technology feature What to do about those immunoprecipitation blues ChIP-seq, DIP-seq and related techniques are informative genome-wide assays, but they don’t always work as planned. Vivien Marx

hen the chromatin immunoprecipitation (ChIP) Wor DNA immunoprecipitation (DIP) blues hit, it’s good to realize you’re not alone. ChIP is part of the winding path of characterizing gene function. To find where transcription factors (TFs) or histone marks bind to genomic loci of interest, labs might choose ChIP-seq, which involves cross-linking, then shearing chromatin, immunoprecipitation with an antibody that recognizes a TF or histone mark of interest, followed by sequencing. DIP plus sequencing (DIP-seq) can be used to locate DNA changes such as methylation. ChIP-seq and DIP-seq have been the “cornerstone of epigenetics research” since the field moved from gene-specific epigenetics to the genome- wide approaches of epigenomics, says Colm Nestor, a researcher at Linköping University. Both techniques are cheap, relatively easy to set up, and widely used “because they work very well—when well controlled, that is,” he says. With ChIP-seq, antibodies are sometimes Immunoprecipitation can be used to locate DNA changes such as methylation or places where transcription not as specific as a lab might have first factors bind, and can involve the struggle with artifacts. Credit: A. Lentini, Linköping University assumed1. Some genomic loci can be ‘sticky’ in too many wrong ways, or masked protein epitopes are not ‘sticky’ when they should be. As Dana Farber Cancer Institute researchers differential binding. His former PhD student sharply defined peaks in open chromatin Xiaole Shirley Liu and Clifford Meyer point Dhawal Jain, now a postdoctoral fellow in regions, where nucleosomes are usually out, some of ChIP-seq’s lurking bias issues the lab of Harvard University researcher lacking. The peaks “were striking to us,” he are a lack of antibody specificity, issues Peter Park, calls ChIP-seq experiments says, but not striking in a good way. with chromatin shearing, cross-linking and essential in functional genomics because They investigated and identified around sequencing2. To battle ChIP-seq and DIP-seq of the insight they deliver on the biological 3,000 loci that had non-specific ChIP artifacts, experimental troubleshooting is roles of TFs. ChIP-seq is his first experiment enrichment3. To validate the experiment, unavoidable. Challenges are not always the of choice for characterizing any novel DNA- they knocked out these two remodeling antibody’s fault, but when labs troubleshoot, binding factor, and it’s crucial to get data factors, and saw similar ChIP-seq profiles. antibodies need to be considered. Here “with high confidence.” Both he and Becker They had found false positive signals: are two cases of immunoprecipitation (IP) got their data confidence rattled. Phantom Peaks, which affect their data headache, and researchers at two antibody When Jain was in the Becker lab, the quality and, possibly, data in many labs and companies comment on ChIP-seq and researchers studied nucleosome remodeling. databases. Most Phantom Peak sites were at DIP-seq challenges. Nucleosomes are complexes of DNA promoters, where many proteins bind with wound around histones, and nucleosome disordered domains, says Becker, and where ChIP-seq data confidence rattled remodelers open transcription ‘windows’ at non-specific antibody-binding is the likely ChIP-seq gives labs a crack at genome-wide certain DNA locations. The remodelers only culprit. These low ChIP peaks typically analysis because “hundreds and thousands transiently interact with DNA, so they are go unnoticed in experiments with strong, of binding sites are displayed in context,” tough to capture and map. Applying ChIP- specific profiles, says Becker. “But as soon says Peter Becker, a researcher at Ludwig seq with antibodies directed at parts of these as your ChIP reaction is inefficient for any Maximilians University in Munich. For remodeling factors, the team expected to reason, and your specific peaks are low, example, the comparison of ChIP-seq see, and saw, broad peaks corresponding to Phantom Peaks show up.” The team advises profiles lets labs generate hypotheses about the remodelers, says Jain. There were also interpreting ChIP-seq peaks with caution.

