Nuclear dynamics maintaining chromatin integrity during DNA replication

项目摘要 人类基因组在3维（3D）空间中组织，具有空间上不同的核区域， 控制基本过程的更高层次的秩序。例如，DNA复制发生在核灶内， 在空间上与含有活性基因启动子和增强子的转录浓缩物分离 元素事实上，复制和转录表现出显着的协调，确保遗传和表观遗传 信息在细胞分裂时是保守的。转录浓缩物是时间动态的， 整个S阶段的重组。人们对这种控制水平是如何实现的知之甚少，因此， 不知道复制和转录如何在空间上保持分离以防止转录-复制冲突 （TRCs），使基因组不稳定。鉴于我们最近的情况，我们在回答这个问题方面处于独特的地位。 发现ATR（共济失调-毛细血管扩张和rad 3相关）的进展，一种DNA损伤检查点激酶， 转录缩合物功能的调节子。ATR在S期凝析油中积累，发出信号， 冷凝物组成的变化，并改变RNA聚合酶II转录周期。此外，急性 ATR的抑制增加了TRC，表明其在转录缩合物中的功能对于协调 复制和转录。有趣的是，我们已经观察到ATR调节的缩合物的一个子集， 定位于组蛋白基因座体，在那里复制依赖性组蛋白被转录， 前体mRNA被加工，将组蛋白生物合成偶联到S期。有趣的是，ATR的丧失 组蛋白产生的多个步骤，并提高与复制停止相关的组蛋白水平， 全球复制灾难标志的出现。这就提出了一个问题： 组蛋白生物合成是ATR信号丢失后复制灾难的关键驱动因素。我们会回答这个问题 质疑和揭示ATR依赖的机制，耦合组蛋白生物合成的核体内 S阶段。我们令人兴奋的进展与ATR信号转导的经典观点形成鲜明对比， 复制应激反应的驱动程序，并暗示ATR是3D空间中核动力学的关键调节因子 在S期。最后，我们将开发一种新的3D染色质构象技术来研究复制是如何进行的。 转录活性区的影响启动子-增强子接触。我们将利用这项技术来阐明 促进复制后重建3D相互作用并确保忠实的机制 3D基因组结构的传递和细胞跨S期的转录同一性。总之，我的 在未来五年的研究计划将导致重要的发现，细胞如何维持 转录状态和基因组结构在S期的高度动态时期。

PROJECT SUMMARY The human genome is organized in 3-dimensional (3D) space, with spatially-distinct nuclear regions creating higher-level order that controls essential processes. For example, DNA replication occurs within nuclear foci that are spatially-separated from transcription condensates that contain active gene promoter and enhancer elements. Indeed, replication and transcription exhibit remarkable coordination ensuring genetic and epigenetic information are conserved upon cell division. Transcription condensates are temporally dynamic and undergo reorganization throughout S phase. How this level of control is achieved is poorly understood, and thus, it is unknown how replication and transcription remain spatially separated to prevent transcription-replication conflicts (TRCs) that destabilize the genome. We are uniquely positioned to answer this question given our recent progress uncovering ATR (ataxia-telangiectasia and rad3-related), a DNA damage checkpoint kinase, as a key regulator of transcription condensate function. ATR accumulates within condensates during S phase, signals a change in condensate composition, and alters the RNA polymerase II transcription cycle. Moreover, acute inhibition of ATR increases TRCs suggesting its function in transcription condensates is critical for coordinating replication and transcription. Interestingly, we have observed a subset of ATR-regulated condensates to co- localize with histone locus bodies, where the replication-dependent histones are transcribed and the resulting pre-mRNAs are processed coupling histone biosynthesis to S phase. Intriguingly, loss of ATR deregulates multiple steps of histone production and elevates histone levels correlating with a shutdown of replication and the appearance of markers of global replication catastrophe. This raises the question as to whether disruption of histone biosynthesis is a key driver of replication catastrophe upon loss of ATR signaling. We will answer this question and uncover the ATR-dependent mechanisms that couple histone biosynthesis within nuclear bodies to S phase. Our exciting progress stands in contrast to the classical view of ATR signaling as predominantly a driver of the replication stress response and implicates ATR as a key regulator of nuclear dynamics in 3D space during S phase. Finally, we will develop a novel 3D chromatin conformation technology to study how replication of transcriptionally-active regions impacts promoter-enhancer contacts. We will use the technology to elucidate the mechanisms that promote re-establishment of 3D interactions post-replication and ensure faithful transmission of 3D genome organization and the transcriptional identity of cells across S phase. In sum, my research program over the next five years will lead to important discoveries as to how cells maintain transcriptional states and genome organization during the highly dynamic period of S phase.