Generation of an Endogenous Dominant Negative Allele of chd5 Using a Novel Homologous Recombination Strategy to Reveal the Role of chd5 in Tumor Suppression and Neural Differentiation

Abstract

CHD5 is a vertebrate specific member of a family of ATP‐dependent chromatin remodeling proteins. Chromodomain/Helicase/DNA‐binding (CHD) proteins play a variety of roles in vertebrate development, and loss of CHD5 causes failure of proper expression in a cohort of genes involved in neural development. CHD5 plays a critical role tumor suppression in humans, and loss of CHD5 contributes to the formation or progression of numerous cancers, in particular neuroblastoma. To understand how CHD5 plays a role in tumor suppression, it is necessary to understand how CHD5 contributes to normal development and how its normal functions are misregulated during tumor progression. Our lab is establishing zebrafish as model system in which to study the role of chd5 dependent processes.We have engineered chd5 knockout fish using CRISPR‐Cas9 technology. The resulting fish do not exhibit any overt developmental phenotypes, and do not produce tumors during adulthood (N=165, aged >16 months). We hypothesize that the closely related remodelers CHD3/4 function redundantly with CHD5, and propose to use a novel knock‐in approach to generate zebrafish carrying a dominant negative mutation in the ATPase domain that has previously been shown to result in strong mutant phenotypes when generated in closely related remodelers.To engineer a dominant negative allele of chd5 we have engineered a homologous recombination (HR) strategy that uses piggyBac transposase technology, which has previously been used in Drosophila, mice, and human cells to engineer scarless genomic modifications. This design uses two‐steps to first, introduce a homologous donor carrying the mutation of interest and a fluorescent marker for easy identification. Second, the HR positive fish will be bred with piggyBac expressing fish lines to seamlessly excise the exogenous sequences, thus leaving only the desired genome modification.Once we have engineered this method into the fish, we will be able to use piggyBac to control when and where we express the dominant negative allele of chd5 so that we can score the resulting fish for developmental and tumor phenotypes. The screening of the dominant negative, knock‐in lines is in progress. The resulting dominant negative lines will give great insight into the role of chd5 in neural development and tumor suppression.Support or Funding InformationFunding made available by: Purdue University Center for Cancer Research, Purdue University Department of Biochemistry, Purdue University College of AgricultureThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.