Zhaohui S. Qin

p53-Mediated Activation of miRNA34 Candidate Tumor-Suppressor Genes

BACKGROUND In response to varied cell stress signals, the p53 tumor-suppressor protein activates a multitude of genes encoding proteins with functions in cell-cycle control, DNA repair, senescence, and apoptosis. The role of p53 in transcription of other types of RNAs, such as microRNAs (miRNAs) is essentially unknown. RESULTS Using gene-expression analyses, reporter gene assays, and chromatin-immunoprecipitation approaches, we present definitive evidence that the abundance of the three-member miRNA34 family is directly regulated by p53 in cell lines and tissues. Using array-based approaches and algorithm predictions, we define genes likely to be directly regulated by miRNA34, with cell-cycle regulatory genes being the most prominent class. In addition, we provide functional evidence, obtained via antisense oligonucleotide transfection and the use of mouse embryonic stem cells with loss of miRNA34a function, that the BCL2 protein is regulated directly by miRNA34. Finally, we demonstrate that the expression of two miRNA34s is dramatically reduced in 6 of 14 (43%) non-small cell lung cancers (NSCLCs) and that the restoration of miRNA34 expression inhibits growth of NSCLC cells. CONCLUSIONS Taken together, the data suggest the miRNA34s might be key effectors of p53 tumor-suppressor function, and their inactivation might contribute to certain cancers.

Bayesian haplotype inference for multiple linked single-nucleotide polymorphisms.

Haplotypes have gained increasing attention in the mapping of complex-disease genes, because of the abundance of single-nucleotide polymorphisms (SNPs) and the limited power of conventional single-locus analyses. It has been shown that haplotype-inference methods such as Clarks algorithm, the expectation-maximization algorithm, and a coalescence-based iterative-sampling algorithm are fairly effective and economical alternatives to molecular-haplotyping methods. To contend with some weaknesses of the existing algorithms, we propose a new Monte Carlo approach. In particular, we first partition the whole haplotype into smaller segments. Then, we use the Gibbs sampler both to construct the partial haplotypes of each segment and to assemble all the segments together. Our algorithm can accurately and rapidly infer haplotypes for a large number of linked SNPs. By using a wide variety of real and simulated data sets, we demonstrate the advantages of our Bayesian algorithm, and we show that it is robust to the violation of Hardy-Weinberg equilibrium, to the presence of missing data, and to occurrences of recombination hotspots.

An Integrated Network of Androgen Receptor, Polycomb, and TMPRSS2-ERG Gene Fusions in Prostate Cancer Progression

Chromosomal rearrangements fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG occur in approximately 50% of prostate cancers, but how the fusion products regulate prostate cancer remains unclear. Using chromatin immunoprecipitation coupled with massively parallel sequencing, we found that ERG disrupts androgen receptor (AR) signaling by inhibiting AR expression, binding to and inhibiting AR activity at gene-specific loci, and inducing repressive epigenetic programs via direct activation of the H3K27 methyltransferase EZH2, a Polycomb group protein. These findings provide a working model in which TMPRSS2-ERG plays a critical role in cancer progression by disrupting lineage-specific differentiation of the prostate and potentiating the EZH2-mediated dedifferentiation program.

A Global Protein Kinase and Phosphatase Interaction Network in Yeast

Budding Yeast Kinome Revealed Covalent modification of proteins by phosphorylation is a primary means by which cells control the biochemical activities and functions of proteins. To better understand the full spectrum of cellular control mechanisms mediated by phosphorylation, Breitkreutz et al. (p. 1043; see the Perspective by Levy et al.) used mass spectrometry to identify proteins that interacted with the complete set of protein kinases from budding yeast and with other molecules, including phosphatases, which influence phosphorylation reactions. The results reveal a network of interacting protein kinases and phosphatases, and analysis of other interacting proteins suggests previously undiscovered roles for many of these enzymes. Phosphorylation reactions in budding yeast reveal the regulatory architecture of a fundamental cellular control system. The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses.

Partition-Ligation–Expectation-Maximization Algorithm for Haplotype Inference with Single-Nucleotide Polymorphisms

We are grateful to Chi-Hse Teng and the two anonymous reviewers for insightful comments. This research was supported in part by the National Science Foundation grants DMS-0094613 and DMS-0104129 and National Institutes of Health grant R01 HG02518-01.

Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer

Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. Progression to CRPC after androgen ablation therapy is predominantly driven by deregulated androgen receptor (AR) signalling. Despite the success of recently approved therapies targeting AR signalling, such as abiraterone and second-generation anti-androgens including MDV3100 (also known as enzalutamide), durable responses are limited, presumably owing to acquired resistance. Recently, JQ1 and I-BET762 two selective small-molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit anti-proliferative effects in a range of malignancies. Here we show that AR-signalling-competent human CRPC cell lines are preferentially sensitive to bromodomain and extraterminal (BET) inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ1 (refs 11, 13). Like the direct AR antagonist MDV3100, JQ1 disrupted AR recruitment to target gene loci. By contrast with MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR-mediated gene transcription, including induction of the TMPRSS2-ERG gene fusion and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft mouse models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer.

Gene Density, Transcription, and Insulators Contribute to the Partition of the Drosophila Genome into Physical Domains

The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.

Coordinated Regulation of Polycomb Group Complexes through microRNAs in Cancer

Polycomb Repressive Complexes (PRC1 and PRC2)-mediated epigenetic regulation is critical for maintaining cellular homeostasis. Members of Polycomb Group (PcG) proteins including EZH2, a PRC2 component, are upregulated in various cancer types, implicating their role in tumorigenesis. Here, we have identified several microRNAs (miRNAs) that are repressed by EZH2. These miRNAs, in turn, regulate the expression of PRC1 proteins BMI1 and RING2. We found that ectopic overexpression of EZH2-regulated miRNAs attenuated cancer cell growth and invasiveness, and abrogated cancer stem cell properties. Importantly, expression analysis revealed an inverse correlation between miRNA and PRC protein levels in cell culture and prostate cancer tissues. Taken together, our data have uncovered a coordinate regulation of PRC1 and PRC2 activities that is mediated by miRNAs.

Insulator function and topological domain border strength scale with architectural protein occupancy

BackgroundChromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions.ResultsBy mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals.ConclusionsWe identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains.

Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer

Beginning with precursor lesions, aberrant DNA methylation marks the entire spectrum of prostate cancer progression. We mapped the global DNA methylation patterns in select prostate tissues and cell lines using MethylPlex-next-generation sequencing (M-NGS). Hidden Markov model-based next-generation sequence analysis identified ∼68,000 methylated regions per sample. While global CpG island (CGI) methylation was not differential between benign adjacent and cancer samples, overall promoter CGI methylation significantly increased from ~12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues, respectively (P-value < 2 × 10(-16)). We found distinct patterns of promoter methylation around transcription start sites, where methylation occurred not only on the CGIs, but also on flanking regions and CGI sparse promoters. Among the 6691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) are cancer-specific, including numerous novel DMRs. A novel cancer-specific DMR in the WFDC2 promoter showed frequent methylation in cancer (17/22 tissues, 6/6 cell lines), but not in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested an epigenetic mechanism for alternate transcription start site utilization, and these modifications segregated into distinct regions when present on the same promoter. Finally, we observed differences in repeat element methylation, particularly LINE-1, between ERG gene fusion-positive and -negative cancers, and we confirmed this observation using pyrosequencing on a tissue panel. This comprehensive methylome map will further our understanding of epigenetic regulation in prostate cancer progression.