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

果蝇基因后见明（hnt）是哺乳动物Ras反应元件结合蛋白-1 （RREB-1）的同源基因，编码一个大的（1894 AA's, 206 KDa）锌指转录因子。hnt的表达被很好地表征；在果蝇的整个发育过程中，它在各种各样的组织中表达。虽然人们对hnt功能丧失表型以及hnt过表达表型有很多了解，但hnt的基本作用仍然难以捉摸。基于我们实验室最近的工作，我们提出，在某些情况下，hnt是Notch信号通路的直接靶点，其首要作用是放大EGFR/Ras/MAPK信号（以下简称EGFR信号）。我们将使用多种方法来研究hnt增强或放大EGFR信号的分子机制。这将涉及候选基因方法，重点关注EGFR效应基因作为假定的靶基因。我们还发现，在某些情况下，hnt的表达本身依赖于EGFR，这表明在hnt表达和EGFR信号传导之间可能存在反馈调节回路。hnt基因的上游调控区很大（~40 Kb）。我们将开发和使用新的基于CRISPR/Cas9的方法，在这个大的上游调控区域中创建缺失、易位和倒置。成功恢复缺失和重排将使我们能够绘制参与egfr依赖性hnt表达的增强子元件。总之，这些方法将用于定义hnt表达放大EGFR信号传导的机制。然而，与此同时，这些实验将被设计成当使用基于CRISPR/Cas9的诱变的多路复用平台时，可以诱导染色体重排的频率和效率的数据。如果成功，基于CRISPR/Cas9的优化方法产生染色体重排将普遍适用，这可能最终促进除黑尾果蝇以外的果蝇物种平衡染色体的恢复。

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.