Abstract: Factors that drive prostate cancer progression remain poorly defined, thus hindering the development of new therapeutic strategies. Disseminated tumors are treated through regimens that ablate androgen signaling, as prostate cancer cells require androgen for growth and survival. However, recurrent, incurable tumors that have bypassed the androgen requirement ultimately arise. This study reveals that the Brm ATPase, a component of selected SWI/SNF complexes, has significant antiproliferative functions in the prostate that protect against these transitions. First, we show that targeted ablation of Brm is causative for the development of prostatic hyperplasia in mice. Second, in vivo challenge revealed that Brm-/- epithelia acquire the capacity for lobe-specific, castration-resistant cellular proliferation. Third, investigation of human specimens revealed that Brm mRNA and protein levels are attenuated in prostate cancer. Fourth, Brm down-regulation was associated with an increased proliferative index, consistent with the mouse model. Lastly, gene expression profiling showed that Brm loss alters factors upstream of E2F1; this was confirmed in murine models, wherein Brm loss induced E2F1 deregulation in a tissue-specific manner. Combined, these data identify Brm as a major effector of serum androgen-induced proliferation in the prostate that is disrupted in human disease, and indicate that loss of Brm confers a proliferative advantage in prostate cancer.

Abstract: To identify biomarkers that discriminate the aggressive forms of prostate cancer, we performed gene expression profiling of prostate tumors using a genetically engineered mouse model that recapitulates the stages of human prostate cancer, namely Nkx3.1; Pten mutant mice. We observed a significant deregulation of the epidermal growth factor and mitogen-activated protein kinase (MAPK) signaling pathways, as well as their major downstream effectors--the activator protein-1 transcription factors c-Fos and c-Jun. Forced expression of c-Fos and c-Jun in prostate cancer cells promotes tumorigenicity and results in activation of extracellular signal-regulated kinase (Erk) MAPK signaling. In human prostate cancer, up-regulation of c-Fos and c-Jun proteins occurs in advanced disease and is correlated with Erk MAPK pathway activation, whereas high levels of c-Jun expression are associated with disease recurrence. Our analyses reveal a hitherto unappreciated role for AP-1 transcription factors in prostate cancer progression and identify c-Jun as a marker of high-risk prostate cancer. This study provides a striking example of how accurate mouse models can provide insights on molecular processes involved in progression and recurrence of human cancer.

Abstract: BACKGROUND: The expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5. RESULTS: We report here that 39 colon tumors from four independent mouse models and 100 human CRCs encompassing all clinical stages shared a striking recapitulation of embryonic colon gene expression. Compared to normal adult colon, all mouse and human tumors over-expressed a large cluster of genes highly enriched for functional association to the control of cell cycle progression, proliferation, and migration, including those encoding MYC, AKT2, PLK1 and SPARC. Mouse tumors positive for nuclear beta-catenin shifted the shared embryonic pattern to that of early development. Human and mouse tumors differed from normal embryonic colon by their loss of expression modules enriched for tumor suppressors (EDNRB, HSPE, KIT and LSP1). Human CRC adenocarcinomas lost an additional suppressor module (IGFBP4, MAP4K1, PDGFRA, STAB1 and WNT4). Many human tumor samples also gained expression of a coordinately regulated module associated with advanced malignancy (ABCC1, FOXO3A, LIF, PIK3R1, PRNP, TNC, TIMP3 and VEGF). CONCLUSION: Cross-species, developmental, and multi-model gene expression patterning comparisons provide an integrated and versatile framework for definition of transcriptional programs associated with oncogenesis. This approach also provides a general method for identifying pattern-specific biomarkers and therapeutic targets. This delineation and categorization of developmental and non-developmental activator and suppressor gene modules can thus facilitate the formulation of sophisticated hypotheses to evaluate potential synergistic effects of targeting within- and between-modules for next-generation combinatorial therapeutics and improved mouse models.

Abstract: BACKGROUND: Transcriptional modules (TM) consist of groups of co-regulated genes and transcription factors (TF) regulating their expression. Two high-throughput (HT) experimental technologies, gene expression microarrays and Chromatin Immuno-Precipitation on Chip (ChIP-chip), are capable of producing data informative about expression regulatory mechanism on a genome scale. The optimal approach to joint modeling of data generated by these two complementary biological assays, with the goal of identifying and characterizing TMs, is an important open problem in computational biomedicine. RESULTS: We developed and validated a novel probabilistic model and related computational procedure for identifying TMs by jointly modeling gene expression and ChIP-chip binding data. We demonstrate an improved functional coherence of the TMs produced by the new method when compared to either analyzing expression or ChIP-chip data separately or to alternative approaches for joint analysis. We also demonstrate the ability of the new algorithm to identify novel regulatory relationships not revealed by ChIP-chip data alone. The new computational procedure can be used in more or less the same way as one would use simple hierarchical clustering without performing any special transformation of data prior to the analysis. The R and C-source code for implementing our algorithm is incorporated within the R package gimmR which is freely available at http://eh3.uc.edu/gimm. CONCLUSION: Our results indicate that, whenever available, ChIP-chip and expression data should be analyzed within the unified probabilistic modeling framework, which will likely result in improved clusters of co-regulated genes and improved ability to detect meaningful regulatory relationships. Given the good statistical properties and the ease of use, the new computational procedure offers a worthy new tool for reconstructing transcriptional regulatory networks.

