Epigenetic silencing of tumor suppressor genes in human cancers is associated with aberrant methylation of promoter region CpG islands and local alterations in histone modifications. However, the mechanisms that drive these events remain unclear. Here, we establish an important role for histone H4 lysine 16 acetylation (H4K16Ac) and the histone acetyltransferase hMOF in the regulation of TMS1/ASC, a proapoptotic gene that undergoes epigenetic silencing in human cancers. In the unmethylated and active state, the TMS1 CpG island is spanned by positioned nucleosomes and marked by histone H3K4 methylation. H4K16Ac was uniquely localized to two sharp peaks that flanked the unmethylated CpG island and corresponded to strongly positioned nucleosomes. Aberrant methylation and silencing of TMS1 was accompanied by loss of the H4K16Ac peaks, loss of nucleosome positioning, hypomethylation of H3K4, and hypermethylation of H3K9. In addition, a single peak of histone H4 lysine 20 trimethylation was observed near the transcription start site. Down-regulation of hMOF or another component of the MSL complex resulted in a gene-specific decrease in H4K16Ac, loss of nucleosome positioning, and silencing of TMS1. Gene silencing induced by H4K16 deacetylation occurred independently of changes in histone methylation and DNA methylation and was reversed on hMOF reexpression. These results indicate that the selective marking of nucleosomes flanking the CpG island by hMOF is required to maintain TMS1 gene activity and suggest that the loss of H4K16Ac, mobilization of nucleosomes, and transcriptional down-regulation may be important events in the epigenetic silencing of certain tumor suppressor genes in cancer. [Cancer Res 2008;68(16):6810-21]

Many human genes exhibit evidence of initiated RNA polymerase II (Pol II) at their promoters, despite a lack of significant full-length transcript. Such genes exhibit promoter-proximal "pausing," wherein initiated Pol II accumulates just downstream of the transcription start site due to a rate-limiting step mediating the transition to elongation. The mechanisms that regulate the escape of Pol II from pausing and the relationship to chromatin structure remain incompletely understood. Recently, we showed that CpG island hypermethylation and epigenetic silencing of TMS1/ASC in human breast cancers are accompanied by a local shift from histone H4 lysine 16 acetylation (H4K16Ac) to H4 lysine 20 trimethylation (H4K20me3). Here, we show that hMOF-mediated H4K16Ac and SUV420H2-mediated H4K20me3 play opposing roles in the regulation of Pol II pausing. We found that H4K16Ac promoted the release of Pol II from pausing through the recruitment of BRD4 and pTEFb. Aberrant methylation of CpG island DNA blocked Pol II recruitment to gene promoters. Whereas the inhibition of DNA methylation allowed for the reassociation and initiation of Pol II at the TMS1 promoter, Pol II remained paused in the presence of H4K20me3. Combined inhibition of H4K20me3 and DNA methylation resulted in the rerecruitment of hMOF and subsequent H4K16Ac, release of Pol II into active elongation, and synergistic reactivation of TMS1 expression. Marking by H4K20me3 was not restricted to TMS1 but also occurred at other genes independently of DNA methylation, where it similarly imposed a block to Pol II promoter escape through a mechanism that involved the local inhibition of H4K16Ac. These data indicate that H4K20me3 invokes gene repression by antagonizing hMOF-mediated H4K16Ac and suggest that overcoming Pol II pausing might be a rate-limiting step in achieving tumor suppressor gene reactivation in cancer therapy.Control of transcription of DNA by RNA polymerase II (Pol II) is regulated at multiple levels, including recruitment of Pol II to the promoter, initiation, elongation, and termination. General transcription factors guide the recruitment and loading of Pol II at gene promoters to form the preinitiation complex. Pol II is activated for transcription initiation by TFIIHmediated phosphorylation of serine 5 in the heptapeptide repeats of the C-terminal domain (CTD) (58). The transition from initiation to elongation is also an important regulatory step. At some genes, Pol II is initiated and begins RNA synthesis but "pauses" 20 to 50 nucleotides downstream of the transcription start site. This has been referred to as Pol II promoter-proximal pausing (19, 42). Initially described for a few inducible genes, such as the Drosophila melanogaster heat shock genes and the c-myc, c-fos, and junB genes in human cells, promoter-proximal pausing of Pol II was thought to represent a mechanism for fast and robust gene activation in response to external stimuli (42). However, it is now evident that Pol II pausing may be more widespread. Genome-wide studies in Drosoph...

