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

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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.