Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Drosophila Ecdysone Receptor Study

ABSTRACT: We used the DamID method to systematically identify the binding sites of Ecdysone Receptor and its heterodimeric partner USP across the whole genome in Drosophila Kc cells. We find that the EcR sites are a subset of the USP sites and that only a proportion are ecdysone regulated from an accompanying ecdysone profiling study. The role of EcR/USP in the ecdysone network appears to be coordinated by the recruitment of many transcription factors as well as signaling molecules. Keywords: DamID, chromatin profiling, DNA microarray In this study we mapped the genomic binding sites of steroid hormone nuclear receptor EcR and USP in Kc167 cells using the DamID method. DamID involves the low level expression of a fusion protein consisting of DNA adenine methyltransferase (Dam) and a chromatin protein of interest. This fusion protein is targeted to the native binding sites of the chromatin protein, where Dam methylates adenines in the surrounding DNA. The methylated DNA fragments were isolated and amplified by selective PCR, labeled with a fluorescent dye and hybridized to whole genome tiling microarrays. In this study,experiments were done with samples obtained from independent experiments and include dye swaps.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Zareen Gauhar

PROVIDER: E-GEOD-9156 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genomic mapping of binding regions for the Ecdysone receptor protein complex.

Gauhar Zareen Z Sun Ling V LV Hua Sujun S Mason Christopher E CE Fuchs Florian F Li Tong-Ruei TR Boutros Michael M White Kevin P KP

Genome research 20090223 6

We determined the physical locations of the heterodimeric Ecdysone receptor/Ultraspiracle (ECR/USP) nuclear hormone receptor complex throughout the entire nonrepetitive genome of Drosophila melanogaster using a cell line (Kc167) that differentiates in response to 20-hydroxyecdysone (20-HE). 20-HE, the natural ligand of this complex, controls major aspects of insect development, including molting, metamorphosis, and reproduction. Direct gene targets of 20-HE signaling were identified by combining ...[more]

PMID: 19237466

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Project description:Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem-cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila Kc cells. We found that Pc associates with large genomic regions of up to ~150kb in size, hereafter referred to as âPc-domainsâ. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 lysine 27 and in general transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains. Keywords: DamID, Chromatin immunoprecipitation, ChIP-chip To study PcG binding profiles we used DamID, which is based on the ability of a chromatin protein fused to E.coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000).

Dataset Information

Drosophila Ecdysone Receptor Study

Publications

Genomic mapping of binding regions for the Ecdysone receptor protein complex.

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