Fish’n ChIPs: Chromatin Immunoprecipitation in the Zebrafish Embryo

Abstract

Chromatin immunoprecipitation (ChIP) is arguably the assay of choice to determine the genomic localization of DNA- or chromatin-binding proteins, including post-translationally modified histones, in cells. The increasing importance of the zebrafish, Danio rerio, as a model organism in functional genomics has recently sparked investigations of ChIP-based genome-scale mapping of modified histones on promoters, and studies on the role of specific transcription factors in developmental processes. ChIP assays used in these studies are cumbersome and conventionally require relatively large number of embryos. To simplify the procedure and to be able to apply the ChIP assay to reduced number of embryos, we re-evaluated the protocol for preparation of embryonic chromatin destined to ChIP. We found that manual homogenization of embryos rather than protease treatment to remove the chorion enhances ChIP efficiency and quickens the assay. We also incorporated key steps from a recently published ChIP assay for small cell numbers. We report here a protocol for immunoprecipitation of modified histones from mid-term blastula zebrafish embryos.

LSD1/KDM1A promotes hematopoietic commitment of hemangioblasts through downregulation of Etv2

“…ChIP analysis was basically carried out as described previously (66,67) using specific antibodies: anti-monoMeK4H3 (ab8895; Abcam), anti-diMeK4H3 (UP07-030; Upstate), and anti-triMeK4H3 (ab8580; Abcam). Two hundred fifty embryos were used for each assay, and cells were sonicated by a Branson Sonifier 250.…”

Regulation of immunity and disease resistance by commensal microbes and chromatin modifications during zebrafish development

“…Embryos/larvae were processed for RNA extraction as described above or for ChIP using the MAGnify Chromatin Immunoprecipitation System (Life Technologies). Briefly, for each immunoprecipitation 200 embryos (30 hpf) or 80 larvae (72 hpf) were cross-linked with 1% formaldehyde for 8 min, and the chromatin was sheared by sonication to an average fragment size of 500-1,000 bp (77). Lysates were cleared by centrifugation and diluted 1:10 in the dilution buffer of the ChIP kit, and immunoprecipitation was performed with 2-4 μg of antibody.…”

“…The antibodies used in this assay were anti-H3 (ab1791; Abcam), anti-H3K4me3 (ab1012; Abcam), anti-H3K9ac (06-942; Millipore), and control mouse and rabbit anti-IgG provided by the MAGnify kit. All these antibodies have been validated in zebrafish previously (77), and the epitopes that they recognize are identical in zebrafish and mammals.…”

Chromatin states of developmentally-regulated genes revealed by DNA and histone methylation patterns in zebrafish embryos

“…Aiming at understanding epigenetic states during zebrafish development, we recently optimized a ChIP assay for zebrafish embryos, resulting in minimal chromatin loss and epitope degradation (Lindeman et al, 2009). We applied this ChIP assay to determine histone PTM enrichment profiles on the promoters of a subset of developmentally-regulated genes in embryos at the mid-late blastula transition (referred to as MBT + ), i.e.…”

“…Of note, these ChIP protocols relied on protease treatment of embryos to remove the chorion prior to isolating chromatin. By re-assessing each step of the ChIP assay, we recently reported that proteases are detrimental to ChIP efficiency in zebrafish embryos, and subsequently improved the procedure (Lindeman et al, 2009). …”