View Program - 63rd Annual Drosophila Research Conference

Spatiotemporal regulation of transcription is essential for directing complex patterns of gene expression during embryogenesis. This is achieved through the action of cis-regulatory elements (CREs), which have been studied extensively in Drosophila with reporter genes that permitted the in vivo dissection of CREs. How these CREs dynamically interact with each other and specific promoters in the context of native chromatin remain as unresolved questions. The Drosophila Hox complex includes the pair-rule gene fushi tarazu (ftz) nested between the homeotic genes Antennapedia and Sex combs reduced (Scr). Interestingly, ftz and Scr CREs are intermixed, but the genes have distinct expression patterns: ftz is expressed in seven stripes during the blastoderm stage while Scr is expressed later in the embryonic labial and prothoracic segments. Our investigation is twofold; one, to assess the necessity of previously identified ftz CREs in the native context of the genome using CRISPR-Cas9 induced deletions; two, to identify the location of potential structural elements in this genomic region through the use of reporter transgenes. To this end, we independently deleted the zebra and upstream CREs, two classic seven-stripe ftz CREs, from the endogenous context of the ftz locus using CRISPR/Cas9. These deletion mutants are homozygous viable and fertile, contrary to expectations from historic rescue experiments. While some of these homozygotes develop all segments, other progeny develop abdominal abnormalities including specific segmental deletions and/or fusions. Quantification of the ftz signal using in situ hybridization chain reaction showed that ftz expression is reduced to 25% of wildtype in stripe four of the zebra deletion mutant. This refines the lower threshold of ftz transcript that is both necessary and sufficient for segmentation to proceed. Similar deletions have been made of several stripe-specific CREs, and these homozygotes are viable and fertile. Their survival demonstrates redundancy of multiple CREs directing ftz expression. Analysis of reporter transgenes generated in both orientations has identified two putative insulators. These regions will be targeted for deletion by CRISPR-Cas9 to determine if removing them causes crosstalk between ftz and Scr. Overall, this work provides insight into how insulators organize specific enhancer-promoter interactions in complex genomic regions, allowing for precise spatiotemporal regulation.

Necessity versus sufficiency: furthering understanding of ftz cis-regulatory elements in Drosophila melanogaster