Dissecting the mechanistic role of multinucleation in breast and trophoblast cancers

剖析多核在乳腺癌和滋养层细胞癌中的机制作用

• 批准号: 10321010
• 负责人: Madeline Keenen
• 金额: $ 8.99万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321010
• 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; Trophoblast Cancer; 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.

项目摘要/摘要 癌症的一个关键驱动因素是细胞内转录网络的全球放松管制。转录输出 依赖于染色质的结构和拓扑结构；被抑制的基因从活跃的基因中隔离到 核膜附近致密的DNA区域。密集的染色体结构域，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Lamin结合受体(LBR)和中间丝蛋白Lamin 因此，经历了转录的全球重排的癌细胞也常常表现出异常的蛋白质。 Lamin B、LBR和HP1的表达。这些蛋白质失调的物理表现是 核膜大小和形状的扭曲。事实上，这种核多形性被用作 肿瘤进展的组织学标志。雌激素受体阳性(ER+)乳腺癌患者， HP1的下调和Lamin B和LBR的上调与早发性高血压的发生密切相关 远端转移。了解支配异染色质组装的生物物理性质 核外周将促进旨在恢复适当基因调控的治疗方法的发展。HP1是 最近发现在体外将DNA和染色质浓缩成液-液相分离(LLP)液滴。这 提示了体内DNA组织的一种潜在机制。为了研究HP1的分子细节- 通过压实和相分离，我利用了DNA窗帘和共聚焦显微镜。我找到了钥匙 多价-LLPs相互作用和DNA紧凑所需的HP1区域。这项初步研究 重点是DNA，目标1的研究生工作将通过评估HP1与DNA的相互作用来建立在这些研究的基础上 复杂的核小体底物。体内的异染色质以均匀分布的核小体和 组蛋白H3赖氨酸9(H3K9me3)的三甲基化。我将制作染色质底物，范围从单色- 核小体到50kb染色质纤维，具有不同的间距和甲基化修饰。我会监控 HP1在这些底物上的结合、齐聚和相分离 一种新的单分子染色质分析方法。目标2中拟议的博士后研究将集中于 关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 在体外进行单分子研究，在细胞内进行超分辨研究。我会用细胞系和 小鼠乳腺癌转移模型研究转移复发的分子机制 ER+乳腺癌中层粘连蛋白B和LBR高表达。这项研究计划将推动我 我的最终目标是领导自己的实验室研究核拓扑是如何与细胞命运决定相关联的 在发展中，以及导致疾病的不当监管。