The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units.

The Drosophila genome encodes four closely related proteins including Insensitive (Insv), Elba1, Elba2 and Elba3 that possess activities in both transcriptional repression and chromatin insulation. The first three proteins are BEN-solo factors, all with a DNA binding BEN domain. The fourth protein Elba3 bridges Elba1 and Elba2 to form a hetero-trimeric complex Elba. Here we report comprehensive investigation on the in vivo functions of these proteins in Drosophila embryos. Insv and Elba bind to many common and distinct genomic loci. Unexpectedly the adapter protein Elba3 can associate with chromatin and repress gene expression independent of Elba1 and Elba2. Our ChIP-nexus analyses show Insv binding to DNA in a symmetric configuration and Elba binding asymmetrical in vivo. In finding that the motifs of known insulator proteins are enriched in Elba and Insv ChIP peaks, we confirmed that Elba collaborates with other insulator proteins to regulate developmental patterning in embryos. To differentiate the insulator function of Elba and Insv from their repressor activity, we determined real-time transcription change in mutant embryos using precise nuclear run-on sequencing (PROseq) assay. elba mutation dampens expression difference between the two Elba-bound neighbor genes. Finally, transgenic reporters confirm insulation activity of Elba-and Insv-bound sites. Together, these findings define Elba and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators in the partition of transcription units.