# Notch-Dependent Expression of the Drosophila Hey Gene Is Supported by a Pair of Enhancers with Overlapping Activities

## TL;DR

This study shows that two enhancer regions work together to control the Notch-dependent expression of the Drosophila Hey gene in neurons and gut cells.

## Contribution

The study identifies two enhancers with overlapping functions that redundantly regulate Notch signaling in the Hey gene.

## Key findings

- Two enhancer regions (HeyUP and HeyIN2) direct overlapping expression patterns of the Hey gene.
- HeyIN2 is Notch-dependent, while HeyUP has both Notch-dependent and independent regulation.
- Deleting all Su(H) binding motifs in both enhancers causes a lethal Hey loss-of-function phenotype.

## Abstract

Drosophila Hey is a basic helix–loop–helix–orange (bHLH-O) protein with an important role in the establishment of distinct identities of postmitotic cells. We have previously identified Hey as a transcriptional target and effector of Notch signalling during the asymmetric division of neuronal progenitors, generating neurons of two types, and we have shown that Notch-dependent expression of Hey also marks a subpopulation of the newborn enteroendocrine (EE) cells in the midgut primordium of the embryo. Here, we investigate the transcriptional regulation of Hey in neuronal and intestinal tissues. We isolated two genomic regions upstream of the promoter (HeyUP) and in the second intron (HeyIN2) of the Hey gene, based on the presence of binding motifs for Su(H), the transcription factor that mediates Notch activity. We found that both regions can direct the overlapping expression patterns of reporter transgenes recapitulating endogenous Hey expression. Moreover, we showed that while HeyIN2 represents a Notch-dependent enhancer, HeyUP confers both Notch-dependent and independent transcriptional regulation. We induced mutations that removed the Su(H) binding motifs in either region and then studied the enhancer functionality in the respective Hey mutant lines. Our results provide direct evidence that although both enhancers support Notch-dependent regulation of the Hey gene, their role is redundant, as a Hey loss-of-function lethal phenotype is observed only after deletion of all their Su(H) binding motifs by CRISPR/Cas9.