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In multi-cellular organisms, gene expression is orchestrated by thousands of transcription factors (TF). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a robust tool to investigate gene expression because this technique profiles in vivo protein-DNA interaction at a genome-wide scale. Eight years after the first ChIP-seq paper, there are limited reports of ChIP-seq experiments in plants, especially for sequence-specific DNA binding TFs This lag greatly prevents our understanding of transcriptional regulation in an entire kingdom. In order to bridge the technical gap, we describe a ChIP-seq procedure that we have successfully applied to dozens of sequence-specific DNA binding TFs. The basic protocol includes procedures to isolate nuclei, sonicate chromatin, immunoprecipitate TF-DNA complex, and recover ChIP-enriched DNA fragments. The support protocol also describes practices to optimize library preparation by a gel-free DNA size selection. Lastly, examples are given to optimize library amplification using real-time PCR.
ChIP-seq is a robust approach to investigate transcriptional regulation in vivo (Ren et al., 2000; Johnson et al., 2007). It is an improvement over ChIP-chip with better signal-to-noise ratio, unambiguous and genome-wide sequence information. ChIP-seq can be applied to any protein that is associated with chromatin, including histones, chromatin remodelers, RNA polymerases, mediators, general and sequence-specific DNA binding transcription factors. Sequence-specific DNA binding TFs are of particular interest because of their large number and functional diversity; yet they are also the most technically challenging to study because of their low protein abundance and rapid turnover in cells. The progress of ChIP-seq in plants has lagged far behind other eukaryotic organisms: a mere 1% of all ChIP-seq samples submitted to Gene Expression Omnibus (GEO) are from plants. Eight years after the publication of the initial ChIP-seq paper, fewer than 30 sequence-specific DNA binding TFs in Arabidopsis have been subjected to ChIP-seq (Heyndrickx et al., 2014; Fan et al., 2014; Pfeiffer et al., 2014). This unit aims to bridge this gap by providing a technical guidance of ChIP-seq experiments in plants.
This protocol describes a step-by-step ChIP procedure. It contains experimental modules for cross-linking samples using formaldehyde, preparation of antibody beads, isolation of nuclei, sonication of chromatin, immunoprecipitation of the TF-DNA complex, and recovery of ChIP-enriched DNA fragments. Arabidopsis etiolated seedlings and floral buds are used as examples here. The protocol can also be applied to many other tissues and plant species. A general overview of ChIP-seq procedures is illustrated in Figure 1, and several key steps are shown in Figure 2.
Use fresh formaldehyde to achieve efficient and reproducible fixation. Avoid handling samples with amine-rich solutions such as Tris buffer before fixation. The amount of samples needed depends on the TF expression level and tissue type. In our hands, 3–4 grams of etiolated seedlings or 1–2 grams of flower buds are sufficient to ChIP many sequence-specific DNA binding TFs. More tissue can be used if the abundance of the TF is very low.
Fixation time can be adjusted according to the thickness of plant tissue. In general, do not cross-link samples for more than 25 minutes for Arabidopsis tissues; otherwise over-fixation may decrease sonication efficiency.
Use uncross-linked sample as a control. A smeared band or bands of higher molecular weight is expected in cross-linked samples.
Sample can be stored for at least a few weeks.
Start this step 6+ hours before sonication.
Choose Dynabeads® protein G or protein A according to their affinity for antibody species and subclasses of IgG.
The antibody is a key factor to a successful ChIP. Based on our experience, GFP tagged TFs ChIPped with a polyclonal GFP antibody (Thermo Fisher Scientific, cat. # A-11122) or FLAG tagged TFs ChIPped with a monoclonal FLAG antibody (Sigma Aldrich, cat. # F1804) usually give a strong signal and clean background. It is also crucial to include “mock IP” controls. If an antibody against a native protein is used in ChIP, mock IP can be carried out by using IgG from the same species in which the antibody was raised. If an antibody against an epitope tag is used, mock IP can either use the same antibody to ChIP wild-type samples, or even better use the same antibody to ChIP transgenic plants expressing the epitope tag alone.
Carry out all procedures at 4 °C from step 11 to step 30. Many buffers contain detergent. Avoid foaming in all steps.
For light-grown samples, repeat steps 17 and 18 as needed if too many chloroplasts are present as evidenced by a deep green pellet.
This is to increase sonication efficiency (F. Turck, personal communication).
Sonication efficiency of Bioruptor® may vary greatly by model. Adjust settings accordingly for best performance. Carry out a western blot to ensure sonication setup does not destroy protein or protein-DNA complex. Wear ear protection as sonication generates high-frequency sound waves that may damage hearing.
The manufacturer of 5 PRIME has a detailed protocol on how to handle Phase Lock Gel®. Handle phenol/chloroform/isoamy alcohol in chemical hood.
Use NaCl instead of NaOAc to prevent precipitation of SDS.
Dried pellet may become transparent.
Unlike input DNA, concentration of ChIPped DNA is often very low. ChIP of low abundance TFs from 3 grams of etiolated Arabidopsis seedlings may frequently enrich less than 5 ng of fragmented DNA.
Many suppliers such as Illumina and New England BioLabs already have detailed protocols to construct ChIP-seq libraries. To avoid redundancy this protocol focuses on how to optimize the size distribution of ChIP-enriched DNA. A tight size distribution of ChIP-enriched DNA is desired. Excluding long fragments improves cluster formation on Illumina sequencer. Tight size distribution of ChIP-enriched DNA also allows more accurate prediction of fragment length and peak calling by analysis packages. A few examples are shown in Figure 3.
The volume of AMPure beads can be increased or decreased for a smaller or larger DNA size cutoff, respectively. We recommend empirically testing DNA size fractionation within the range from 0.65 volume to 0.85 volume of beads.
