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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 [...]

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]

DNA methylation at cytosine is a major epigenetic mark, heavily implicated in controlling key cellular processes such as development and differentiation, cellular memory, or carcinogenesis. Bisulfite treatment in conjunction with next generation sequencing has been a powerful tool for studying this modification in a quantitative manner in the context of the whole genome and with a single nucleotide resolution. This chapter describes a protocol for bisulfite sequencing adapted to a single-cell format that allows for capturing the methylation signal from up to 50% CpG nucleotides in each cell.

Many of the key cellular processes including establishing the cell's identity are governed by chromatin proteins. Mapping their binding on the level of a single cell would give us important insights into a new dimension of cellular heterogeneity. However, ChIP-seq, the main method to study protein-DNA interaction in the chromatin context, has proven very challenging to scale down. ChIPmentation is a modification of ChIP-seq, in which the Tn5 transposase is used to introduce sequencing adapters in one step. This allows to significantly reduce the required input material. ChIPmentation is a robust and versatile approach and even though it has not yet achieved single-cell resolution, we believe that it is a very promising starting point for further downscaling.