Dissecting the molecular mechanism of heterochromatin misregulation at the nuclear periphery in cancer

剖析癌症核周边异染色质失调的分子机制

• 批准号: 10021632
• 负责人: Madeline Keenen
• 金额: $ 4.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021632
• 关键词: Architecture; Atomic Force Microscopy; Behavior; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Biology; Biophysical Process; Biophysics; Breast Cancer Patient; Breast Cancer cell line; Breast cancer metastasis; CRISPR interference; Cancer cell line; Cell Line; Cell Nucleus; Cell physiology; Cells; Characteristics; Chromatin; Chromatin Fiber; Chromatin Structure; Chromosomes; Complex; Confocal Microscopy; Coupled; DNA; DNA-Protein Interaction; Development; Disease; Distal; Down-Regulation; Electron Microscopy; Estrogen receptor positive; Exhibits; Fluorescence Microscopy; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Genome; Goals; Heterochromatin; Histologic; Histone H3; In Vitro; Individual; Injections; Integral Membrane Protein; Intermediate Filament Proteins; Label; Lamin Type B; Lamins; Length; Liquid substance; Lysine; MCF7 cell; Malignant Neoplasms; Mechanics; Mediating; Mediator of activation protein; Membrane; Methylation; Modification; Molecular; Monitor; Morphology; Mus; Neoplasm Metastasis; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Pleomorphism; Nucleosomes; Organism; Output; Patient-Focused Outcomes; Phase; Physiological; Play; Pleomorphism; Protein Region; Proteins; Recurrence; Research; Research Project Grants; Resolution; Role; Severities; Shapes; Sum; System; T47D; Techniques; Tertiary Protein Structure; Testing; Time; Tumor Markers; Up-Regulation; Variant; Work; biophysical properties; cancer cell; diagnostic biomarker; disease phenotype; experimental study; heterochromatin-specific nonhistone chromosomal protein HP-1; histone modification; in vivo; lamin B receptor; malignant breast neoplasm; migration; mouse model; neoplastic cell; novel; overexpression; physical model; programs; protein expression; public health relevance; receptor binding; reconstitution; recruit; restoration; single molecule; targeted cancer therapy; therapy development; tumor progression

Project Summary/Abstract A key driver of cancer is the global deregulation of transcription networks in the cell. Transcriptional output is dependent on chromatin structure and topology; repressed genes are sequestered from active genes into regions of compacted DNA near the nuclear membrane. Dense chromosomal domains, termed heterochromatin, are associated with Heterochromatin Protein-1 (HP1) and are tethered to the periphery via an interaction with the nuclear transmembrane protein, Lamin Binding Receptor (LBR), and the intermediate filament protein, Lamin B. Consequently, cancer cells that undergo global rewiring of transcription also often exhibit aberrant protein expression of Lamin B, LBR and HP1. The physical manifestation of the misregulation of these proteins is distortion in the size and shape of the nuclear envelope. Indeed, this nuclear pleomorphism is used as a histological marker of tumor progression. In estrogen receptor positive (ER+) breast cancer patients, downregulation of HP1 and upregulation of Lamin B and LBR is strongly correlated with earlier occurrence of distal metastasis. Understanding the biophysical properties that govern the assembly of heterochromatin at the nuclear periphery will facilitate the development of therapies aimed at restoring proper gene regulation. HP1 was recently found to concentrate DNA and chromatin into liquid-liquid phase separated (LLPS) droplets in vitro. This suggests a potential mechanism of DNA organization in vivo. To investigate the molecular details of HP1- mediated compaction and phase separation, I utilized DNA curtains and confocal microscopy. I identified key regions of HP1 required for multivalent—LLPS interactions and DNA compaction. This preliminary research focused on DNA, and the graduate work in Aim 1 will build on these studies by evaluating HP1 interactions with complex nucleosomal substrates. Heterochromatin in vivo is distinguished by evenly spaced nucleosomes and the trimethylation of histone H3 lysine 9 (H3K9me3). I will make chromatin substrates that range from mono- nucleosomes to 50kb chromatin fibers with variations in spacing and methylation modification. I will monitor the binding, oligomerization and phase separation of HP1 on these substrates by a combination of bulk biochemical assays and a novel single molecule chromatin assay. The proposed postdoctoral research in Aim 2 will focus on determining a physical model of the nuclear periphery. I will reconstitute the interactions between chromatin and the lamina with single molecule studies in vitro and super resolution studies in cells. I will use cell lines and mouse models of breast cancer metastasis to determine the molecular mechanism guiding metastatic recurrence of ER+ breast cancer patients with high expression of Lamin B and LBR. This research program will propel me toward my ultimate goal of leading my own lab studying how nuclear topology is coupled to cell fate determination in development, and the misregulation that leads to disease.

项目概要/摘要 癌症的一个关键驱动因素是细胞内转录网络的整体失调。转录输出 取决于染色质结构和拓扑；被抑制的基因被从活跃的基因中隔离出来 核膜附近的压缩 DNA 区域。致密染色体结构域，称为异染色质， 与异染色质蛋白 1 (HP1) 相关，并通过与 核跨膜蛋白、核纤层蛋白结合受体 (LBR) 和中间丝蛋白、核纤层蛋白 B. 因此，经历转录全局重连的癌细胞也经常表现出异常蛋白 Lamin B、LBR 和 HP1 的表达。这些蛋白质失调的物理表现是 核膜的大小和形状发生扭曲。事实上，这种核多形性被用作 肿瘤进展的组织学标志物。在雌激素受体阳性（ER+）乳腺癌患者中， HP1 的下调以及 Lamin B 和 LBR 的上调与早期发生密切相关 远端转移。了解控制异染色质组装的生物物理特性 核外围将促进旨在恢复适当基因调控的疗法的开发。 HP1 是 最近发现可以在体外将 DNA 和染色质浓缩到液-液相分离 (LLPS) 液滴中。这 提出了体内 DNA 组织的潜在机制。研究 HP1- 的分子细节 为了介导压实和相分离，我利用了 DNA 帘和共焦显微镜。我确定了关键 多价-LLPS 相互作用和 DNA 压缩所需的 HP1 区域。本次初步研究 重点关注 DNA，目标 1 的研究生工作将通过评估 HP1 与 复杂的核小体底物。体内异染色质的特征是均匀分布的核小体和 组蛋白 H3 赖氨酸 9 (H3K9me3) 的三甲基化。我将制作从单染色质底物到 核小体到 50kb 染色质纤维，间距和甲基化修饰各不相同。我会监控 通过大量生化的组合，HP1 在这些底物上的结合、寡聚化和相分离 测定和新型单分子染色质测定。目标 2 中拟议的博士后研究将集中于 确定核外围的物理模型。我将重建染色质之间的相互作用 以及在体外进行单分子研究和在细胞中进行超分辨率研究的层板。我将使用细胞系和 乳腺癌转移小鼠模型以确定指导转移复发的分子机制 Lamin B 和 LBR 高表达的 ER+ 乳腺癌患者。这个研究计划将推动我 我的最终目标是领导自己的实验室研究核拓扑结构如何与细胞命运决定相结合 发育过程中的失调，以及导致疾病的失调。