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Their list of Phantom Peaks is part of their Protein of interest High-occupancy site paper as a resource for others. To rule out Phantom Peaks, they recommend knockout Epitope 1 Epitope 2 or knockdown experiments. Sometimes these are not feasible if the factor under investigation is essential, says Jain. Input controls can be helpful, says Becker. Even when labs have established antibody Antibody 1 specificity through western blots (WB) and ChIP immunofluorescence (IF), non-specific binding can be a problem. The team ran into just such issues. “To add more to it, oftentimes, the antibody that works in one validation assay does not work in the other,” says Jain. Labs then try to validate in either Antibody 2 ChIP WB or IF and try to reproduce the ChIP-seq profile with an orthogonal antibody. But the orthogonally generated ChIP-seq profiles do not necessarily remove Phantom Peak Real peak: factor binds Phantom Peak: false positive data artifacts. They also found enrichment of a ChIP-seq signal with completely unrelated antibodies, such as anti-GFP. Overall, Jain says he sees a growing awareness about such ‘hyper-ChIPable’ regions of the genome that ChIP-seq data can be littered with ‘Phantom Peaks’ that do not correspond to the immunoprecipitated lead to false positive data. One issue with the protein of interest. The use of two orthogonal antibodies for pulldown can be a failsafe. But that does nucleosome-remodeling factors they were not always work. For example, the antibodies might bind non-specifically to proteins occupying the open studying, says Becker, notably ACF1 and chromatin. Credit: D. Jain, Harvard Medical School; P. Becker, LMU Munich; E. Dewalt/Springer Nature RCF1, is that they do not cross-link well to chromatin given their transient interactions with DNA. Fundamentally, the antibodies in their experiment were fine, he says, but there (5hmC) antibody does the same at highly has been the standard control, says Nestor, was non-specific binding. Since that first modified regions of the genome. referring to the chromatin sample pre-IP. As work, they have tethered the protein ACF1 In their analysis of published DIP-seq a postdoctoral fellow, he remembers being to DNA with a fused DNA-binding domain studies, they saw that low-abundance advised to avoid IgG controls because they and could “ChIP it” just fine, he says. The genomic modifications, such as add too much noise to experiments. But the issue was essentially noise related to non- 5-formlycytosine and 5-carboxylcytosine, researchers say input is a highly inconsistent specific antibody-binding. have the highest false positive rates. control and that the 5-modC landscape They had been looking at just those in mammalian genomes has been greatly DIP-seq peculiarities modifications to explore the role of DNA overestimated by DIP-seq. Although Nestor Antibody-based techniques are useful for methylation in human T cells. They noticed and others use computational tools to filter investigating newly discovered epigenetic “peculiar” binding patterns in their DIP the DIP-seq data, he is concerned about changes such as N6-methyladenine, since immunoglobulin G (IgG) controls. As much large errors creeping into datasets. it’s not hard to raise antibodies against new as 95% of published work does not include Another challenge is bacterial modifications, says Linköping University’s an IgG control, they found. “As a researcher contamination. Cell cultures, often infected Nestor. “Consequently, researchers are that has consistently used DIP-seq without an with Mycoplasma and Escherichia coli, spared the considerable hassle of having to appropriate IgG control, I am in no position show high levels of the modification N6- develop modification-specific methods each to point fingers at others,” says Nestor. methyladenine (6mA), which is rare in time we detect a novel epigenetic mark in The team validated their results with mammals and frequent in microbes. 6mA mammals.” He has been using DIP-seq for embryonic stem cells lacking 5mC and “is still an enigma,” says Nestor. It’s hard to a decade, immunoprecipitating methylated 5hmC modifications. The antibodies still detect, no one technique detects it reliably DNA chunks and sequencing them. Nestor, bound to STR sites. Perhaps single-strand in mammals, and it remains a puzzle to be his PhD student Antonio Lentini, and others DNA is binding to IgG antibodies. Lentini explored. He wonders “what the function on the team didn’t set out to find a major speculates that the STR binding may offer of this incredibly shy member of the DNA DIP-seq flaw, but that’s what happened. functional clues and that antibodies might modification club could be.” Andrew According to the team, between 50% and be engineered to inhibit this binding. “Until Chalmers, who is at the University of Bath, 99% of enriched regions in DIP-seq data