Proteogenomic Characterization of Patient-Derived Xenografts Highlights the Role of REST in Neuroendocrine Differentiation of Castration-Resistant Prostate Cancer.

PURPOSE: An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood.Experimental Design: We applied mass spectrometry-based proteomics to a unique set of 17 prostate cancer patient-derived xenografts (PDX) to characterize the effects of castration in vivo, and the proteome differences between NEPC and prostate adenocarcinomas. Genome-wide profiling of REST-occupied regions in prostate cancer cells was correlated to the expression changes in vivo to investigate the role of the transcriptional repressor REST in castration-induced NEPC differentiation. RESULTS: An average of 4,881 proteins were identified and quantified from each PDX. Proteins related to neurogenesis, cell-cycle regulation, and DNA repair were found upregulated and elevated in NEPC, while the reduced levels of proteins involved in mitochondrial functions suggested a prevalent glycolytic metabolism of NEPC tumors. Integration of the REST chromatin bound regions with expression changes indicated a direct role of REST in regulating neuronal gene expression in prostate cancer cells. Mechanistically, depletion of REST led to cell-cycle arrest in G1, which could be rescued by p53 knockdown. Finally, the expression of the REST-regulated gene secretagogin (SCGN) correlated with an increased risk of suffering disease relapse after radical prostatectomy. CONCLUSIONS: This study presents the first deep characterization of the proteome of NEPC and suggests that concomitant inhibition of REST and the p53 pathway would promote NEPC. We also identify SCGN as a novel prognostic marker in prostate cancer.

Epigenetic inheritance through self-recruitment of the polycomb repressive complex 2.

Maintenance of gene expression through epigenetic mechanisms such as DNA- and histone-methylation is essential for preserving cellular identity and function. Multiplication of eukaryotic cells requires that the DNA content of the cell is duplicated through replication, which is coupled to incorporation of de novo synthesized core histones into nucleosomal structures. One of the challenging questions in biology is to explain how the organism ensures that regulatory epigenetic marks, once established, are transferred from one cell generation to the next. Based on studies in our laboratory, we have recently proposed a model for how the methylated lysine 27 of histone H3 (H3K27) can be stably transmitted through the cell division cycle. We found that the Polycomb Repressive Complex 2 (PRC2), which is responsible for di- and trimethylation of H3K27 (H3K27me2/me3), binds to its own site of methylation. Moreover, our results suggested that maintenance of transcriptional repression by PRC2 requires the binding of the PRC2 complex to H3K27me3/me2. Based on these two key observations we propose that PRC2 is able to copy the mark from an old parental H3 molecule to a newly synthesized H3 molecule as DNA replication proceeds. In addition, our results support a model for how the H3K27me3 mark could be preserved in the interphase of the cell cycle, where other events such as histone exchange and demethylation could counteract PRC2 function. Here we discuss the implications of our results in further detail.

A model for transmission of the H3K27me3 epigenetic mark.

Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division. Here we provide a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells. We show that the PRC2 complex binds to the H3K27me3 mark and colocalizes with this mark in G1 phase and with sites of ongoing DNA replication. Efficient binding requires an intact trimeric PRC2 complex containing EZH2, EED and SUZ12, but is independent of the catalytic SET domain of EZH2. Using a heterologous reporter system, we show that transient recruitment of the PRC2 complex to chromatin, upstream of the transcriptional start site, is sufficient to maintain repression through endogenous PRC2 during subsequent cell divisions. Thus, we suggest that once the H3K27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, leading to methylation of H3K27 on the daughter strands during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure and transcriptional programs.

Coordinated regulation of transcriptional repression by the RBP2 H3K4 demethylase and Polycomb-Repressive Complex 2.

Polycomb group (PcG) proteins regulate important cellular processes such as embryogenesis, cell proliferation, and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions. The Polycomb-Repressive Complex 2 (PRC2) is highly conserved during evolution, and its intrinsic histone H3 Lys 27 (K27) trimethylation (me3) activity is essential for PcG-mediated transcriptional repression. Here, we show a functional interplay between the PRC2 complex and the H3K4me3 demethylase Rbp2 (Jarid1a) in mouse embryonic stem (ES) cells. By genome-wide location analysis we found that Rbp2 is associated with a large number of PcG target genes in mouse ES cells. We show that the PRC2 complex recruits Rbp2 to its target genes, and that this interaction is required for PRC2-mediated repressive activity during ES cell differentiation. Taken together, these results demonstrate an elegant mechanism for repression of developmental genes by the coordinated regulation of epigenetic marks involved in repression and activation of transcription.

Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus.

The Polycomb group (PcG) proteins are essential for embryogenesis, and their expression is often found deregulated in human cancer. The PcGs form two major protein complexes, called polycomb repressive complexes 1 and 2 (PRC1 and PRC2) whose function is to maintain transcriptional repression. Here, we demonstrate that the chromodomain-containing protein, CBX8, which is part of one of the PRC1 complexes, regulates proliferation of diploid human and mouse fibroblasts through direct binding to the INK4A-ARF locus. Furthermore, we demonstrate that CBX8 is limiting for the regulation of INK4A-ARF, and that ectopic expression of CBX8 leads to repression of the Ink4a-Arf locus and bypass of senescence, leading to cellular immortalization. Gene expression and location analysis demonstrate that besides the INK4A-ARF locus, CBX8 also regulates a number of other genes important for cell growth and survival. On the basis of these results, we conclude that CBX8 is an essential component of one of the PRC1 complexes, which directly regulate the expression of numerous target genes, including the INK4A-ARF locus, involved in cell-fate decisions.

The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells.

The p16INK4A and p14ARF proteins, encoded by the INK4A-ARF locus, are key regulators of cellular senescence, yet the mechanisms triggering their up-regulation are not well understood. Here, we show that the ability of the oncogene BMI1 to repress the INK4A-ARF locus requires its direct association and is dependent on the continued presence of the EZH2-containing Polycomb-Repressive Complex 2 (PRC2) complex. Significantly, EZH2 is down-regulated in stressed and senescing populations of cells, coinciding with decreased levels of associated H3K27me3, displacement of BMI1, and activation of transcription. These results provide a model for how the INK4A-ARF locus is activated and how Polycombs contribute to cancer.

RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3.

Methylation of histones has been regarded as a stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of histone demethylases has challenged the stable nature of histone methylation. Here we demonstrate that the JARID1 proteins RBP2, PLU1, and SMCX are histone demethylases specific for di- and trimethylated histone 3 lysine 4 (H3K4). Consistent with a role for the JARID1 Drosophila homolog Lid in regulating expression of homeotic genes during development, we show that RBP2 is displaced from Hox genes during embryonic stem (ES) cell differentiation correlating with an increase of their H3K4me3 levels and expression. Furthermore, we show that mutation or RNAi depletion of the C. elegans JARID1 homolog rbr-2 leads to increased levels of H3K4me3 during larval development and defects in vulva formation. Taken together, these results suggest that H3K4me3/me2 demethylation regulated by the JARID1 family plays an important role during development.

The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3.

Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.

Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions.

The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes.