a robust chromatin immunoprecipitation protocol for studying transcription factor–dna interactions...

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© 2014 Nature America, Inc. All rights reserved. PROTOCOL 2180 | VOL.9 NO.9 | 2014 | NATURE PROTOCOLS INTRODUCTION Wood is the most widely used renewable feedstock for materials and energy 1–4 . The formation of wood involves differentiation of secondary xylem cells from the vascular cambium (VC), followed by cell-wall thickening 5 (Fig. 1). Similar to many com- plex growth and developmental processes, wood formation is controlled by genetic regulatory hierarchies formed by TF-DNA interactions 6,7 . Little is known about these interactions, and more knowledge is needed to better understand and improve wood formation to meet the escalating demand for materials and energy. TF-DNA interactions in vivo can be identified at the genome level by ChIP, which was first developed to probe specific DNA-protein interactions associated with chromatin structures in Drosophila 8 . In ChIP, an anti-TF antibody is used to enrich for chromatin that carries a TF and its interacting DNAs 8 . For plant species, ChIP has been applied mainly to Arabidopsis 9–11 . The ChIP technique has never been reported for any woody species, thus limiting our capability for understanding DNA (chromatin)-based developmental processes in these economi- cally important species. Here we describe a robust anti-TF antibody–based ChIP protocol that was systematically optimized for identifying TF-DNA interactions during wood formation, using SDX (Fig. 1g–i) of Populus trichocarpa 7 . We have also used this protocol for studying histone modifications associated with wood formation in P. trichocarpa. Overview of ChIP Although there are many procedural improvements in our proto- col compared with the other methods (see details in ‘Development of the protocol’), the underlying principles of ChIP remain the same. The key steps and time frame of our protocol are illustrated in Figure 2. Briefly, the SDX is readily obtained by scraping the tissue from the surface of a freshly debarked stem segment from a greenhouse- or field-grown P. trichocarpa (Fig. 1f–i). The SDX is first cross-linked to stabilize TF-DNA interactions before the nuclei are isolated and chromatin is extracted from the cross- linked SDX. The chromatin DNA is then sheared and an aliquot of the fragmented DNA is used as the input control. The remain- ing DNA fragments are divided into two equal fractions; one is used for immunoprecipitation with the anti-TF antibody (the ChIP sample; Fig. 2), and the other for immunoprecipitation with preimmune serum (the mock control; Fig. 2). The cross-linking of the TF-DNA complexes from input, mock and ChIP samples are then reversed and the DNA is purified. Purified DNA can then be analyzed by PCR to determine the enrichment of genomic regions of interest, or by sequencing to identify all direct targets at the genome level. Development of the protocol Initially, we carried out ChIP analysis on P. trichocarpa SDX using Arabidopsis procedures developed by us 9 or those by Kaufmann et al. 10 and others 11–13 , but we failed to enrich the target DNAs. All of these procedures gave a very low SDX nuclear or chromatin yield, leading to poor or no immunoprecipitation. Factors that impede nuclear isolation, chromatin extraction and immunopre- cipitation in ChIP for woody tissues or cells were unknown, so we systematically identified factors adversely affecting these proc- esses. We found that thick-walled cells and high levels of phenolics and polysaccharides in differentiating xylem (DX) 5,14 are major factors that adversely affect many key steps in ChIP protocols optimized for tissues or cells of non-woody plants 9–13 (Fig. 2). We then tested and modified conditions for specific processes A robust chromatin immunoprecipitation protocol for studying transcription factor–DNA interactions and histone modifications in wood-forming tissue Wei Li 1,2 , Ying-Chung Lin 1,2 , Quanzi Li 1–3 , Rui Shi 2 , Chien-Yuan Lin 2 , Hao Chen 2 , Ling Chuang 2 , Guan-Zheng Qu 1 , Ronald R Sederoff 2 & Vincent L Chiang 1,2,4 1 State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. 2 Forest Biotechnology Group, Department of Forestry & Environmental Resources, North Carolina State University, Raleigh, North Carolina, USA. 3 College of Forestry, Shandong Agricultural University, Taian, Shandong, China. 4 Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina, USA. Correspondence should be addressed to V.L.C. ([email protected]). Published online 21 August 2014; doi:10.1038/nprot.2014.146 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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2180 | VOL.9 NO.9 | 2014 | nature protocols

IntroDuctIonWood is the most widely used renewable feedstock for materials and energy1–4. The formation of wood involves differentiation of secondary xylem cells from the vascular cambium (VC), followed by cell-wall thickening5 (Fig. 1). Similar to many com-plex growth and developmental processes, wood formation is controlled by genetic regulatory hierarchies formed by TF-DNA interactions6,7. Little is known about these interactions, and more knowledge is needed to better understand and improve wood formation to meet the escalating demand for materials and energy. TF-DNA interactions in vivo can be identified at the genome level by ChIP, which was first developed to probe specific DNA-protein interactions associated with chromatin structures in Drosophila8. In ChIP, an anti-TF antibody is used to enrich for chromatin that carries a TF and its interacting DNAs8. For plant species, ChIP has been applied mainly to Arabidopsis9–11. The ChIP technique has never been reported for any woody species, thus limiting our capability for understanding DNA (chromatin)-based developmental processes in these economi-cally important species.

Here we describe a robust anti-TF antibody–based ChIP protocol that was systematically optimized for identifying TF-DNA interactions during wood formation, using SDX (Fig. 1g–i) of Populus trichocarpa7. We have also used this protocol for studying histone modifications associated with wood formation in P. trichocarpa.

Overview of ChIPAlthough there are many procedural improvements in our proto-col compared with the other methods (see details in ‘Development of the protocol’), the underlying principles of ChIP remain the same. The key steps and time frame of our protocol are illustrated

in Figure 2. Briefly, the SDX is readily obtained by scraping the tissue from the surface of a freshly debarked stem segment from a greenhouse- or field-grown P. trichocarpa (Fig. 1f–i). The SDX is first cross-linked to stabilize TF-DNA interactions before the nuclei are isolated and chromatin is extracted from the cross-linked SDX. The chromatin DNA is then sheared and an aliquot of the fragmented DNA is used as the input control. The remain-ing DNA fragments are divided into two equal fractions; one is used for immunoprecipitation with the anti-TF antibody (the ChIP sample; Fig. 2), and the other for immunoprecipitation with preimmune serum (the mock control; Fig. 2). The cross-linking of the TF-DNA complexes from input, mock and ChIP samples are then reversed and the DNA is purified. Purified DNA can then be analyzed by PCR to determine the enrichment of genomic regions of interest, or by sequencing to identify all direct targets at the genome level.

Development of the protocol Initially, we carried out ChIP analysis on P. trichocarpa SDX using Arabidopsis procedures developed by us9 or those by Kaufmann et al.10 and others11–13, but we failed to enrich the target DNAs. All of these procedures gave a very low SDX nuclear or chromatin yield, leading to poor or no immunoprecipitation. Factors that impede nuclear isolation, chromatin extraction and immunopre-cipitation in ChIP for woody tissues or cells were unknown, so we systematically identified factors adversely affecting these proc-esses. We found that thick-walled cells and high levels of phenolics and polysaccharides in differentiating xylem (DX)5,14 are major factors that adversely affect many key steps in ChIP protocols optimized for tissues or cells of non-woody plants9–13 (Fig. 2). We then tested and modified conditions for specific processes

A robust chromatin immunoprecipitation protocol for studying transcription factor–DNA interactions and histone modifications in wood-forming tissueWei Li1,2, Ying-Chung Lin1,2, Quanzi Li1–3, Rui Shi2, Chien-Yuan Lin2, Hao Chen2, Ling Chuang2, Guan-Zheng Qu1, Ronald R Sederoff2 & Vincent L Chiang1,2,4

1State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. 2Forest Biotechnology Group, Department of Forestry & Environmental Resources, North Carolina State University, Raleigh, North Carolina, USA. 3College of Forestry, Shandong Agricultural University, Taian, Shandong, China. 4Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina, USA. Correspondence should be addressed to V.L.C. ([email protected]).

Published online 21 August 2014; doi:10.1038/nprot.2014.146

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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to reduce or eliminate problems associated with these adverse factors. Modifications conferring substantial improvements on the nuclear or chromatin yield and target DNA enrichment were identified and incorporated into the optimized protocol. Essentially, all major steps needed modifications, and repeated experiments demonstrated that these modifications were cru-cial to the success of the protocol for wood-forming tissue. Modifications from the previous protocols are listed in Table 1 and outlined below.

Cross-linking. The thick and hard wall in wood-forming cells impedes the penetration of the cross-linking buffer. Therefore, cross-linking must be carried out under vacuum to ensure penetration. The optimal procedure for SDX is vacuum (5 min)/release/mix at room temperature (RT; 23–25 °C) six times (Supplementary Fig. 1).

Nuclear isolation. Simple mechanical treatments of tissues or cells optimized for non-woody plants9–13 gave very low nuclear and chromatin yields, leading to low or no DNA enrichment. We developed a specific grinding and homog-enization step for nuclear isolation from wood-forming cells (Supplementary Fig. 2).

Chromatin extraction. Chromatin extraction from nuclei of wood-forming cells is difficult mainly owing to the high quanti-ties of phenolics, polysaccharides and other resinous materials in a woody tissue. We tried the detergents normally used9–13 and found that a stronger detergent (1% (wt/vol) SDS) in nuclear lysis buffer can greatly facilitate solubilization of these resinous materials and thus enhance chromatin extraction.

DNA fragmentation. Methods for chromatin DNA fragmen-tation are quite different, depending on the DNA shearing devices and the reaction time for DNA-protein cross-linking. In our case, the chromatin must be sonicated to generate 0.2–2-kb DNA fragments.

Immunoprecipitation. The antibody-antigen interactions can be disrupted by high concentrations (>0.1%, wt/vol) of ionic detergents such as SDS or sarkosyl. Because we used 1% (wt/vol) SDS in the nuclear lysis buffer, the SDS concentration must be diluted to 0.1% (wt/vol) using a dilution buffer to minimize its interference with immunoprecipitation.

DNA purification. Two phenol/chloroform extractions must be applied to purify the DNA to ensure proper PCR efficiency and high-quality DNA for sequencing. Although ChIP proto-cols for Arabidopsis and other non-woody plants9–13 failed on

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Figure 1 | Cell types in the stem of P. trichocarpa and the collection of wood-forming tissue. (a–c) A stem segment from a 6-month-old greenhouse-grown P. trichocarpa (a) and the cross-section view of the intact stem (b), as well as of the stem with the bark partially separated from the wood (c). (d,e) Transverse semi-thin sections of the stem (d) and of the stem with bark partially separated from the wood (e). The bark includes vascular cambium (VC), phloem (P) and phloem fiber (PF) cells. The xylem or wood includes differentiating xylem (DX) cells, which mature into thick-walled xylem fiber (XF), vessel (V) and ray (R) cells. When the bark is separated or removed from the wood, all VC, P and PF cells remain attached to the bark (e), whereas DX cells stay with XF, V and R cells on the wood (e). (f,g) To collect the wood-forming cells, or the DX cells, the stem is first debarked (f) and the stem surface is scraped with a razor blade to generate stem strips (g) containing pure DX cells. (h,i) Transverse semi-thin cross-sections of a debarked stem before (h) and after (i) scraping, showing ~10–15 layers of DX cells (space between dotted lines in i), are scraped off for protein-DNA cross-linking. Cross-sections (d,e,h and i) were stained with Fast Green and Safranin O showing mature XF, V and R cells in red and DX cells in light brown and blue. White scale bars (a–c,f,g), 1 cm; black scale bars (d,e,h,i), 50 µm.

Tissue collection (Step 1: 10–15 min)

Cross-linking (Steps 2–5: 40–45 min)

Nuclear isolation (Steps 6–13: 100–110 min)

Chromatin extraction (Step 14: 5–10 min)

DNA fragmentation (Steps 15 and 16: 30–35 min)

Reference Immunoprecipitation(Steps 17–28: 17–18 h including overnight)

Reverse cross-linking (Step 29: 6–7 h or overnight)

DNA detection (Steps 38 and 39A: 2 h)

Mock ChIPInput

DNA purification (Steps 30–37: 2–3 h)

(Steps 38 and 39B: 3 h)

Figure 2 | Key steps involved in the woody tissue–optimized ChIP protocol.

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P. trichocarpa SDX, our optimized protocol provides a very good 8–10-fold specific enrichment of direct targets of the TF and high-quality DNA of ~150 ng/g of SDX.

Advantages and disadvantages of the methodThe advantage of our protocol is its application to wood- forming tissue. Although our protocol is probably applicable to many tissue types, it is to date the only reported ChIP system for wood-forming tissue7. A common ChIP-based approach to map in vivo TF-DNA interactions in model plant species is the use of a transgenic tagged TF to engineer these interactions, which can then be immunoprecipitated through the tag13. This tagged-TF

approach is not yet suitable for woody plants because no TF mutants are known for these species; mutants are required to validate the functional equivalence between the tagged TF and the corresponding endogenous TF through complementation. The advantage of our protocol is that our anti-TF antibody–based ChIP system does not rely on tagged transgenics and TF mutants. Our approach allows more exclusive enrichment of the genomic regions that are bound to the specific endogenous TF, revealing directly the native TF-DNA interactions.

A disadvantage of our protocol compared with other proto-cols9–13 is that more tissue (~5 g) and processing time are needed to obtain adequate amounts of chromatin because of the adverse

taBle 1 | Comparison of ChIP protocols in wood-forming tissue and non-woody tissue.

other protocols9–13 results from sDX tissue using other protocols9–13

our protocol7 results from sDX tissue using our protocol7

Tissue collection Non-woody tissue9–13 SDX, the wood forming tissue, can be readily identified and collected

Cross-linking Involve treatment of the tissue in cross-linking buffer for 10 min under vacuum at room temperature9,11–13, or under vacuum for 15 min on ice, followed by releasing the vacuum and mixing the contents, and then repeating the treatment (vacuum for 15 min/release/mix) one more time10

Obtained 1–2-fold enrichment of ChIP DNA over non-specifically precipitated DNA

Perform the treatment of vacuum (5 min)/release/mix at room temperature, six times

Obtained ~5-fold enrichment of ChIP DNA over non-specifically precipitated DNA

Nuclear isolation Grind tissue with a mortar and pestle, and homogenize by vortex9–13

Obtained ~5 mg of wet weight nuclei per g of SDX

Grind tissue with an analytical mill, homogenize with an ULTR-TURRAX homogenizer and agitate for 10 min at 4 °C

Obtained ~9 mg of wet weight nuclei per g of SDX

Chromatin extraction Extraction buffer contains 1% (wt/vol) SDS9,12, 1% (wt/vol) SDS and Triton X-100 (ref. 11), 0.5% (wt/vol) Sarkosyl10 or 1% (vol/vol) Triton X-100 (ref. 13)

Obtained <10 µg of DNA per g of SDX using 1% (vol/vol) Triton X-100 or 0.5% (wt/vol) Sarkosyl

Extraction buffer contains 1% (wt/vol) SDS9,12

Obtained ~25 µg of DNA per g of SDX using 1% (wt/vol) SDS

DNA fragmentation Time of sonication depends on shearing devices and time of fixation9–13

Not tested using other shearing devices

Branson Sonifier 250, 10% power for 12 times (10 s each)

Obtained a large number of DNAs in the 0.2–2-kb range

Immunoprecipitation Various types of IP buffer9–13 Obtained 30–60 ng of ChIP DNA per g of SDX

A new dilution buffer (0.25% (vol/vol) Triton X-100, 1.2 mM EDTA, 10 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM PMSF, 1 µg/ml pepstatin A and 1 µg/ml leupeptin) for immunoprecipitation

Obtained ~80 ng of ChIP DNA per g of SDX

DNA purification Purify with phenol, chloroform and isoamyl alcohol once9,11–13, or use a PCR purification kit10,13

No or faint PCR bands Phenol, chloroform and isoamyl alcohol twice

Strong and clear PCR bands

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cell properties of the wood-forming tissue. Similarly to all ChIP protocols, our approach relies on the availability of a highly spe-cific antibody against the protein of interest and is dependent on the expression level of the protein.

Applications of the method Our protocol is robust and has been applied to the establish-ment of a PtrSND1-B1 (a specific NAC TF)-directed hierarchical genetic regulatory network in wood formation in P. trichocarpa7,15. Interactions between DNA and other TFs, such as two other P. trichocarpa NACs, PtrSND1-A2 and PtrVND6-C1, were also identified using antibodies against these TFs (W.L., Y.-C.L. and V.L.C., unpublished data). We have also used this protocol to establish profiles of histone modifications, such as tri-methylated Lys 4 of histone H3 (H3K4me3), di-methylated Lys 9 of histone H3 (H3K9me2) and acetylated Lys 9 of histone H3 (H3K9ac), of many TF-encoding genes expressed in the wood-forming tissue of P. trichocarpa under normal and stressed conditions (W.L., Y.-C.L. and V.L.C., unpublished data). Representative results of these applications are discussed in ANTICIPATED RESULTS.

