Investigating the role of the transcription factor Hindsight in the regulation of EGFR/Ras/MAPK signaling: new applications for CRISPR/Cas9 genome editing

事后调查转录因子在 EGFR/Ras/MAPK 信号传导调节中的作用：CRISPR/Cas9 基因组编辑的新应用

• 批准号: RGPIN-2020-05610
• 负责人: Reed, Bruce
• 金额: $ 2.62万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740679
• 关键词: Investigating; role; transcription; factor; Hindsight

The Drosophila gene hindsight (hnt), which is the homolog of mammalian Ras Responsive Element Binding protein - 1 (RREB-1), encodes a large (1894 AA's, 206 KDa) Zinc-finger transcription factor. The expression of hnt is well characterized; it is expressed in a staggering variety of tissues throughout Drosophila development. While much is known regarding hnt loss-of-function phenotypes, as well as hnt overexpression phenotypes, the fundamental role of hnt remains elusive. Based on recent work from our lab, we propose that the overarching role of hnt, which in certain contexts is a direct target of the Notch signaling pathway, is to amplify EGFR/Ras/MAPK signaling (hereafter referred to as EGFR signaling). We will use a variety of approaches to investigate the molecular mechanism by which hnt functions to enhance or amplify EGFR signaling. This will involve a candidate gene approach, focusing on the EGFR effector gene pointed as a putative hnt target gene. We have also found that expression of hnt is itself EGFR-dependent in certain contexts, and this suggests the possibility of a feedback regulatory loop between hnt expression and EGFR signaling. The upstream regulatory region of the hnt gene is large (~40 Kb). We will develop and use novel CRISPR/Cas9 based approaches to create deletions as well as translocations and inversions throughout this large upstream regulatory region. Successful recovery of deletions and rearrangements will allow us to map enhancer elements involved in EGFR-dependent hnt expression. Overall, these approaches will be used to define the mechanism by which hnt expression amplifies EGFR signaling. At the same time, however, these experiments will be designed to generate data on the frequency and efficiency with which chromosomal rearrangements can be induced when using a multiplexing platform for CRISPR/Cas9 based mutagenesis.  If successful, the optimization of CRISPR/Cas9 based methods to generate chromosomal rearrangements will be generally applicable, and this may eventually facilitate the recovery of balancer chromosomes in Drosophila species other than Drosophila melanogaster.

果蝇基因事后（HNT）是哺乳动物Ras响应元件结合蛋白-1（RREB-1）的同源物，编码大型（1894 AA，206 kDa）锌指转录因子。 Hnt的表达很好。它在果蝇发育过程中以惊人的多种组织表达。尽管Hnt功能障碍表型以及HNT过表达表型知之甚少，但HNT的基本作用仍然难以捉摸。基于我们实验室的最新工作，我们建议Hnt的总体作用在某些情况下是Notch信号传导途径的直接目标，就是扩大EGFR/RAS/MAPK信号传导（以下称为EGFR信号传导）。我们将使用多种方法来研究HNT功能增强或扩增EGFR信号传导的分子机制。这将涉及一种候选基因方法，重点是指向推定的HNT靶基因的EGFR效应基因。我们还发现，Hnt的表达本身在某些情况下依赖于EGFR，这表明HNT表达和EGFR信号之间有反馈调节环的可能性。 HNT基因的上游调节区域很大（约40 kb）。我们将开发和使用基于CRISPR/CAS9的新型方法来通过这个大型上游监管区域创建缺失以及易位和反转。成功恢复缺失和重排将使我们能够绘制涉及EGFR依赖性HNT表达的增强子元素。总体而言，这些方法将用于定义HNT表达放大器EGFR信号传导的机制。但是，同时，这些实验将设计为生成有关在基于CRISPR/CAS9的诱变时，可以诱导染色体重排的频率和效率的数据。如果成功，通常适用于基于CRISPR/CAS9产生染色体重排的方法，这最终可能有助于果蝇除果蝇Melanogaster以外的果蝇物种中恢复均衡染色体。