1 2 3 4 5 7 8of Drosophila melanogaster. Interestingly, genes embedded in pericentric heterochromatin of D. melanogaster may occupy different genomic loci, euchromatic or heterochromatic, due to repositioning in the course of evolution of Drosophila species. By surveying factors that contribute to the normal functioning of the relocated genes in distant Drosophila species, i.e. D. melanogaster and D. virilis, we identify certain insulator proteins (e.g.BEAF-32) that facilitate the expression of heterochromatic genes in spite of the repressive environment.Despite the repressive environment, dozens of essential genes were identified in the pericentric heterochromatin of D. melanogaster (13)(14)(15)(16). Interestingly, genes embedded in pericentric heterochromatin in D. melanogaster may occupy distinct genomic loci, euchromatic and heterochromatic, in other Drosophila species (17). For instance, two adjacent genes RpL15 and Dbp80 located in the pericentric region of chromosome 3L in D. melanogaster reside in a euchromatic region in D. pseudoobscura (18). A similar pattern was demonstrated for genes light and Yeti located in pericentric regions in D. melanogaster, while in D. virilis they are found within euchromatin on the same chromosomal elements (19,20). Recently, it was shown that most of the pericentric genes found at both arms of chromosome 2 of D. melanogaster are located in euchromatic loci in the D. virilis genome (21). However, although repositioning of genes between euchromatin and heterochromatin during genome evolution is not unusual in the Drosophilidae lineage, the "immunity" of heterochromatic genes to the transcriptionally repressive environment remains paradoxical and unexplained. Thus, it is not clear whether these loci have undergone adaptation to heterochromatic environment or had some intrinsic properties permitting local adaptation.Previously, it was shown that molecular organization of promoter regions is largely conserved between heterochromatic and euchromatic genes, indicating that adaptation to heterochromatin probably does not require major changes in regulatory sequences (20). However, expression of heterochromatic genes requires the methylated H3K9 mark (22,23), and the ability to repositioning during evolution is a characteristic feature of gene clusters that show close association with HP1a protein (21).Chromatin insulator elements and associated proteins were originally defined by their ability to protect transgenes from PEV, exerting a block to cis spreading of a chromatin state (24, 25). To date, a set of insulators have been identified in Drosophila, including BEAF-32 (Boundary element associated factor of 32 kDa), dCTCF (Drosophila homolog of CTCF), Su(Hw) (Suppressor of hairy wing), Zw5 (Zeste-white-5), GAF (GAGA factor) and recently expression of heterochromatic genes and make them predisposed for evolutionary repositioning into transcriptionally repressive genomic environments. Results Evolutionary repositioning of Myb and Ranbp16 genes between euchromatin and heterochromatin...