Our optimized ChIP protocol should be applicable to other woody species. Very recently, we have applied our ChIP protocol to demonstrate histone H3 Lys 9 acetylation (H3K9ac) of a cellulose synthase gene (LpCesA1) in loblolly pine (Pinus taeda) (W.L., Y.-C.L. and V.L.C., unpublished data). ChIP is particularly powerful when combined with full genome–based next- generation sequencing because the combination allows map-ping of TF-DNA interactions and characterization of histone modifications in vivo at the whole-genome level16–20. Our proto-col is very timely because the genomes of many tree species, such as Norway spruce (Picea abies)21, loblolly pine (Pinus taeda)22 and a basal angiosperm, Amborella trichopoda, have just recently been sequenced23, and genome sequences of many other tree species are expected to be completed in the near future. Genomes of these and previously sequenced woody species, such as P. trichocarpa24 and Eucalyptus grandis25, provide a foundation for extensive com-parative analyses of the evolution of genetic regulatory hierar-chies and epigenetic modifications associated with growth and development, such as wood formation. Wood is an indispensable component of the bio-based economy and industries worldwide, and the demand for wood continues to increase1–4. The study of the fundamental structure of genetic regulatory hierarchies in wood formation will reveal the genetic mechanisms used by plants to produce biomass, leading to improved productivity. These important fundamental studies have not previously been possible. Our optimized ChIP system should help advance these studies.

Experimental designTarget protein adundance. The abundance of the protein of interest is crucial for a successful ChIP experiment. Because the abundance of many TFs is very low26–28, it is therefore advisable to verify before a ChIP experiment whether the target protein can be detected in the tissue of interest by western blotting. Alternatively, transcript abundance, as quantified by, e.g., RNA sequencing or quantitative reverse-transcription PCR (qRT-PCR), can be an indication of protein abundance. The SDX is an excellent tissue for ChIP because a large number of the TFs in the P. trichocarpa genome annotated for cell-wall formation are abundantly and

specifically expressed in this tissue, as determined by transcript abundance6,7,29.

Amount of starting material. The amount of starting material is important for a ChIP experiment because DNA-associated pro-teins are expressed at levels that can vary greatly. The transcript level of the target protein can usually be used to adjust the amount of tissue needed for the ChIP experiment. In our case, at least ~5 g of freshly isolated SDX is needed for ChIP of PtrSND1-B1, which has a transcript abundance of 827 copy numbers per ng of total SDX RNA quantified by qRT-PCR6,7. More tissue is needed for low-abundance proteins. Usually 5–10 g of SDX is recommended for target proteins with transcript levels between 800 and 400 copy numbers per ng of SDX RNA, respectively.

Collection of woody tissue. The DX, the wood-forming tissue, can be readily collected in a large quantity from a tree stem after debarking. A partially debarked stem cross-section of a 6-month-old greenhouse-grown P. trichocarpa is shown in Figure 1 to illus-trate the types and distribution of cells and tissues. Between the bark and DX, there are 3–5 thin layers of soft and juicy cells, the VC5 (Fig. 1d,e). Because of this juicy VC zone, the bark on actively growing trees (in a greenhouse or in the field) can be very readily and cleanly peeled off the DX (Fig. 1f). After debarking, VC and phloem that includes phloem fiber cells are attached to the bark. The surface of the debarked stem is covered with the DX cells (Fig. 1h), which originate from the VC and differentiate into mature xylem fiber, vessel and ray cells5 (Fig. 1d). Collectively, these three mature cells comprise the secondary xylem, or wood.

In Populus species (P. tremula × tremuloides), actively differenti-ating xylem cells are located within ~400 µm centripetal distance from VC30. In P. trichocarpa, this distance is equivalent to ~16 layers of DX cells (Fig. 1h). The DX (or SDX) cells on the surface of the debarked stem can be collected by scraping the debarked stem with a razor blade6,31–34 (Fig. 1g–i). We usually can col-lect ~10–15 layers of SDX cells, as demonstrated by histological analysis of the stem cross-sections before and after scraping. The fresh SDX should be scraped onto a piece of aluminum foil on ice before transferring it to the cross-linking buffer.

Cross-linking. Cross-linking fixes the protein (antigen) of inter-est to its chromatin-binding site, and the extent of cross-linking can markedly affect DNA enrichment. If the protein of interest is not sufficiently cross-linked to the DNA, the ChIP experiment will not work. However, excessive cross-linking may reduce anti-body accessibility to antigen or mask the epitopes, and it may also reduce DNA fragmentation efficiency. The efficiency of cross-linking depends on a number of interrelated parameters, including time, cross-linker concentration and temperature; for example, lower temperatures require longer cross-linking times. Thus, optimization of the cross-linking step is crucial to the suc-cess of the experiment. We have determined that, owing to the thick and hard cell walls of woody tissues, cross-linking of SDX cells must be carried out with multiple differential pressure treat-ments at RT to increase the penetration of the cross-linking agents into the cells (Table 1).

Nuclear isolation and chromatin extraction. It is important to extract sufficient chromatin (~20 µg of DNA/g of SDX) for

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immunoprecipitation to achieve high DNA enrichment. In our optimized ChIP protocol, we include a specific grinding and homogenization step and a stronger ionic detergent (1% (wt/vol) SDS) in the nuclear lysis buffer (Table 1); this has resulted in good and reproducible chromatin yields of ~25 µg of DNA/g of SDX. If necessary, the final chromatin yields can be increased by extend-ing the incubation of homogenized SDX cells in nuclear isolation buffer from 10 min up to a maximum of 20 min.

Antibody. One of the most crucial components of a success-ful ChIP experiment is the antibody used to capture a specific DNA-associated protein. The antibody must be highly specific to the target protein and also effective in binding for immu-noprecipitation. An antibody recognizes and binds to the epitope of the protein antigen. Many TFs have closely sequence-related family members6,35,36, which may have similar epitope sequences and are therefore difficult to distinguish by antibodies produced from the full-size protein. In addition, if an epitope resides in the DNA-binding domain, it may be masked by the DNA-protein complex, particularly after cross-linking, leading to reduced availability of the epitope to the antibody and thus diminished or even no DNA enrichment after immunoprecipi-tation. To circumvent these difficulties, we produce antibodies using immunogens derived from protein-specific polypeptides that avoid the DNA-binding domain, so that the antibody can both distinguish sequence-related members and bind outside of the DNA-binding domain6,7,37,38. If possible, for each target TF protein two distinct types of such peptide-based antibodies should be made to increase the success rate of ChIP. Before a ChIP experiment, the specificity of the antibody should be veri-fied6,7,37,38 (Fig. 3). A range of antibody concentrations must be tested for each ChIP experiment to determine the optimal concentration for a high level of DNA enrichment compared with the background.

Experimental controls. Both positive and negative controls are crucial for ChIP assays. Positive controls are used to dem-onstrate that the entire ChIP experiment is working. A known direct TF-DNA binding system is a good positive control. We have validated PtrSND1-B1-DNA interactions at 76 P. trichocarpa

genome loci using a highly specific anti–PtrSND1-B1 antibody6,7. Therefore, any of these PtrSND1-B1-DNA pairs can be a good positive control for ChIP assays on SDX tissue. We have routinely used the PtrSND1-B1-PtrMYB021 system as a positive control for our ChIP experiments on SDX because the binding site on the PtrMYB021 gene promoter has been well characterized using electrophoretic mobility shift assays6,7.

Negative controls are needed to establish a background level, and may be represented by a ChIP experiment without a specific antibody. We always use preimmune serum as the negative control (the mock ChIP reaction) to reveal the background nonspecific DNA enrichment7. Input DNAs isolated from the cross-linked tis-sue without being treated with preimmune serum or anti-serum but fragmented under the same conditions as the immunoprecipi-tation DNAs are always used for DNA detection to specify that the PCR is functioning. In addition, for PCR-based DNA detection, an unrelated gene locus, such as that encoding actin or tubulin, needs to be included as a negative PCR control to demonstrate the specificity of enrichment. For DNA sequence–based detec-tion (ChIP-seq), a reference DNA sequencing control is needed. In general, either the input DNAs or DNAs from a mock ChIP reaction can be used as the reference for this purpose. However, mock ChIP DNAs are normally present in a quantity insufficient for a sequencing library. More often, the input DNAs are used as the reference because such DNAs can usually be recovered in amounts adequate for library construction.

A1Anti-GSTanti–PtrSND1-B1

70 kDa70 kDa

A2 B1 B2

Figure 3 | Western blot to determine the specificity of the antibody used in our ChIP assay7. PtrSND1-B1 and its other family members, PtrSND1-A1 (A1), PtrSND1-A2 (A2) and PtrSND1-B2 (B2), in the genome were tested. Purified A1, A2, B1 and B2 Escherichia coli recombinant proteins fused with a glutathione S-transferase (GST) tag were probed with the anti-GST and anti–PtrSND1-B1 antibodies, respectively. These four PtrSND1 member proteins could be recognized by the monoclonal anti-GST antibodies, but only PtrSND1-B1 could be detected by anti–PtrSND1-B1-peptide antibodies, demonstrating the specificity of this anti-PtrSND1-B1 antibody. Figure 3 was reproduced from Lin et al.7 with permission from http://www.plantcell.org, copyright American Society of Plant Biologists.

MaterIalsREAGENTS

Plant material (P. trichocarpa stem-differentiating xylem) Plants are grown as described in Reagent SetupPremier peat moss (Wyatt-Quarles, cat. no. PM-0078P)Sunshine MVP (Wyatt-Quarles, cat. no. GP-92079)Osmocote (Hummert International, cat. no. 19-6-12)Miracle-Gro (Hummert International, cat. no. 65-3633)Sucrose (Sigma-Aldrich, cat. no. S5390)Formaldehyde solution 37% (wt/wt) (Sigma-Aldrich, cat. no. 252549) ! cautIon Formaldehyde solution is toxic. Handle it with care. Wear gloves and eye protection.Glycine (Fisher Scientific, cat. no. AC12007-0010)Tris base (Promega, cat. no. H5135)Hydrochloric acid (HCl; Sigma-Aldrich, cat. no. 258148) ! cautIon HCl is corrosive. Handle it with care. Wear gloves and eye protection.PMSF (Sigma-Aldrich, cat. no. P7626)Pepstatin A (Sigma-Aldrich, cat. no. P5318)Leupeptin (Sigma-Aldrich, cat. no. L2884)

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DMSO (Sigma-Aldrich, cat. no. D8418)Isopropyl alcohol (Sigma-Aldrich, cat. no. I9516)β-Mercaptoethanol (Sigma-Aldrich, cat. no. M3148) ! cautIon β-Mercaptoethanol is toxic. Handle it with care. Wear gloves and eye protection.Miracloth (Fisher Scientific, cat. no. NC9147303)Magnesium chloride (MgCl2; Sigma-Aldrich, cat. no. M8266)Triton X-100 (Sigma-Aldrich, cat. no. T8787)EDTA disodium salt (Sigma-Aldrich, cat. no. E5134)Sodium hydroxide (NaOH; Sigma-Aldrich, cat. no. S5881) ! cautIon NaOH is corrosive. Handle it with care. Wear gloves and eye protection.SDS (Sigma-Aldrich, cat. no. L3771) ! cautIon SDS is carcinogenic. Handle it with care. Wear gloves and eye protection.Sodium chloride (NaCl; Sigma-Aldrich, cat. no. S3014)Dynabeads protein G (Life Technologies, cat. no. 10003D)Lithium chloride (LiCl; Sigma-Aldrich, cat. no. L4408)NP-40 detergent solution (Thermo Scientific, cat. no. 28324)Sodium deoxycholate (Sigma-Aldrich, cat. no. D6750)

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Sodium bicarbonate (NaHCO3; Sigma-Aldrich, cat. no. S8875)Proteinase K (Thermo Scientific, cat. no. EO0491)RNase A solution (Promega, cat. no. A7973)UltraPure phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol) (Invitrogen, cat. no. 15593-031) ! cautIon This reagent is toxic. Handle it with care. Wear gloves and eye protection.Ethanol (VWR, cat. no. 89125-170)Sodium acetate (Sigma-Aldrich, cat. no. S2889)Glacial acetic acid (Fisher Scientific, cat. no. A38SI-212) ! cautIon It is corrosive. Handle it with care. Wear gloves and eye protection.Glycogen (Invitrogen, cat. no. 10814-010)Ethidium bromide (Sigma-Aldrich, cat. no. E8751) ! cautIon It is a powerful mutagen. Handle it with care. Wear gloves and eye protection.GoTaq DNA polymerase (Promega, cat. no. M3005)dNTP Mix (Promega, cat. no. C1141)Faststart Univ SG master (Roche, cat. no. 04913850001)DNA chips 1,000 (Agilent Technologies, cat. no. 5067-1504)High-sensitivity DNA chips (Agilent Technologies, cat. no. 5067-4626)Double-distilled autoclaved water (ddH2O)

EQUIPMENTSingle-edged blades (Electron Microscopy Science, single-edge carbon steel, cat. no. 71960)Aluminum foil roll (VWR, cat. no. 89068-737)Vacuum pump (KNF Laboport, Mini-pump, Z288268)Vacuum chamber (OMEGA, cat. no. LAB-420200000) Conical tubes (50 ml; Worldwide, cat. no. 41021039)Analytical Mill (IKA, model A11 basic)ULTRA-TURRAX homogenizer (IKA, model T18 Basic)Vortexer (Fisher Scientific, Vortex Genie 2)Rotator (Ted Pella, PELCO R2 rotator with 1051 heads shown in the 55°)Microcentrifuge (Denville Scientific, MiniMouse II)High-speed refrigerated centrifuge (Sigma-Aldrich, cat. no. 4-16K)Swing-out rotor (Sigma-Aldrich, cat. no. 11150)Round bucket for swing-out rotor (Sigma-Aldrich, cat. no. 13350)Refrigerated microcentrifuge (Eppendorf, cat. no. 5415R)Microcentrifuge tubes (1.5 and 2.0 ml)Branson Sonifier (Fisher Scientific, Branson Sonifier S-250D)Magnetic stand (Promega, MagneSphere Technology magnetic separation stand)Hot water bath (Fisher Scientific, Isotemp 215 dual-chamber hot water bath)Electrophoresis system (Embi Tec, EP-2100)Vacuum dryer (Eppendorf, cat. no. AG22331)NanoDrop (Thermo Scientific, NanoDrop 2000)Bioanalyzer (Agilent, Agilent 2100 Bioanalyzer)Standard PCR machine (GMI, MJ Research PTC-100 thermal cycler)Standard 200-µl PCR tubes (BioExpress, cat. no. T-3035)Real-time PCR machine (Agilent Technologies, Mx3000p qPCR system)qPCR plates (Agilent Technologies, Mx3000p 96-well plates, cat. no. 401333)qPCR plate caps (Agilent Technologies, 8× strip, cat. no. 401425)

REAGENT SETUPSucrose, 2 M Dissolve 68.46 g of sucrose in 56 ml of ddH2O. Mix and heat to completely dissolve the sucrose. Bring the volume to 100 ml. Autoclave and store the solution at 4 °C for up to 3 months.Tris-HCl, 1 M, pH 6.5 or 8.0 Dissolve 121.1 g of Tris base in 800 ml of ddH2O. Adjust the pH of the solution to 6.5 or 8.0 with HCl. Bring the volume to 1 liter. Autoclave and store Tris-HCl at RT for up to 12 months.PMSF, 1 M Dissolve 174 mg of PMSF in 1 ml of isopropyl alcohol. Store the solution at 20 °C. Stock solutions are stable for at least 6 months at 20 °C. crItIcal Add this reagent to buffers just before use because PMSF has a short half-life in aqueous solutions.Pepstatin A, 1 mg/ml Dissolve 1 mg of pepstatin A in 1 ml of 10% (vol/vol) acetic acid in DMSO. Store the solution at 20 °C. Stock solutions are stable for up to 6 months at 20 °C. crItIcal Add this reagent to buffers before use.Leupeptin, 1 mg/ml Dissolve 1 mg of leupeptin in 1 ml of ddH2O. Store it at 20 °C. Stock solutions are stable for up to 6 months at 20 °C. crItIcal Add this reagent to buffers before use.

