Dissecting histone regulation of the DNA-damage checkpoint in proliferating cells

剖析增殖细胞中 DNA 损伤检查点的组蛋白调节

• 批准号: 10271752
• 负责人: Amanda A Amodeo
• 金额: $ 26.24万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10271752
• 关键词: Activity Cycles; Affinity; Behavior; Biochemical; Biosensor; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleus; Cell Size; Cell Survival; Cell model; Cells; Centers of Research Excellence; Chemicals; Chromatin; Collaborations; Communities; Consensus; Cytoplasm; DNA; DNA Damage; DNA Markers; DNA damage checkpoint; Data; Development; Diploidy; Disease; Drosophila genus; Embryo; Embryonic Development; Ensure; Exposure to; Extramural Activities; Funding; Genetic Transcription; Genomic Instability; Genomics; Growth; Histone H3; Histones; In Vitro; Institutes; Ionizing radiation; Ions; Libraries; Measurement; Measures; Mediating; Mentors; Microscopy; Mind; Modeling; Molecular; Molecular Target; Mutation; Nuclear; Oncogenic; Pathway interactions; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Proliferating; Regulation; Repression; Research; Research Personnel; Role; Series; Signal Pathway; Signal Transduction; Site; Substrate Cycling; Tail; Testing; Time; Tissues; Titrations; Translating; Variant; Wing; Work; arm; blastocyst; blastomere structure; carcinogenesis; data acquisition; experimental study; fly; genome integrity; hydroxyurea; imaginal disc; in vivo; inhibitor/antagonist; interest; irradiation; kinetic model; mutant; novel; offspring; predictive modeling; replication stress; response; tumor

Cell sizes vary across several orders of magnitude both within and between species. Yet, nuclear volumes and genomic content typically scale with cell size leading to a robustly consistent ratio of nucleus to cytoplasm (N/C ratio). One exception to this N/C ratio rule is the early embryo of many externally developing species which contains all the material to produce the resultant offspring but starts with only a single diploid nucleus. These embryos immediately undertake a series of reductive cleavage divisions to restore more typical N/C ratios. These cleavage divisions halt and transcription starts at the Mid-Blastula Transition (MBT). A longstanding hypothesis has been that the embryo measures the N/C ratio to time the MBT through titration of some maternally provided factor. We and others have proposed that histones, which are maternally provided in vast excess of DNA in the early embryo are one such factor. However, how the molecular mechanism by which histone availability is translated into a cell cycle signal has remained a mystery. Recently, we discovered that histone H3 promote cell cycle progression and inhibit the DNA damage kinase, Chk1. Chk1, in turn is required to stop the cell cycle at the MBT. Here we propose to test the hypothesis that histone H3 is a direct Chk1 competitive inhibitor in the early embryo and extend this hypothesis to other contexts of DNA damage. In Specific Aim 1 we will directly measure the in vitro affinities and in vivo concentrations of the relevant species to construct a kinetic model of H3 dependent Chk1 activity at the MBT. We will then test this model by measuring Chk1 activity and cell cycle behavior in response to a variety of H3 perturbations using fluorescent biosensors. In Specific Aim 2 we will extend these findings to Chk1 mediated DNA damage response triggered by irradiation, chemical damaging agents, and oncogenic replication stress in both the embryo and wing imaginal disc. Successful completion of this work will answer the longstanding question of how cells in the early embryo senses its N/C ratio and provide a potential new regulatory arm to the DNA- damage response pathway that may have implications for carcinogenesis. The BioMT COBRE will benefit the project by providing essential core infostructure, strong mentoring support, and providing a community of other like-minded junior investigators for scientific discussion and collaboration. In addition, the BioMT COBRE will support the acquisition of the data necessary to extend my work beyond the early embryo and into the wing disc and provide sufficient preliminary data to be competitive for extramural funding. Project funding will also help to expand the community of biologists interested in molecular targeting at Dartmouth to help nucleate a mutually supportive research cluster. .

细胞的大小在物种内部和物种之间都有几个数量级的差异。然而，核容量 基因组含量通常会随着细胞大小的增加而增加，导致核质比例保持稳定。 (N/C比率)。N/C比规则的一个例外是许多外部发育物种的早期胚胎 它包含了产生后代所需的所有材料，但只从一个二倍体细胞核开始。 这些胚胎立即进行一系列还原的卵裂分裂，以恢复更典型的N/C 比率。这些卵裂分裂在囊胚中期(MBT)停止，转录开始。一个 长期以来的假设是，胚胎通过滴定MBT来测量N/C比来计时 一些母亲提供的因素。我们和其他人提出，组蛋白是由母体提供的 早期胚胎中DNA的大量过剩就是其中一个因素。然而，分子机制是如何通过 组蛋白的可获得性如何转化为细胞周期信号仍然是一个谜。最近，我们 发现组蛋白H3促进细胞周期进程并抑制DNA损伤激酶Chk1。通道1，英寸 需要转动才能在MBT处停止细胞周期。在这里，我们建议验证组蛋白H3是一个 在早期胚胎中直接使用Chk1竞争抑制物，并将这一假说扩展到DNA的其他背景 损坏。在特定的目标1，我们将直接测量体外亲和力和体内浓度 相关物种在MBT上构建依赖于H3的Chk1活性的动力学模型。然后我们将对此进行测试 通过测量Chk1活性和细胞周期行为来响应各种H3扰动 荧光生物传感器。在特定目标2中，我们将把这些发现扩展到chk1介导的dna损伤。 辐射、化学损伤剂和致癌复制应激引发的反应 胚胎和翅的成像盘。这项工作的成功完成将回答长期存在的问题 早期胚胎中的细胞如何感知其N/C比率，并为DNA提供潜在的新调控臂-- 可能与癌症发生有关的损伤反应途径。BioMT Cobre将使 通过提供基本的核心信息结构、强大的指导支持和提供其他社区 志同道合的初级调查人员进行科学讨论和合作。此外，BioMT Cobre将 支持获得必要的数据，以便将我的工作从早期胚胎扩展到机翼 光盘，并提供足够的初步数据，使其在外部资金方面具有竞争力。项目资金也将 帮助扩大达特茅斯大学对分子靶向感兴趣的生物学家社区，以帮助形成 相互支持的研究集群。 。