of chromatin-remodelling complexes for oestrogen receptor-alpha.

The female sex steroid hormone oestrogen stimulates both cell proliferation and cell differentiation in target tissues. These biological actions are mediated primarily through nuclear oestrogen receptors (ERs). The ligand‐dependent transactivation of ERs requires several nuclear co‐regulator complexes; however, the cell‐cycle‐dependent associations of these complexes are poorly understood. By using a synchronization system, we found that the transactivation function of ERα at G2/M was lowered. Biochemical approaches showed that ERα associated with two discrete classes of ATP‐dependent chromatin‐remodelling complex in a cell‐cycle‐dependent manner. The components of the NuRD‐type complex were identified as G2/M‐phase‐specific ERα co‐repressors. Thus, our results indicate that the transactivation function of ERα is cell‐cycle dependent and is coupled with a cell‐cycle‐dependent association of chromatin‐remodelling complexes.

Class I histone deacetylase inhibitors inhibit the retention of BRCA1 and 53BP1 at the site of DNA damage

BRCA1 and 53BP1 antagonistically regulate homology‐directed repair (HDR) and non‐homologous end‐joining (NHEJ) of DNA double‐strand breaks (DSB). The histone deacetylase (HDAC) inhibitor trichostatin A directly inhibits the retention of 53BP1 at DSB sites by acetylating histone H4 (H4ac), which interferes with 53BP1 binding to dimethylated histone H4 Lys20 (H4K20me2). Conversely, we recently found that the retention of the BRCA1/BARD1 complex is also affected by another methylated histone residue, H3K9me2, which can be suppressed by the histone lysine methyltransferase (HKMT) inhibitor UNC0638. Here, we investigate the effects of the class I HDAC inhibitors MS‐275 and FK228 compared to UNC0638 on histone modifications and the DNA damage response. In addition to H4ac, the HDAC inhibitors induce H3K9ac and inhibit H3K9me2 at doses that do not affect the expression levels of DNA repair genes. By contrast, UNC0638 selectively inhibits H3K9me2 without affecting the levels of H3K9ac, H3K56ac or H4ac. Reflecting their effects on histone modifications, the HDAC inhibitors inhibit ionizing radiation‐induced foci (IRIF) formation of BRCA1 and BARD1 as well as 53BP1 and RIF1, whereas UNC0638 suppresses IRIF formation of BRCA1 and BARD1 but not 53BP1 and RIF1. Although HDAC inhibitors suppressed HDR, they did not cooperate with the poly(ADP‐ribose) polymerase inhibitor olaparib to block cancer cell growth, possibly due to simultaneous suppression of NHEJ pathway components. Collectively, these results suggest the mechanism by that HDAC inhibitors inhibit both the HDR and NHEJ pathways, whereas HKMT inhibitor inhibits only the HDR pathway; this finding may affect the chemosensitizing effects of the inhibitors.

Purification and identification of estrogen receptor alpha co‐regulators in osteoclasts

Mature osteoclasts are multinuclear, macrophage‐like cells derived from hematopoietic stem cells in the bone marrow. Several transcription factors regulating osteoclast differentiation have been identified. However, the molecular basis of transcriptional regulation in osteoclasts at epigenetic levels is largely unknown. In fact, no osteoclast‐specific transcriptional co‐regulators have been characterized. Recently, selective ablation of estrogen receptor alpha (ERα) in mature osteoclasts derived from female mice (ERαΔoc/Δoc) exhibited trabecular bone loss due to induced apoptosis via upregulated expression of Fas ligand mRNA. In general, the component composition of the ERα‐associated co‐activator complex and its expression levels are distinct among tissues. However, ERα transcriptional co‐regulators in mature osteoclasts remain unclear. In the present study, we achieved large‐scale cultivation of mature, multinucleated osteoclasts and established a purification system for ERα‐associated proteins. In addition to co‐regulators previously found in other ERα target cells, several unexpected factors were found such as CAP‐H. The mRNA expression level of CAP‐H was high during osteoclast differentiation. These results demonstrate the existence of osteoclast‐specific transcriptional co‐regulators supporting ERα function.

Liganded ERα Stimulates the E3 Ubiquitin Ligase Activity of UBE3C to Facilitate Cell Proliferation

Estrogen receptor (ER)α is a well-characterized ligand-dependent transcription factor. However, the global picture of its nongenomic functions remains to be illustrated. Here, we demonstrate a novel function of ERα during mitosis that facilitates estrogen-dependent cell proliferation. An E3 ubiquitin ligase, UBE3C, was identified in an ERα complex from estrogen-treated MCF-7 breast cancer cells arrested at mitosis. UBE3C interacts with ERα during mitosis in an estrogen-dependent manner. In vitro, estrogen dramatically stimulates the E3 activity of UBE3C in the presence of ERα. This effect was inhibited by the estrogen antagonist tamoxifen. Importantly, estrogen enhances the ubiquitination of cyclin B1 (CCNB1) and destabilizes CCNB1 during mitosis in a manner dependent on endogenous UBE3C. ERα, UBE3C, and CCNB1 colocalize in prophase nuclei and at metaphase spindles before CCNB1 is degraded in anaphase. Depletion of UBE3C attenuates estrogen-dependent cell proliferation without affecting the transactivation function of ERα. Collectively, these results demonstrate a novel ligand-dependent action of ERα that stimulates the activity of an E3 ligase. The mitotic role of estrogen may contribute to its effects on proliferation in addition to its roles in target gene expression.

Retraction: ‘Switching of chromatin‐remodelling complexes for oestrogen receptor‐α’

The female sex steroid hormone oestrogen stimulates both cell proliferation and cell differentiation in target tissues. These biological actions are mediated primarily through nuclear oestrogen receptors (ERs). The ligand‐dependent transactivation of ERs requires several nuclear co‐regulator complexes; however, the cell‐cycle‐dependent associations of these complexes are poorly understood. By using a synchronization system, we found that the transactivation function of ERα at G2/M was lowered. Biochemical approaches showed that ERα associated with two discrete classes of ATP‐dependent chromatin‐remodelling complex in a cell‐cycle‐dependent manner. The components of the NuRD‐type complex were identified as G2/M‐phase‐specific ERα co‐repressors. Thus, our results indicate that the transactivation function of ERα is cell‐cycle dependent and is coupled with a cell‐cycle‐dependent association of chromatin‐remodelling complexes.