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Glycine, 2 M Dissolve 1.5 g of glycine in 8 ml of ddH2O and adjust the volume to 10 ml. Store the solution at 4 °C for up to 3 months. Before use, allow the stock solution to reach RT.MgCl2, 1 M Dissolve 9.52 g of MgCl2 in ddH2O and bring the volume to 100 ml. Autoclave and store the solution at RT for up to 12 months.EDTA, 0.5 M, pH 8.0 Add 18.6 g of EDTA to 80 ml of ddH2O. While stirring, add ~2 g of NaOH slowly to the solution to obtain a pH of 8.0. Bring the volume to 100 ml. Autoclave and store the solution at RT for up to 12 months.SDS, 20% (wt/vol) Dissolve 20 g of SDS in 80 ml of ddH2O. Mix and heat the solution to 68 °C to completely dissolve SDS. Make up the total volume to 100 ml with ddH2O. Store the solution at RT for up to 6 months.NaCl, 5 M Dissolve 146.1 g of NaCl in 400 ml of ddH2O and adjust the volume to 500 ml. Autoclave and store the solution at RT for up to 12 months.LiCl, 4 M Dissolve 16.96 g of LiCl in 40 ml of ddH2O and adjust the volume to 100 ml. Autoclave and store the solution at 4 °C for up to 6 months.Sodium acetate, 3 M, pH 5.2 Dissolve 40.8 g of sodium acetate trihydrate in 70 ml of ddH2O. Adjust the pH of the solution to 5.2 with glacial acetic acid. Bring the solution to a final volume to 100 ml. Filter it through a 0.22-µm filter and store it at RT for up to 6 months.Cross-linking buffer Cross-linking buffer contains 0.4 M sucrose, 10 mM Tris-HCl (pH 8.0), 5 mM β-mercaptoethanol, 1 mM PMSF, 1 µg/ml pepstatin A, 1 µg/ml leupeptin and 1% (wt/vol) formaldehyde. crItIcal Freshly prepare the buffer using stock solutions in the amount required, and add protein inhibitors to the buffer just before use.Buffer 1 Buffer 1 contains 0.4 M sucrose, 10 mM Tris-HCl (pH 8.0), 5 mM β-mercaptoethanol, 1 mM PMSF, 1 µg/ml pepstatin A and 1 µg/ml leupeptin. crItIcal Always freshly prepare the buffer using stock solutions in the amount required, and keep it on ice until use. Add protease inhibitors to the buffer before use.Buffer 2 Buffer 2 contains 0.25 M sucrose, 10 mM Tris-HCl (pH 8.0), 5 mM β-mercaptoethanol, 10 mM MgCl2, 1% (vol/vol) Triton X-100, 1 mM PMSF, 1 µg/ml pepstatin A and 1 µg/ml leupeptin. crItIcal Always freshly prepare the buffer using stock solutions in the amount required and keep it on ice until use. Add protease inhibitors to the buffer before use.Buffer 3 Buffer 3 contains 1.7 M sucrose, 10 mM Tris–HCl (pH 8.0), 5 mM β-mercaptoethanol, 2 mM MgCl2, 0.15% (vol/vol) Triton X-100, 1 mM PMSF, 1 µg/ml pepstatin A and 1 µg/ml leupeptin. crItIcal Always freshly prepare the buffer using stock solutions in the amount required, and keep it on ice until use. Add protease inhibitors to the buffer before use.Lysis buffer Lysis buffer contains 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% (wt/vol) SDS, 1 mM PMSF, 1 µg/ml pepstatin A and 1 µg/ml leupeptin. crItIcal Freshly prepare the solution using stock solutions in the amount required for every experiment, and keep it on ice until use; add protease inhibitors to the buffer before use. SDS should be added just before use.Dilution buffer Dilution buffer contains 0.25% (vol/vol) Triton X-100, 1.2 mM EDTA, 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM PMSF, 1 µg/ml pepstatin A and 1 µg/ml leupeptin. Prepare the buffer using stock solutions on the day of use, and keep it at 4 °C until required; add protease inhibitors to the buffer just before use.High-salt wash buffer High-salt wash buffer contains 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 500 mM NaCl and 0.25% (vol/vol) Triton X-100. Prepare the buffer using stock solutions on the day of use and keep it at 4 °C until required.LiCl buffer LiCl buffer contains 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 25 mM LiCl, 0.5% (wt/vol) NP-40 and 0.25% (wt/vol) sodium deoxycholate. Prepare the buffer using stock solutions on the day of use and keep it at 4 °C until required.TE buffer TE buffer contains 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. Stock solutions are stable for months at RT. Precool the buffer on ice before use.Elution buffer Dissolve 0.042 g of NaHCO3 in 4.75 ml of ddH2O, and then add 250 µl of 20% (wt/vol) SDS (final concentration: 1% (wt/vol) SDS and 0.1 M NaHCO3). crItIcal Freshly prepare the solution and prewarm it at 65 °C before use.

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Proteinase/RNase buffer Proteinase/RNase buffer contains 150 mM EDTA (pH 8.0), 615 mM Tris-HCl (pH 6.5), 14 mg/ml proteinase K and 0.31 µg/µl RNase A. crItIcal Freshly prepare the buffer using stock solutions in the amount required, and keep it on ice until use.Ethidium bromide, 10 mg/ml Dissolve 100 mg of ethidium bromide in 10 ml of ddH2O. Store it in a light-tight container at 4 °C for up to 6 months.Tris-acetate-EDTA (TAE) buffer, 50× stock solution Dissolve 242 g of Tris base in 600 ml of ddH2O. Add 57.1 ml of glacial acetic acid and 100 ml of 0.5 M EDTA. Bring the solution to a final volume of 1 liter with ddH2O. Store it at RT for up to 12 months.

Agarose gel, 2% (wt/vol) Dissolve 2 g of agarose in 100 ml of 1× TAE buffer by heating the solution to boiling in the microwave, and then add 5 µl of ethidium bromide (10 mg/ml) to the dissolved agarose and mix it. Pour it immediately into trays and then cool it.Growth of P. trichocarpa Plants are grown as described previously6,7. Briefly, cut branches of P. trichocarpa plants (genotype Nisqually-1), and then root them in water for 2 weeks in a greenhouse under 16 h of light (at a light intensity of ~150 µE/m2/s) and 8 h of dark at 17–26 °C. After rooting, trans-fer the plants to pots containing one-half Premier peat moss and one-half Sunshine MVP. Maintain the plants in the greenhouse by watering every day and fertilizing once a month (Osmocote and Miracle-Gro) for 3–9 months.

proceDuretissue collection ● tIMInG 10–15 min1| Cut a stem from a 3–9-month-old tree, peel off the bark, scrape the wood-forming tissue (SDX) strips from the stem surface longitudinally using a single-edged blade and immediately put them on foil on ice (Fig. 1). The amount of starting material depends on the expression level of the protein of interest in the tissue. Collect at least 5 g of SDX for each experiment for studying TF-DNA interactions and histone modifications.

cross-linking ● tIMInG 40–45 min2| Add at least 5 g of freshly collected SDX to 37 ml of cross-linking buffer in a 100-ml beaker. Cross-link the tissue for a total of 30 min under vacuum at RT. During the 30 min, perform vacuum treatment for 5 min, release the vacuum and the mix tissue. Repeat this treatment of vacuum (5 min/release/mix) five additional times. crItIcal step The cell walls of woody tissues are thick and hard. Therefore, to maximize the penetration of the cross-linking agents into the cells, cross-linking must be carried out with multiple differential pressure treatments of SDX cells at RT. Temperature affects the efficiency of cross-linking. For SDX tissue, we recommend performing the cross-linking reaction at RT.

3| Quench the cross-linking reaction by adding 2.5 ml of 2 M glycine and incubating it for 10 min under vacuum. During this 10 min of vacuum treatment, after 5 min (halfway through), release the vacuum, mix the tissue and reapply vacuum for another 5 min.

4| Rinse SDX three times with precooled ddH2O. Dry the SDX between paper towels. crItIcal step Remove the excess water from the cross-linked tissue as thoroughly as possible; otherwise, it is very difficult to grind it to a fine powder. However, the SDX should remain slightly damp to the touch.

5| Freeze the cross-linked SDX in liquid nitrogen and either proceed immediately to Step 6 to grind the tissue or store it at −80 °C. pause poInt The cross-linked tissue can be stored at 80 °C for up to 2 months.

nuclear isolation ● tIMInG 100–110 min crItIcal Perform all steps at 4 °C, keep the samples on ice and precool all buffers unless otherwise indicated.6| Grind the cross-linked SDX to a fine powder in liquid nitrogen using an analytical mill, and transfer the powder from the cold mill to an ice-cold 50-ml conical tube. Add 37 ml of cold buffer 1 to the sample. crItIcal step For successful nuclear isolation, it is important to grind the cross-linked tissue to a fine powder.

7| Homogenize the mixture in buffer 1 using an ULTRA-TURRAX Homogenizer. Vortex the sample and agitate it for 10 min at 4 °C on a rotating wheel. crItIcal step Complete homogenization of the mixture is crucial in order to isolate the nuclei efficiently.

8| Pass the mixture gradually through three layers of Miracloth into a new ice-cold 50-ml conical tube, and squeeze the Miracloth to collect all of the liquid. crItIcal step The Miracloth should be squeezed to avoid substantial cell loss.

9| Centrifuge the solution in the 50-ml conical tube at 1,800g for 10 min at 4 °C.

10| Decant and discard the supernatant, resuspend the pellet completely in 2 ml of buffer 2, and transfer the solution to a 2.0-ml microcentrifuge tube.? trouBlesHootInG

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11| Centrifuge at 16,000g for 10 min at 4 °C.

12| Repeat Steps 10 and 11 once more.

13| Fully resuspend the pellet in 500 µl of buffer 3. Layer the solution onto another 500 µl of buffer 3 in a new 1.5-ml microcentrifuge tube and centrifuge the sample for 1 h at 16,000g at 4 °C. crItIcal step The pellet may be difficult to resuspend. It must be fully resuspended in buffer 3 by pipetting the suspension up and down at least ten times.? trouBlesHootInG

chromatin extraction and Dna fragmentation ● tIMInG 40–45 min14| Remove the supernatant and resuspend the pellet (chromatin) in 350 µl of lysis buffer. Save 10 µl of the resuspended chromatin and store it at 20 °C for DNA gel analysis at Step 16 (Box 1). crItIcal step Including 1% (wt/vol) SDS in lysis buffer can greatly facilitate solubilization of these resinous materials and thus enhance chromatin extraction. The pellet should be gently resuspended to avoid creating any foam. The 10 µl of the chromatin solution saved as a control is used to check sonication efficiency.

