Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor–derived xenografts (PDX) revealed that AR–negative SCPC (AR^?SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR–positive castration-resistant adenocarcinomas (AR⁺ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR^? and AR⁺ PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR^?SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR^?SCPC cell lines. We conclude that the epigenome of AR^? is distinct from that of AR⁺ castration-resistant prostate carcinomas, and that the AR^? phenotype can be reversed with epigenetic drugs.     

Mechanism of SUMOylation-Mediated Regulation of Type I IFN Expression

Type I interferons (IFN) are cytokines that bridge the innate and adaptive immune response, and thus play central roles in human health, including vaccine efficacy, immune response to cancer and pathogen infection, and autoimmune disorders. Post-translational protein modifications by the small ubiquitin-like modifiers (SUMO) have recently emerged as an important regulator of type I IFN expression as shown by studies using murine and cellular models and recent human clinical trials. However, the mechanism regarding how SUMOylation regulates type I IFN expression remains poorly understood. In this study, we show that SUMOylation inhibition does not activate IFNB1 gene promoter that is regulated by known canonical pathways including cytosolic DNA. Instead, we identified a binding site for the chromatin modification enzyme, the SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1), located between the IFNB1 promoter and a previously identified enhancer. We found that SETDB1 regulates IFNB1 expression and SUMOylation of SETDB1 is required for its binding and enhancing the H3K9me3 heterochromatin signal in this region. Heterochromatin, a tightly packed form of DNA, has been documented to suppress gene expression through suppressing enhancer function. Taken together, our study identified a novel mechanism of regulation of type I IFN expression, at least in part, through SUMOylation of a chromatin modification enzyme.     

Synergistic chromatin repression of the tumor suppressor gene RARB in human prostate cancers

DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the co-existence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor β (RARβ), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARβ promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARβ during prostate tumorigenesis.     

Epidaurus: aggregation and integration analysis of prostate cancer epigenome

Integrative analyses of epigenetic data promise a deeper understanding of the epigenome. Epidaurus is a bioinformatics tool used to effectively reveal inter-dataset relevance and differences through data aggregation, integration and visualization. In this study, we demonstrated the utility of Epidaurus in validating hypotheses and generating novel biological insights. In particular, we described the use of Epidaurus to (i) integrate epigenetic data from prostate cancer cell lines to validate the activation function of EZH2 in castration-resistant prostate cancer and to (ii) study the mechanism of androgen receptor (AR) binding deregulation induced by the knockdown of FOXA1. We found that EZH2''s noncanonical activation function was reaffirmed by its association with active histone markers and the lack of association with repressive markers. More importantly, we revealed that the binding of AR was selectively reprogramed to promoter regions, leading to the up-regulation of hundreds of cancer-associated genes including EGFR. The prebuilt epigenetic dataset from commonly used cell lines (LNCaP, VCaP, LNCaP-Abl, MCF7, GM12878, K562, HeLa-S3, A549, HePG2) makes Epidaurus a useful online resource for epigenetic research. As standalone software, Epidaurus is specifically designed to process user customized datasets with both efficiency and convenience.     

CPEB1, a histone-modified hypomethylated gene,is regulated by miR-101 and involved in cell senescence in glioma

Epigenetic mechanisms have important roles in carcinogenesis. We certified that the mRNA translation-related gene cytoplasmic polyadenylation element-binding protein 1 (CPEB1) is hypomethylated and overexpressed in glioma cells and tissues. The knockdown of CPEB1 reduced cell senescence by regulating the expression or distribution of p53 in glioma cells. CPEB1 is also regulated directly by the tumor suppressor miR-101, a potential marker of glioma. It is known that the histone methyltransferase enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) are direct targets of miR-101. We demonstrated that miR-101 downregulated the expression of CPEB1 through reversing the methylation status of the CPEB1 promoter by regulating the presence on the promoter of the methylation-related histones H3K4me2, H3K27me3, H3K9me3 and H4K20me3. The epigenetic regulation of H3K27me3 on CPEB1 promoter is mediated by EZH2 and EED. EZH2 has a role in the regulation of H3K4me2. Furthermore, the downregulation of CPEB1 induced senescence in a p53-dependent manner.     

Direct interaction between DNA methyltransferase DIM-2 and HP1 is required for DNA methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genomic stability in mammals, plants, and fungi. Genetics studies of Neurospora crassa have revealed that a DNA methyltransferase (DIM-2), a histone H3K9 methyltransferase (DIM-5), and heterochromatin protein 1 (HP1) are required for DNA methylation. We explored the interrelationships of these components of the methylation machinery. A yeast two-hybrid screen revealed that HP1 interacts with DIM-2. We confirmed the interaction in vivo and demonstrated that it involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1. Both regions are essential for proper DNA methylation. We also determined that DIM-2 and HP1 form a stable complex independently of the trimethylation of histone H3K9, although the association of DIM-2 with its substrate sequences depends on trimethyl-H3K9. The DIM-2/HP1 complex does not include DIM-5. We conclude that DNA methylation in Neurospora is largely or exclusively the result of a unidirectional pathway in which DIM-5 methylates histone H3K9 and then the DIM-2/HP1 complex recognizes the resulting trimethyl-H3K9 mark via the chromo domain of HP1.     

Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation,motor function,and neuropathology in a Huntington’s disease mouse model

The present study describes evaluation of epigenetic regulation by a small molecule as the therapeutic potential for treatment of Huntington’s disease (HD). We identified 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) as a novel SETDB1/ESET inhibitor using a combined in silico and in vitro cell based screening system. APQ reduced SETDB1 activity and H3K9me3 levels in a HD cell line model. In particular, not only APQ reduced H3K9me3 levels in the striatum but it also improved motor function and neuropathological symptoms such as neuronal size and activity in HD transgenic (YAC128) mice with minimal toxicity. Using H3K9me3-ChIP and genome-wide sequencing, we also confirmed that APQ modulates H3K9me3-landscaped epigenomes in YAC128 mice. These data provide that APQ, a novel small molecule SETDB1 inhibitor, coordinates H3K9me-dependent heterochromatin remodelling and can be an epigenetic drug for treating HD, leading with hope in clinical trials of HD.