then, our best option is to be aware of recommends that when scientists select might be false positives. They found non- these issues and control for them,” he says. antibodies for experiments, including ChIP- specific enrichment of areas of the genome Avoiding false positives and correcting for seq, they look at previous experiments with called short tandem repeats (STRs)4. Other this observed off-target binding, the team these reagents. Chalmers co-founded CiteAb, known sources of bias include the fact that says, is best handled by normalizing DNA- an online resource for finding published the 5-methylcytosine (5mC) antibody labs modification enrichment to an IgG control, literature and company information about mainly use preferentially enriches genomic which increases the signal-to-noise ratio antibodies. The “five pillars paper” on antibody regions of low CG content and the most threefold, and labs can detect more subtle validation is not perfect, he says, but is a good frequently used 5-hydroxymethylcytosine changes in DNA modifications. ‘Input’ framework for discussing and analyzing

290 Nature Methods | VOL 16 | APRIL 2019 | 289–292 | www.nature.com/naturemethods technology feature performed validation5. Often, the planned interact with DNA, such as histones. But experiment will not be among those previously transient interactions, or those mediated done, but the available data and hard-earned through a protein complex, may not be lessons from others can be useful guides. well captured this way. More optimized, intermediate to long-range cross-linkers Knockout/knockdown might be better. “Using these, either singly Experiments with antibodies blend on-target or in combination with formaldehyde, may and off-target effects, says Velibor Savic, be a critical step in boosting cross-linking who leads the research and development efficacy to the level usable for subsequent teams at the antibody company Abcam. steps,” he says. A protein can be cross- Experimentalists need to reduce off-target linked to the DNA it interacts with and signals, also in ChIP, he says. ChIP-seq is there can be non-specific cross-linking sensitive, which can render off-target signals with nearby DNA sequences, which would more visible than other techniques. That be randomized across the cell population antibodies from mouse, goat and sheep bind gives knockdowns and knockouts critical and blend with the background signal, says better to protein G, he says. Labs should use importance in ChIP-seq. “With this,” he Savic. But unexpected peaks in sequencing beads tested and validated for ChIP-seq. “I says, “it is easier to tease out target from depend on the immunoprecipitated target. always recommend using magnetic beads, not off-target signals and identify the correct With non-random interactions such as those only for convenience, but also because they positive peaks.” with sequence-specific promoters, a DNA typically yield lower background than agarose segment can be ‘pulled down’ with the target or Sepharose beads,” he says. protein even when it is not directly bound to Given differing methods for chromatin it. Shearing and sonication quality are other fragmentation and DNA library prep, important factors. Some structures will cross- says Fry, ChIP-seq experiments have an link or sonicate better or worse, depending inherently high non-specific background on chromatin condensation, compaction, signal with fragments that “come along for protein–DNA interaction and many other the ride.” They stick to the protein, beads factors, he says. These might end up in the or tubes even when the antibody is highly IP reaction as fragments larger than 1 kb of specific and binds strongly to the protein DNA with an elevated chance of off-target of interest. “This is why it is important to pulldown. And “sonication-resistant” regions always include a ‘negative control’ in the can deliver false positive results. experiment,” he says. ChIP protocols differ mainly in the method For ChIP-qPCR, he and his team used to fragment chromatin, says Chris Fry, recommend using normal IgG as a negative who directs epigenetics and ChIP product control to measure non-specific enrichment. Even transient interactions can have a development at the antibody company Cell For ChIP-seq, in his view, the preferred profound effect. “Such interaction may be Signaling Technology. Sonication-based negative control is input chromatin. As a poorly captured via ChIP,” says Savic. Results methods subject the chromatin to harsh, positive control for both ChIP-qPCR and might blend into background because only denaturing conditions involving high heat, ChIP-seq, labs can look at genomic regions some of the interaction sites are occupied at much detergent and strong shearing force that known to bind the protein of interest or a given time. It can help to explore ways to can damage antibody epitopes and chromatin use the antibody against