Advancing Epigenetics Towards Systems Biology

Chromatin Immunoprecipitation Assay for Early Zebrafish Embryos (Prot 59)

Leif C. Lindeman1, Philippe Collas1

Introduction

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 (Prot 59) 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).

A widely used method for identifying occupancy of the genome by modified histones is chromatin immunoprecipitation (ChIP). However, a challenge with ChIP assays from early-stage zebrafish embryos is access to chromatin, due to a thick glycoprotein chorion which protects the embryo, the large amount of yolk (which supports embryo development) and the low nuclearcytoplasmic ratio in embryonic cells particularly prior to the MBT. In addition, we have found that the use of protease (pronase) to remove the chorion in order to isolate embryonic cells and prepare chromatin, as carried out in earlier protocols (Hart et al., 2007; Havis et al., 2006; Vastenhouw et al., 2010; Wardle et al., 2006), is detrimental to the efficiency of ChIP (Lindeman et al., 2009), notably by resulting in the degradation in modified histone epitopes such as H3K27me3. We have alleviated the use of protease to isolate embryonic cells, and present here our ChIP protocol for early stage zebrafish embryos (Lindeman et al., 2009). This protocol enables the examination of modified histones by quantitative PCR (ChIP-qPCR) or by hybridization of ChIP DNA to microarrays (ChIP-chip) as early as the 256-cell stage (Lindeman et al., 2011).

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Leif C. Lindeman1, Philippe Collas1

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
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Leif C. Lindeman