Function and mechanism of chromatin boundaries in Drosophila Hox gene complexes
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Chromatin boundaries or insulators partition the genome into structurally and functionally autonomous domains. The mechanism of boundary function is unclear. Based on the observations that the gypsy retrotransposon, which contains the su(Hw) insulator, and the su(Hw) insulator proteins, SUHW and MOD(MDG4), cluster near the nuclear periphery, it was proposed that the function of su(Hw) depends on its localization to the nuclear periphery. Using fluorescent in situ hybridization (FISH), we show that transgenes containing the su(Hw) insulator do not preferentially localize near the nuclear periphery. Immunostainings show that SUHW and MOD(MDG4) are not restricted to the nuclear periphery. Additionally, the enhancer-blocking activity of suHw remains intact during heat shock, which disrupts the cluster of insulator proteins at the nuclear periphery. Our data show that su(Hw) can function in the nuclear interior, possibly through interactions with other insulators or nuclear structures. To understand how endogenous insulators regulate gene expression, I studied the SF-1 insulator in the Antennapedia complex (ANT-C). Using a cell-based insulator assay, we show that Ebx/NURF301 and Iswi modulate the activity of multiple Drosophila insulators including SF-1, su(Hw), Fab-7 and Fab-8. Reduced levels of Ebx/NURF301 or Iswi disrupts the enhancer-blocking activity of SF1 and su(Hw) in transgenic embryos. Our findings provide the first evidence that a nucleosome-remodeling complex participates in the enhancer-blocking mechanism of insulators. To gain insights into the role of insulators in Hox gene regulation, we studied the genomic organization of Drosophila Hox complexes using Chromosome Conformation Capture (3C) and FISH. First, we find that SF-1 interacts with multiple regions in ANT-C, and show that these regions contain novel insulators. Second, we find that the lab and Antp gene in ANT-C are closely associated with each other inside the nucleus despite the large linear distance. Third, we find an interaction between promoter regions of Antp in ANT-C and Abd-B in BX-C. This interaction occurs in posterior tissues, where both genes are expressed, or in tissues where both genes are ectopically activated due to mutations in the Polycomb group proteins. This is the first documented example of a developmentally regulated, long-range physical interaction between Hox clusters.