Nuclear dynamics maintaining chromatin integrity during DNA replication

项目总结 人类基因组是在三维(3D)空间中组织的，空间上不同的核区域创建了 控制基本过程的更高级别的秩序。例如，DNA复制发生在核焦点内， 与含有活性基因启动子和增强子的转录凝聚体在空间上分离 元素。事实上，复制和转录表现出显著的协调，确保了遗传和表观遗传 信息在细胞分裂时是保守的。转录缩合物在时间上是动态的，并经历 整编贯穿S阶段。人们对这种程度的控制是如何实现的知之甚少，因此， 未知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)破坏基因组的稳定。鉴于我们最近的情况，我们处于独特的地位来回答这个问题 DNA损伤检查点激酶ATR(共济失调-毛细血管扩张和RAD3相关)的研究进展 转录缩合物的调节功能。在S阶段，ATR在凝析油中积累，发出信号 凝结物组成的变化，并改变RNA聚合酶II的转录周期。此外，急性 抑制ATR可增加TRCs，提示其在转录缩合物中的作用是协调的关键 复制和转录。有趣的是，我们观察到ATR调节的凝析油的子集到CO- 用组蛋白节点体定位，在那里复制依赖的组蛋白被转录，并产生 Pre-mRNAs是将组蛋白的生物合成偶联到S相的过程。耐人寻味的是，ATR的损失放松了监管 组蛋白产生的多个步骤，并提高组蛋白水平与关闭复制和 全球复制灾难的标志的出现。这就提出了一个问题，即是否会扰乱 组蛋白的生物合成是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 质疑并揭示核体内偶联组蛋白生物合成的ATR依赖机制 到了S阶段。我们令人兴奋的进展与ATR信号主要是一种 复制应激反应的驱动因素，并暗示ATR是3D空间中核动力学的关键调节因子 在S阶段。最后，我们将开发一种新的3D染色质构象技术来研究如何复制 转录活性区域的变化会影响启动子-增强子的接触。我们将利用这项技术来澄清 促进复制后3D交互重新建立并确保忠实的机制 S期3D基因组组织与细胞转录特性的传递。总而言之，我的 未来五年的研究计划将导致细胞如何维持的重要发现 S生长期高动态时期的转录状态和基因组组织。

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.