Androgen-induced rhox homeobox genes modulate the expression of AR-regulated genes

Rhox5, the founding member of the reproductive homeobox on the X chromosome (Rhox) gene cluster, encodes a homeodomain-containing transcription factor that is selectively expressed in Sertoli cells, where it promotes the survival of male germ cells. To identify Rhox5-regulated genes, we generated 15P-1 Sertoli cell clones expressing physiological levels of Rhox5 from a stably transfected expression vector. Microarray analysis identified many genes altered in expression in response to Rhox5, including those encoding proteins controlling cell cycle regulation, apoptosis, metabolism, and cell-cell interactions. Fifteen of these Rhox5-regulated genes were chosen for further analysis. Analysis of Rhox5-null male mice indicated that at least 9 of these are Rhox5-regulated in the testes in vivo. Many of them have distinct postnatal expression patterns and are regulated by Rhox5 at different postnatal time points. Most of them are expressed in Sertoli cells, indicating that they are candidates to be directly regulated by Rhox5. Transfection analysis with expression vectors encoding different mouse and human Rhox family members revealed that the regulatory response of a subset of these Rhox5-regulated genes is both conserved and redundant. Given that Rhox5 depends on AR for expression in Sertoli cells, we examined whether some Rhox5-regulated genes are also regulated by androgen receptor (AR). We provide several lines of evidence that this is the case, leading us to propose that RHOX5 serves as a key intermediate transcription factor that directs some of the actions of AR in the testes

Loss of androgen signaling in mesenchymal sonic hedgehog responsive cells diminishes prostate development, growth, and regeneration.

Prostate embryonic development, pubertal and adult growth, maintenance, and regeneration are regulated through androgen signaling-mediated mesenchymal-epithelial interactions. Specifically, the essential role of mesenchymal androgen signaling in the development of prostate epithelium has been observed for over 30 years. However, the identity of the mesenchymal cells responsible for this paracrine regulation and related mechanisms are still unknown. Here, we provide the first demonstration of an indispensable role of the androgen receptor (AR) in sonic hedgehog (SHH) responsive Gli1-expressing cells, in regulating prostate development, growth, and regeneration. Selective deletion of AR expression in Gli1-expressing cells during embryogenesis disrupts prostatic budding and impairs prostate development and formation. Tissue recombination assays showed that urogenital mesenchyme (UGM) containing AR-deficient mesenchymal Gli1-expressing cells combined with wildtype urogenital epithelium (UGE) failed to develop normal prostate tissue in the presence of androgens, revealing the decisive role of AR in mesenchymal SHH responsive cells in prostate development. Prepubescent deletion of AR expression in Gli1-expressing cells resulted in severe impairment of androgen-induced prostate growth and regeneration. RNA-sequencing analysis showed significant alterations in signaling pathways related to prostate development, stem cells, and organ morphogenesis in AR-deficient Gli1-expressing cells. Among these altered pathways, the transforming growth factor β1 (TGFβ1) pathway was up-regulated in AR-deficient Gli1-expressing cells. We further demonstrated the activation of TGFβ1 signaling in AR-deleted prostatic Gli1-expressing cells, which inhibits prostate epithelium growth through paracrine regulation. These data demonstrate a novel role of the AR in the Gli1-expressing cellular niche for regulating prostatic cell fate, morphogenesis, and renewal, and elucidate the mechanism by which mesenchymal androgen-signaling through SHH-responsive cells elicits the growth and regeneration of prostate epithelium

Leveraging Epidemiology to Improve Risk Assessment.

The field of environmental public health is at an important crossroad. Our current biomonitoring efforts document widespread exposure to a host of chemicals for which toxicity information is lacking. At the same time, advances in the fields of genomics, proteomics, metabolomics, genetics and epigenetics are yielding volumes of data at a rapid pace. Our ability to detect chemicals in biological and environmental media has far outpaced our ability to interpret their health relevance, and as a result, the environmental risk paradigm, in its current state, is antiquated and ill-equipped to make the best use of these new data. In light of new scientific developments and the pressing need to characterize the public health burdens of chemicals, it is imperative to reinvigorate the use of environmental epidemiology in chemical risk assessment. Two case studies of chemical assessments from the Environmental Protection Agency Integrated Risk Information System database are presented to illustrate opportunities where epidemiologic data could have been used in place of experimental animal data in dose-response assessment, or where different approaches, techniques, or studies could have been employed to better utilize existing epidemiologic evidence. Based on the case studies and what can be learned from recent scientific advances and improved approaches to utilizing human data for dose-response estimation, recommendations are provided for the disciplines of epidemiology and risk assessment for enhancing the role of epidemiologic data in hazard identification and dose-response assessment

Genome-wide association study identifies common and low-frequency variants at the AMHgene locus that strongly predict serum AMH levels in males

