Differential Regulation and Roles of A-type Lamins in Early G1

G1 早期 A 型核纤层蛋白的差异调节和作用

• 批准号: 10386791
• 负责人: Karen Lynn Reddy
• 金额: $ 58.13万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386791
• 关键词: 3-Dimensional; Aging; Anaphase; Architecture; Binding Proteins; Cardiomyopathies; Cell Cycle; Cell Nucleus; Cells; Chromatin; Chromosomes; Coupled; Daughter; Dependence; Disease; Ensure; Fibroblasts; Filament; Genes; Genetic Transcription; Genome; Genomic Segment; Heterochromatin; Histones; Human; Image; Individual; Insulin Resistance; Intermediate Filaments; Interphase; Knowledge; Lamin Type A; Lamins; Link; Lipodystrophy; Malignant Neoplasms; Membrane Proteins; Messenger RNA; Mitosis; Mitotic; Modeling; Molecular Conformation; Mus; Muscular Dystrophies; Mutation; Nuclear; Nuclear Envelope; Nuclear Lamina; Nuclear Pore Complex; Paint; Pathway interactions; Positioning Attribute; Prophase; Protein Isoforms; Proteins; Proteome; Proteomics; RNA Splicing; Regulation; Reporting; Resolution; Role; Scientist; System; Tertiary Protein Structure; Testing; Tissues; Tracer; Work; chromatin immunoprecipitation; connectome; emerin; epigenomics; fascinate; gene network; high resolution imaging; insight; lamin C; metabolic phenotype; novel; novel therapeutics; predictive modeling; recruit; telophase

Summary Lamins A, C, B1 and B2 form nuclear intermediate filaments as major components of the dynamic genome- associated nucleoskeleton. Lamins associate with nuclear envelope (NE) membrane proteins, together forming nuclear ‘lamina’ networks. Lamins and key partners (LEM-domain proteins and BANF1) are essential during exit from mitosis to ensure that chromosomes are coalesced, captured and properly organized within the daughter nucleus. During interphase, nuclear lamina networks have fascinating roles in the higher-order architecture of transcriptionally-inactive regions of the genome (heterochromatin). Silent regions of each chromosome, known as Lamina Associated Domains (LADs), are typically located near the NE. There are clear correlations between LAD organization, epigenomic regulation, and the functional three-dimensional (3D) folding of the genome. A-type lamins (encoded by LMNA) have key roles in LAD organization. LMNA gives rise to two major somatic isoforms, lamin A and lamin C, by alternative mRNA splicing. Because the first 566 residues of human lamin A and lamin C are identical, they were long thought to function redundantly. However new reports show lamin A and lamin C form separate filaments, associate differentially with nuclear pore complexes and have distinct metabolic phenotypes. We discovered lamin C is required for LADs to associate with the NE during interphase. Furthermore, lamin C is specifically and strikingly nucleoplasmic during telophase and early-G1, in stark contrast to lamin A at the nascent NE. Although lamin C is not LAD- associated in early-G1, we found lamin C associates with LADs as they return to their ‘tethered’ positions at the NE. We propose lamin C is required for LAD recruitment to the NE, and will test this hypothesis in cells specifically downregulated for lamin C or lamin A. We can detect distinct yet overlapping proteomes in unsynchronized cells, comprising emerin and LAP2beta at the nuclear membrane, lamins and soluble partners (‘connectome’), and a novel LAD-associated proteome. We hypothesize that lamin C specifically interacts with LADs or LAD-associated proteins during exit from mitosis as a pathway to re-establish the tissue-specific positioning of silent chromatin (LADs) at the NE. Our models predict distinct proteomes for lamin C vs lamin A during mitotic exit, distinct changes in the LAD proteome during mitotic exit, and perturbed LAD organization or LAD recruitment to the NE in cells that lack lamin C during mitotic exit. We will test these models by super- resolution imaging of lamins and LADs in single cells, directed proteomics, genome organization mapping and functional studies in cells downregulated for either lamin C, lamin A or validated proteins identified in this work. This work is expected to fill major gaps in understanding how genome architecture is established after mitosis, and functional differences between lamin A and lamin C that may also be relevant to the mechanisms of diseases linked to LMNA.

总结 核纤层蛋白A、C、B1和B2形成核中间丝，作为动态基因组的主要组分- 相关核骨架核纤层蛋白与核膜（NE）膜蛋白结合，共同形成 核"纤层"网络。核纤层蛋白和关键伙伴（LEM结构域蛋白和BANF1）在 退出有丝分裂，以确保染色体合并，捕获和正确组织内 子核在分裂间期，核纤层网络在更高层次上起着重要的作用 基因组转录非活性区域的结构（异染色质）。每个人的沉默区域 染色体，称为板层相关结构域（LAD），通常位于NE附近。有 LAD组织、表观基因组调控和功能三维（3D）之间存在明确的相关性 基因组的折叠A型核纤层蛋白（由LMNA编码）在LAD组织中具有关键作用。LMNA产生 两个主要的体细胞同种型，核纤层蛋白A和核纤层蛋白C，通过选择性mRNA剪接。因为第一个566 人核纤层蛋白A和核纤层蛋白C的残基是相同的，它们长期被认为是冗余的功能。然而 新的报道显示核纤层蛋白A和核纤层蛋白C形成独立的细丝， 复合物并具有不同的代谢表型。我们发现纤层蛋白C是前降支联合 与NE在间期。此外，核纤层蛋白C在细胞分裂期间特异性且惊人地具有核质性。 末期和早期G1，形成鲜明对比，核纤层蛋白A在新生NE。尽管核纤层蛋白C不是LAD- 在G1早期相关，我们发现核纤层蛋白C与LAD相关，因为它们在G1时返回到它们的"拴系"位置。 的NE。我们提出核纤层蛋白C是LAD向NE募集所必需的，并将在细胞中检验这一假设 特异性下调核纤层蛋白C或核纤层蛋白A。我们可以检测到不同但重叠的蛋白质组， 非同步化细胞，包括核膜上的emerin和LAP 2 β、核纤层蛋白和可溶性配偶体 （"连接体"）和一种新的LAD相关蛋白质组。我们假设核纤层蛋白C特异性地与 LAD或LAD相关蛋白在退出有丝分裂过程中作为重建组织特异性的途径， 沉默染色质（LAD）在NE的定位。我们的模型预测了核纤层蛋白C与核纤层蛋白A的不同蛋白质组 在有丝分裂退出过程中，LAD蛋白质组在有丝分裂退出过程中发生明显变化，并扰乱LAD组织或 在有丝分裂退出过程中缺乏核纤层蛋白C的细胞中LAD向NE的募集。我们将通过超级测试这些模型- 单细胞核纤层蛋白和LAD的分辨率成像，定向蛋白质组学，基因组组织作图和 核纤层蛋白C、核纤层蛋白A或本工作中鉴定的经验证的蛋白质下调的细胞中的功能研究。 这项工作有望填补理解有丝分裂后基因组结构如何建立的主要空白， 以及核纤层蛋白A和核纤层蛋白C之间的功能差异，这些差异也可能与核纤层蛋白A和核纤层蛋白C的机制有关。 与LMNA有关的疾病。