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Chromatin Immunoprecipitation: Optimization, Quantitative Analysis and Data Normalization

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Chromatin Immunoprecipitation: Optimization, Quantitative Analysis and Data Normalization

Max Haring et al. Plant Methods.

Abstract

Background: Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality.

Results: We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches.

Conclusion: Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization.

Figures

Figure 1
Figure 1
Outline of the ChIP-QPCR procedure. This outline represents the ChIP procedure as described in the text. IP, ChIP sample; NoAb, No-antibody control.
Figure 2
Figure 2
Crosslinking efficiency analysis. Leaf material was crosslinked in buffer containing increasing amounts of formaldehyde. Samples were decrosslinked (+DC) or not (-DC), and DNA isolated using phenol/chloroform extraction followed by ethanol precipitation. While DNA is efficiently isolated from samples that were not crosslinked (lanes indicated with 0×), decrosslinking is required for the isolation of DNA from crosslinked samples (lanes indicated with 1× and 3×). Over-crosslinking strongly decreases DNA isolation efficiency (lanes indicated with 3×). DC, decrosslinking; 0×, 1×, 3×, relative concentration of formaldehyde; M, lambda DNA cut with PstI.
Figure 3
Figure 3
Titration of chromatin relative to a fixed amount of antibody. (a) ChIP titration experiment using an antibody against acetylated H4. Dilution of the chromatin has no effect on the precipitation efficiency. (b) ChIP titration experiment using an antibody against an invariant domain of H3 (H3core). Dilution of the chromatin improves the precipitation efficiency, suggesting that the antibody recognizing the H3core is sensitive to inhibitory factors present in the chromatin sample. ChIP-QPCR was performed as described in this paper. Chromatin was isolated from leaves of 2 week-old plants, sonicated and used in 1:1, 1:2 and 1:4 dilutions for ChIP. The input samples are diluted by the same factors as the chromatin samples. Results are represented as %IP, the error bars indicate the standard error. ChIP samples are represented by closed bars, while open bars indicate the signals from the 'no-antibody' serum controls. Each data point represents the average of two different chromatin samples, each analyzed in duplo.
Figure 4
Figure 4
Outline for setting up QPCR. This outline represents setting up the QPCR analysis, as described in the text. Abbreviations: TF: transcription factor.
Figure 5
Figure 5
ChIP analysis of control sequences. (a) Location of amplicons used in ChIP-QPCR analysis. The boxed regions indicate part of the coding sequence of maize actin 1 (genbank #J01238) and the reverse transcriptase sequence of the maize copia TY1 type retrotransposon (genbank #AF398212). The amplified sequences are indicated by bars. (b) ChIP-QPCR analysis of the actin 5' untranslated leader (UTR). (c) ChIP-QPCR analysis of the actin exon 2 fragment. (d) ChIP-QPCR analysis of the copia sequence (copia). ChIP-QPCR is performed as described in this paper. Input chromatin was isolated from leaves of 4 weeks old plants (young leaves) and from husks of 3 months old plants (husk leaves). The ChIP results obtained by 4 independent replicate experiments are represented as percentage of input (%IP), the error bars indicate the standard error. The ChIP signals are represented by closed bars, and open bars indicate the signals from the no-antibody control (NoAb).

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