A genome-wide, gene-by-gene siRNA-based knockdown screen has been developed to identify novel factors and networks that maintain epigenetic gene silencing in human cancer cells. This screen is based on the principle that siRNA knockdown of specific epigenetic silencing factors will lead to reactivation of silent genes. A human cancer cell reporter system was devised whereby reactivation of an epigenetically silent green fluorescent protein (GFP) gene provides a high throughput readout. This approach was validated using a pre-selected siRNA set that is enriched for epigenetic regulators. Screens using the pre-selected and genome-wide siRNA sets have revealed that knockdown of individual factors is sufficient for reactivation, and that specific networks can be identified. Unknown factor hits are being analyzed using bioinformatics methods to predict function, and by chromatin immunoprecipitation to confirm direct roles for the factors at silent loci. These screens have the potential to identify novel cellular pathways that mark chromatin for epigenetic silencing, and thus reveal new targets for epigenetic therapy of cancer and other diseases. Recent studies have focused on the detailed characterization of several novel epigenetic silencing factors identified by siRNA screening.

A prominent feature of tumors, and melanomas in particular, is phenotypic and functional heterogeneity.  Recent studies have addressed two models for heterogeneity in melanoma: i) rare Cancer Stem Cells (CSCs), or Tumor Initiating Cells (TICs),  give rise to heterogeneous tumors, ii) heterogeneity arises through cell plasticity, i.e., epigenetic-based phenotypic switching. Melanoma tumors and cell lines frequently contain a large fraction of TICs, counter to the cancer stem cell model. Melanoma cells have also been demonstrated to form three-dimensional stem cell-like bodies in culture.

We have developed a robust system to monitor melanoma cell plasticity in culture. In this system, melanoma cells can be tracked as they enter and exit a stem cell-like state, and genes that drive these processes can be identified.  The biological relevance of these factors can then be assessed in human melanoma tissues. In culture, melanoma cells are able to rapidly assemble into three-dimensional stem cell-like bodies. Microarray experiments revealed accompanying dramatic up-regulation of a small set of stem cell and developmental genes.  To identify the factors that control the acquisition of stem cell properties, we used siRNA knockdown of candidates, and monitored formation of stem cell-like bodies. This study has identified candidate factors that are drivers or passengers in cancer cell plasticity, and therefore may serve as novel targets or biomarkers.  A novel factor that was identified using this approach was found to be expressed in melanoma tissues.

Integrated retroviral DNA is subject to epigenetic gene silencing in human cells, resulting in a latent viral state, or loss of vector transgene expression. It is clear that such silencing is maintained by the cellular epigenetic machinery, however, very little in known about how such cellular machinery initiates the silencing process on newly integrated retroviral DNA.

We previously identified an antiviral mechanism that affects the initiation of epigenetic silencing of foreign retroviral DNA in human cells, and is dependent on the large, multifunctional, and ubiquitous scaffolding protein, Daxx. We showed that Daxx binds to the incoming viral DNA-protein complex, and acts as an adapter to recruit epigenetic factors. Our subsequent investigations have uncovered several early and late events in the silencing process. We found that siRNA-based knockdown of Daxx prior to infection transiently abrogated silencing. Furthermore, DNA methylation of viral DNA is detectable within several days post infection, suggesting that this modification is important for initiating silencing. Lastly, we obtained evidence that Daxx can recruit the repressive DNA methyltransferases (DNMTs), and siRNA-mediated knockdown of Daxx resulted in loss of viral DNA methylation and reactivation of the silent viral DNA. These findings have important implications for how human cells can respond to repress foreign DNA expression. Silencing of viral genomes by epigenetic mechanisms can contribute to pathogenesis by promoting a latent viral state, as in the case of HIV/AIDS. Because the epigenetic marks that mediate gene silencing are reversible, there is intense interest in devising therapeutic strategies to reactivate silent genes, and to reverse viral latency in a controlled manner.

Over the past two decades, therapeutic compounds have been developed that promote reactivation of epigenetically silent tumor suppressor genes and selectively kill, or drive differentiation of cancer cells. More recently, compounds that affect epigenetic processes have been isolated as Bioactive Food Components (BFCs). In some cases, these compounds mimic known epigenetic drugs. The presence of these BFCs in specific diets may underlie their cancer preventative properties, and also provide the basis for development of new therapeutics. To identify the epigenetic activities of BFCs, we have used our robust cell-based system in which both an epigenetically silent GFP reporter gene, and a resident silent gene are reactivated in response to epigenetic drug treatment. We are applying this system to characterize known BFCs compounds and to identify new phytochemicals that reactivate epigenetically silent genes.