Mechanism of cell lethality following loss of gene expression.

项目摘要 本项目的目标是确定转录抑制导致细胞死亡的机制。 该领域的共识模型假定，转录抑制后的细胞死亡是由于转录因子的丢失导致的。 特定的mRNA种类和随后的蛋白质损失。通过靶向这样的核心细胞过程， 抑制被认为是压倒细胞控制并导致不可避免的细胞死亡。这种死亡过程，定义为 作为意外细胞死亡（ACD），不受细胞控制，也不是使用定义的效应物引起的 分子。与传统模型相反，我们发现， 转录抑制由先前未描述的调节的凋亡信号引起。此外，我们发现， RNA Pol II降解，而不是mRNA产生的损失，导致细胞死亡。我们的数据显示 新模型，其中Pol II的降解诱导信号离开细胞核并被细胞核接收。 线粒体启动凋亡。为了鉴定转录后调节促凋亡信号的基因， 在抑制之后，我们进行了全基因组CRISPR筛选。全基因组CRISPR筛选通常无法识别 死亡调控基因，使得很难阐明细胞死亡的机制。为了克服这个问题，我们开发了 一种新的实验策略，使我们能够识别基因的敲除调节细胞死亡率 转录抑制后。根据我们的筛选结果，在目标1中，我们将检验以下假设： 选择性剪接调节子PTBP 1促进调节性前mRNA的改变的剪接和核输出， 并且这种活性是转录抑制后细胞死亡所必需的。我们将使用活细胞显微镜 确定PTBP 1核出口的功能作用。我们将使用SLAM-seq和RIP-seq来定量PTBP 1 转录抑制后的活性。我们的筛选还确定BCL 2L 12为关键的细胞凋亡效应子 转录抑制基因。在目的2中，我们将检验BCL 2L 12在以下情况下激活凋亡的假设： 以同种型特异性方式抑制转录。我们将进行一系列的功能遗传学研究 实验来表征BCL 2L 12在凋亡反应中的作用。通过描述一种新的机制， 模型，转录抑制诱导细胞死亡，我们将提高我们的理解如何 有效地使用转录抑制剂治疗。最终，我们希望我们的工作将提高我们的能力， 预测哪些患者对转录抑制剂的反应最好，并帮助确定新的治疗策略。

PROJECT SUMMARY The goal of this project is to determine the mechanism by which cell death results from transcriptional inhibition. The consensus model in the field posits that cell death following transcriptional inhibition results from the loss of specific mRNA species and subsequent loss of protein. By targeting such a core cellular process, transcriptional inhibition is thought to overwhelm cellular control and lead to unavoidable cell death. This death process, defined as Accidental Cell Death (ACD), is not controlled by the cell and does not result from the use of defined effector molecules. Contrary to the conventional model, we found that, rather than induce ACD, cell death following transcriptional inhibition results from a previously undescribed regulated apoptotic signal. Furthermore, we found that RNA Pol II degradation, rather than loss of mRNA production, resulted in cell death. Our data suggests a new model, whereby degradation of Pol II induces a signal that leaves the nucleus and is received by the mitochondria to initiate apoptosis. To identify genes that regulate a pro-apoptotic signal following transcriptional inhibition, we performed a genome-wide CRISPR screen. Genome-wide CRISPR screens often fail to identify death regulatory genes, making it difficult to elucidate mechanisms of cell death. To overcome this, we developed a novel experimental strategy that allowed us to identify genes whose knockout modulated the cell death rate following transcriptional inhibition. Based on the results of our screen, in Aim 1 we will test the hypothesis that the alternative splicing regulator PTBP1 facilitates altered splicing and nuclear export of regulatory pre-mRNA, and that this activity is required for cell death following transcriptional inhibition. We will use live cell microscopy to establish the functional role of PTBP1 nuclear export. We will use SLAM-seq and RIP-seq to quantify PTBP1 activity following transcriptional inhibition. Our screen also identified BCL2L12 as the critical apoptotic effector gene for transcriptional inhibition. In Aim 2, we will test the hypothesis that BCL2L12 activates apoptosis following transcriptional inhibition in an isoform-specific manner. We will perform a series of functional genetics experiments to characterize the role of BCL2L12 in the apoptotic response. By describing a new mechanistic model by which transcriptional inhibition induces cell death, we will improve our understanding of how to effectively use transcriptional inhibitors therapeutically. Ultimately, we hope our work will improve our ability to predict which patients will best respond to transcriptional inhibitors and help identify novel treatment strategies.