Phosphorylation of histone H3T6 by PKCβI controls demethylation at histone H3K4

Abstract

Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation1,2. 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 (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)4. 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.

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Figure 1: PKCα, β I and β II specifically phosphorylate H3T6.
Figure 2: Phosphorylation of H3T6 blocks demethylation at H3K4 by LSD1.
Figure 3: PKCβ I phosphorylates H3T6 and controls demethylation at H3K4 during AR-dependent gene expression.
Figure 4: PKCβ I , H3T6ph and H3K4me2 levels positively correlate with the malignancy of prostate cancer, and PKCβ I controls androgen-dependent tumour cell proliferation and cancer growth in tumour xenografts.

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Acknowledgements

We thank U. Elsäßer-Beile, K. Alt, U. Wetterauer, W. Schulze-Seelmann, R. Schneider, T. Waldmann, L. Heukamp, J. M. G. Higgins and N. Potier for providing reagents. We are obliged to A. Rieder, F. Pfefferle, L. Walz, L. Spady and J. Lauterwasser for technical assistance. This work was supported by grants of the Deutsche Krebshilfe and the Deutsche Forschungsgemeinschaft (Reinhart Koselleck-Projekt, SFB 746/P2, Schu 688/9-1, Dr. Hans Messner Stiftung, Dr. Oetker-Stiftung and SFB 832/Z1) to R.S. and R.B.

Author Contributions E.M., R.B. and R.S. designed this study, analysed the data and edited the manuscript. E.M., H.G., J.M.M. and T.G. performed experiments and edited the manuscript. A.I. performed mass spectrometry analysis. C.B.-H. synthesized reagents. D.P., P.K., K.H., N.F., N.K., S.J. and J.K. performed experiments.

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Correspondence to Roland Schüle.

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Metzger, E., Imhof, A., Patel, D. et al. Phosphorylation of histone H3T6 by PKCβI controls demethylation at histone H3K4. Nature 464, 792–796 (2010). https://doi.org/10.1038/nature08839

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