15| Sonicate the chromatin solution 12 times on 10% power using a Branson Sonifier 250 for 10 s each time with a 1-min interval on ice between sonications. Mix the chromatin solution gently by tapping the tube before each sonication. Generate DNA fragments of ~0.2–2 kb, as shown in Figure 4. crItIcal step Care should be taken to avoid creating any foam, which decreases the efficiency of DNA fragmentation. To avoid overheating the sample, the tube should be kept on ice throughout sonication.

16| Centrifuge the chromatin sample at 16,000g for 10 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube and centrifuge the sample one more time. Take an aliquot of 10 µl of supernatant to check the sonication efficiency. The remaining supernatant can be used immediately for preclearing (Step 17) or stored at 80 °C before use. crItIcal step Make sure that there is no cell debris in the supernatant; otherwise, it will interfere with immunoprecipitation.? trouBlesHootInG pause poInt The chromatin can be stored at −80 °C for ~2 months.

Immunoprecipitation ● tIMInG 17–18 h, including overnight incubation17| Save 50 µl of the supernatant as an input control and store it at 20 °C for reverse cross-linking at Step 29, and then dilute 200 µl of the remaining supernatant tenfold by adding 1.8 ml of dilution buffer in a new 2.0-ml microcentrifuge tube. crItIcal step Input control is important for DNA detection to confirm that the PCR is working, or as a negative control for ChIP-seq. Input sample from either Step 17 or after preclearing in Step 21 can be used as the control. crItIcal step The supernatant should be diluted ten times with the dilution buffer to reduce the concentration of SDS from 1 to 0.1% (wt/vol). Too much (>0.1%) SDS may disrupt the antibody-antigen interactions.

18| Add 5 µl of preimmune sera to the chromatin solution and incubate the mixture for 1 h at 4 °C on a rotating wheel. This is the preclearing step.

crItIcal step Salmon sperm–sheared DNA or other DNA should not be used for preclearing; they will interfere with the ChIP-seq experiment and may give false sequencing reads. crItIcal step For studying histone modification in our ChIP experiments, there are no preimmune sera, so we omit this preclearing step and increase the incubation time to 2 h at Step 20. Alternatively, IgG from the same source as the antibodies could be used in place of preimmune serum for preclearing.

Box 1 | Checking sonication efficiency ● tIMInG 1.5 h 1. Take the 10-µl samples from Steps 14 and 16, and add 10 µl of 0.5 M NaCl to each sample; heat the samples at 95 °C for 30 min to reverse cross-linking.2. Run the reverse cross-linked samples on a 2% (wt/vol) agarose gel in 1× TAE buffer. The sample from Step 16 should have a large number of DNAs in the 0.2–2-kb range compared with the aliquot from Step 14.

(FU)

400

35

10,3

80300200

Flu

ores

cenc

e

1000

35 150 300 500 1,000 10,380 (bp)

Figure 4 | An example of electropherogram analysis of P. trichocarpa SDX chromatin after sonication7. Agilent Bioanalyzer analysis is showing a large number of DNAs in the 0.2–2-kb range. Peaks at 35 and 10,380 are lower and upper markers, respectively.

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19| Prepare 150 µl of Dynabeads protein G as follows. Rinse the beads by fully resuspending them in 1 ml of dilution buffer by inverting the tube several times. Place the tube on the magnet stand for 1 min at 4 °C, and then, with the tube still on the stand, discard the supernatant by aspiration with a pipette. Repeat this rinsing process two more times. After the final rinse, remove the tube from the magnet stand, and resuspend the beads in 150 µl of dilution buffer. crItIcal step The beads should be equilibrated with the dilution buffer before use to increase the binding efficiency of the Dynabeads–protein G in the dilution buffer.

20| Add 50 µl of Dynabeads–protein G from Step 19 to the chromatin solution from Step 18 and incubate for 1 h at 4 °C on a rotating wheel. Keep the remaining 100 µl of beads on ice until required at Step 24. crItIcal step If no preclearing step (Step 18) was carried out (e.g., if you are studying histone modification), add the Dynabeads to the diluted chromatin solution from Step 17 and incubate the tube for 2 h.

21| Place the tube on the magnet stand for 1 min at 4 °C and collect the supernatant in a new 2.0-ml microcentrifuge tube. Centrifuge at 16,000g for 10 min at 4 °C to remove debris and the residual beads, and transfer the supernatant to another 2.0-ml microcentrifuge tube.

22| Equally split the 2.0 ml of supernatant into two 1.5-ml microcentrifuge tubes. One is the ChIP sample and the other is used as the mock control. crItIcal step The 2.0 ml of supernatant contains ~70 µg of chromatin. As a successful immunoprecipitation requires at least 20 µg of chromatin to obtain a substantial enrichment (more than fivefold) of the target DNA over nonspecific sequences, the 2.0 ml of supernatant can be split equally into three 1.5-ml microcentrifuge tubes (666 µl each) to analyze two modifications with one mock control.

23| Add antibodies to the ChIP sample and add preimmune sera to the mock control. Incubate the sample overnight at 4 °C on a rotating wheel. crItIcal step The amount of antibody required per ChIP experiment should be empirically determined. Typically, 1–10 µg of antibodies is used. If the exact concentration of antibodies is unavailable, a range of antibody concentrations can be tested in ChIP experiments to determine the antibody concentration that gives optimal high levels of enrichment. crItIcal step To map histone modifications, we use antibodies against specific amino acid modifications, such as di- and trimethylated Lys 4 of histone H3, or acetylated Lys 9 of histone H3. If no preimmune serum is available (as is the case for these histone antibodies), either do not add antibody to the mock control or add an equal amount of IgG from the same source as the antibodies.

24| Add 50 µl of Dynabeads–protein G from Step 19 to the ChIP sample and 50 µl to the mock control; mix them completely by inverting each tube several times. Incubate the mixture for 2 h at 4 °C on a rotating wheel.

25| Place each tube on the magnet stand for 1 min at 4 °C and discard the supernatant by aspiration with a pipette while the tube remains on the magnet stand.

26| Add 1 ml of dilution buffer to each tube, and then fully resuspend the beads by gently inverting each tube several times. Place each tube on the magnet stand for 1 min at 4 °C and discard the supernatant by aspiration with a pipette while the tube remains on the magnet stand. Repeat this wash step once more.

27| Wash the beads with 1 ml of each of the following buffers: once with high-salt wash buffer, once with LiCl wash buffer and twice with TE buffer. These wash steps are performed as follows: add the wash buffer, and then mix the beads and solution completely by gently inverting each tube several times. Incubate each tube for 5 min at 4 °C on a rotating wheel. Place each tube on the magnet stand for 1 min at 4 °C and discard the supernatant by aspiration with a pipette while the tube remains on the magnet stand. crItIcal step For studying histone modification, washing with high-salt wash buffer and LiCl wash buffer should be extended from 5 to 10 min.

28| Add 250 µl of prewarmed (65 °C) elution buffer to each tube, completely resuspend the beads by tapping and incubate at 65 °C for 15 min, by mixing at 5-min intervals. Place each tube on the magnet stand for 1 min at RT and collect the supernatant in a new 1.5-ml microcentrifuge tube while the tube remains on the magnet stand. Repeat this elution step once more. crItIcal step The elution buffer should be freshly prepared and prewarmed before use.

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reverse cross-linking ● tIMInG 6–7 h or overnight29| Combine the two eluates corresponding to the same sample (from Step 28; total volume will be 500 µl). At the same time, add 450 µl of elution buffer to 50 µl of input sample (from Step 17). Add 20 µl of 5 M NaCl to each tube and incubate the mixture at 65 °C for 6 h, or overnight, to reverse cross-linking.

Dna purification ● tIMInG 2–3 h30| Add 32.5 µl of protease/RNase buffer to each tube (from Step 29) and incubate the mixture at 45 °C for 1 h.

31| Add 550 µl of phenol/chloroform/isoamyl alcohol to each tube and vortex briefly.

32| Centrifuge each sample in a microcentrifuge at 13,800g for 15 min at 4 °C and transfer the supernatant to a 2.0-ml microcentrifuge tube. Repeat Steps 31 and 32 once more.

33| Add 1.25 ml of 100% ethanol, 50 µl of 3 M sodium acetate (pH 5.2) and 4 µl of glycogen (20 mg/ml) to each tube; incubate the tubes for 1 h or overnight at −80 °C to precipitate the DNA.

