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A robust chromatin immunoprecipitation protocol for studying transcription factor–DNA interactions and histone modifications in wood-forming tissue

Nature Protocols volume 9, pages 2180–2193 (2014)Cite this article

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Abstract

Woody cells and tissues are recalcitrant to standard chromatin immunoprecipitation (ChIP) procedures. However, we recently successfully implemented ChIP in wood-forming tissue of the model woody plant Populus trichocarpa. Here we provide the detailed ChIP protocol optimized for wood-forming tissue that we used in those studies. By using stem-differentiating xylem (SDX; a wood-forming tissue), we identified all steps that were ineffective in standard ChIP protocols and systematically modified them to develop and optimize a robust ChIP protocol. The protocol includes tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis. The protocol takes 2.5 d to complete and allows a robust 8–10-fold enrichment of transcription factor (TF)–bound genomic fragments (∼150 ng/g of SDX) over nonspecific DNAs. The enriched DNAs are of high quality and can be used for subsequent PCR and DNA-seq analyses. We used this protocol to identify genome-wide specific TF-DNA interactions during wood formation and histone modifications associated with regulation of wood formation. Our protocol, which may be suitable for many tissue types, is so far the only working ChIP system for wood-forming tissue.

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Figure 1: Cell types in the stem of P. trichocarpa and the collection of wood-forming tissue.
Figure 2: Key steps involved in the woody tissue–optimized ChIP protocol.
Figure 3: Western blot to determine the specificity of the antibody used in our ChIP assay7.
Figure 4: An example of electropherogram analysis of P. trichocarpa SDX chromatin after sonication7.
Figure 5: ChIP-PCR of TF-DNA interactions in P. trichocarpa wood-forming tissue7.
Figure 6: ChIP-qPCR of histone modification in P. trichocarpa wood-forming tissue.

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Acknowledgements

This work was supported by the Office of Science (Biological and Environmental Research), US Department of Energy Grant DE-SC000691 (to V.L.C.). We also thank the support of the North Carolina State University Jordan Family Distinguished Professor Endowment.

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Authors and Affiliations

  1. State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China

    Wei Li, Ying-Chung Lin, Quanzi Li, Guan-Zheng Qu & Vincent L Chiang

  2. Department of Forestry & Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA

    Wei Li, Ying-Chung Lin, Quanzi Li, Rui Shi, Chien-Yuan Lin, Hao Chen, Ling Chuang, Ronald R Sederoff & Vincent L Chiang

  3. College of Forestry, Shandong Agricultural University, Taian, Shandong, China

    Quanzi Li

  4. Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina, USA

    Vincent L Chiang

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Contributions

W.L. and Y.-C.L. designed and performed experiments, analyzed data and wrote the paper; Q.L. designed the experiments and antibodies, and edited the manuscript; H.C. validated the specificity of antibodies; R.S., C.-Y.L. and L.C. performed the imaging experiments; R.R.S. and G.-Z.Q. analyzed the data and edited the manuscript; V.L.C. supervised and designed the experiments, analyzed the data and wrote the paper.

Corresponding author

Correspondence to Vincent L Chiang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Optimization of the cross-linking time.

5 g freshly collected SDX were merged into Crosslinking Buffer, and crosslinking performed by vacuum (5 min)/release/mix at room temperature, three times (15 min), six times (30 min) or eight times (40 min). A positive direct target PtrMYB021 of PtrSND1-B16,7 was used. PtrACTIN was used as a negative control. The binding of PtrSND1-B1 to the promoter of PtrMYB021 or PtrACTIN was determined by ChIP using 5 μl of anti-PtrSND1-B1 antibodies followed by detection using ChIP-PCR. Vacuum (5 min)/release/mix at room temperature, six times (30 min) was determined to be optimal. Input, Mock and Anti-B1 are PCR reactions using the chromatin preparations before immunoprecipitation, immunoprecipitated with preimmune serum and immunoprecipitated with anti-PtrSND1-B1 antibody, respectively. Three independent biological replicates of ChIP assays were performed, and the results of one biological replicate are shown. The primers used are described in ref. 7.

Supplementary Figure 2 Microscopic image of isolated nuclei.

Examples of P. trichocarpa SDX nuclei stained with DAPI before adding the nuclei lysis buffer. The scale bar is equivalent to 100 μm.

Supplementary information

Supplementary Figure 1

Optimization of the cross-linking time. (PDF 123 kb)

Supplementary Figure 2

Microscopic image of isolated nuclei. (PDF 116 kb)

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Li, W., Lin, YC., Li, Q. et al. A robust chromatin immunoprecipitation protocol for studying transcription factor–DNA interactions and histone modifications in wood-forming tissue. Nat Protoc 9, 2180–2193 (2014). https://doi.org/10.1038/nprot.2014.146

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