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

核层在染色质组织和基因表达中的作用

• 批准号: 8099826
• 负责人: Marcos Estecio
• 金额: $ 17.18万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099826
• 关键词: Address; Adenine; Apoptosis; Architecture; Binding; Biological; Biomedical Research; Body Regions; Cell Line; Cell division; Cell physiology; Cells; Cellular Structures; Characteristics; Chromatin; Code; Collection; Coupled; DNA; DNA Methylation; DNA Microarray Chip; DNA biosynthesis; Data; Disease; Engineering; Enzymes; Epigenetic Process; Epithelium; Event; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genome; Genome Mappings; Goals; Histocompatibility Testing; Histone Deacetylase; Histones; Intercistronic Region; LNCaP; Lamin B1; Lamin Type A; Lamins; Maintenance; Malignant Neoplasms; Mediating; Methods; Microarray Analysis; Modeling; Modification; Normal Cell; Nuclear Envelope; Nuclear Lamina; PC3 cell line; Pattern; Plasmids; Post-Translational Protein Processing; Prostate; Protein Binding Domain; Proteins; Protocols documentation; Publishing; Research Project Grants; Research Proposals; Resolution; Role; Staging; Structural Protein; Structure; Tail; Techniques; Technology; Transfection; VCaP; base; cancer cell; chromatin immunoprecipitation; cohort; developmental disease; gene repression; genome-wide analysis; histone methyltransferase; histone modification; human disease; neoplastic cell; promoter; stable cell line; tumor

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.

描述（由申请方提供）：核纤层蛋白A/C和B1蛋白以网状结构组织，该网状结构覆盖核膜的核内部分，并与其他结构蛋白沿着形成核纤层。这种结构在关键的细胞功能中具有活性，例如DNA复制、细胞分裂和凋亡。染色质与核纤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，将高阶染色质结构域区室化。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纤层蛋白结合，染色质与核纤层的相互作用被认为是组蛋白标记的必要事件。由于核纤层在维持正常细胞功能中的重要作用及其在组蛋白标记中的影响，我们假设染色质与核纤层结合模式的变化可能存在于肿瘤细胞中并影响表观遗传状态。目前，DamID是唯一公布的方法，揭示lamina相关结构域（LAD）在高分辨率;这种方法是基于引入工程质粒编码的融合核纤层蛋白B1-大坝蛋白，然后通过DNA微阵列分析甲基化腺嘌呤。因此，该方法只能应用于稳定的细胞系。我们开发并验证了一种研究染色质与核纤层结合（LAD-Seq）的方法，该方法独立于工程质粒转染，适用于原代未培养细胞。我们的方法采用了修改后的染色质免疫沉淀协议，结合大规模并行Solexa 1G测序技术。在我们的初步研究中，LAD-Seq已被应用于培养的正常细胞和癌细胞，并且该技术已被证明是高度特异性的，并且对于染色质与核纤层结合的全基因组分析非常有用。在这个项目中，我们将应用LAD-Seq研究染色质与一组培养的正常细胞和癌细胞中的核纤层蛋白A/C和核纤层蛋白B1的结合，并评估其与其他表观遗传调控标记的相关性。该研究项目产生的数据将使我们对正常和癌细胞基因组组织的理解取得重大进展，并将有助于理解核纤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来解决来自各种组织类型和疾病阶段的大量原代正常细胞和癌细胞的生物学假设。 公共卫生关系：核纤层是一种重要的细胞结构，具有染色质组织、表观遗传标记和基因调控等功能。在这个提议中，我们将在原代正常细胞和癌细胞中分析基因组-核纤层相互作用、组蛋白尾翻译后修饰和DNA甲基化。由于在许多人类疾病中发现了纤层蛋白的改变，包括发育障碍和癌症，我们开发的方法和随后的正常细胞和癌细胞的分析将在生物医学研究中产生广泛的影响。