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.
Highlights
Like enhancers and promoters, insulators or boundary elements are a specialized class of regulatory DNA sequences
Using mutant BEAF-32B rescue transgenes, we show that the middle region and the BESS domain are essential
Details of molecular mechanisms are unclear, support for these models comes from chromosome conformation capture experiments that have found that genomes are divided into topologically associating domains (TADs) that often have insulator binding proteins located at TAD boundaries [12,13,14]
Summary
Insulators or boundary elements are a specialized class of regulatory DNA sequences. Insulators are defined by their ability to function in transgene assays to limit enhancer-promoter communication when placed between a promoter and an enhancer [1, 2], and to protect bracketed transgenes from chromosomal position effects [3, 4]. Based on these activities, it is thought that insulators separate genes into domains such that intra-domain. Details of molecular mechanisms are unclear, support for these models comes from chromosome conformation capture experiments that have found that genomes are divided into topologically associating domains (TADs) that often have insulator binding proteins located at TAD boundaries [12,13,14]
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