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hindsight(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个氨基酸，206 KDa)锌指转录因子。Hnt的表达具有很好的特征；它在整个果蝇发育过程中的各种组织中都有表达。虽然关于Hnt功能丧失表型以及Hnt过表达表型已知很多，但Hnt的基本作用仍然难以捉摸。根据我们实验室最近的工作，我们认为在某些情况下是Notch信号通路的直接靶点的Hnt的首要作用是放大EGFR/RAS/MAPK信号(以下称为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。这将涉及一种候选基因方法，重点放在EGFR效应基因上，该基因被认为是一个假定的hnt靶基因。我们还发现，在某些情况下，Hnt的表达本身是EGFR依赖的，这表明在Hnt表达和EGFR信号之间存在反馈调控环的可能性。HNT基因的上游调控区很大(~40kb)。我们将开发和使用基于CRISPR/Cas9的新方法，在这个庞大的上游监管区域创造缺失、易位和倒置。缺失和重排的成功恢复将使我们能够定位参与EGFR依赖的hnt表达的增强子元件。总而言之，这些方法将被用来定义hnt表达放大EGFR信号的机制。然而，与此同时，这些实验将被设计成产生有关使用多路复用平台进行基于CRISPR/Cas9的突变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的染色体重排方法的优化将普遍适用，这最终可能有助于恢复除黑腹果蝇以外的果蝇物种的平衡染色体。

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.