SpDamID: Marking DNA Bound by Protein Complexes Identifies Notch-Dimer Responsive Enhancers [array]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Raphael Kopan
PROVIDER: E-GEOD-70388 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Development of an alternative method to ChIP for the identification of DNA bound by transcriptional complexes assayed using Nimblegen arrays
2015-08-01 | GSE70388 | GEO
Project description:Development of an alternative method to ChIP for the identification of DNA bound by transcriptional complexes assayed using next-generation sequencing Next-generation sequencing data from sites identified by different Notch complexes using SpDamID-seq and compared against FAIRE and ChIP data
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Project description:Development of an alternative method to ChIP for the identification of DNA bound by transcriptional complexes assayed using next-generation sequencing
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Project description:We performed a genome-scale ChIP-chip comparison of two modifications (trimethylation of lysine 9 and trimethylation of lysine 27) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the PRC2 complex is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the PRC2 complex, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 co-repressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5kb promoter arrays. We found that a large number of promoters of zinc finger transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38 array tiling set, identifying ~7000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 zinc fingers, especially those containing KRAB domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1. Keywords: Human whole genome tiling array Amplicons were applied either to ENCODE arrays, 5 kb promoter arrays, or to the human genome tiling array set consisting of 38 arrays (see www.nimblegen.com for details). The labeling and hybridization of DNA samples for ChIP-chip analysis was performed by NimbleGen Systems, Inc, except that ENCODE arrays were hybridized at UC Davis. Briefly, each DNA sample (1 μg) was denatured in the presence of 5'-Cy3- or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water. Then, 13ug of the Cy5-labeled ChIP sample and 13ug of the Cy3- labeled total sample were mixed, dried down, and resuspended in 40 μl of NimbleGen Hybridization Buffer (NimbleGen Systems) plus 1.5 ug of human COT1 DNA. After denaturation, hybridization was carried out in a MAUI Hybridization System (BioMicro Systems) for 18 h at 42°C. The arrays were washed using NimbleGen Wash Buffer System (NimbleGen Systems), dried by centrifugation, and scanned at 5-μm resolution using the GenePix 4000B scanner (Axon Instruments). Fluorescence intensity raw data were obtained from scanned images of the oligonucleotide tiling arrays using NIMBLESCAN 2.0 extraction software (NimbleGen Systems). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. Then, the biweight mean of this log2 ratio was subtracted from each point; this procedure is approximately equivalent to mean-normalization of each channel. Total RNA was prepared from 5x106 Ntera cells using RNAeasy Kit (Qiagen) following the manufacturer’s instructions. RNA quality was ensured using the Agilent Systems Bioanalyzer. The RNA was hybridized to human whole genome expression microarrays from Nimblegen, which contain probes for every human gene based on genome build HG18 from the UCSC database (more details at http://www.nimblegen.com). 10 ug total RNA were used to synthesize cDNA using the SuperScript Double-Stranded cDNA synthesis kit (Invitrogen). The labeling of RNA samples, array hybridization and preliminary RNA expression analysis (data normalization) was performed by NimbleGen Systems, Inc.
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Project description:The oil palm fruit abnormality, mantled, is a somaclonal variant arising from tissue culture that drastically reduces yield, and has largely halted efforts to clone elite hybrids for breeding and oil production. Widely regarded as epigenetic, mantling had defied explanation, and has become an icon of unsustainability in environmentally sensitive tropical plantation crops. We identified the MANTLED gene using Epigenome Wide Association analysis of genetically identical palms from multiple clonal lineages. Hypomethylation of a LINE retrotransposon related to rice Karma, found in the intron of the homeotic gene DEFICIENS, is common to all mantled clones and is correlated with alternative splicing and loss of small RNA. DNA methylation is regained in spontaneous revertants accounting for non-Medelian inheritance of the Good Karma and Bad Karma epialleles. Thus epigenetic regulation of transposable elements results in somaclonal variation and provides a means to cull mantled nursery palms before committing limiting plantation resources to clonal propagation. DNA methylation profiling was performed using the McrBC DNA methylation dependent fractionation and microarray hybridization method as described in Lippman, Z., Gendrel, A. V., Colot, V. & Martienssen, R. Profiling DNA methylation patterns using genomic tiling microarrays. Nat Methods 2, 219-224 (2005). DNA methylation profiling was performed on 54 parthenocarpic mantled ramet, 43 normal ramet and 1 ortet adult leaf samples. For each sample, two independent fractionations of total genomic DNA and DNA methylation-depleted DNA were performed. Each total DNA/methylation-depleted DNA pair was differentially labeled and hydrized to a custon Nimblegen microarray in a dye-swapped design (4 array hybridizations per sample).
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Project description:Early zebrafish embryo development proceeds first from a maternally transcribed and stored mRNAs, and zygotic gene activation (ZGA) is initiated at the mid-blastula transition (MBT; 1000-cell stage), 3.3 h post-fertilization. Very little is known on how the zygotic genome is programmed for transcriptional activation at the MBT. To start addressing this issue, we have mapped by ChIP-chip genome-wide promoter histone methylation (H3K4me3, H3K9me3, H3K27me3, H3K36me3) and RNA Pol II profiles before ZGA (256-cell stage; 2.5 hpf), during ZGA (MBT; 3.5 hpf)) and after ZGA (Post-MBT; 5.3 hpf) . We used a custom 2.1M probe HD promoter array (Nimblegen) for ChIP and input DNA hybridization. Peak detection was done using MA2C with P=10e-4 as cutoff. ChIP-chip experiments were performed from chromatin prepared by sonication after formaldehyde cross-linking, from embryos are the indicated developmental stages and ChIP DNA was hybridized onto the aforementioned Nimbegen promoter arrays.
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Project description:We developed Split DamID (SpDamID), a protein complementation version of DamID, to mark genomic DNA bound in vivo by interacting or juxtapositioned transcription factors. Inactive halves of DAM (DNA adenine methyltransferase) were fused to protein pairs to be queried. Either direct interaction between proteins or proximity enabled DAM reconstitution and methylation of adenine in GATC. Inducible SpDamID was used to analyze Notch-mediated transcriptional activation. We demonstrate that Notch complexes label RBP sites broadly across the genome and show that a subset of these complexes that recruit MAML and p300 undergo changes in chromatin accessibility in response to Notch signaling. SpDamID differentiates between monomeric and dimeric binding, thereby allowing for identification of half-site motifs used by Notch dimers. Motif enrichment of Notch enhancers coupled with SpDamID reveals co-targeting of regulatory sequences by Notch and Runx1. SpDamID represents a sensitive and powerful tool that enables dynamic analysis of combinatorial protein-DNA transactions at a genome-wide level.
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