Replication stress in laminopathies: causes and consequences

核纤层蛋白病中的复制应激：原因和后果

• 批准号: 9762783
• 负责人: Susana Gonzalo Hervas
• 金额: $ 33.33万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762783
• 关键词: Affect; Aging; Architecture; Attention; Binding; Biological Assay; Breast; Calcitriol; Cardiovascular Diseases; Cells; Characteristics; Chromatin; Clinical Management; Colon; Cytoplasm; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Degenerative Disorder; Deterioration; Diet; Disease; Down-Regulation; Electron Microscopy; Ensure; Fiber; Functional disorder; Gene Expression; Gene Mutation; Genetic; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Human; In Vitro; Inflammatory Response; Interferons; Knowledge; Lamin Type A; Lamins; Link; Longevity; Lung; Malignant - descriptor; Malignant Neoplasms; Mechanical Stress; Mediating; Molecular; Mus; Mutation; Names; Nuclear; Nuclear Lamin; Nuclear Matrix; Nucleic Acids; Ovarian; Pathology; Pathway interactions; Patients; Pattern recognition receptor; Pharmacology; Phenotype; Play; Pre-Clinical Model; Premature aging syndrome; Process; Progeria; Proteins; Regulation; Rejuvenation; Research; Role; STAT1 gene; Scientific Advances and Accomplishments; Source; Syndrome; Teenagers; Testing; Translating; Up-Regulation; Vitamin D; analog; base; cellular pathology; disease phenotype; effective therapy; experimental study; fitness; genome integrity; healthspan; improved; in vivo; induced pluripotent stem cell; infancy; insight; leukemia; mouse model; mutant; new technology; normal aging; nuclease; outcome forecast; paricalcitol; pathogen; reconstitution; recruit; repaired; replication stress; response; scaffold; senescence; single molecule; targeted treatment

Abstract The spatial organization of the genome has emerged as an additional level of regulation of genome function and integrity. Lamins provide a scaffold for the compartmentalization of genome functions, being important for nuclear architecture, response to mechanical stress, chromatin organization, and DNA transcription, replication and repair. These findings, and the association of lamins mutations with degenerative disorders, premature aging, and cancer, provide evidence for these proteins operating as “caretakers of the genome”. However, the mechanisms whereby lamins regulate genome function and stability remain poorly understood. Unveiling these mechanisms is key to identify therapies that ameliorate the progression of lamin-related diseases in patients. Our proposal combines molecular, cellular, and organismal studies, to identify new mechanisms contributing to the pathology of laminopathies, focusing on Hutchinson Gilford Progeria Syndrome (HGPS), a premature aging disease caused by a mutant lamin A protein called “progerin”. We present evidence for lamins playing a direct role in DNA replication. Lamins depletion reduces recruitment of factors that protect stalled forks, leading to nuclease-mediated fork degradation, and replication stress (RS)-induced genomic instability. Progerin expression elicits a more robust effect on DNA replication, causing replication fork stalling, in addition to fork deprotection and degradation. RS in progerin-expressing cells is accompanied by upregulation of the cGAS/ STING cytosolic DNA sensing pathway, and activation of a STAT1-regulated IFN-like response. This response has received much attention lately due to its involvement in malignant transformation and senescence/aging. Importantly, treatments that ameliorate HGPS cellular phenotypes, especially calcitriol, reduces RS, represses the IFN-like response, and increases reprogramming efficiency, a paradigm of rejuvenation. Here, we will use new technologies such as single-molecule replication assays (DNA fibers), iPOND (Isolation of Proteins On Nascent DNA) and electron microscopy to identify molecular mechanisms whereby lamins loss and progerin expression hinder DNA replication (Aim 1). In addition, we will determine the cause-and-effect relationship between RS and activation of the cGAS/STING pathway and the STAT1-regulated IFN-like response, and the consequences of these alterations for cellular fitness (Aim 2). Moreover, we will test whether the broad beneficial effects of calcitriol in cells in vitro translate into amelioration of phenotypes in vivo using mouse models of laminopathies (Aim 3). If successful, our study will fill gaps in our knowledge about mechanisms whereby lamins ensure proper DNA replication, advance laminopathies’ research by identifying new pathways contributing to cellular and organismal deterioration, and provide evidence for the benefits of calcitriol in preclinical models, which will serve as proof-of-concept for its utilization in human patients. Our findings are expected to advance scientific knowledge and change paradigms in the clinical management of HGPS, having potential applicability to other laminopathies, and ultimately normal aging and cancer.

摘要 基因组的空间组织已经成为基因组功能调节的另一个层次 和正直核纤层蛋白为基因组功能的区室化提供了支架，对于 核结构，对机械应力的反应，染色质组织和DNA转录，复制 和修复。这些发现，以及核纤层蛋白突变与退行性疾病的关联， 衰老和癌症，为这些蛋白质作为“基因组管理者”发挥作用提供了证据。但 核纤层蛋白调节基因组功能和稳定性的机制仍然知之甚少。揭开这些 因此，确定核纤层蛋白相关疾病的机制是确定改善患者中核纤层蛋白相关疾病进展的疗法的关键。 我们的建议结合了分子、细胞和生物体的研究，以确定新的机制， 核纤层蛋白病的病理学，重点是哈钦森吉尔福德早衰综合征（HGPS），一种过早衰老 由突变的核纤层蛋白A引起的疾病，称为“早老蛋白”。我们提出的证据表明，lamins发挥直接 在DNA复制中的作用核纤层耗竭减少了保护停滞分叉的因子的募集，导致 核酸酶介导的叉降解和复制应激（RS）诱导的基因组不稳定性。progerin 表达对DNA复制有更强的影响，除了叉外，还引起复制叉停滞。 脱保护和降解。早老蛋白表达细胞中的RS伴随着cGAS/mRNA的上调。 STING细胞溶质DNA传感途径，以及STAT1调节的IFN样应答的激活。此响应 由于其参与恶性转化和衰老/老化，近来受到了很多关注。 重要的是，改善HGPS细胞表型的治疗，特别是骨化三醇，减少RS，抑制 IFN样反应，并增加重编程效率，一个复兴的范例。 在这里，我们将使用新的技术，如单分子复制测定（DNA纤维），iPOND（分离 新生DNA上的蛋白质）和电子显微镜，以确定核纤层蛋白损失的分子机制， 和早老蛋白表达阻碍DNA复制（目的1）。另外，我们将确定 RS与cGAS/STING通路的激活和STAT1调节的IFN样 反应，以及这些改变对细胞适应性的后果（目标2）。此外，我们将测试 骨化三醇在体外细胞中的广泛有益作用是否转化为体内表型的改善 使用核纤层蛋白病的小鼠模型（Aim 3）。如果成功的话，我们的研究将填补我们在以下方面的知识空白： 核纤层蛋白确保正确DNA复制的机制，通过识别核纤层蛋白， 新的途径有助于细胞和有机体的恶化，并提供证据的好处， 钙三醇在临床前模型中的应用，这将作为其在人类患者中应用的概念验证。我们 研究结果有望推动科学知识的发展，并改变临床管理的范式。 HGPS，具有潜在的适用性，其他核纤层蛋白病，并最终正常老化和癌症。