histone H3 as a stabilize the interactions. Using knockdown integrity. These conditions tend to not agree ‘universal’ positive control, which will, he says, or functional knockout of the target would with TFs and cofactors, he says. Labs can try enrich for any genomic locus if the chromatin address both the off-target binding and lower detergent concentrations or cross-link was intact and the IP worked as planned. “It non-specific interactions. “In most cases, with different formaldehyde concentrations. always amazes me how many people perform this would nicely define the locations of For successful ChIP, chromatin fragment their ChIP experiments without any positive the target protein within the experimental length and integrity matter, says Fry. Many or negative controls,” says Fry. sample and the patterns of background in labs over-sonicate chromatin in the belief the absence of it,” he says. ChIP delivers a that shorter fragments are more suitable for Antibody strategies static picture of the average target-to-DNA ChIP-seq. “However, this over-sonication Enrichment in the wrong places, says interaction profile in a very dynamic system, damages the chromatin and often results in Fry, is one reason that he recommends he says. “As much as one can’t judge a movie complete loss of target protein and target loci using a highly validated antibody for from one still image, one can’t draw dynamic enrichment,” he says. ChIP experiments. Labs can ask vendors conclusions from a single ChIP experiment,” Enzymatic digestion with micrococcal about validation for ChIP or ChIP-seq he says. The journey to functional insight nuclease cuts the linker region between and then closely study the data for levels only begins with ChIP-seq data, says Savic. nucleosomes and fragments the chromatin of enrichment and the signal-to-noise in more gently, and is likely more amenable to those assays. A “dirty” WB or incorrect Cross-linking ChIP for TFs and cofactors, says Fry. One localization in IF or immunohistochemistry Cross-linking, says Savic, is “one of the most “pushback” he hears from customers is that (IHC) warns of potential non-specific critical variables” that need optimization the method is biased toward open chromatin. binding in ChIP assays, he says. With in any ChIP program. Formaldehyde is That is not the case, he says. Sonication histone-modification antibodies, Fry says the standard cross-linker, but as a short- might bias more toward open chromatin than labs should look for data that support range cross-linker it has limitations. It’s best enzymatic fragmentation. Labs have to choose specificity to the studied modification such suited for linking DNA and proteins and for between protein A and protein G beads for as histone peptide array data. This assay localizing proteins that directly and strongly IP. Both work well for rabbit antibodies, but comprehensively determines antibody

Nature Methods | VOL 16 | APRIL 2019 | 289–292 | www.nature.com/naturemethods 291 technology feature specificity to a modification site and for IF or IHC, an antibody might work in helps reveal the effect of neighboring ChIP, since these assays involve cell fixation modifications on antibody binding. Using and epitope recognition of a protein in its proprietary histone peptide arrays, Cell more native conformation. If an antibody Signaling scientists test the specificity of shows incorrect localization in IF or IHC or the histone methyl-lysine, acetyl-lysine both, then cross-reactivity in a ChIP assay and methyl-arginine antibodies, and “we becomes more likely, he says. “Unfortunately, will provide data for any antibody upon not all ‘validation’ is created equally,” he says. customer request.” For planning a ChIP For example, some companies use the “ChIP experiment, Fry recommends carefully Grade” label but don’t provide any validation selecting protocols and reagents. “Histone data to support it. proteins, which are abundant and bind very strongly to DNA, are very forgiving and Antibody next relatively easy to ChIP,” he says. TFs are Hiroshi Sasaki, a postdoctoral fellow at less abundant, bind less stably to DNA and Harvard University’s Wyss Institute, battles are more difficult to ChIP. Transcription noise and false positives in his single-cell cofactors that often do not directly contact super-resolution imaging of chromosomes. DNA and may belong to large protein ChIP-seq is powerful for investigating complexes are the most difficult target chromatin state genome-wide and provides proteins to CHIP. Avoiding false positives and correcting for much data to statistically analyze, he says. Commenting on DIP-seq flaws, Savic off-target binding is best handled by normalizing Many labs have begun using ‘pulldown’ says epitopes tend to be larger targets DNA-modification enrichment to an IgG assays in single-cell biology, where than one DNA modification. Histone- control, says the Nestor lab. Credit: