Chromatin immunoprecipitation in early Xenopus laevis embryos

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful method for analyzing the interaction of regulatory proteins with genomic loci, but has been difficult to apply to studies on early embryos due to the limiting amount of genomic material in these samples. Here, we present a comprehensive technique for performing ChIP on blastula and gastrula stage Xenopus embryos. We also describe methods for optimizing crosslinking and chromatin shearing, verifying antibody specificity, maximizing PCR sensitivity, and quantifying PCR results, allowing for the use of as few as 50 early blastula stage embryos (approximately 5x10(4) cells) per experimental condition. Finally, we demonstrate the predicted binding of endogenous beta-catenin to the nodal-related 6 promoter, binding of tagged Fast-1/FoxH1 to the goosecoid promoter, and binding of tagged Tcf3 to the siamois and nodal-related 6 promoters as examples of the potential application of ChIP to embryological investigations. Developmental Dynamics 238:1422-1432, 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1. Crosslinking and Sonication Optimization

(A) Minimally-sonicated, gastrula stage (stage 10) DNA from a 1% formaldehyde/1×PBS time course was resolved by 2% agarose gel electrophoresis. Onset of crosslinking is observed by the recovery of sonication-resistant, high molecular weight DNA (>1kb). (B) 60-minute crosslinked chromatin from gastrula stage embryos was fully sonicated for one to four rounds of 20 seconds each and resolved by 1.2% agarose gel electrophoresis. Three rounds of sonication balances optimal yield (90%) with maximal shearing (<1kb) of the crosslinked chromatin.

Figure 2. Chromatin immunoprecipitation in blastula-stage Xenopus embryos

Schematic representations of the Xnr6a and Xnr1 genomic loci (A) demonstrate the locations of predicted Tcf/Lef consensus sequences relative to the ChIP PCR amplicons. (B) These primer sets amplify standard genomic DNA by PCR with similar efficiencies, with a limit of detection at approximately 30 haploid genomes. (C) Blastula-stage embryos (Stage 9) were processed for ChIP using either anti-acetylated histone H3, or anti-β-catenin antisera (with the corresponding negative controls). The PCR products were visualized by 3% agarose gel electrophoresis and ethidium bromide staining. Co-immunoprecipitation of associated genomic DNA is observed when the PCR signal is greater for the experimental IP (AcH3, β-catenin) than in the control IP (IgG, Serum). Note that the Xnr1 (-221) product occasionally amplifies as a doublet (seen in panel C): this represents genetic variation at this locus within our colony.

Figure 3. Controls for antibody specificity

(A) The specificity of the β-catenin antiserum was confirmed by performing ChIP on blastula-stage (stage 9) embryos and competing with an excess of immunizing peptide (lane 2: pre-incubation of antibody with peptide; lane 3, addition of peptide directly to sheared chromatin sample). Following IP, samples were processed as described in the text and separated by 8% SDS-PAGE. Immunoprecipitated β-catenin was analyzed by a standard western blot using the β-catenin antiserum. (B) Peptide competition of the β-catenin ChIP and (C) knockdown of β-catenin by morpholino injection (20ng/cell at the 2-cell stage) confirms the specificity of the interaction between β-catenin and the Xnr6 promoter. The competitions in (B) and (C) were performed by pre-incubation of the peptide with the antiserum for 1 hour before addition to the sheared chromatin sample.

Figure 4. Quantitative PCR analysis of ChIP

Quantitative PCR for the Goosecoid promoter (gray bars) and Ef1α (white bars) normalized to uninjected embryo control (A) or quantified as a percentage of input DNA (B). Graphs represent average relative quantification for four independent experiments. An average of 45 one-cell embryos were injected with myc-Fast1 (250pg) alone or in combination with Xnr1 (50pg) and harvested at gastrula stage (stage 10.5) for ChIP analysis according to this protocol. QPCR was performed using SYBR green and relative quantification was performed using the ΔΔC(t) method. Error bars shown represent standard error.

Figure 5. Effect of crosslinking times on ChIP

Anti-Myc-Tag ChIP was performed on sets of 37 gastrula stage (stage 10), non-injected or Myc-Tcf3 injected (50pg) embryos, which were crosslinked for 15, 30, 45, or 60 minutes, as indicated. Samples were analyzed by QPCR for enrichment of Xmlc2 (-118), Xnr6 (-118), and Siamois (-303), represented as % Input recovery. Crosslinking times between 45 and 60 minutes are required for the specific enrichment of the promoters for both Siamois (black bars) and Xnr6 (hatched bars) by Myc-Tcf3 ChIP compared to the Xmlc2 promoter (gray bars). All non-injected (negative control) ChIPs yielded a comparatively low signal and are represented as white bars. Recovery of input material was similar for each time point.