Posttranslational modifications of histones, such as methylation, regulate chromatin structure and gene expression. Recently, lysine-specific demethylase 1 (LSD1), the first histone demethylase, was identified. LSD1 interacts with the androgen receptor and promotes androgen-dependent transcription of target genes by ligand-induced demethylation of mono- and dimethylated histone H3 at Lys 9 (H3K9) only. Here, we identify the Jumonji C (JMJC) domain-containing protein JMJD2C as the first histone tridemethylase regulating androgen receptor function. JMJD2C interacts with androgen receptor in vitro and in vivo. Assembly of ligand-bound androgen receptor and JMJD2C on androgen receptor-target genes results in demethylation of trimethyl H3K9 and in stimulation of androgen receptor-dependent transcription. Conversely, knockdown of JMJD2C inhibits androgen-induced removal of trimethyl H3K9, transcriptional activation and tumour cell proliferation. Importantly, JMJD2C colocalizes with androgen receptor and LSD1 in normal prostate and in prostate carcinomas. JMJD2C and LSD1 interact and both demethylases cooperatively stimulate androgen receptor-dependent gene transcription. In addition, androgen receptor, JMJD2C and LSD1 assemble on chromatin to remove methyl groups from mono, di and trimethylated H3K9. Thus, our data suggest that specific gene regulation requires the assembly and coordinate action of demethylases with distinct substrate specificities.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor 3 (ERR3) complexed with a steroid receptor coactivator-1 (SRC-1) peptide reveals a transcriptionally active conformation in absence of any ligand. The structure explains why estradiol does not bind ERRs with significant affinity. Docking of the previously reported ERR antagonists, diethylstilbestrol and 4-hydroxytamoxifen, requires structural rearrangements enlarging the ligand binding pocket that can only be accommodated with an antagonist LBD conformation. Mutant receptors in which the ligand binding cavity is filled up by bulkier side chains still interact with SRC-1 in vitro and are transcriptionally active in vivo, but are no longer efficiently inactivated by diethylstilbestrol or 4-hydroxytamoxifen. These results provide structural and functional evidence for ligand-independent transcriptional activation by ERR3.

Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.

GTPases of the Rho family are transducers of extracellular signals and control cellular processes such as organization of the actin cytoskeleton, motility, adhesion and gene regulation. The Rho signalling pathway is activated, for example, by bioactive sphingolipids such as sphingosine-1-phosphate (SPP) or by overexpression of Rho family members in tumorigenesis and metastases. Here, we show that stimulation of the Rho signalling pathway induces translocation of the transcriptional LIM-only coactivator FHL2 to the nucleus and subsequent activation of FHL2-and androgen receptor-dependent genes. Interestingly, prostate tumours overexpress Rho GTPases and display altered cellular localization of FHL2 concomitant with tumour dedifferentiation. SPP-induced FHL2 activation is mediated by Rho GTPases, but not by the GTPases Cdc42, Rac1 or Ras, and depends on Rhokinase. In addition, Rho signalling in¯uences other transcriptional coactivators, thus pointing to a general regulatory role for Rho GTPases in cofactor function. In summary, our data propose a yet undescribed signalling pathway in which the coactivator FHL2 acts as a novel molecular transmitter of the Rho signalling pathway, thereby integrating extracellular cues into altered gene expression. Keywords: androgen receptor/FHL2/nuclear translocation/small GTPases/transcriptional coactivator IntroductionThe transcriptional coactivator FHL2 is a LIM-only member of the LIM protein superfamily. LIM proteins are de®ned by the presence of one or multiple copies of the signature LIM domain. The LIM domain is a cysteine-rich motif with the consensus sequence CX 2 CX 16±23 HX 2 -CX 2 CX 2 CX 16±21 CX(C,H,D) that coordinately binds two zinc atoms and mediates protein±protein interactions (Bach, 2000). Based on the number of LIM domains and the presence or absence of additional motifs, LIM proteins are grouped into different classes (Bach, 2000). One class comprises LIM-only proteins that are composed solely of up to ®ve LIM domains. A newly identi®ed subclass of LIM-only proteins contains four and a half LIM domains and are designated as FHL proteins. The ®ve members of the FHL subclass (FHL1±4 and ACT) display a restricted expression pattern in tissues such as striated muscle, heart, prostate, ovary and testis Madgwick, 1996, 1999a,b;Chan et al., 1998;Fimia et al., 1999;Mu Èller et al., 2000). Very little is known about the biological function of FHL proteins but, recently, ACT, FHL3 and FHL2 were characterized as coactivators for the transcription factors CREM/CREB and androgen receptor (AR), respectively (Fimia et al., 1999(Fimia et al., , 2000Mu Èller et al., 2000). FHL2 contains a strong, autonomous transactivation function and binds speci®cally to the AR in vitro and in vivo (Mu Èller et al., 2000). In an agonist-and activation function-2 (AF-2)-dependent manner, FHL2 selectively increases the transcriptional activity of the AR (Mu Èller et al., 2000).An emerging question in transcriptional regulation is whether extracellular signals might be transmitted into altere...

