Genomewide analysis of Insulator protein binding sites in Drosophila embryos at E0-12

Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP-chip the genome-wide binding sites of 6 insulator-associated proteins – dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF – to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all previously known insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw) respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters, divide differentially expressed, alternative, and divergent promoters, act as chromatin boundaries, are associated with chromosomal breakpoints among species, and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units, and a framework for understanding insulator function during the development and evolution of Drosophila. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf ChIP-chip experiments were performed in Triplicate on Affymetrix arrays and compared to Input samples for the following datasets: E-0-12H: CTCF-C, CTCF-N, CP-190, BEAF-32, Su(Hw)-1, Su(Hw)-2, Mod(mdg4), GAF, H3K4me3, H3K27me3 and PolII. In addition ChIP experiments in S2 cells and Kc cells have been performed for H3K4me3 and CTCF. In addition, total RNA extracts have been performed in Embryos 0-12h and hybridized on Affymetrix Tiling arrays.