The Role of the Nuclear Lamina in Chromatin Organization and Gene Expression in N

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DESCRIPTION (provided by applicant): Lamin A/C and B1 proteins are organized in a mesh-like structure that coats the intranuclear portion of the nuclear envelope and, along with other structural proteins, forms the nuclear lamina. This structure has activity in key cellular functions, such as DNA replication, cell division and apoptosis. Chromatin binding to the nuclear lamina dictates the genome architecture, bringing spatially close co-regulated genes and, through the action of insulators, compartmentalizes higher-order chromatin domains. Moreover, many enzymes with histone deacetylase and methylase activities are bound to lamin proteins, and the interaction of chromatin with the nuclear lamina is proposed as a necessary event to histone marking. Due to the prominent role of the nuclear lamina in maintenance of normal cell function and its impact in histone marking, we hypothesize that changes in the pattern of chromatin binding to the nuclear lamina may be present in neoplastic cells and influence epigenetic states. Currently, DamID is the only published method to reveal lamina-associated domains (LADs) in high resolution; this method is based in the introduction of an engineered plasmid coding a fusion Lamin B1-Dam protein, followed by DNA microarray analysis of methylated adenines. Thus, this method can only be applied to stable cell lines. We developed and validated a method to study the chromatin binding to the nuclear lamina (LAD-Seq) that is independent from engineered plasmids transfection and applicable to primary, uncultured cells. Our method employs a modified chromatin immunoprecipitation protocol coupled with massively parallel Solexa 1G sequencing technology. In our preliminary studies, LAD-Seq has been applied to cultured normal and cancer cells, and this technique has proven to be highly specific and very useful for the genome-wide analysis of the chromatin binding to the nuclear lamina. In this project, we will apply LAD-Seq to study chromatin binding to Lamin A/C and Lamin B1 in a panel of cultured normal and cancer cells, and evaluate its correlation to other epigenetic regulatory marks. The data generated in this research project will enable major advances in our understanding of the genome organization in normal and cancer cells, and will aid in understanding the role of the nuclear lamina in gene regulation and how multiple layers of epigenetic control are intertwined. Our long term goal is to use Lam-Seq to address biological hypotheses in a large cohort of primary normal and cancer cells from various tissue types and disease stages. PUBLIC HEALTH RELEVANCE: The nuclear lamina is an important cellular structure with function in chromatin organization, epigenetic marking and gene regulation. In this proposal, we will profile genome-nuclear lamina interactions, histone tail posttranslational modifications and DNA methylation in primary normal and cancer cells. Because alterations of lamina proteins are found in many human diseases, including developmental disorders and cancer, our developed method and subsequent profiling of normal and cancer cells will have broad impact in biomedical research.