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: 10170189
• 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/10170189
• 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; Prognosis; Proteins; Regulation; Rejuvenation; Research; Role; STAT1 gene; Scientific Advances and Accomplishments; Source; Stimulator of Interferon Genes; 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; 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作为基因组功能的额外调节水平而出现 和正直。层蛋白为基因组功能的划分提供了一个支架，对 核结构、对机械应激的反应、染色质组织和DNA转录、复制 并进行修复。这些发现，以及Lamins突变与退行性疾病的关联，过早 衰老和癌症为这些蛋白质作为“基因组看守人”的运作提供了证据。然而， Lamins调节基因组功能和稳定性的机制仍然知之甚少。揭开这些面纱 机制是确定改善患者层粘连蛋白相关疾病进展的治疗方法的关键。 我们的建议结合了分子、细胞和生物体的研究，以确定有助于 椎板病变的病理学，重点是Hutchinson Gilford Progeria综合征(HGPS)，一种早衰 由突变层粘连蛋白引起的疾病称为“孕激素”。我们提出的证据表明，Lamins扮演着直接的 在DNA复制中的作用。板层耗尽减少了保护失速叉子的因素的招募，导致 核酸酶介导的分叉降解，以及复制应激(RS)诱导的基因组不稳定性。进展性 表达对DNA复制的影响更强，除了分叉外，还会导致复制分叉停滞 去保护和退化。孕激素表达细胞中的RS伴随着cGAS/2的上调 刺痛胞浆DNA传感通路，以及激活STAT1调节的干扰素样反应。这一反应 由于它参与了恶性转化和衰老/衰老，近年来受到了人们的广泛关注。 重要的是，改善HGPS细胞表型的治疗，特别是骨化三醇，可以减少RS，抑制 干扰素样反应，并增加重新编程的效率，这是一种年轻化的范例。 在这里，我们将使用新技术，如单分子复制分析(DNA纤维)、iPOND(分离 蛋白质在新生DNA上的变化)和电子显微镜以确定板层丢失的分子机制 而孕激素表达阻碍DNA复制(目标1)。此外，我们将确定因果关系 RS与cGAS/STIN通路激活及STAT1调节的干扰素样蛋白的关系 反应，以及这些变化对细胞健康的影响(目标2)。此外，我们还将测试 骨化三醇在体外对细胞的广泛有益作用是否转化为体内表型的改善 使用小鼠椎板病变模型(目标3)。如果成功，我们的研究将填补我们对 层粘连蛋白确保适当的DNA复制的机制，通过识别 促进细胞和机体退化的新途径，并提供了证据表明 临床前模型中的骨化三醇，这将作为其在人类患者中应用的概念验证。我们的 这些发现有望促进科学知识的进步并改变临床治疗的范式。 HGPS，具有潜在的适用性，可用于其他椎板病，最终导致正常衰老和癌症。