Applications of chromatin immunoprecipitation-basedepigenomic tools in nutritional studies

Jiejun Wu and Tim H-M Huang

© 2008 International Life Sciences Institute

Recent studies support an important role of epigeneticvariations in linking early nutritional involvement toadult susceptibility to disease. The details of molecularpathways associated with this susceptibility later in life arenot clear. Current applications of epigenomics tools,such as chromatin immunoprecipitation (ChIP)-basedapproaches, in the studies of both DNA-binding factorsand histone modifications have produced comprehensiveand intriguing results. This rapid development of ChIP-based technologies makes it possible to examine diet-related epigenetic alterations on a genome-wide scale.

EPIGENETICS: A POSSIBLE LINK BETWEENNUTRITION AND DISEASE

From both human epidemiologic data and mousemodels, increasing evidence shows a strong relationshipbetween early nutritional stimuli and chronic diseases inadulthood, including cardiovascular disease, type 2 dia-betes, obesity, and cancer.1–3 Early nutritional perturba-tion can influence the epigenetic status of the genome andlead to later changes in phenotype.4,5 Therefore, epige-netic variations, which might be modified by differentialdietary intake, comprise the potential mechanistic linkbetween early nutritional perturbation and adult suscep-tibility to certain diseases.

The word “epigenetics” was originally coined to cat-egorize those phenomena that could not be explained bygenetic principles.6 Currently, epigenetics is defined as thestudy of those phenomena that can change gene expres-sion and cell phenotype without altering the genotype,i.e., the DNA sequence.7,8 The link between epigeneticsand nutrition may become clearer if epigenetics is viewedin the context of the three layers of regulatory networks

(Figure 1). In this model, the core layer is the DNAsequence, which contains protein-coding genes, regula-tory units, and even some non-coding elements ofunknown functions. The genetic information in all cellsof one organism is the same, and therefore the differentialexpression of cells has to be based on more adjustablesystems. The outer layer of the regulatory networkincludes various signal transduction pathways and othermetabolic processes. The core layer contains relativelypermanent information, which can be stably transferredfrom parents to offspring, whereas the main feature of theouter layer is the flexibility to control the temporal andspatial expression of the genome. Between these twolayers is an intermediate, epigenetic region, the definitionof which is still in evolution but at least includes DNAmethylation and histone modifications. Different fromthose of the permanent DNA sequence and the tempo-rary outer layer, the epigenetic information contains sig-natures that are both stable enough to be inherited bycells and yet be reversible without altering the DNAsequences. Therefore, this three-layer model of regulatorynetworks supports the current notion that early nutri-tional influence imposes heritable epigenetic changeswithout altering DNA sequences, and that these changesare stable enough to result in susceptibility to diet-relatedchronic diseases later in adult life. Meanwhile, the revers-ibility of epigenetics also justifies the application of epi-genetic therapies to clinical interference of these diseases.

However, more issues need to be addressed to provethe role of epigenetics in diet-related chronic diseases.For example, cross-talk between DNA methylation andhistone modifications is well-known, but it is unclearwhether histone modifications alone, or the cross-talk, arealso involved in the development of diet-related diseases.9

In addition, due to the genome-wide effects of both

Affiliation: J Wu and TH-M Huang are with the Human Cancer Genetics Program, Department of Molecular Virology, Immunology andMedical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

Correspondence: TH-M Huang, Human Cancer Genetics Program, The Ohio State University, 460 West 12th Avenue, Columbus, OH 43210,USA. E-mail: tim.huang@osumc.edu, Phone: +1-614-688-8277, Fax: +1-614-688-3981.

doi:10.1111/j.1753-4887.2008.00068.xNutrition Reviews® Vol. 66(Suppl. 1):S49–S53 S49

nutrition and epigenetic regulation, applications of epige-nomic approaches may be an efficient way to delineate thepathway from early nutrition to long-lasting epigeneticchanges and to adult susceptibility to certain diseases.

