Zebrafish (Danio rerio) is well established as a model organism to study embryogenesis. Practical advantages of using zebrafish are that hundreds of synchronized embryos can easily be collected, embryos are transparent, development is rapid and external, and its genome is sequenced. The 9th assembly of the zebrafish genome (Zv9) reports 1.41 billion base pairs with ~24,000 protein-coding genes. Information on the Danio rerio genome assembly can be found at http://www.sanger.ac.uk/Projects/D_rerio/Zv9_assembly_information.shtml.
A unique feature of zebrafish (and of anamniote vertebrates) is a developmental period of several hours after fertilization in the quasi-absence of on-going transcription. In zebrafish, this developmental period lasts for 3.3 h during which the embryo undergoes 10 rounds of synchronous Chromatin Immunoprecipitation Assay for Early Zebrafish Embryos (Prot59) cell divisions. Zygotic genome activation (ZGA) occurs at the ~1,000-cell stage, at the mid-blastula transition (MBT) (Tadros and Lipshitz, 2009) (see http://www.neuro.uoregon.edu/k12/Table%201.html for a description of zebrafish developmental stages). This 3.3 h pre-MBT period provides a unique opportunity to identify epigenetic processes, including enrichment in post-translationally modified histones, associated with the establishment of the embryonic gene expression program (Lindeman et al., 2011). [...]
1 Stem Cell Epigenetics Laboratory (Collas lab), Institute of Basic Medical Sciences, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway
Corresponding author: Leif C. Lindeman
RNA polymerase II (RNAPII) is responsible for the transcription of protein-coding genes, in addition to a large number of non-coding RNAs. The C-terminal domain (CTD) of its largest subunit consists of multiple heptad repeats (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7; 52 copies in mammals) that are targeted for a wide range of post-translational modifications, providing a platform for interaction with chromatin modifiers and RNA processing machinery (Brookes and Pombo, 2009). Ser5 residues become phosphorylated during transcription initiation and Ser2 residues during productive transcription elongation. [...]
1 Genome Function Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.
2 Present address: Cancer Research Technology Discovery Laboratory, Wolfson Institute for Biomedical Research, The Cruciform Building, Gower Street, London, WC1E 6BT, UK.
Corresponding author: Ana Pombo, MRC Clinical Sciences Centre, Imperial College School of Medicine,Hammersmith Hospital Campus, Du Cane Road, London W12 0NN,UK.
The technique of chromatin immunoprecipitation (ChIP) has proven to be a powerful tool, allowing the detection of protein-DNA interactions in living cells. Hybridization of ChIP samples to DNA microarrays (i.e. the ChIP-chip assay) allows a global analysis of binding sites for transcription factors and components of the transcriptional machinery, as well as of chromatin modification patterns. However, a single ChIP sample does not yield enough DNA for hybridization to a genomic tiling array. Therefore, we have adapted the standard protocol for whole genome amplification using the Sigma GenomePlex WGA kit to amplify our ChIP sample (O'Geen et al., 2006). Using Oct4 ChIP-chip assays as an example, we have compared the quality of ChIP-chip data derived from 1) WGA amplified ChIP samples, 2) a pool of 10 ChIP samples without further amplification, and 3) linker-mediated PCR (LMPCR) amplification of ChIP samples. Based on the low background, reproducibility, and the fact that a single WGA amplified ChIP sample can provide sufficient material for several array hybridizations, we recommend the WGA protocol for ChIP-chip analyses. We have successfully tested our new ChIP amplification protocol on a variety of different factors (E2F family members, KAP1, CtBP2, ZNF217) as well as on histone modifications (H3me3K9, H3me3K27, H3me3K4) (Krig et al., 2007; O'Geen et al., 2007). Another benefit of the WGA amplification method is the ability to perform a second round of amplification from the initial WGA product if a higher DNA yield is required. We have applied the re-amplification protocol to KAP1 amplicons that were hybridized to a whole genome tiling array set consisting of 38 arrays (O'Geen et al., 2007). Detailed protocols for ChIP assays from mammalian cells and tissue samples, as well as preparation of amplicons can be found on the Farnham Lab website.
University of California Davis - UC Davis Genome Center - Genome and Biomedical Sciences Facility - 451 East Health Sciences Drive - Davis, CA 95616-8816, USA
Cross-linking Chromatin Immunoprecipitation (ChIP) has become a popular method to detect the in vivo binding of proteins to DNA. In general the protein/DNA complex is fixed with formaldehyde, a cross-linking agent that, because of its short spacer arm (about 2 A), generates reversible covalent links, mainly between proteins and DNA. Subsequently, chromatin is fragmented by sonication and DNA protein complexes are selectively immunoprecipitated with antibodies raised against proteins of interest. Covalent links are reversed and proteins are removed from DNA through phenol/chloroform extraction. Following DNA purification, specific DNA regions supposed to be engaged with proteins of interest are analyzed by semi-quantitative PCR or real time PCR. Although very powerful, this method may present some limits when applied to nuclear proteins that, although being tightly associated with chromatin DNA, are not directly bound to it. For example, recent studies have shown that c-Myc can repress transcription of the p21CIP/WAF gene without binding DNA directly but through interaction with different transcription factors such as Sp1 and Miz-1 that recognize specific DNA sequences in the p21CIP/WAF promoter (Gartel et al., 2001 and Wu e al., 2003). [...]
University of Bologna - Department of Biology - University of Bologna - Via Francesco Selmi 3 - 40126 Bologna, Italy
ChIP (chromatin immunoprecipitation) is a powerful tool that allows one to determine whether and where a protein or protein modification is associated with chromatin in vivo. The technique starts with a formaldehyde treatment of cells to crosslink protein-protein and protein-DNA complexes. After cross-linking, the cells are lysed and crude extracts are sonicated to shear the DNA. Proteins together with crosslinked DNA are immunoprecipitated. Protein-DNA crosslinks in the immunoprecipitate and input (non-immunoprecipitated whole cell extract) are then reversed and the DNA fragments are purified. Real-time quantitative PCR can then be used to amplify the region where either a protein or protein modification is present. DNA fragments of this genomic locus should be enriched in the immunoprecipitate compared to that in the input (which represents all portions of the genome). This protocol has been successfully used in the Gasser lab to study proteins at replication forks and chromosomal DNA double-strand breaks (DSBs) in budding yeast (Saccharomyces cerevisiae) (Cobb et al., 2003; van Attikum et al., 2004).
Friedrich Miescher Institute for Biomedical Research - Maulbeerstrasse 66 - 4058 Basel, Switzerland