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: 10549720
• 负责人: Madeline Keenen
• 金额: $ 2.36万
• 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 2023-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10549720
• 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; Chromosome Segregation; 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; Gene Structure; Genes; Genetic Transcription; Genome; Goals; Heterochromatin; Histologic; Histone H3; In Vitro; Individual; Injections; Integral Membrane Protein; Intermediate Filament Proteins; Invaded; Label; Lamin Type B; Lamins; Length; Liquid substance; Lysine; MCF7 cell; Malignant Neoplasms; Mechanics; Mediating; Mediator; Membrane; Metastatic/Recurrent; 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; Postdoctoral Fellow; Proliferating; Protein Region; Proteins; Research; Research Project Grants; Role; Severities; Shapes; Sum; System; T47D; Techniques; Tertiary Protein Structure; Testing; Time; Trophoblast Cancer; Tumor Markers; Up-Regulation; Variant; Visualization; Work; biophysical properties; cancer cell; diagnostic biomarker; disease phenotype; experimental study; gene repression; heterochromatin-specific nonhistone chromosomal protein HP-1; histone methylation; histone modification; in vivo; lamin B receptor; malignant breast neoplasm; micronucleus; 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; ultra high resolution

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 癌症的关键驱动力是细胞中转录网络的全球放松管制。转录输出 取决于染色质结构和拓扑；从活性基因隔离为代表基因 核膜附近压实DNA的区域。致密的染色体结构域，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，层粘连蛋白结合受体（LBR）和中间丝蛋白层粘连蛋白 B.因此，经历全局转录的癌细胞也经常表现出异常蛋白 Lamin B，LBR和HP1的表达。这些蛋白质不正常的身体表现是 核包膜大小和形状的变形。实际上，这种核多态性被用作 肿瘤进展的组织学标记。在雌激素受体阳性（ER+）乳腺癌患者中 HP1的下调和层粘连蛋白B和LBR的上调与早期出现密切相关 远端转移。了解控制异染色质在 核周围将有助于开发旨在恢复适当基因调节的疗法。 HP1是 最近发现，将DNA和染色质浓缩到体外液态液相（LLP）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 介导的压实和相分离，我利用了DNA窗帘和共聚焦显微镜。我确定了密钥 多价 - LLP相互作用和DNA压实所需的HP1区域。这项初步研究 专注于DNA，AIM 1中的研究生工作将通过评估HP1相互作用与 复杂的核体底物。体内异染色质通过均匀间隔的核症和 组蛋白H3赖氨酸9（H3K9me3）的三甲基化。我将使染色质底物范围从单声道 核小体至50KB染色质纤维，其间距和甲基化修饰方面有变化。我将监视 通过散装生化的结合，HP1在这些底物上的结合，寡聚和相位分离 测定和新型的单分子染色质测定。 AIM 2中拟议的博士后研究将重点 确定核周围的物理模型。我将重建染色质之间的相互作用 以及在细胞中进行单分子研究和超级分辨率研究的单分子研究。我将使用单元线， 乳腺癌转移的小鼠模型，以确定分子机制指导转移性复发 ER+乳腺癌患者的层lamin B和LBR高表达。该研究计划将推动我 为了实现我自己的实验室的最终目标，研究核拓扑如何与细胞脂肪的确定结合在一起 在发育中，以及导致疾病的错误调节。