Various methods have been used to analyze DNAmethylation and histone modifications. The frequentlyused methods include methylation-specific PCR (MS-PCR), combined bisulfite restriction analysis (COBRA),bisulfite sequencing, and chromatin immunoprecipita-tion PCR (ChIP-PCR). MS-PCR, COBRA, and bisulfitesequencing are all based on bisulfite conversion of DNA,which can discriminate methylated cytosine and unm-ethylated cytosine. In MS-PCR, two pairs of specificprimers are adopted to detect the two convertedsequences from methylated and unmethylated DNA. InCOBRA, a specific restriction endonuclease recognizesthe difference between bisulfite-converted sequencesof unmethylated and methylated DNA. Alternatively,cloning followed by sequencing can also be applieddirectly to the bisulfite-converted DNA to check themethylation status, which is called bisulfite sequencing.More detailed profiling of methods for DNA methylationanalysis can be found in other publications.10 ChIP-PCRis generally used to check histone modifications associ-ated with DNA, as discussed with details in the nextsection.

The current state of epigenomics makes it possible toexamine epigenetic status on a genome-wide scale. Thetwo well-characterized epigenetic markers are DNAmethylation and histone modification. Most genome-wide strategies for DNA methylation are based on either

methylation-sensitive restriction enzyme digestion ofDNA or bisulfite conversion of DNA.11 Representativetools include differential methylation hybridization,restriction landmark genomic scanning and methylatedDNA immunoprecipitation.12–14 ChIP-chip, which com-bines chromatin immunoprecipitation with microarray,is a widely used assay for screening genome-wide histonemodifications.15 To overcome the limit of coverage ofthese approaches, massive sequencing is introduced toperform large-scale bisulfite sequencing and ChIPsequencing.16–19 The latest sequencing technology hasadded the possibility of realizing the blueprint of thehuman epigenomic project.20,21 Due to the rapid develop-ment of this technology, we introduce here the brief prin-ciples of ChIP-chip and focus on its recent progress.

ChIP-BASED EPIGENOMIC TOOLS

Chromatin immunoprecipitation is a technique used toexplore the in vivo interactions between DNA and pro-teins (Figure 2). Briefly, cells or tissues are treated withformaldehyde so that interacting DNA and protein arecrosslinked in vivo. DNA bound by protein is thensheared, typically by sonication to ~0.2–2 kb. The DNA-protein complexes are then immunoprecipitated withspecific antibodies against the protein of interest. Afterimmunoprecipitation, the crosslinking of complexes isreversed and the protein-bound DNA is isolated. Thetraditional strategy is to apply PCR to examine knowncandidate genes regulated by this nucleoprotein complex.ChIP-PCR is limited by the number of targets that can

Figure 1 The three-layer regulatory model and the role of epigenetics in mediating diet-related diseases. The three-layer regulatory networks are composed of permanent genetic information, stable but reversible epigenetic information, andflexible pathways. The epigenetic marks include: Me, methylation; Ac, acetylation; Pi, phosphorylation; Ub, ubiquitination. Thedark arrows show possible nutrition pathways that lead to changed phenotypes.

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feasibly be examined. To overcome this limitation, ChIP-chip has been developed combining ChIP and microarraytechniques together (Figure 2). With ChIP-chip, the puri-fied DNA is labeled with fluorescence dyes and hybrid-ized to microarrays. In parallel, genomic DNA withoutimmunoprecipitation, or another reference DNA, is usedas a control. The hybridized slide is scanned and ana-lyzed. Comparison of immunoprecipitated DNA with thecontrol helps to profile the binding positions of specificproteins across the genome.