A. Lentini, improving signal-to-noise is tough. Most modification antibodies may be specific Linköping University single-cell data suffer from non-specific to a particular modification, such as lysine signals that can’t be cancelled out as they tri-methylation, but the antibody recognizes can in population assays. Sasaki and his around eight amino acids with the modified positive peaks, more antibody produces colleagues see non-specific signals in target among them. The same holds for more false positives. Boosting a signal with their single-molecule localization imaging nucleotide modifications. “It is safe to a mix of antibodies raised against the same with antibody labeling and mislocalized expect that the composition of the flanks protein can help, he says. If raised against antibodies, but there is noise with oligo would affect the binding of the antibody to non-overlapping protein segments, these probes, too. All issues, including the a certain extent,” says Savic. Potentially, he antibodies can interact with the target specificity of antibodies and oligo probes, says, using additional antibodies to the same at the same time, which can translate to lot-to-lot stability, protocol-to-protocol target with different affinities to flanking improved retention during IP-bead washing optimization and experimental validation, sequences would alleviate the problem. and better signal-to-noise ratios. But he “are getting more and more critical in Scientists shopping for ChIP antibodies, he advises caution: a polyclonal antibody is modern biology,” he says. says, should pick the antibody that will bind “usually raised only against the peptide Neither antibody selection nor validation to the target in the cross-linked chromatin, of limited size, and therefore may in fact is easy, says Nestor. There is also a limit to recognize it in its native format and remain act equivalently to monoclonal in ChIP the number of validation experiments a bound to beads and to the target throughout assays.” That’s when it helps to know the researcher can be expected to do before the precipitation and wash. “Ideally, all four immunizing peptide to confirm coverage scientific community will accept results. variables would be tested before embarking of the protein target’s distinctive part. The DIP-seq caveats he and his group found were on a larger ChIP-seq experimental use of multiple antibodies does not apply “not with the antibodies themselves, per se, program,” he says. to histone modifications, where only one but in how they controlled the experiments antibody can bind per target because only in which they used those antibodies,” More is not better one target exists per protein. says Nestor. Labs can consider antibody- Validation should always include antibody free techniques and check independent titration. “Customers still tell me they Validated or not replication in other labs. “But as we found, need as much as 5 to 10 μg of antibody per Some antibody companies such as Abcam if the error is pervasive enough, it can hide immunoprecipitation in their ChIP assay,” and Cell Signaling Technology validate in plain sight, through the consistency of the says Fry. “This is simply not true. And more antibodies for ChIP. When buying a non- error across studies,” he says. “So I suppose, is not always better.” For many antibodies, validated antibody, a lab needs to test and caveat emptor.” ❐ both polyclonal and monoclonal, he and his optimize the antibody for the ChIP assay, team find the optimal range of antibody is says Fry, for example, to titrate the antibody Vivien Marx 0.5–2 μg per IP. Adding too much antibody and look at enrichment of known target Technology editor for Nature Methods. can decrease target enrichment. genes using ChIP-qPCR, to use controls and e-mail: [email protected] Adding more antibody to troubleshoot to consider testing more than one antibody will deliver variable results, says Savic. It to a target protein or protein complex. Just Published online: 28 March 2019 might appear a promising way to raise because an antibody is not validated for ChIP https://doi.org/10.1038/s41592-019-0365-3 signal; “however, it may also increase the doesn’t mean it will fail ChIP, he says. “It may References signal rising from off-target interactions.” just not have been tested, and you will be the 1. Park, P. J. Nat. Rev. Genet. 10, 669–680 (2009). Off-target signals might blend with the first to test it,” he says. A scientist can consult 2. Meyer, C. A. & Liu, X. S. Nat. Rev. Genet. 11, 709–721 (2014). 3. Jain, D. et al. Nucleic Acids Res. 43, 6959–6968 (2015). background noise, but once the off-targets IP data for indications that an antibody is 4. Lentini, A. et al. Nat. Methods 15, 499–504 (2018). become more prominent and resemble more likely to work in ChIP. If validated 5. Uhlen, M. et al. Nat. Methods 13, 823–827 (2016).

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