Phosphorylation of histone H3T6 by PKCbeta(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 (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.

Ciliary neurotrophic factor as a transient negative regulator of rod development in rat retina.

PURPOSE: Ciliary neurotrophic factor (CNTF) has been shown to inhibit the developmental expression of rod differentiation markers in rat retinal cultures. The present study was undertaken to investigate whether CNTF transiently inhibits rod differentiation or induces irreversible changes in the developmental fate of rod precursors. METHODS: The effects of CNTF on rod differentiation were monitored in organotypic slice cultures from early postnatal rat retinas by quantification of opsin-immunoreactive cells. The in vitro formation of the photoreceptor layer was analyzed by light and electron microscopy. The developmental expression of the CNTF receptor in photoreceptors was determined by immunoblot analysis. RESULTS: CNTF did not interfere with the generation of rod precursors, their morphologic differentiation, or the formation of the outer nuclear layer. Inhibition of rod differentiation was reversible. In the continuous presence of CNTF the number of opsin-positive cells increased at a normal rate but with a delay of 3 to 4 days. Developing rods became resistant to CNTF, both in vivo and in vitro, and this correlated temporally with the downregulation of CNTF receptor expression. Receptor downregulation was inhibited by CNTF in a dose-dependent manner. At higher CNTF concentrations with sustained receptor expression, the CNTF-induced decrease in opsin expression was accompanied by an increase in the expression of a bipolar cell marker in rod precursors located in the photoreceptor layer. CONCLUSIONS: These data indicate that, although rod precursors exhibit some phenotypic plasticity in the presence of CNTF, the factor does not induce a switch in the developmental fate of rod precursors but plays a role as a transient and reversible negative regulator of rod differentiation.

The coactivator of transcription CREB-binding protein interacts preferentially with the glycosylated form of Stat5.

The signal transducer and activator of transcription (Stat) gene family comprises seven members with similarities in their domain structure and a common mode of activation. Members of this gene family mediate interferon induction of gene transcription and the response to a large number of growth factors and hormones. Extracellular ligand binding to transmembrane receptors causes the intracellular activation of associated tyrosine kinases, phosphorylation of Stat molecules, dimerization, and translocation to the nucleus. Prolactin-induced phosphorylation of Stat5 is a key event in the development and differentiation of mammary epithelial cells. In addition to the crucial phosphorylation at tyrosine 694, we have identified an O-linked N-acetylglucosamine (O-GlcNAc) as another secondary modification essential for the transcriptional induction by Stat5. This modification was only found on nuclear Stat5 after cytokine activation. Similar observations were made with Stat1, Stat3, and Stat6. Glycosylation of Stat5, however, does not seem to be a prerequisite for nuclear translocation. Mass spectrometric analysis revealed a glycosylated peptide in the N-terminal region of Stat5. Replacement of threonine 92 by an alanine residue (Stat5a-T92A) strongly reduced the prolactin induction of Stat5a glycosylation and abolished transactivation of a target gene promoter. Only the glycosylated form of Stat5 was able to bind the coactivator of transcription CBP, an essential interaction for Stat5-mediated gene transcription.