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: 10612726
• 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/10612726
• 关键词: 3-Dimensional; Acceleration; Aging; Anaphase; Architecture; Binding Proteins; Cardiomyopathies; Cell Cycle; Cell Nucleus; Cells; Chromatin; Chromosomes; Coupled; Daughter; Dependence; Disease; Fibroblasts; Filament; Gene Structure; Genes; Genetic Transcription; Genome; Genomic Segment; Heterochromatin; Histones; Human; Individual; Insulin Resistance; Intermediate Filaments; Interphase; Knowledge; Lamin Type A; Lamins; Link; Lipodystrophy; Malignant Neoplasms; Maps; Membrane Proteins; Messenger RNA; Mitosis; Mitotic; Modeling; Molecular Conformation; Mus; Muscular Dystrophies; Mutation; Nuclear; Nuclear Envelope; Nuclear Lamina; Nuclear Pore Complex; Nucleoplasm; Paint; Pathway interactions; Positioning Attribute; Prophase; Protein Isoforms; Proteins; Proteome; Proteomics; RNA Splicing; Regulation; Reporting; Role; Scientist; System; Tertiary Protein Structure; Testing; Tissues; Tracer; Visualization; Work; cell fixing; chromatin immunoprecipitation; connectome; emerin; epigenomics; fascinate; gene network; high resolution imaging; insight; lamin C; metabolic phenotype; novel; novel therapeutics; predictive modeling; recruit; superresolution imaging; 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.

摘要 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成部分- 相关的核骨骼。与核膜(NE)膜蛋白相关的层，共同形成 核‘层’网络。层粘连蛋白和关键伙伴(LEM结构域蛋白和BANF1)在 退出有丝分裂，以确保染色体在有丝分裂中结合、捕获和适当组织 女儿核。在间期，核层网络在更高的阶中扮演着令人着迷的角色 基因组转录非活跃区的结构(异染色质)。每个区域都是静默区 染色体通常位于NE附近，称为板层相关结构域(LAD)。确实有 LAD组织、表观基因组调控和功能三维(3D)之间的明确相关性 基因组的折叠。A-型层蛋白(由LMNA编码)在LAD组织中起关键作用。LMNA带来了 到两个主要的体细胞亚型，层蛋白A和层蛋白C，通过选择性的mRNA剪接。因为前566名 人层粘连蛋白A和C的残基是相同的，长期以来人们认为它们的功能是冗余的。然而， 新报告显示层蛋白A和层蛋白C形成不同的细丝，与核孔有不同的联系 并具有不同的代谢表型。我们发现层蛋白C是LAD关联所必需的 在间期与NE发生碰撞。此外，层蛋白C是特异的和惊人的核质。 末期和早期-G1期，与新生NE的层蛋白A形成鲜明对比。尽管层蛋白C不是LAD- 在早期-G1，我们发现层蛋白C与LAD在回到他们的‘拴住’位置时相关联 东北地区。我们认为层蛋白C是LAD向NE募集所必需的，并将在细胞中验证这一假说 层蛋白C或层蛋白A的特异性下调。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LAP2β、板层和可溶性伙伴 (‘Connectome’)和一个新的LAD相关蛋白质组。我们假设层粘连蛋白C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 静默染色质(LADS)在NE的定位。我们的模型预测了层蛋白C和层蛋白A的不同蛋白质组 在有丝分裂退出期间，LAD蛋白质组在有丝分裂退出期间发生了明显的变化，LAD组织或 在有丝分裂退出过程中缺乏层蛋白C的细胞中，LAD向NE募集。我们将对这些模型进行超级- 单细胞中层粘连蛋白和低密度脂蛋白的分辨率成像，定向蛋白质组学，基因组组织图谱和 在细胞中的功能研究下调的层蛋白C，层蛋白A或验证的蛋白质在这项工作中确定。 这项工作有望填补在理解有丝分裂后基因组结构如何建立方面的主要空白， 层蛋白A和层蛋白C之间的功能差异也可能与 与LMNA有关的疾病。