Characterization of the Drosophila BEAF-32A and BEAF-32B Insulator Proteins

Abstract

Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene. They differ in their N-terminal 80 amino acids, which contain single DNA-binding BED fingers. The remaining 200 amino acids are identical in the two proteins. The structure and function of the middle region of 120 amino acids is unknown, while the C-terminal region of 80 amino acids has a putative leucine zipper and a BESS domain and mediates BEAF-BEAF interactions. Here we report a further characterization of BEAF. We show that the BESS domain alone is sufficient to mediate BEAF-BEAF interactions, although the presence of the putative leucine zipper on at least one protein strengthens the interactions. BEAF-32B is sufficient to rescue a null BEAF mutation in flies. Using mutant BEAF-32B rescue transgenes, we show that the middle region and the BESS domain are essential. In contrast, the last 40 amino acids of the middle region, which is poorly conserved among Drosophila species, is dispensable. Deleting the putative leucine zipper results in a hypomorphic mutant BEAF-32B protein. Finally, we document the dynamics of BEAF-32A-EGFP and BEAF-32B-mRFP during mitosis in embryos. A subpopulation of both proteins appears to remain on mitotic chromosomes and also on the mitotic spindle, while much of the fluorescence is dispersed during mitosis. Differences in the dynamics of the two proteins are observed in syncytial embryos, and both proteins show differences between syncytial and later embryos. This characterization of BEAF lays a foundation for future studies into molecular mechanisms of BEAF function.

4C-seq characterization of Drosophila BEAF binding regions provides evidence for highly variable long-distance interactions between active chromatin

“…There are two 32 kDa BEAF proteins (BEAF-32A and BEAF-32B, hereafter referred to as 32A and 32B) made from alternative promoters of the same gene [ 14 ]. The two isomers of BEAF differ in their N-termini by 80 amino acids which contain single DNA-binding BED zinc fingers, and interact via a BESS domain located in their C-termini [ 15 – 17 ]. The number of BEAF subunits in a BESS-mediated complex is unknown, and might be variable.…”

The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex

“…A P-element based transgenic fly line with an insulated FLAG-BEAF-32B-EGFP gene expressed from its endogenous promoter ( Avva and Hart 2016 ) on the X chromosome was used to collect 4- to 20-h embryos. These flies also had the wild-type BEAF gene.…”

“…Polybromo sequences were PCR amplified from a cDNA (Drosophila Genomics Resource Center FI03643) and cloned into the EcoRI site of the GAL4 activation domain (AD) plasmid pOAD. Cloning BEAF sequences into the GAL4 DNA-binding domain (DBD) plasmid pOBD2 was previously described ( Avva and Hart 2016 ), except the long leucine zipper (LLZ) sequence which was PCR amplified and inserted into the pOBD2 EcoRI site. Note that there are 2 BEAF isoforms made from 1 gene, BEAF-32A and BEAF-32B ( Hart et al 1997 ).…”

“…All subregions of BEAF tested are common to both isoforms. Y2H assays were conducted using standard methods as previously described ( Avva and Hart 2016 ). Between 50 and 100 colonies containing both plasmids (growth on plates lacking tryptophan and leucine) for each plasmid combination were transferred onto plates to score for reporter gene expression (growth on medium also lacking adenine and histidine, with X-α-Gal to score α-galactosidase expression; all colonies that grew also showed α-galactosidase expression).…”

Functional dissection of the developmentally restricted BEN domain chromatin boundary factor Insensitive

“…Adult abdominal cuticles of homozygous enclosed 3–4-day-old flies were prepared essentially as described in Mihaly et al [46] and mounted in Hoyer’s solution. Squashed salivary gland specimens were prepared and stained following standard protocols [59, 60]. Primary antibodies were rabbit polyclonal anti-Insv at 1:1000 dilution and rat polyclonal anti-CP190 at 1:1500 dilution.…”