Abstract
Chromatin boundaries facilitate independent gene regulation by insulating genes from the effects of enhancers or organized chromatin. However, the mechanisms of boundary action are not well understood. To investigate whether boundary function depends on a higher order of chromatin organization, we examined the function of several Drosophila melanogaster insulators in cells with reduced chromatin-remodeling activities. We found that knockdown of NURF301 and ISWI, key components of the nucleosome-remodeling factor (NURF), synergistically disrupted the enhancer-blocking function of Fab7 and SF1 and augmented the function of Fab8. Mutations in Nurf301/Ebx and Iswi also affected the function of these boundaries in vivo. We further show that ISWI was localized on the endogenous Fab7 and Fab8 insulators and that NURF knockdown resulted in a marked increase in the nucleosome occupancy at these insulator sites. In contrast to the effect of NURF knockdown, reduction in dMi-2, the ATPase component of the Drosophila nucleosome-remodeling and deacetylation (NuRD) complex, augmented Fab7 and suppressed Fab8. Our results provide the first evidence that higher-order chromatin organization influences the enhancer-blocking activity of chromatin boundaries. In particular, the NURF and NuRD nucleosome-remodeling complexes may regulate Hox expression by modulating the function of boundaries in these complexes. The unique responses by different classes of boundaries to changes in the chromatin environment may be indicative of their distinct mechanisms of action, which may influence their placement in the genome and selection during evolution.