Supernatant contains DNA fragment smaller than 500 bp.
The purpose is to remove leftover ethanol from previous wash steps.
If TapeStation is not available or if DNA concentration is too low, size-fractionate input sample from step 42 of the basic protocol and run a gel to infer the size of ChIP-enriched DNA. SYBRgold will interfere with DNA migration in the gel (Figure 3B vs Figure 3C). Therefore, we recommend running samples in ethidium bromide gel, or stain gel with SYBRgold after the run.
* Add fresh before experiments. Same for extraction buffers II and III, nuclei lysis buffer and ChIP dilution buffer. Handle β-mercaptoethanol in chemical hood.
Chromatin immunoprecipitation has been used to study protein-DNA interactions for almost 40 years (Jackson, 1978). The technique has evolved multiple times by the advances of DNA detection methods. In the early days, ChIPped DNA were examined by low throughput approaches such as Southern blot or PCR (Solomon et al., 1988; Orlando and Paro, 1993; Hecht et al., 1996). Understanding protein-DNA interactions at a genome-wide scale only became possible after the invention of ChIP-chip by Ren et al. (Ren et al., 2000) and Iyer et al. (Iyer et al., 2001), via the development of high-density DNA microarrays (Schena et al., 1995). ChIP-chip has facilitated numerous breakthroughs in biology, including elucidation of transcriptional regulatory networks in Saccharomyces cerevisiae (Lee et al., 2002) and characterization of DNA regulatory sequences in human (Consortium et al., 2007). On the other hand, ChIP-chip suffers from all the limitations of microarray technology. For instance, the design of array depends on a priori knowledge of genome sequence; high background signal makes it difficult to detect weak protein-DNA interactions; cross-hybridization is problematic, especially for highly homologous or repetitive regions; to obtain micrograms of DNA for hybridization, ChIPped DNA needs to be amplified by several orders of magnitude and the process may introduce biases. In 2007, Johnson et al. addressed these issues by combining ChIP with high-throughput DNA sequencing technologies (Johnson et al., 2007). With the help of sophisticated statistical analysis (Landt et al., 2012), ChIP-seq has become the most prevalent method to confidently identify protein-DNA interaction in vivo. More recently, Rhee et al. developed an improved ChIP-seq protocol called ChIP-exo (Rhee and Pugh, 2011). ChIP-exo utilizes 5'-to-3' exonuclease to trim DNA to the precise protein binding location. Exonuclease also cleans up ChIP background by digesting naked DNA. However, the extra steps associated with exonuclease digestion often result in greater sample loss and subsequently low library complexity. As an answer to this problem, He et al.'s ChIP-nexus protocol improves the ligation efficiency during library preparation, and tracks DNA over-amplification by unique, randomized barcodes (He et al., 2015).
Several factors contribute to the difficulty of performing ChIP-seq in plants. First, unlike animal cells in which nuclei can be extracted by mild detergents, extraction of plant nuclei usually requires vigorous physical disruption because of cell walls. This is arguably the step that causes the greatest sample loss in a ChIP procedure and prevents parallel handling of samples. Secondly, plant tissues often contain high level of phenolic compounds and polysaccharides, which may be problematic for PCR amplification during library preparation. Thirdly, there is limited selection of ChIP-grade antibodies in plants, and as a consequence researchers have to spend months to generate transgenic lines to express epitope-tagged proteins before ChIP-seq experiments can be carried out. Besides this protocol, several other laboratories have also published detailed procedures of ChIP-chip or ChIP-seq for Arabidopsis (Kaufmann et al., 2010; Reimer and Turck, 2010). We recommend readers combine knowledge of all protocols to decide the best practice.
ChIP-seq is a long procedure. We include multiple quality control steps in the protocols to ensure its success. Formaldehyde fixation is the first key step in the experiment. Both under- and over-fixation will result in inefficient ChIP of transcription factors. Therefore, we suggest using fresh formaldehyde, applying accurate control of cross-link time, and measuring the level of cross-link by western blot (step 5, basic protocol). Ideally, sonication should shear DNA to a consistent, tightly distributed size smaller than 500 bp whereas still preserving the protein-DNA complexes. The size distribution of sheared DNA can be examined by TapeStation (step 43, basic protocol), and can be further tightened by a bead-based size selection (support protocol 1, Figure 3). Antibody quality is crucial for successful ChIP. We suggest monitoring the effectiveness of IP by examining the presence of TF-of-interest after IP, wash, and final elution (step 33, basic protocol). Finally, over-amplification may bias library composition. We suggest determining the number of PCR cycles by real-time PCR to avoid over-amplification of ChIP-seq libraries (Figure 4).
Using the basic protocol, 3 grams of etiolated seedlings is expected to yield more than 10 μg of input DNA. Depending on the TF and antibody, the ChIP-enriched DNA can be 5 ng or less. Light-grown samples especially flowers will usually yield more DNA. In most cases, twelve or fewer cyclers of PCR is sufficient to amplify enough DNA for sequencing.
Once the samples are harvested and cross-linked, it takes 4 – 6 days to complete ChIP. The most time consuming steps in the procedure are IP (usually overnight, can be shortened to a few hours if the immunoprecipitated protein is very abundant), reverse cross-linking (6 hours to overnight), ethanol precipitation of reverse cross-linked DNA (3 hours to overnight), and adaptor ligation (overnight).
We thank F.Turck, K.N.Chang, S.C.Huang, H.Qiao, U. Padmale, N. Krogan, M. Zander, M. Lewsey, and M. Urich for providing useful discussions on ChIP and DNA size fractionation. L.S. was supported by Salk Pioneer postdoc fellowship. This work was supported by grants from NSF (MCB-1024999 to J.R.E.). J.R.E. is an investigator of the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation.