The role of UBR5 in the PRC1-mediated transcriptional repression at damaged chromatin

科学抽象 基因组的完整性不断受到各种形式的遗传毒性胁迫的威胁，包括辐射或 内源性代谢产物活跃增殖细胞中的DNA损伤导致DNA停滞 复制聚合酶以及RNA聚合酶，其可导致复制叉停滞或转录抑制。 逮捕，分别。了解细胞反应以监测和执行聚合酶停止将 让我们更好地了解压力细胞，如癌细胞，如何应对挑战。 含有BMI 1的Polycomb复合物（PRC 1; Polycomb Repressive Complex 1）是一种重要的 发育过程中表观遗传沉默程序的调节因子，部分通过诱导H2 AK 119 与抑制的RNA聚合酶II（Pol II）相关的单泛素化（H2 AK 119-Ub）。最近， BMI 1在DNA损伤反应中的作用已引起人们的广泛关注，但BMI 1在DNA损伤反应中的作用机制和作用机制是什么 在这个过程中是不清楚的。 在我们的初步研究中，我们确定UBR 5是一个下游因子，其染色质募集是 由含有BMI 1的PRC 1复合物调节。而转录在紫外线诱导的损伤中受到抑制， 位点，PRC 1成员或UBR 5单独耗竭， 这表明UBR 5与PRC 1在诱导基因沉默中以线性途径起作用 在病变处。我们发现UBR 5与BMI 1以及组蛋白伴侣复合物FACT相互作用， UBR 5与FACT组分SPT 16以BMI 1依赖性方式共定位于UV诱导的病变。我们 进一步提供了UBR 5直接泛素化SPT 16的证据。我们的证据表明， BMI 1-UBR 5轴拮抗UV损伤后的FACT依赖性Pol II延长。 基于这些数据，我们提出了一个假设，即UBR 5是PRC 1的关键下游效应子。 介导的基因沉默。具体的目的是确定：1）PRC 1和组蛋白修饰在 UBR 5募集，2）BMI 1和UBR 5在DNA损伤或损伤后调节转录抑制中的作用， 3）OTUD 5去泛素化酶在UBR 5和PRC 1诱导的细胞凋亡中的作用 在损伤处的转录抑制。 这些研究的成功结果将使UBR 5成为一个关键的下游介质。 PRC 1复合物介导的转录调控。拟议的工作将填补重要的差距， Polycomb复合体在基因沉默中的功能在很大程度上是未知的， 更广泛的转录、DNA损伤反应和癌症发展领域。

Scientific Abstract Genome integrity is constantly threatened by various forms of genotoxic stresses, including radiation or endogenous metabolic products. Damaged DNA lesions in actively proliferating cells cause stalling of DNA replication polymerases as well as RNA polymerases, which can lead to replication fork arrest or transcriptional arrest, respectively. Understanding the cellular response to monitor and execute the polymerase stalling will give us better insight on how stressed cells, such as cancer cells, deal with the challenges. The BMI1-containing Polycomb complex (PRC1; Polycomb Repressive Complex 1) is an important regulator of epigenetic silencing programs during development, in part by inducing H2AK119 monoubiquitination (H2AK119-Ub) associated with repressed RNA Polymerase II (Pol II). Recently, the role of BMI1 in DNA damage response has gained much attention, but in what aspect and mechanism it participates in the process is unclear. In our preliminary studies, we identified UBR5 as a downstream factor whose chromatin recruitment is regulated by the BMI1-containing PRC1 complex. While transcription is repressed at UV-induced damaged sites on chromatin, depletion of the PRC1 members or UBR5 alone de-repressed transcription elongation at the damaged sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at the lesions. We found that UBR5 interacts with BMI1 as well as histone chaperone complex FACT, and that UBR5 co-localizes with FACT component SPT16 to the UV-induced lesions in a BMI1-dependent manner. We further provide evidence that UBR5 directly ubiquitinates SPT16. Our evidence suggests a model that the BMI1-UBR5 axis antagonizes the FACT-dependent Pol II elongation upon UV insults. Based on these data, we propose a hypothesis that UBR5 is a key downstream effector of the PRC1- mediated gene silencing. The specific aims are to determine: 1) role of PRC1 and histone modification in UBR5 recruitment, 2) role of BMI1 and UBR5 in regulating transcriptional repression under DNA damaged or unperturbed conditions, 3) role of OTUD5 deubiquitinating enzyme in the UBR5 and PRC1-induced transcriptional repression at the lesions. The successful outcome of these studies will establish UBR5 as a key downstream mediator of the transcriptional regulation mediated by the PRC1 complex. The proposed work will fill the gap of important yet largely unknown function of the Polycomb complex in gene silencing, and will have an impact that advances the broader area of transcription, DNA damage response, and cancer development.