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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin immunopreciptitation of Drosophila DNA using antibodies against RMA polymerase II CTD repeats

ABSTRACT: Crosslinked chromatin from wild type flies was sonicated into small fragments, and used in ChIP reactions using a monoclonal antibody, 8WG16, against RNA polymerase II CTD repeats. Input material was removed prior to chIP, and a control chIP was incubated with normal rabbit IgG. The Input and IPed DNA was purified, amplified, and hybridized to Affymetrix Drosophila genomic tiling arrays.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Cizhong Jiang

PROVIDER: E-MEXP-1520 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Nucleosome organization in the Drosophila genome.

Mavrich Travis N TN Jiang Cizhong C Ioshikhes Ilya P IP Li Xiaoyong X Venters Bryan J BJ Zanton Sara J SJ Tomsho Lynn P LP Qi Ji J Glaser Robert L RL Schuster Stephan C SC Gilmour David S DS Albert Istvan I Pugh B Franklin BF

Nature 20080413 7193

Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. Here we produce a high-resolution reference map of H2A.Z and bulk nucleosome locations across the genome of the fly Drosophila melanogaster and compare it to that from the yeast Saccharomyces cerevisiae. Like Saccharomyces, Drosophila nucleosomes are organized around active transcription start sites in a canonical -1, nucleosome-fre ...[more]

PMID: 18408708

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Project description:ChIP-chip was performed using chromatin isolated from formaldehyde crosslinked, staged embryos, and affinity purified antibodies against 21 transcription factors (including: BCD, HB, KR, KNI, GT, CAD, HKB, TLL, D, FTZ, HRY, PRD, RUN, SLP1, DA, DL, MAD, MED, SHN, SNA, TWI) that belong to 11 DNA binding domain families, specify distinct developmental fates; as well as the general transcription factor TFIIB, and RNA POLII. Affinity purification of the antibodies was performed with E.coli expressed His-tag fusions of the whole protein (designated as 3, e.g. anti-DL 3), the N-terminal portion of the full length protein (designiated as 1, e.g. anti-BCD 1), or the C-terminal portion of the protein (designated as 2, e.g. anti-BCD 2). While for most factors, we only used one antibody in the ChIP-chip study, for some factors, two or more antibodies that recognize non-overlapping parts of each of the proteins were used. The chromatin immunoprecitation and hybridization for each antibody were carried out in duplicates, and in each chromatin immunoprecitation and hybridization series, two mock IP controls and two input DNA controls were also generated. The microarray used in this study is the Affymetrix Drosophila genomic DNA tiling array, which has about 3 million oligo pairs (perfect match-mismatch), covering the whole euchromatic sequences of the fly genome at a resolution of about 35 bp.

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Project description:The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target-selection and gene activation in each context. To investigate, we partitioned Drosophila chromatin into different states, based on histone modifications, establishing the preferred chromatin conditions for binding of CSL, the Notch pathway transcription factor. While most histone modifications were unchanged by CSL binding or Notch activation, rapid changes in H3K56 acetylation occurred at Notch regulated-enhancers. This modification required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation are a conserved indicator of enhancer activation, also occurring at mammalian Notch-regulated Hey1 and at Drosophila ecdysone-regulated genes. This core histone modification may therefore underpin the changes in chromatin accessibility needed to promote transcription following signaling activation. H3K56ac profile of control cells (KP) and of NICD overexpression cells (KN). In total 6 samples, with 2 input files (from 2 different conditions) and 2 replicates of H3K56ac ChIP samples of each condition.

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