Effective blocking of the white enhancer requires cooperation between two main mechanisms suggested for the insulator function.

Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the eye-specific enhancer. The results showed that one copy of the gypsy or Fab-7 insulator failed to block the eye enhancer in most of genomic sites, whereas a chromatin loop formed by two gypsy insulators flanking either the eye enhancer or the reporter completely blocked white stimulation by the enhancer. However, strong enhancer blocking was achieved due not only to chromatin loop formation but also to the direct interaction of the gypsy insulator with the eye enhancer, which was confirmed by the 3C assay. In particular, it was observed that Mod(mdg4)-67.2, a component of the gypsy insulator, interacted with the Zeste protein, which is critical for the eye enhancer-white promoter communication. These results suggest that efficient enhancer blocking depends on the combination of two factors: chromatin loop formation by paired insulators, which generates physical constraints for enhancer-promoter communication, and the direct interaction of proteins recruited to an insulator and to the enhancer-promoter pair.

Functional role of dimerization and CP190 interacting domains of CTCF protein in Drosophila melanogaster.

BACKGROUND: Insulators play a central role in gene regulation, chromosomal architecture and genome function in higher eukaryotes. To learn more about how insulators carry out their diverse functions, we have begun an analysis of the Drosophila CTCF (dCTCF). CTCF is one of the few insulator proteins known to be conserved from flies to man. RESULTS: In the studies reported here we have focused on the identification and characterization of two dCTCF protein interaction modules. The first mediates dCTCF multimerization, while the second mediates dCTCF-CP190 interactions. The multimerization domain maps in the N-terminus of the dCTCF protein and likely mediates the formation of tetrameric complexes. The CP190 interaction module encompasses a sequence ~200 amino acids long that spans the C-terminal and mediates interactions with the N-terminal BTB domain of the CP190 protein. Transgene rescue experiments showed that a dCTCF protein lacking sequences critical for CP190 interactions was almost as effective as wild type in rescuing the phenotypic effects of a dCTCF null allele. The mutation did, however, affect CP190 recruitment to specific Drosophila insulator elements and had a modest effect on dCTCF chromatin association. A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the null and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies. Finally, we show that elimination of maternally contributed dCTCF at the onset of embryogenesis has quite different effects on development and Abd-B regulation than is observed when the homozygous mutant animals develop in the presence of maternally derived dCTCF activity. CONCLUSIONS: Our results indicate that dCTCF-CP190 interactions are less critical for the in vivo functions of the dCTCF protein than the N-terminal dCTCF-dCTCF interaction domain. We also show that the phenotypic consequences of dCTCF mutations differ depending upon when and how dCTCF activity is lost.