Ikuko Yamaoka

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.

Distinct function of 2 chromatin remodeling complexes that share a common subunit, Williams syndrome transcription factor (WSTF)

A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF−/− mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF−/− neonates and ≈10% of WSTF+/− neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF−/− embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF−/− mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter, but the effects were reversed by overexpression of WINAC components. Likewise in WSTF−/− MEF cells, recruitment of Snf2h, an ISWI ATPase, to PCNA and cell survival after DNA damage were both defective, but were ameliorated by overexpression of WICH components. Thus, the present study provides evidence that WSTF is shared and is a functionally indispensable subunit of the WICH complex for DNA repair and the WINAC complex for transcriptional control.

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.

Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes

Changes in the environment of a cell precipitate extracellular signals and sequential cascades of protein modification and elicit nuclear transcriptional responses. However, the functional links between intracellular signaling-dependent gene regulation and epigenetic regulation by chromatin-modifying proteins within the nucleus are largely unknown. Here, we describe novel epigenetic regulation by MAPK cascades that modulate formation of an ATP-dependent chromatin remodeling complex, WINAC (WSTF Including Nucleosome Assembly Complex), an SWI/SNF-type complex containing Williams syndrome transcription factor (WSTF). WSTF, a specific component of two chromatin remodeling complexes (SWI/SNF-type WINAC and ISWI-type WICH), was phosphorylated by the stimulation of MAPK cascades in vitro and in vivo. Ser-158 residue in the WAC (WSTF/Acf1/cbpq46) domain, located close to the N terminus of WSTF, was identified as a major phosphorylation target. Using biochemical analysis of a WSTF mutant (WSTF-S158A) stably expressing cell line, the phosphorylation of this residue (Ser-158) was found to be essential for maintaining the association between WSTF and core BAF complex components, thereby maintaining the ATPase activity of WINAC. WINAC-dependent transcriptional regulation of vitamin D receptor was consequently impaired by this WSTF mutation, but the recovery from DNA damage mediated by WICH was not impaired. Our results suggest that WSTF serves as a nuclear sensor of the extracellular signals to fine-tune the chromatin remodeling activity of WINAC. WINAC mediates a previously unknown MAPK-dependent step in epigenetic regulation, and this MAPK-dependent switching mechanism between the two functionally distinct WSTF-containing complexes might underlie the diverse functions of WSTF in various nuclear events.

Testis-specific protein on Y chromosome (TSPY) represses the activity of the androgen receptor in androgen-dependent testicular germ-cell tumors

Testis-specific protein on Y chromosome (TSPY) is an ampliconic gene on the Y chromosome, and genetic interaction with gonadoblastoma has been clinically established. However, the function of the TSPY protein remains to be characterized in physiological and pathological settings. In the present study, we observed coexpression of TSPY and the androgen receptor (AR) in testicular germ-cell tumors (TGCTs) in patients as well as in model cell lines, but such coexpression was not seen in normal testis of humans or mice. TSPY was a repressor for androgen signaling because of its trapping of cytosolic AR even in the presence of androgen. Androgen treatment stimulated cell proliferation of a TGCT model cell line, and TSPY potently attenuated androgen-dependent cell growth. Together with the finding that TSPY expression is reduced in more malignant TGCTs in vivo, the present study suggests that TSPY serves as a repressor in androgen-induced tumor development in TGCTs and raises the possibility that TSPY could be used as a clinical marker to assess the malignancy of TGCTs.

Coactivation of Estrogen Receptor β by Gonadotropin-Induced Cofactor GIOT-4

ABSTRACT Estrogen exerts its diverse effects through two subtypes of estrogen receptors (ER), ERα and ERβ. Each subtype has its own distinct function and expression pattern in its target tissues. Little, however, is known about the transcriptional regulatory mechanism of ERβ in the major ERβ-expressing tissues. Using biochemical methods, we identified and described a novel ERβ coactivator. This protein, designated GIOT-4, was biochemically purified from 293F cells. It coactivated ERβ in ovarian granulosa cells. GIOT-4 expression was induced by stimulation with follicle-stimulating hormone (FSH). GIOT-4 recruited an SWI/SNF-type complex in a ligand-independent manner to ERβ as an ER subtype-specific physical bridging factor and induced subsequent histone modifications in the ERβ target gene promoters in a human ovarian granulosa cell line (KGN). Indeed, two ERβ-specific target genes were upregulated by FSH at a specific stage of a normal ovulatory cycle in intact mice. These findings imply the presence of a novel regulatory convergence between the gonadotropin signaling cascade and ERβ-mediated transcription in the ovary.

Retraction: A reduction state potentiates the glucocorticoid response through receptor protein stabilization

The intracellular redox state regulates all biological processes including gene expression. The glucocorticoid receptor (GR), a hormone‐dependent transcription factor, is affected by the redox state. GR translocation from the cytoplasm to the nucleus is regulated by oxidative stress. The molecular mechanism of how the redox state affects GR transcriptional regulation, however, has not been clarified. We identified a deoxidizing agent, cobalt chloride (CoCl2), that potentiates the GR transcriptional effects by stabilizing endogenously expressed GR protein as well as exogenously over‐expressed one without affecting GR mRNA level. Consequent GR protein stabilization enhanced co‐factor recruitments on the target gene promoters. These results support the existence of a novel redox‐dependent mechanism of GR transcriptional regulation mediated by receptor protein stabilization.

Retraction: DNA demethylation in hormone-induced transcriptional derepression

This corrects the article DOI: 10.1038/nature08456

DNA demethylation for hormone-induced transcriptional derepression

This corrects the article DOI: 10.1038/nature08456

Retraction for Kouzu-Fujita et al., Coactivation of Estrogen Receptor β by Gonadotropin-Induced Cofactor GIOT-4

Madoka Kouzu-Fujita, Yoshihiro Mezaki, Shun Sawatsubashi, Takahiro Matsumoto, Ikuko Yamaoka, Tetsu Yano, Yuji Taketani, Hirochika Kitagawa, Shigeaki Kato Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Obstetrics and Gynecology, University of Tokyo, Bunkyo-ku, Tokyo, Japan; ERATO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan