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

描述（由申请人提供）：层粘连蛋白A/C和B1蛋白被组织成网状结构，覆盖在核膜的核内部分，与其他结构蛋白一起形成核层。这种结构在关键的细胞功能中具有活性，如DNA复制、细胞分裂和细胞凋亡。染色质与核层的结合决定了基因组的结构，带来了空间上接近的共调节基因，并通过绝缘体的作用划分了高阶染色质结构域。此外，许多具有组蛋白去乙酰化酶和甲基化酶活性的酶与核层蛋白结合，染色质与核层的相互作用被认为是组蛋白标记的必要事件。由于核层在维持正常细胞功能方面的重要作用及其对组蛋白标记的影响，我们假设肿瘤细胞中可能存在染色质与核层结合模式的变化并影响表观遗传状态。目前，DamID是唯一已发表的高分辨率揭示层叠相关结构域（LADs）的方法；该方法是基于引入编码融合Lamin B1-Dam蛋白的工程质粒，然后对甲基化腺嘌呤进行DNA微阵列分析。因此，这种方法只能应用于稳定的细胞系。我们开发并验证了一种独立于工程质粒转染的方法来研究染色质与核层的结合（LAD-Seq），该方法适用于原代未培养的细胞。我们的方法采用改进的染色质免疫沉淀方案，结合大规模并行Solexa 1G测序技术。在我们的初步研究中，LAD-Seq已应用于培养的正常细胞和癌细胞，该技术已被证明是高度特异性的，对于核层染色质结合的全基因组分析非常有用。在本项目中，我们将应用LAD-Seq技术研究正常细胞和癌细胞中染色质与Lamin A/C和Lamin B1的结合，并评估其与其他表观遗传调控标记的相关性。本研究项目产生的数据将使我们对正常细胞和癌细胞基因组组织的理解取得重大进展，并将有助于理解核层在基因调控中的作用以及多层表观遗传控制如何相互交织。我们的长期目标是使用Lam-Seq来解决来自不同组织类型和疾病阶段的原发性正常细胞和癌细胞的生物学假设。

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.