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 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.

Impairment of prostate cancer cell growth by a selective and reversible lysine‐specific demethylase 1 inhibitor

“…17 One milligram peptide of histone H3 (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] carrying one or two methyl groups at lysine 4 were incubated with 2 lg of GST-LSD1 in the absence or presence of Namoline. The reaction mixture was incubated in demethylation buffer for 4 hr at 37 C and analyzed by mass spectroscopy.…”

“…Next, we investigated the effect of Namoline on the expression of endogenous AR target genes shown to be involved in prostate cancer. 17 qRT-PCR analysis of LNCaP cells demonstrates that Namoline severely impairs androgen-induced expression of genes such as FKBP5, TMPRSS2, ELK4, MAK, NKX3.1, IGF1R, MAF, GREB1, KLK2 and PSA (Fig. 3b and Supporting Information Fig.…”

Chemical “Diversity” of Chromatin Through Histone Variants and Histone Modifications

“…Recent studies have now identified new connections between other signal transduction pathways and histone phosphorylation. For example, PKC-β 1 phosphorylates H3 threonine 6, which then opposes demethylation of H3K4 by LSD1 during androgen stimulation [126]. This mark is elevated in prostate carcinomas, and inhibition of PKC-β 1 interferes with AR-induced cell proliferation in cell culture and cancer progression of tumor xenografts [126].…”

Transcriptional Regulation of the Human Thromboxane A2 Receptor Gene by Wilms’ Tumour (WT)

“…Significantly in the context of PCa, through detailed mechanistic studies, we recently discovered that both the TPα and the TPβ isoforms directly interact with and regulate signalling by protein kinase C-related kinase/ protein kinase novel (PRK/PKN) (48), a family of 3 AGC kinases that act immediately downstream of phosphatidylinositol 3′kinases, and are strongly, yet differentially, implicated in several cancers (49)(50)(51) and in B-cell development (52). Indeed, in addition to acting as Rho GTPase effectors, activation of the PRKs (e.g., PRK1) in response to androgen receptor (AR) signalling within the prostate catalyses phosphorylation of histone (H)3 at Thr11 (H3pThr11) which, in turn, serves as a specific epigenetic marker, and gatekeeper, of androgen-induced chromatin remodelling and transcriptional activation (48,(53)(54)(55). Hence, owing to their ability to regulate RhoA-/C-mediated responses, including metastatic processes, combined with their epigenetic priming of tumour cells, members of the PRK family are key chemotherapeutic targets particularly in castrate-resistant prostate cancer, the metastatic lethal form of PCa that occurs following androgen deprivation therapy (53,55,56).…”

“…Indeed, in addition to acting as Rho GTPase effectors, activation of the PRKs (e.g., PRK1) in response to androgen receptor (AR) signalling within the prostate catalyses phosphorylation of histone (H)3 at Thr11 (H3pThr11) which, in turn, serves as a specific epigenetic marker, and gatekeeper, of androgen-induced chromatin remodelling and transcriptional activation (48,(53)(54)(55). Hence, owing to their ability to regulate RhoA-/C-mediated responses, including metastatic processes, combined with their epigenetic priming of tumour cells, members of the PRK family are key chemotherapeutic targets particularly in castrate-resistant prostate cancer, the metastatic lethal form of PCa that occurs following androgen deprivation therapy (53,55,56).…”