Background DNA replication‐related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication‐ and proliferation‐related genes in Drosophila. While the effects on the mutation in the DNA replication‐related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild‐type DREF activity in developing tissues have hitherto remained unclear.Results We over‐expressed DREF in the wing imaginal disc using a GAL4‐UAS targeted expression system in Drosophila. Over‐expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF‐induced notching wing phenotype. Furthermore, this was also the case with co‐expression of baculovirus P35, a caspase inhibitor. In addition, over‐expression of the 32 kDa boundary element‐associated factor (BEAF‐32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF‐induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF‐32 gene, exerted enhancing effects. To our knowledge, this is the first evidence for a genetic interaction between DREF and BEAF‐32.Conclusion The DREF‐induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc.