The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation

engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal disks; small IDE-reporter transgenes are expressed in the anterior compartment, the opposite of what is expected. Our data show that the En protein binds to en IDEs, and we suggest that En directly represses IDE function. We identified two en IDEs, O and S. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from the endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether our data show that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic regulators. Further, the endogenous locus imparts a stability to en function not seen even in a large transgene, reflecting the importance of both positive and negative epigenetic influences that act over relatively large distances in chromatin. Author SummaryGenes that control development are often used at different times and places in a developing embryo. Transcription of these important genes must be tightly regulated; therefore, these genes often have large arrays of regulatory DNA. In Drosophila, discrete fragments of DNA (enhancers) can be identified that turn genes on in patterns in the early embryo. In cells where the genes are transcriptionally ON, there are active modifications on chromatin, setting later enhancers in a transcription-permission environment. In cells where the genes are OFF, repressive chromatin marks keep later enhancers inactive. In this paper we studied two late enhancers of the Drosophila en gene. We show that the correct activity of these enhancers is dependent on being next to other, earlier acting en enhancers. Our data also show that En can repress its own expression, likely directly by acting on these late enhancers. The chromatin-regulated activity of these en late enhancers is similar to what was described for a late enhancer of another Drosophila developmental gene, Ubx. We suggest that this mode of regulation is likely to be important for many late-acting developmental enhancers in many different organisms.