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Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation (1,2). 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) (1,3). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9)2. 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 (PKC[β.sub.I], also known as PKCβ) 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, PKC[β.sub.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 PKC[β.sub.I] abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKC[β.sub.I] requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1) (4). Notably, increased levels of PKC[β.sub.I] and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKC[β.sub.I] blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xeno-grafts 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., We previously showed that during AR-dependent gene activation, LSD1 co-operates with a trimethyl demethylase, the Jumonji C (JMJC) (5) domain-containing protein JMJD2C (also known as KDM4C), in removing repressive histone [...]

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities. For this array, we expressed 266 human kinases in four different S. cerevisiae strains and profiled ectopic growth as a proxy for kinase activity across 33 conditions. More than half of the kinases showed an activity‐dependent phenotype across many conditions and in more than one strain. We then employed the kinase array to identify the kinase(s) that can modulate protein–protein interactions (PPIs). Two characterized, phosphorylation‐dependent PPIs with unknown kinase–substrate relationships were analyzed in a phospho‐yeast two‐hybrid assay. CK2α1 and SGK2 kinases can abrogate the interaction between the spliceosomal proteins AAR2 and PRPF8, and NEK6 kinase was found to mediate the estrogen receptor (ERα) interaction with 14‐3‐3 proteins. The human kinase yeast array can thus be used for a variety of kinase activity‐dependent readouts. Synopsis: A large array of human kinases is developed in yeast, covering the activities of more than 50% of the known human kinome and including many difficult‐to‐study kinases. The array is used to identify human kinases modulating phosphorylation‐dependent protein interactions. Growth phenotypes of 266 human protein kinases expressed in yeast are systematically assayed across four laboratory strains and 73 growth conditions.The human kinase yeast array contains 150 highly active human kinases and is a versatile tool for studying human kinase activities, including low abundant, tissue‐specific and "dark" kinases with largely unknown functions.The array is used in a phospho‐yeast two‐hybrid assay to identify kinases regulating phosphorylation‐dependent protein–protein interactions.SGK2 and CK2α1 are shown to modulate phospho‐dependent interactions between spliceosomal proteins AAR2 and PRPF8, while NEK6 and PAK5 modulate the association between ERα and 14‐3‐3. [ABSTRACT FROM AUTHOR]

NIR (Novel INHAT Repressor) is a potent transcriptional corepressor that is not blocked by histone deacetylase inhibitors, capable of silencing both basal and activator-driven transcription. A tandum affinity purification approach identified the tumor suppressor p53 as a NIR-interacting partner and it was observed that upon recruitment by p53, NIR represses transcription of both p53 dependent reporters and endogenous target genes.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]