Anti-Müllerian hormone (AMH) is an essential messenger of sexual differentiation in the foetus and is an emerging biomarker of postnatal reproductive function in females. Due to a paucity of adequately sized studies, the genetic determinants of circulating AMH levels are poorly characterized. In samples from 2815 adolescents aged 15 from the ALSPAC study, we performed the first genome-wide association study of serum AMH levels across a set of ∼9 M ‘1000 Genomes Reference Panel’ imputed genetic variants. Genetic variants at the AMH protein-coding gene showed considerable allelic heterogeneity, with both common variants [rs4807216 (PMale = 2 × 10−49, Beta: ∼0.9 SDs per allele), rs8112524 (PMale = 3 × 10−8, Beta: ∼0.25)] and low-frequency variants [rs2385821 (PMale = 6 × 10−31, Beta: ∼1.2, frequency 3.6%)] independently associated with apparently large effect sizes in males, but not females. For all three SNPs, we highlight mechanistic links to AMH gene function and demonstrate highly significant sex interactions (PHet 0.0003–6.3 × 10−12), culminating in contrasting estimates of trait variance explained (24.5% in males versus 0.8% in females). Using these SNPs as a genetic proxy for AMH levels, we found no evidence in additional datasets to support a biological role for AMH in complex traits and diseases in men

A Systems Biology Approach to Epigenetic Gene Regulation

The ability to control when, and how much of the genetic code is being expressed is the underlying principle behind gene regulation. Control of gene production is able to influence a cell's phenotype by determining which structural components of the cell's observable traits (shape, growth, and behavior) are made. In multicellular organism’s different cell types are able to arise from the same genetic code due to a difference in the patterns of genes being expressed. Essentially anywhere in the process of gene expression from transcription, RNA processing, translation, and post-translational modifications of the protein is subject to regulation. As transcription is the first step in the process of gene expression, it is the first level of regulation for influencing the cell phenotype. The actions of transcription factors, histone modifiers, and other proteins work together to influence RNA polymerase's ability to complete the process of transcription. The actions of transcription factors are able to influence transcription by controlling the ability of RNA polymerase to be recruited to the start of a protein coding region and histone modifiers can rearrange the histones of the chromatin causing entire regions of a chromosome to become exposed or sequestered. These transcriptional regulators are able to work in a combinatorial fashion with one another to either activate and/or repress wide repertoires of transcriptional targets. Mapping out a network of interactions between these transcriptional regulators in gene expression programs allows researchers to understand how each protein is able to influence the phenotype of the cell, and how mutations to any of these transcriptional regulators are able to drive the cell into a diseased state. In the case of cancer, changes in the mechanisms of gene regulation brought on by mutations to these transcriptional regulators may drive the cell's hyper proliferative state. With the creation of next generation sequencing researchers are now better able to define where regulation is taking place in the genome, and how much it is able to influence gene expression. This gives researchers the ability to build these gene regulatory networks and evaluate their impact on gene expression. The subsequent chapters of this dissertation are a reflection of my published work investigating the contribution of oncogenic processes to gene regulatory networks in cancer through the study of hyperactivating somatic mutation of a histone modifier, changes in transcription factor response element specificity, epigenetic regulation of transcription factor signaling, and a transcription factor coactivation network

Modelling the transcriptional regulation of androgen receptor in prostate cancer

Transcription of genes and production of proteins are essential functions of a normal cell. If disturbed, misregulation of crucial genes leads to aberrant cell behaviour and in some cases, leads to the development of diseased states such as cancer. One major transcriptional regulation tool involves the binding of transcription factor onto enhancer sequences that will encourage or repress transcription depending on the role of the transcription factor. In prostate cells, misregulation of the androgen receptor(AR), a key transcriptional regulator, leads to the development and maintenance of prostate cancer. Androgen receptor binds to numerous locations in the genome, but it is still unclear how and which other key transcription factors aid and repress AR-mediated transcription. Here I analyzed the data that contained the transcriptional activity of 4139 putative AR binding sites (ARBS) in the genome with and without the presence of hormone using the STARR-seq assay. Only a small fraction of ARBS showed significant differential expression when treated with hormone. To understand the underlying essential factors behind hormone-dependent behaviour, we developed both machine learning and biophysical models to identify active enhancers in prostate cancer cells. We also identify potentially crucial transcription factors for androgen-dependent behaviour and discuss the benefits and shortcomings of each modelling method

“Topological Significance” Analysis of Gene Expression and Proteomic Profiles from Prostate Cancer Cells Reveals Key Mechanisms of Androgen Response

The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer.We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of "topological significance". This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a "second phase" response, not directly dependent on the initial androgen stimulus.We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an interconnected network module containing many alternative routes from inputs to outputs. If the whole network is involved, a precisely formulated combination therapy may be required to fight the tumor growth effectively