34| Centrifuge each sample at 13,800g for 15 min at 4 °C.

35| Discard the supernatant, wash the pellet with 500 µl of 70% (vol/vol) ethanol, and centrifuge again at 13,800g for 10 min at 4 °C.

36| Discard the supernatant and vacuum-dry the pellet at 30 °C for 5 min.

37| Dissolve the DNA in 40 µl of ddH2O. DNA can be used immediately for subsequent analysis (ChIP-PCR as described in Step 39 or ChIP-seq) or it can be stored at −80 °C. pause poInt The DNA can be stored at −80 °C for ~2 months.

Dna detection ● tIMInG 2–3 h38| Measure the DNA concentration using a NanoDrop and a BioAnalyzer DNA chip according to the manufacturer’s instructions.? trouBlesHootInG

a ATG

500 1

(001) PtrSND1-L-2 (006) Integrase-typeDNA-binding protein

(031) Pectin lyase-likesuperfamily protein

(083) Oxygenasesuperfamily protein

(120) Flavin-bindingmonooxygenase

(169) Serine carboxy-peptidase-like 48

(053) Methylesteraseinhibitor superfamily

(007) Zn-finger protein,RING/FYVE/PHD type

(008) Zn-finger protein,RING/U-box type

(009) Zn-finger protein,RING/U-box type

(011) Zn-finger protein,C3H-type

(002) PtrMYB002

(003) PtrMYB021

(004) PtrMYB50

(005) MYB-like 102

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ock

Anti-B

1

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tM

ock

Anti-B

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ock

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(088) Cupredoxinsuperfamily protein

(172) Thioredoxinsuperfamily protein

(131) POPTR_0002s18880.1

(132) POPTR_0002s18880.2

(133) POPTR_0002s18880.3

(028) Peroxidasesuperfamily protein

(071) Thaumatinsuperfamily protein

(035) UDP-Glucosyltransferase 85A2

(040) Rhamnogalactu-ronate lyase protein

(045) Arabinogalactanprotein

(066) TIR-NBS-LRRprotein

(087) CytochromeP450, family

(010) Zn finger familyprotein, C2H2-like

(012) Zn finger familyprotein, C3H-type

(013) Zn finger familyprotein, TTF-type

(014) Zn finger familyprotein

(017) PtrLAC18

(023) PtrLAC40

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ock

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Figure 5 | ChIP-PCR of TF-DNA interactions in P. trichocarpa wood-forming tissue7. (a) A simplified gene structure to indicate the locations of the amplified promoter sequences. The thick line corresponds to a gene promoter that drives its gene represented by the rectangle. The arrowheads show the promoter sequence location for primer design. (b) ChIP-PCR assays of direct targets of PtrSND1-B1 using chromatin from differentiating xylem and anti–PtrSND1-B1 antibody. ChIP-PCR assays validated that the randomly selected 15 inferred directed target genes of PtrSND1-B1 are authentic. (c) ChIP-PCR assays of indirect targets of PtrSND1-B1 using chromatin from differentiating xylem and anti–PtrSND1-B1 antibody. We also used our ChIP protocol to test 18 randomly selected indirect target genes of PtrSND1-B1, and found no enrichment of PtrSND1-B1 in the promoters of 17 of these 18 genes. (d) PtrACTIN was used as a negative control. No enrichment of PtrSND1-B1 was detected in the promoter region of PtrACTIN. Input, Mock and Anti-B1 are PCRs 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. All primers used can be found in Lin et al.7. Figure 5 was adapted from Lin et al.7 with permission from http://www.plantcell.org, copyright American Society of Plant Biologists.

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39| Perform conventional PCR (option A) or qPCR (option B) to determine the level of enrichment of the target DNA (Figs. 5 and 6).? trouBlesHootInG(a) conventional pcr (i) Set up a 25-µl reaction mix as tabulated below.

component amount/reaction (ml) Final concentration

Primer F (5 µM) 2.0 0.4 µM

Primer R (5 µM) 2.0 0.4 µM

5× Green GoTaq reaction buffer 5.0 1×

dNTP mix, 10 mM each 0.5 0.2 mM each dNTP

GoTaq DNA polymerase (5 U/µl) 0.125 0.625 U

DNA (Step 38) 2.0

ddH2O 13.375

(ii) Perform PCR using the following conditions.

cycle number Denature anneal extend

1 94 °C, 5 min — —

2–33 94 °C, 30 s 55 °C, 30 s 72 °C, 30 s

34 — – 72 °C, 5 min

Analyze the amplified input, mock, immunoprecipitate DNA on a 2% (wt/vol) agarose gel in 1× TAE buffer, as shown in Figure 5. The input control has a strong and clear PCR band, and the mock control has no PCR signal. The immunopre-cipitation sample has a clear PCR band for a TF’s direct target and no signal for an indirect target.

(B) qpcr ● tIMInG 2.5 h (i) Dilute the DNA from Step 38 tenfold (5 µl of DNA in 45 µl of ddH2O).

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Figure 6 | ChIP-qPCR of histone modification in P. trichocarpa wood-forming tissue. (a) The relative density of histone H3 at the PtrACTIN locus in P. trichocarpa SDX. (b) The relative H3K9 acetylation levels at the PtrACTIN locus in P. trichocarpa SDX. ChIP was performed with antibodies against H3 (anti–histone H3 antibody (Abcam, cat. no. ab1791)) (a) and H3K9ac (anti–histone H3 (acetyl K9) antibody (Abcam, cat. no. ab10812)) (b), respectively, using chromatin isolated from P. trichocarpa SDX. The ChIP signal was quantified as relative to input DNA. The precipitates without using any antibody served as a negative control (mock). As expected, histone 3 is enriched at the PtrACTIN locus (a). There is also a high enrichment of H3K9ac at the PtrACTIN locus (b). We have performed these experiments with at least ten biological replicates (n = 10), i.e., SDX from ten individual wild-type P. trichocarpa trees grown in a greenhouse. Here we show the results from four such biological replicates. The value shown for each biological replicate is the average of three ChIP-qPCR technical replicates with the standard error represented by the error bar. Asterisks indicate values that are significantly different with P < 0.0001 by a Student’s t test. The primers used for ChIP-qPCR of P. trichocarpaV2.2 gene POPTR_0019s02630.1 are PtrActin-F 5′-TGTTGCCCTTGACTATGAGCAGGA-3′ and PtrActin-R 5′-ACGGAATCTCTCAGCTCCAATGGT-3′.

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taBle 2 | Troubleshooting table.

step problem possible reason solution

10 Low cell yield Not enough starting tissue Collect more tissue (Step 1)

Cells are not completely released Make sure that the tissue is ground to a fine powder and that the mixture of the powder with buffer 1 is completely homogenized (Steps 6 and 7)

13 The pellet of nuclei is brown

Oxidation of phenolics, which are enriched in wood-forming tissue

Quickly collect tissue into cross-linking buffer or add polyvinylpolypyrrolidone 2.5% (wt/vol) in buffer 1 (Step 1 or 6)

16 Visible insoluble materials in the chromatin solution

Cell debris is not completely removed Centrifuge the chromatin solution a few more times at 4 °C and transfer it to a new tube (Step 16)

38 Low yield of ChIP DNA Immunoprecipitation efficiency is low Use an antibody with high efficiency for immunoprecipitation, or make sure that the protein A or G used in the immunoprecipitation step has the best binding strength to the antibody (Steps 23 and 24)

(ii) Set up triplicate 15-µl reactions as tabulated below. Also set up the triplicate reactions for positive (i.e., a known interaction) and negative (i.e., a known non-interaction) controls. crItIcal step Negative controls, such as those for genes encoding actin or tubulin, should be used to check whether the observed enrichment is specific to the protein of interest.

component amount/reaction (ml) Final concentration

Primer F (5 µM) 0.6 0.2 µM

Primer R (5 µM) 0.6 0.2 µM

2× SYBR Green mixa 7.5 1×

Diluted DNA (Step 39B(i)) 6.3aThis SYBR Green mix contains ROX as a reference dye, which minimizes fluorescence intensity variation owing to differences in sample volume.

(iii) Run the reactions on a qPCR machine with the following program:

segment 1 segment 2 Dissociation/melt segment

Temperature 95 °C 95 °C, 60 °C 95 °C, 55 °C, 95 °C

Time 10 min 30 s, 1 min 1 min, 30 s, 30 s

Cycles 1 45 1

(iv) After the data are acquired, normalize the amount of the precipitated DNA to the input sample and calculate the percentage of immunoprecipitation compared with the input as a measure of the ChIP enrichment. Usually more than fivefold enrichment indicates a successful ChIP experiment. crItIcal step The Ct (cycle threshold) is defined as the number of cycles required for the fluorescence signal to cross the threshold. Ct levels are inversely proportional to the amount of the DNA molecule in the sample. If the Ct value obtained from the qPCR initially using the settings suggested above is >32, more sample DNA should be used for PCR.