Abstract: In this report, we have investigated the relationship between androgen levels and prostate tumorigenesis in Nkx3.1; Pten mutant mice, a genetically engineered mouse model of human prostate cancer. By experimentally manipulating serum levels of testosterone in these mice for an extended period (i.e., 7 months), we have found that prolonged exposure of Nkx3.1; Pten mutant mice to androgen levels that are 10-fold lower than normal (the "Low-T" group) resulted in a marked acceleration of prostate tumorigenesis compared with those exposed to androgen levels within the reference range (the "Normal-T" group). We found that prostate tumors from the Low-T mutant mice share a similar gene expression profile as androgen-independent prostate tumors from these mutant mice, which includes the deregulated expression of several genes that are up-regulated in human hormone-refractory prostate cancer, such as Vav3 and Runx1. We propose that exposure to reduced androgens may promote prostate tumorigenesis by selecting for molecular events that promote more aggressive, hormone-refractory tumors.

Abstract: Transcriptional activation is often associated with chromatin remodeling. However, little is known about the dynamics of remodeling of nucleosome arrays in vivo. Upon induction of Saccharomyces cerevisiae PHO5, a novel kinetic assay of DNA methyltransferase accessibility showed that nucleosomes adjacent to the histone-free upstream activating sequence (UASp1) are disrupted earlier and at higher frequency in the cell population than are those more distal. Individually cloned molecules, each representing the chromatin state of a full promoter from a single cell, revealed multiple promoter classes with either no remodeling or variable numbers of disrupted nucleosomes. Individual promoters in the remodeled fraction were highly enriched for contiguous blocks of disrupted nucleosomes, the majority of which overlapped the UAS region. These results support a probabilistic model in which chromatin remodeling at PHO5 spreads from sites of transactivator association with DNA and attenuates with distance.

Abstract: Cytosine-5 DNA methyltransferases (C5 DMTases) are effective reagents for analyzing chromatin and footprinting DNA-bound factors in vivo. Cytosine methylation in accessible regions is assayed positively by the PCR-based technique of bisulfite sequencing. In this article, we outline two complementary uses for the DNA methyltransferase CviPI (M.CviPI, GC specificity) in probing chromatin organization. First, we describe the use of the naturally occurring, free enzyme as a diffusible probe to map changes in nucleosome structure and to footprint factor interactions at cis-regulatory sequences. In a second application, termed targeted gene methylation (TAGM), the DMTase is targeted via in-frame fusion to a DNA-binding factor. The rapid accumulation of DNA methylation enables highly sensitive detection of factor binding. Both strategies can be applied with any C5 DMTase, such as M.SssI, which also possesses a short-recognition specificity (CG). A description of methods for constructing C5 DMTase-expressing strains of Saccharomyces cerevisiae and analyzing chromatin regions is provided. We also include comprehensive protocols for the isolation and bisulfite treatment of genomic DNA as well as the subsequent bisulfite sequencing steps. Data demonstrating the efficacy of both DMTase probing techniques, theoretical considerations, and experimental analyses are presented at GAL1 and PHO5.

Abstract: We report a technique, named targeted gene methylation (TAGM), for identifying in vivo protein-binding sites in chromatin. M.CviPI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing. Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34- fold occur proximal to native Pho4-binding sites. Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure. In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation. Notably, substantial amounts of methylation are targeted by Pho4-M.CviPI under repressive conditions when most of the transactivator is excluded from the nucleus. Thus, TAGM enables rapid detection of DNA-protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level. Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.

Abstract: Androgens, particularly testosterone and its potent metabolite 5-α-dihydrotestosterone, serve as critical mediators in the development and maintenance of the male reproductive and non-reproductive systems. Androgen-dependent signaling is conveyed by the androgen receptor (AR), which is a member of the nuclear receptor superfamily. AR binding of androgen stimulates its ability to bind DNA and regulate gene transcription. The importance of the AR in human physiology is exemplified by the fact that disruption of this key receptor is causative for androgen insensitivity syndrome (AIS) and spinal and bulbar muscular atrophy (SBMA), also known as Kennedy’s disease. In contrast, AR activity is essential for benign prostatic hyperplasia (BPH) and the development and progression of prostate cancer. The advent of microarray technology has provided significant contributions toward the understanding of the underlying mechanisms that govern the AR function in these physiological and pathophysiological conditions. The aim of this chapter is to summarize the recent advances using genomics to study the role of androgens in developmental and reproductive processes, AIS, Kennedy’s disease, and prostate cancer. As the body of work relative to prostate cancer is large, a gene list comparison was performed with prostate cancer studies that utilized a well-characterized human prostatic adenocarcinoma cell line treated with physiological concentrations of androgen. The consensus targets were then evaluated on their physiological relevance as well as on their potential as direct AR targets. In addition, alternate models of AR action in prostate cancer were examined. These studies provide evidence for AR-dependent regulation of other identified genes involved in metabolism, transcription, and signaling pathways. Together, these collective observations reveal insightful information concerning androgen action in human health and disease.

Abstract: Promoter transactivation is often associated with the disruption or remodeling of nucleosomes in chromatin. The budding yeast PHO5 promoter, which drives expression of the major secreted acid phosphatase, is a proven model system for investigating gene regulatory mechanisms in the context of chromatin. Under conditions of high external phosphate concentration, PHO5 transcription is repressed through an upstream signaling cascade and an array of nucleosomes that are well positioned and hinder access to crucial regulatory elements. Much recent progress has been made in elucidating the molecular steps that lead to promoter activation upon phosphate starvation. Inhibition of an upstream repressive signaling cascade enables DNA binding by site-specific activator proteins, which, in turn, lead to the direct recruitment of two histone modifying and two ATP-dependent chromatin remodeling complexes. Using a novel single-molecule assay for chromatin remodeling in vivo, we have shown that the number of nucleosomes disrupted from cell-to-cell is highly variable. Moreover, nucleosomes adjacent to activator binding sites are disrupted earlier and at higher frequency than are those more distal. The results suggest that the localized recruitment of chromatin modifying and remodeling complexes leads to nucleosome disruption that spreads from sites of transactivator association with DNA and attenuates with distance.