The Hedgehog (Hh) pathway was first defined by its role in segment polarity in the Drosophila melanogaster embryonic epidermis and has since been linked to many aspects of vertebrate development and disease. In humans, mutation of the Patched1 (PTCH1) gene, which encodes an inhibitor of Hh signaling, lead to tumors of the skin and pediatric brain. Despite the high level of conservation between the vertebrate and invertebrate Hh pathways, studies in Drosophila have yet to find direct evidence that ptc limits organ size. Here we report identification of Drosophila ptc in a screen for mutations that require a synergistic apoptotic block in order to drive overgrowth. Developing imaginal discs containing clones of ptc mutant cells immortalized by the concurrent loss of the Apaf-1-related killer (Ark) gene are overgrown due, in large part, to the overgrowth of wild type portions of these discs. This phenotype correlates with overexpression of the morphogen Dpp in ptc,Ark double-mutant cells, leading to elevated phosphorylation of the Dpp pathway effector Mad (p-Mad) in cells surrounding ptc,Ark mutant clones. p-Mad functions with the Hippo pathway oncoprotein Yorkie (Yki) to induce expression of the pro-growth/anti-apoptotic microRNA bantam. Accordingly, Yki activity is elevated among wild type cells surrounding ptc,Ark clones and alleles of bantam and yki dominantly suppress the enlarged-disc phenotype produced by loss of ptc. These data suggest that ptc can regulate Yki in a non-cell autonomous manner and reveal an intercellular link between the Hh and Hippo pathways that may contribute to growth-regulatory properties of the Hh pathway in development and disease.

Understanding the molecular nature of human cancer is essential to the development of effective and personalized therapies. Several different molecular signal transduction pathways drive tumorigenesis when deregulated and respond to different types of therapeutic interventions. The Hippo signaling pathway has been demonstrated to play a central role in the regulation of tissue and organ size during development. The deregulation of Hippo signaling leads to a concurrent combination of uncontrolled cellular proliferation and inhibition of apoptosis, two key hallmarks in cancer development. The molecular nature of this pathway was first uncovered in Drosophila melanogaster through genetic screens to identify regulators of cell growth and cell division. The pathway is strongly conserved in humans, rendering Drosophila a suitable and efficient model system to better understand the molecular nature of this pathway. In the present study, we review the current understanding of the molecular mechanism and clinical impact of the Hippo pathway. Current studies have demonstrated that a variety of deregulated molecules can alter Hippo signaling, leading to the constitutive activation of the transcriptional activator YAP or its paralog TAZ. Additionally, the Hippo pathway integrates inputs from a number of growth signaling pathways, positioning the Hippo pathway in a central role in the regulation of tissue size. Importantly, deregulated Hippo signaling is frequently observed in human cancers. YAP is commonly activated in a number of in vitro and in vivo models of tumorigenesis, as well as a number of human cancers. The common activation of YAP in many different tumor types provides an attractive target for potential therapeutic intervention.

Prostate cancer is the most common cancer diagnosed and the second leading cause of cancer-related deaths in men in the United States. The etiological factors that give rise to prostate cancer are not known. Therefore, it is not possible to develop primary intervention strategies to remove the causative agents from the environment. However, secondary intervention strategies with selenium (Se) compounds and other agents represent a viable option to reduce the morbidity and mortality of prostate cancer. In this review, we discuss ongoing clinical trials. In addition, we discuss preclinical mechanistic studies that provide insights into the biochemical and molecular basis for the anti-carcinogenic activity of both inorganic and organic forms of Se.

DNA methyltransferase inhibitors are currently the standard of care for myelodysplastic syndrome and are in clinical trials for leukemias and solid tumors. However, the molecular basis underlying their activity remains poorly understood. Here, we studied the induction and long-term stability of gene reactivation at three methylated tumor suppressor loci in response to the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-azaCdR) in human breast cancer cells. At the TMS1/ASC locus, treatment with 5-azaCdR resulted in partial DNA demethylation, the reengagement of RNA polymerase II (Pol II), and a shift from a repressive chromatin profile marked with H3K9me2 and H4K20me3 to an active profile enriched in H3ac and H3K4me2. Using a single-molecule approach coupling chromatin immunoprecipitation with bisulfite sequencing, we show that H3ac, H3K4me2, and Pol II selectively associated with the demethylated alleles, whereas H3K9me2 preferentially marked alleles resistant to demethylation. H4K20me3 was unaffected by DNA demethylation and associated with both unmethylated and methylated alleles. After drug removal, TMS1 underwent partial remethylation, yet a subset of alleles remained stably demethylated for over 3 months. These alleles remained selectively associated with H3K4me2, H3ac, and Pol II and correlated with a sustained low level of gene expression. TMS1 alleles reacquired H3K9me2 over time, and those alleles that became remethylated retained H3ac. In contrast, CDH1 and ESR1 were remethylated and completely silenced within ∼1 week of drug removal, and failed to maintain stably unmethylated alleles. Our data suggest that the ability to maintain Pol II occupancy is a critical factor in the long-term stability of drug-induced CpG island demethylation.

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.

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