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

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.本研究证实果蝇32 kDa边界元件关联因子（Boundary Element-Associated Factors）BEAF-32A与BEAF-32B兼具染色质结构域绝缘子元件（chromatin domain insulator element）功能与启动子（promoter）功能，二者还可能通过持续结合有丝分裂染色体（mitotic chromosomes）发挥表观遗传记忆（epigenetic memory）作用。两种蛋白由同一基因编码，仅在N端（N-terminal）80个氨基酸序列存在差异，该区域包含单个DNA结合BED指结构域（DNA-binding BED fingers）；其余200个氨基酸序列在两种蛋白中完全一致。其中间120个氨基酸区域的结构与功能目前尚不明确，而C端（C-terminal）80个氨基酸区域含有推定的亮氨酸拉链（leucine zipper）与BESS结构域（BESS domain），可介导BEAF蛋白间的相互作用。本研究进一步对BEAF开展了表征分析，证实仅BESS结构域即可介导BEAF蛋白间的相互作用，但若至少一个蛋白带有推定的亮氨酸拉链，则可增强该相互作用。BEAF-32B单独即可挽救果蝇的BEAF基因无效突变，通过突变型BEAF-32B挽救转基因载体，我们证实中间区域与BESS结构域是功能必需的；相比之下，中间区域最后40个在果蝇物种间保守性较差的氨基酸残基则是非必需的。删除推定的亮氨酸拉链会得到低活性的BEAF-32B突变蛋白。最后，我们记录了胚胎有丝分裂过程中BEAF-32A-增强型绿色荧光蛋白（Enhanced Green Fluorescent Protein，EGFP）与BEAF-32B-单体红色荧光蛋白（monomeric Red Fluorescent Protein，mRFP）的动态变化。两种蛋白均有一个亚群似乎仍结合在有丝分裂染色体与有丝分裂纺锤体（mitotic spindle）上，而大部分荧光信号在有丝分裂过程中发生弥散。在合胞体胚胎（syncytial embryos）中可观察到两种蛋白的动态差异，且两种蛋白在合胞体胚胎与晚期胚胎中均表现出特性差异。本次BEAF的表征分析为后续解析BEAF功能的分子机制奠定了基础。