CTCF is essential for insulator function in vertebrates, where it plays an active role in regulating imprinted gene expression. In Drosophila, dCTCF has likewise been shown to be involved in the insulator function of boundary elements [28, 37]. Our results show that parent-specific expression from an imprinted domain in Drosophila is dependent on dCTCF function. Maintenance of expression from maternally inherited Dp(1;f)LJ9 mini-X chromosome is highly sensitive to dCTCF; even a modest decrease in dCTCF mRNA alters the maternal imprint so that it resembles the paternal imprint.
The effect of dCTCF on maternal-specific expression is limited to the maintenance of imprint. The presence of mutant dCTCF in either the maternal or paternal parents, when the imprint is being established, does not affect the imprint in the progeny. These results are strikingly similar to the role of CTCF in mammalian imprinting, where CTCF assists in the postfertilization formation of an imprinted region, but is dispensable for the establishment of an imprint [11, 12, 38].
Furthermore, the requirement for dCTCF for maintenance of the maternal Dp(1;f)LJ9 imprint is specific and does not represent a ubiquitous role for dCTCF in regulating heterochromatic silencing. Not only is the paternal Dp(1;f)LJ9 imprint unaffected by mutant dCTCF, but other variegating Drosophila reporter genes respond differently to mutant dCTCF. Thus, the association of dCTCF expression with the maintenance of the maternal Dp(1;f)LJ9 imprint boundary demonstrates a distinct function for dCTCF in imprinted gene expression.
In mammals, maternally imprinted regions that bind CTCF rely critically on this binding to insulate the imprinted loci and establish distinct chromatin domains. Our results show that a reduction in dCTCF levels disrupts the maternal imprint boundary on the Drosophila Dp(1;f)LJ9 mini-X chromosome, and consequently the marker gene, garnet, is silenced. Variegated silencing of garnet from Dp(1;f)LJ9
PAT inheritance is a consequence of heterochromatin formation, nucleated from the paternal imprint control region, spreading in cis [29, 32]. The absence of an effect upon the introduction of dCTCF mutant alleles to Dp(1;f)LJ9
PAT suggests that dCTCF binding and boundary function occurs only on the maternal chromosome. Thus, it is conceivable that a reduction in dCTCF levels enables the spreading of heterochromatin on the maternal Dp(1;f)LJ9
MAT in a manner similar to that of the paternal Dp(1;f)LJ9
PAT. This would suggest that dCTCF defines the boundary of a distinct maternal-specific imprinted chromatin domain required to maintain maternal-specific gene expression on the X chromosome.
The model organism Encyclopedia of DNA Elements (modENCODE) project provides detailed mapping of regulatory elements throughout the Drosophila genome . Large-scale profiling of dCTCF insulator sites from early embryo modENCODE data reveals several candidate dCTCF insulator sites present proximal to the predicted heterochromatic breakpoint of the Dp(1;f)LJ9 mini-X chromosome. These dCTCF insulator sites, located between the centric heterochromatic imprinting center and the imprint marker gene garnet, could account for the sensitivity of the maternal imprint to dCTCF expression. If dCTCF were bound only when the X chromosome was transmitted maternally, mutations to dCTCF would disrupt insulator function and lead to maternal silencing of the imprint marker gene. Although such binding remains to be tested, it is similar to the function of CTCF at mammalian imprinted regions.
That the structure of CTCF and its role as an insulator, barrier, and transcriptional regulator is conserved between mammals and insects have been well established [23, 27, 28]. However, the finding that CTCF maintains its function in regulating the imprinting of diverse genes in such phylogenetically distinct organisms is remarkable. CTCF is a versatile DNA binding factor; subsets of its zinc fingers are adept at binding diverse DNA sequences, and the rest of the protein is able to maintain common regulator interactions and insulator function . This feature may explain how CTCF can regulate imprinting in organisms as diverse as insects and mammals, in which the imprinted target sequences are different.
Previously, the evolutionary origin of imprinting has been extrapolated from the conservation of imprinting among specific genes. Such studies have led to the proposal that mammalian imprinting is of relatively recent origin and restricted to eutherian mammals [41, 42]. However, studies showing that the molecular mechanism of imprinting is highly conserved have suggested a much more ancient origin [7, 30, 43]. Mammalian imprint control elements inserted into transgenic Drosophila act as discrete silencing elements [44, 45] and can retain posttranscriptional silencing mechanisms involving noncoding RNA . Whereas these transgenic imprinting elements lose their parent-specific functions, the retention of epigenetic silencing mechanisms suggests an ancient and conserved origin of imprinting mechanisms. Our finding that CTCF has a role in the maintenance of maternal imprints in insects, as it does in mammals, supports the possibility of evolutionary conservation for both CTCF function and the mechanisms of genomic imprinting.