? trouBlesHootInGTroubleshooting advice can be found in table 2.

(continued)

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taBle 2 | Troubleshooting table (continued).

step problem possible reason solution

The DNA and protein are not sufficiently cross-linked

Make sure that the formaldehyde and vacuum infiltration step is working well (Step 2)

Excessive cross-linking Make sure that the formaldehyde concentration is 1% (wt/vol) or optimize the cross-linking time (Step 2)

39 No PCR signals PCR does not work Make sure that the PCR program and reaction components are appropriate (Step 39)

RNA contamination in the DNA template Treat the DNA with RNase (Step 37)

Protein or phenol contamination in the DNA template

Make sure that only the upper phase is collected, excluding the intermediate and lower phases (Step 32)

DNA-protein complexes are not reversed Make sure that reverse cross-linking conditions are correct, or optimize the cross-linking time (Step 29 or 2)

The enrichment of ChIP DNA is low compared with that of mock control

The antibody is not specific to the protein of interest

Use a highly specific antibody to the protein of interest (Step 23)

Too much nonspecific binding Make sure that preclearing with preimmune sera is performed, or adjust the stringency of the wash solutions and the number of washes (Steps 18–21 or 26 and 27)

The chromatin is not sheared to ~0.2–2 kb

Make sure to have good sonication of the chromatin and optimize the sonication conditions (Step 15)

The expression of the protein of interest is extremely low in the tissue

Make sure that the expression pattern of the intended protein is determined before starting a ChIP experiment, and collect more specific tissue in which the protein is expressed (Step 1)

● tIMInGDay 1Step 1, tissue collection: 10–15 minSteps 2–5, cross-linking: 40–45 minSteps 6–13, nuclear isolation: 100–110 minSteps 14–16, chromatin extraction and DNA fragmentation: 40–45 minSteps 17–23, immunoprecipitation: 13–14 h, performed overnightBox 1, checking sonication efficiency: 1.5 hDay 2Steps 24–28, immunoprecipitation: 3–4 hStep 29, reverse cross-linking: 6–7 h or overnightDay 3Steps 30–37, DNA purification: 2–3 hSteps 38 and 39, DNA detection: 2–3 h

antIcIpateD resultsA high chromatin yield at Step 16 and, particularly, a high specific DNA enrichment at Step 39 are indicators of an effective ChIP protocol. By using this protocol, a chromatin yield of ~25 µg of DNA per g of SDX should be expected. A similar yield is anticipated for other woody species. Chromatin yields lower than 10 µg/g of source tissue usually will not give useful DNA enrichment. By using this protocol with a specific TF antibody on wood-forming tissue, 8–10-fold enrichment of TF-bound genomic fragments over nonspecifically precipitated DNAs should be achieved, as we find for NAC-related TFs associated with

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wood formation (Fig. 5). Usually, a fivefold enrichment gives good-quality DNA for reliable identification by ChIP-qPCR or ChIP sequencing13. Much higher enrichment can be obtained for histone-DNA associations. For example, our protocol demonstrated that H3K9ac is over 50-fold enriched at the PtrACTIN locus in wood-forming tissue of P. trichocarpa (Fig. 6).

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

acknoWleDGMents 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.

autHor 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.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Sarkanen, K.V. Renewable resources for the production of fuels and chemicals. Science 191, 773–776 (1976).

2. Chiang, V.L. From rags to riches. Nat. Biotechnol. 20, 557–558 (2002).3. Ragauskas, A.J. et al. The path forward for biofuels and biomaterials.

Science 311, 484–489 (2006).4. Hinchee, M. et al. Short-rotation woody crops for bioenergy and biofuels

applications. In Vitro Cell Dev. Biol. Plant 45, 619–629 (2009).5. Evert, R.F. Esau’s Plant Anatomy: Meristems, Cells, and Tissues of the Plant

Body: Their Structure, Function, and Development 3rd edn. (John Wiley & Sons, 2006).

6. Li, Q. et al. Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa. Proc. Natl. Acad. Sci. USA 109, 14699–14704 (2012).

7. Lin, Y.-C. et al. SND1 transcription factor–directed quantitative functional hierarchical genetic regulatory network in wood formation in Populus trichocarpa. Plant Cell 25, 4324–4341 (2013).

8. Solomon, M.J., Larsen, P.L. & Varshavsky, A. Mapping protein–DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53, 937–947 (1988).

9. Li, W. et al. DNA methylation and histone modifications regulate de novo shoot regeneration in Arabidopsis by modulating WUSCHEL expression and auxin signaling. PLoS Genet. 7, e1002243 (2011).

10. Kaufmann, K. et al. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat. Protoc. 5, 457–472 (2010).

11. Saleh, A., Alvarez-Venegas, R. & Avramova, Z. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat. Protoc. 3, 1018–1025 (2008).

12. Bowler, C. et al. Chromatin techniques for plant cells. Plant J. 39, 776–789 (2004).

13. Zhu, J.Y., Sun, Y. & Wang, Z.Y. Genome-wide identification of transcription factor-binding sites in plants using chromatin immunoprecipitation followed by microarray (ChIP-Chip) or sequencing (ChIP-seq). Plant Signalling Networks: Methods and Protocols (eds. Wang, Z. & Yang, Z.) 173–188 (Humana Press, 2012).

14. Freudenberg, K. Biosynthesis and constitution of lignin. Nature 183, 1152–1155 (1959).

15. Lockhart, J. Breaking Down the complex regulatory web underlying lignin biosynthesis. Plant Cell 25, 4282–4282 (2013).

16. Kim, T.H. & Ren, B. Genome-wide analysis of protein-DNA interactions. Annu. Rev. Genomics Hum. Genet. 7, 81–102 (2006).

17. Farnham, P.J. Insights from genomic profiling of transcription factors. Nat. Rev. Genet. 10, 605–616 (2009).

18. Zhou, V.W., Goren, A. & Bernstein, B.E. Charting histone modifications and the functional organization of mammalian genomes. Nat. Rev. Genet. 12, 7–18 (2011).

19. Margueron, R., Trojer, P. & Reinberg, D. The key to development: interpreting the histone code? Curr. Opin. Genet. Dev. 15, 163–176 (2005).

20. Pfluger, J. & Wagner, D. Histone modifications and dynamic regulation of genome accessibility in plants. Curr. Opin. Plant Biol. 10, 645–652 (2007).

21. Nystedt, B. et al. The Norway spruce genome sequence and conifer genome evolution. Nature 497, 579–584 (2013).

22. Neale, D.B. et al. Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies. Genome Biol. 15, R59 (2014).

23. Amborella Genome Project. The Amborella genome and the evolution of flowering plants. Science 342, 1241089 (2013).

24. Tuskan, G.A. et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 1596–1604 (2006).

25. Myburg, A. et al. The Eucalyptus grandis Genome Project: Genome and transcriptome resources for comparative analysis of woody plant biology. BMC Proc. 5, 20 (2011).

26. Müller, C.W. Transcription factors: global and detailed views. Curr. Opin. Struct. Biol. 11, 26–32 (2001).

27. Chen, K. & Rajewsky, N. The evolution of gene regulation by transcription factors and microRNAs. Nat. Rev. Genet. 8, 93–103 (2007).

28. Hobert, O. Gene regulation by transcription factors and microRNAs. Science 319, 1785–1786 (2008).

29. Lu, S. et al. Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa. Proc. Natl. Acad. Sci. USA 110, 10848–10853 (2013).

30. Courtois-Moreau, C.L. et al. A unique program for cell death in xylem fibers of Populus stem. Plant J. 58, 260–274 (2009).

31. Hu, W. et al. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat. Biotechnol. 17, 808–812 (1999).

32. Li, L. et al. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc. Natl. Acad. Sci. USA 100, 4939–4944 (2003).

33. Lu, S. et al. Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 17, 2186–2203 (2005).

34. Shi, R. et al. Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes. Plant Cell Physiol. 51, 144–163 (2010).

35. Mochida, K. et al. TreeTFDB: An integrative database of the transcription factors from six economically important tree crops for functional predictions and comparative and functional genomics. DNA Res. 20, 151–162 (2013).

36. Jin, J., Zhang, H., Kong, L., Gao, G. & Luo, J. PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res. 42, D1182–D1187 (2014).

37. Chen, H. et al. Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis. Proc. Natl. Acad. Sci. USA 108, 21253–21258 (2011).

38. Chen, H. et al. Monolignol pathway 4-coumaric acid:coenzyme A ligases in Populus trichocarpa: novel specificity, metabolic regulation, and simulation of coenzyme A ligation fluxes. Plant Physiol. 161, 1501–1516 (2013).