부산대학교 도서관

Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers, brk5' and brk3', that drive similar but not identical expression of the gene brinker (brk) in early Drosophila embryos. Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5' deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3' deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions. Author summary: Genes expressed during development are often regulated by multiple cis-regulatory sequences, non-coding DNA, also known as enhancer sequences. Many instances have been found where two or more distinct enhancer sequences support similar spatiotemporal outputs relating to a single gene. These enhancers have generally been considered redundantly acting, or overlapping in activity, though few have been studied functionally. We created deletions of coacting enhancer pairs associated with the genes brinker (brk) and short-gastrulation (sog) from the genome of D. melanogaster fruit fly using CRISPR-Cas9 genome editing. Surprisingly, opposite phenotypes relating to some target genes are associated with the enhancer deletions. Deletion of one enhancer generally exhibits phenotypes in early embryo patterning similar to respective gene mutants; whereas, in contrast, deletion of the other presents unique phenotypes including change in cell number for a particular tissue in the embryo, the amnioserosa. In summary, this study shows that coacting enhancers driving similar expression outputs can support distinct, and sometimes complementary, functions to differentially impact the development of embryos and that the individual mutation of these enhancers can provide insight into new gene functions. [ABSTRACT FROM AUTHOR]