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 ((5m)CpG) 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 (5m)CpG 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.
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.
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.
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 ATPdependent 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.
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 ERa at G2/M was lowered. Biochemical approaches showed that ERa 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 ERa co-repressors. Thus, our results indicate that the transactivation function of ERa is cell-cycle dependent and is coupled with a cellcycle-dependent association of chromatin-remodelling complexes.
Estrogen exerts its diverse effects through two subtypes of estrogen receptors (ER), ER␣ and ER. Each subtype has its own distinct function and expression pattern in its target tissues. Little, however, is known about the transcriptional regulatory mechanism of ER in the major ER-expressing tissues. Using biochemical methods, we identified and described a novel ER coactivator. This protein, designated GIOT-4, was biochemically purified from 293F cells. It coactivated ER 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 ER as an ER subtype-specific physical bridging factor and induced subsequent histone modifications in the ER target gene promoters in a human ovarian granulosa cell line (KGN). Indeed, two ER-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 ER-mediated transcription in the ovary.Estrogen plays important roles in many target organs, including the female reproductive organs, the central nervous system, and bone. Estrogen exerts its diverse biological actions through binding to and activating one of two nuclear estrogen receptor (ER) subtypes (ER␣ or ER) (12,22,35,40). ERs are members of the nuclear receptor (NR) gene superfamily. ERs, bound to and activated by estrogen, bind to specific DNA sequences called estrogen-responsive elements (ERE) to induce target genes (14,21).Like the other NR members, the ER requires the cooperation of distinct classes of coregulators and multiprotein coregulator complexes in order to initiate estrogen-mediated chromatin reorganization (16,46). These complexes appear to modify the chromatin configuration in a highly regulated manner by controlling nucleosomal rearrangement and enzymecatalyzed modifications of histone tails. By altering chromatin structure, the coregulator complexes facilitate bridging between NRs and basal transcription factors, along with RNA polymerase II, thereby controlling transcription. As for the nucleosomal rearrangement, two major classes of chromatinmodifying complexes that coregulate NRs have been well-characterized. One class is the histone-modifying complexes, including discrete subfamilies of transcription coregulatory complexes (2,29,36). The best-characterized NR coregulator complexes possess either histone acetylase or histone deacetylase activities. Recently, histone methylases/demethylases have also been shown to be significant NR coregulators. The other class of coregulator complexes is ATP-dependent chromatinremodeling complexes. These complexes use ATP hydrolysis to rearrange nucleosomal arrays in a noncovalent manner to facilitate or prevent the access of NRs to nucleosomal DNA (5,17,33). These ATP-dependent chromatin-remodeling complexes have been classified into three subfamilies based on the major catalytic ...
Several lanes of the ChIP analyses in this Letter were inadvertently duplicated or erroneously created during figure assembly. We now provide corrected figure panels for Figs 1f, 2c, 2f, 2g and 3h and Supplementary Figs S8, S9a, S9b, S11, S13b, S18 and S28. Our results and conclusions are not affected by these errors, but we apologise for the careless mistakes made.During initial preparation of the figure panels, the panels for the negative controls (with no obvious signals), inputs and some data were inappropriately duplicated and mixed up. The experiments were performed several times, so a set of data with adequate negative controls and inputs from one experimental round could be found. We have replaced the previous set of data in these panels with a correct set, and confirmed that these corrections do not affect the original claims in the published text. Representative ChIP data are displayed and significance was statistically assessed from several independently repeated experiments. We have also repeated the experiments and obtained the same results as those in the published figures.In Fig. 1f, lane 5 of anti-AcH3, lane 1 of anti-5-MetC, and the antiIgG panel of a previous data set were left blank without placing of gel images. In Fig. 2c, the panels of distal regions (23,907/23,442; left panels) and the input panels (2230/1130) were duplicated. The predicted data of anti-5MetC was inadvertently mixed up. In Figs 2f, 2g and 3h, the input panels were duplicated.Further errors in the Supplementary Figures of the original Letter are described and corrected in the Supplementary Information of this Corrigendum, and the supporting raw data is also presented.
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 (CoCl 2 ), 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.
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