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

基本信息

批准号：
8233985
负责人：
Marcos Estecio
金额：
$ 20.62万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8233985
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 public health relevance stable cell line tumor

项目摘要

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（由申请人提供）：核纤层蛋白 A/C 和 B1 蛋白组织成网状结构，覆盖核膜的核内部分，并与其他结构蛋白一起形成核纤层。该结构在关键细胞功能中具有活性，例如 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结构，使共同调节的基因在空间上接近，并通过绝缘体的作用，划分更高阶的染色质结构域。此外，许多具有组蛋白脱乙酰酶和甲基化酶活性的酶与核纤层蛋白结合，并且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。由于核层在维持正常细胞功能中的突出作用及其对组蛋白标记的影响，我们假设染色质与核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 是唯一已发表的以高分辨率揭示层相关域 (LAD) 的方法；该方法基于引入编码融合核纤层蛋白 B1-Dam 蛋白的工程质粒，然后进行甲基化腺嘌呤的 DNA 微阵列分析。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层结合的方法 (LAD-Seq)，该方法独立于工程质粒转染，适用于原代、未培养的细胞。我们的方法采用改良的染色质免疫沉淀方案，并结合大规模并行 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，并且该技术已被证明具有高度特异性，对于染色质与核纤层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 研究一组培养的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。该研究项目产生的数据将使我们对正常细胞和癌细胞的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交织在一起。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大量原代正常细胞和癌细胞的生物学假设。公共卫生相关性：核纤层是一种重要的细胞结构，在染色质组织、表观遗传标记和基因调控中发挥作用。在本提案中，我们将分析原代正常细胞和癌细胞中的基因组-核纤层相互作用、组蛋白尾部翻译后修饰和 DNA 甲基化。由于核纤层蛋白的改变存在于许多人类疾病中，包括发育障碍和癌症，因此我们开发的方法以及随后对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。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv(16) subtype.

DOI：
10.1111/bjh.13299
发表时间：
2015-05
期刊：
British journal of haematology
影响因子：
6.5
作者：
Estécio MR;Maddipoti S;Bueso-Ramos C;DiNardo CD;Yang H;Wei Y;Kondo K;Fang Z;Stevenson W;Chang KS;Pierce SA;Bohannan Z;Borthakur G;Kantarjian H;Garcia-Manero G
通讯作者：
Garcia-Manero G