Exposure to environmental cues such as cold or nutritional imbalance requires white adipose tissue (WAT) to adapt its metabolism to ensure survival. Metabolic plasticity is prominently exemplified by the enhancement of mitochondrial biogenesis in WAT in response to cold exposure or β3-adrenergic stimulation. Here we show that these stimuli increase the levels of lysine-specific demethylase 1 (LSD1) in WAT of mice and that elevated LSD1 levels induce mitochondrial activity. Genome-wide binding and transcriptome analyses demonstrate that LSD1 directly stimulates the expression of genes involved in oxidative phosphorylation (OXPHOS) in cooperation with nuclear respiratory factor 1 (Nrf1). In transgenic (Tg) mice, increased levels of LSD1 promote in a cell-autonomous manner the formation of islets of metabolically active brown-like adipocytes in WAT. Notably, Tg mice show limited weight gain when fed a high-fat diet. Taken together, our data establish LSD1 as a key regulator of OXPHOS and metabolic adaptation in WAT.

No abstract

The estrogen-related receptor (ERR) ␥ behaves as a constitutive activator of transcription. Although no natural ligand is known, ERR␥ is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERR␥ ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERR␥ results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ER␣ LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERR␥ does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERR␥ and ER␣ reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERR␥ relative to ER␣. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERR␥ and reveal structural differences with ERs that explain why ERR␥ does not bind estradiol or raloxifene and will help to design new selective antagonists.The estrogen-related receptors ERR␣, 1 ERR␤, and ERR␥ (NR3B1, -2, and -3) (1) form a subfamily of orphan nuclear receptors that share significant amino acid homology with the estrogen receptors ER␣ and ER␤ (NR3A1 and -2) (2, 3). Because of the high conservation in the DNA binding domain, ERRs and ERs have overlapping DNA binding selectivity (4 -6) and, accordingly, may co-regulate target genes in tissues in which they are co-expressed. ERR subfamily members have for example been shown to modulate the expression of ER target genes in bone (7,8) or breast tissue (9, 10). Importantly, overexpression of ERR␣ and ERR␥ in samples from breast cancer patients correlates with unfavorable and favorable biomarkers, respectively (11). Therefore, these receptors might serve as prognostic markers themselves or even be targets for endocrine therapy in human breast cancer.Despite their significant homology with ERs in the ligand binding domain (LBD), ERRs do not (or only very weakly) respond to estradiol (E2) (2, 12). Furthermore, whereas ERs are ligand-activated receptors, ERRs are constitutively active (13-16), and a structural study confirmed that the ERR␥ LBD can adopt a transcriptionally active conformation and interact with the steroid receptor coactivator 1 (SRC-1) in the absence of any ligand (12). Together, these observations suggest that ERRs are ligand-independent activators of transcription whose activation potential may rather be determined by the presence of transcriptional coactivators (17)(18)(1...

NGFI-B is a ligand-independent orphan nuclear receptor of the NR4A subfamily that displays important functional differences with its homolog Nurr1. In particular, the NGFI-B ligand-binding domain (LBD) exhibits only modest activity in cell lines in which the Nurr1 LBD strongly activates transcription. To gain insight into the structural basis for the distinct activation potentials, we determined the crystal structure of the NGFI-B LBD at 2.4-Å resolution. Superimposition with the Nurr1 LBD revealed a significant shift of the position of helix 12, potentially caused by conservative amino acids exchanges in helix 3 or helix 12. Replacement of the helix 11-12 region of Nurr1 with that of NGFI-B dramatically reduces the transcriptional activity of the Nurr1 LBD. Similarly, mutation of Met 414 in helix 3 to leucine or of Leu 591 in helix 12 to isoleucine (the corresponding residues found in NGFI-B) significantly affects Nurr1 transactivation. In comparison, swapping the helix 11-12 region of Nurr1 into NGFI-B results in a modest increase of activity. These observations reveal a high sensitivity of LBD activity to changes that influence helix 12 positioning. Furthermore, mutation of hydrophobic surface residues in the helix 11-12 region (outside the canonical co-activator surface constituted by helices 3, 4, and 12) severely affects Nurr1 transactivation. Together, our data suggest that a novel co-regulator surface that includes helix 11 and a specifically positioned helix 12 determine the cell type-dependent activities of the NGFI-B and the Nurr1 LBD.