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

细胞大小在物种内部和物种之间都有几个数量级的变化。然而， 并且基因组内容通常随细胞大小而缩放，导致细胞核与细胞质的比例稳定一致 （N/C比率）。这个N/C比例规则的一个例外是许多外部发育物种的早期胚胎 它含有产生后代的所有物质，但仅从一个二倍体细胞核开始。 这些胚胎立即进行一系列的还原卵裂分裂以恢复更典型的N/C 比率。这些切割分裂停止，转录开始于囊胚中期转换（MBT）。一 长期以来的假设是，胚胎通过滴定N/C来测量N/C比率以确定MBT的时间。 一些母性因素。我们和其他人提出，由母体提供的组蛋白 在早期胚胎中大量过量的DNA就是这样一个因素。然而，分子机制是如何通过 哪种组蛋白的可用性被翻译成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Chk 1。Chk 1，in 在MBT处停止细胞周期需要转动。在这里，我们提出测试的假设，组蛋白H3是一个 在早期胚胎中直接Chk 1竞争性抑制剂，并将这一假设扩展到其他DNA背景 损害在具体目标1中，我们将直接测量化合物的体外亲和力和体内浓度。 相关物种，以构建在MBT的H3依赖性Chk 1活性的动力学模型。然后我们将测试这个 通过测量Chk 1活性和细胞周期行为来响应各种H3扰动， 荧光生物传感器在具体目标2中，我们将这些发现扩展到Chk 1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， 胚和翅盘。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提供潜在的新调节臂， 损伤反应途径，可能与致癌作用有关。BioMT COBRE将使 项目通过提供必要的核心信息结构，强大的指导支持，并提供一个社区的其他 志同道合的初级研究人员进行科学讨论和合作。此外，BioMT COBRE将 支持获取必要的数据，将我的工作扩展到早期胚胎和翅膀 光盘，并提供足够的初步数据，以竞争校外资助。项目资金也将 有助于扩大达特茅斯对分子靶向感兴趣的生物学家社区， 相互支持的研究集群。 .

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. .