Eric Metzger

LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription

Gene regulation in eukaryotes requires the coordinate interaction of chromatin-modulating proteins with specific transcription factors such as the androgen receptor. Gene activation and repression is specifically regulated by histone methylation status at distinct lysine residues. Here we show that lysine-specific demethylase 1 (LSD1; also known as BHC110) co-localizes with the androgen receptor in normal human prostate and prostate tumour. LSD1 interacts with androgen receptor in vitro and in vivo, and stimulates androgen-receptor-dependent transcription. Conversely, knockdown of LSD1 protein levels abrogates androgen-induced transcriptional activation and cell proliferation. Chromatin immunoprecipitation analyses demonstrate that androgen receptor and LSD1 form chromatin-associated complexes in a ligand-dependent manner. LSD1 relieves repressive histone marks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repression of androgen receptor target genes. Furthermore, we identify pargyline as an inhibitor of LSD1. Pargyline blocks demethylation of H3-K9 by LSD1 and consequently androgen-receptor-dependent transcription. Thus, modulation of LSD1 activity offers a new strategy to regulate androgen receptor functions. Here, we link demethylation of a repressive histone mark with androgen-receptor-dependent gene activation, thus providing a mechanism by which demethylases control specific gene expression.

Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression

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.

Androgen Receptor Coactivators Lysine-Specific Histone Demethylase 1 and Four and a Half LIM Domain Protein 2 Predict Risk of Prostate Cancer Recurrence

Prostate cancer biology varies from locally confined tumors with low risk for relapse to tumors with high risk for progression even after radical prostatectomy. Currently, there are no reliable biomarkers to predict tumor relapse and poor clinical outcome. In this study, we correlated expression patterns of the androgen receptor (AR) coactivators lysine-specific histone demethylase 1 (LSD1) and four and a half LIM-domain protein 2 (FHL2), AR, Gleason score, Gleason grade, and p53 expression in clinically organ confined prostate cancers with relapse after radical prostatectomy. Our data reveal that high levels of LSD1, nuclear expression of the FHL2 coactivator, high Gleason score and grade, and very strong staining of nuclear p53 correlate significantly with relapse during follow-up. No correlation exists with relapse and the expression of AR and cytoplasmic expression of FHL2. To confirm these data, we did quantitative reverse transcription-PCR and Western blot analyses in a subset of tumor specimens. Consistently, both LSD1 mRNA and protein levels were significantly up-regulated in high-risk tumors. We previously identified LSD1 and FHL2 as nuclear cofactors interacting specifically with the AR in prostate cells and showed that both stimulate androgen-dependent gene transcription. Our present study suggests that LSD1 and nuclear FHL2 may serve as novel biomarkers predictive for prostate cancer with aggressive biology and point to a role of LSD1 and FHL2 in constitutive activation of AR-mediated growth signals.

FHL2, a novel tissue-specific coactivator of the androgen receptor

The control of target gene expression by nuclear receptors requires the recruitment of multiple cofactors. However, the exact mechanisms by which nuclear receptor–cofactor interactions result in tissue‐specific gene regulation are unclear. Here we characterize a novel tissue‐specific coactivator for the androgen receptor (AR), which is identical to a previously reported protein FHL2/DRAL with unknown function. In the adult, FHL2 is expressed in the myocardium of the heart and in the epithelial cells of the prostate, where it colocalizes with the AR in the nucleus. FHL2 contains a strong, autonomous transactivation function and binds specifically to the AR in vitro and in vivo. In an agonist‐ and AF‐2‐dependent manner FHL2 selectively increases the transcriptional activity of the AR, but not that of any other nuclear receptor. In addition, the transcription of the prostate‐specific AR target gene probasin is coactivated by FHL2. Taken together, our data demonstrate that FHL2 is the first LIM‐only coactivator of the AR with a unique tissue‐specific expression pattern.

Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy.

Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA-mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene-specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy.

A TFTC/STAGA Module Mediates Histone H2A and H2B Deubiquitination, Coactivates Nuclear Receptors, and Counteracts Heterochromatin Silencing

Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.

Phosphorylation of histone H3T6 by PKCβ I controls demethylation at histone H3K4

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 (PKCβI, also known as PRKCβ) 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, PKCβI co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCβI abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCβI requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCβI and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCβI 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.

The transcriptional coactivator FHL2 transmits Rho signals from the cell membrane into the nucleus

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 Rho‐kinase. In addition, Rho signalling influences 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.

Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation

Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.

A novel inducible transactivation domain in the androgen receptor: implications for PRK in prostate cancer

In addition to the classical activation by ligands, nuclear receptor activity is also regulated by ligand‐independent signalling. Here, we unravel a novel signal transduction pathway that links the RhoA effector protein kinase C‐related kinase PRK1 to the transcriptional activation of the androgen receptor (AR). Stimulation of the PRK signalling cascade results in a ligand‐dependent superactivation of AR. We show that AR and PRK1 interact both in vivo and in vitro. The transactivation unit 5 (TAU‐5) located in the N‐terminus of AR suffices for activation by PRK1. Thus, TAU‐5 defines a novel, signal‐inducible transactivation domain. Furthermore, PRK1 promotes a functional complex of AR with the co‐activator TIF‐2. Importantly, PRK signalling also stimulates AR activity in the presence of adrenal androgens, which are still present in prostate tumour patients subjected to testicular androgen ablation therapy. Moreover, PRK1 activates AR even in the presence of the AR antagonist cyproterone acetate that is used in the clinical management of prostate cancer. Since prostate tumours strongly overexpress PRK1, our data support a model in which AR activity is controlled by PRK signalling.