ChIP-chip has been widely used in genome-widestudies of DNA-bound protein factors and histone modi-fications.15 The difference in application between ChIP-chip and ChIP-PCR is analogous to that of satellite andmanual surveys in the field of geology. A high-throughput method, ChIP-chip not only producesnumerous data, it also offers a wider view of studiedtargets that cannot be achieved by candidate geneapproaches. As an example, a recent application of ChIP-chip discovered a diverse pattern of histone modificationsin relation to DNA methylation in mouse leukemia cells,which is contrary to previous established concepts andprovides new clues for the understanding of epigenetic

regulation in mammalian genomes.22 ChIP-chip withvarious types of arrays also shows the distribution ofsome DNA-bound transcription factors. For instance, inyeast, an overwhelming majority of the binding targets ofheat shock transcription factors are detected by ChIP-chip in the upstream regions of open-reading framesrather than in intergenic regions that are not upstreamof any open-reading frame.23 However, in the humangenome, contrary to previous assumptions, some well-studied transcriptional factors (e.g., p53, c-MYC, andestrogen receptor) have a wider distribution pattern ofbinding and are not limited to the intergenic regionsupstream of DNA-coding regions.24,25 For example, arevised model of chromatin loop has been introduced tointerpret the transcription regulation mediated by theestrogen receptor, which may also be suitable for explain-ing the regulation mechanisms of other widely distrib-uted DNA- binding transcription factors.26 The successof ChIP-chip in these fields, including the profiling ofhistone modifications and transcription-factor bindingsites, provide convincing evidence that once applied innutrition studies, unbiased ChIP-chip screening mayproduce unexpected discoveries and resolve some previ-ously unanswered questions.

The potential of ChIP-chip encourages researchersto modify this tool and produce various ChIP-based func-tional genomic or epigenomic approaches. The afore-mentioned methylated DNA immunoprecipitationstrategy, applies an antibody specific for methylatedcytosines to immunocapture methylated DNA fragments,which are then hybridized to microarray for genome-wide methylation analysis.14 The limitation of ChIP-chipcomes partly from the coverage of arrays. To overcomethis limitation and increase the coverage of genome-wideprofiling, ChIP-SAGE is created, combining ChIP withmodified serial analysis of gene expression to exploreprotein-DNA interactions.27 With ChIP-SAGE, DNA isfirst pulled down with a regular ChIP assay. The immu-noprecipitated DNA is processed with a SAGE protocolinstead of microarray hybridization to create a SAGElibrary with ChIP DNA inserted as concatenates. Then,sequencing is applied to the SAGE library to detect ChIPDNA so that all the DNA fragments bound with proteinfactor of interest will be counted. Similar to ChIP-SAGE,with the chromatin immunoprecipitation paired-end tag(ChIP-PET) technique, only the two ends of DNA frag-ments from the ChIP assay are used to construct a PETlibrary, and the sequencing of both ends of ChIP DNAfragments achieve increased resolution.28

Recently, new high-throughput resequencing tech-niques, combined with ChIP assay (ChIP-seq), have madeit possible to perform true whole-genome analysis ofprotein-DNA interactions and epigenomic profiles. Oneexample of these whole-genome sequencing techniques is

Figure 2 The ChIP-chip procedure. The method combinesChIP assay with a microarray technique – more detailedsteps are described in the text.

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the Solexa system, which covalently attaches single mol-ecules to a solid planar surface for in situ amplificationand parallel sequencing.29 ChIP-seq was first applied toanalyze the binding positions of a transcription factor,called neuron restrictive silencer factor (NRSF/REST), inthe human genome.17 The results showed that ChIP-seqhas high sensitivity and specificity and increased resolu-tion and coverage. It detected about 30% more bindingsites of NRSF/REST than ChIP-chip, and more detaileddistribution patterns of NRSF/REST were also described.Similarly, ChIP-seq showed high performance in analyz-ing another transcription factor, STAT1, in interferon-g-stimulated HeLa cells.18 The capability of ChIP-seq makesit a powerful new epigenomic tool. With this tool, mul-tiple histone modifications have been analyzed in mouseand human genomes. The results produce a combinato-rial map of different histone modifications acrossgenomes.19,30 It will likely be discovered that gene regula-tion is affected by integrated multiple histone marksinstead of a single mark. Therefore, comprehensive analy-ses of these marks with efficient epigenomic tools, likeChIP-seq, will be critical in order to fully understand theactivities of mammalian chromatin.

CONCLUSIONS

The use of epigenomic tools offers significant advantagesfor nutritional studies. Epigenetic reprogramming,whether affected by nutritional interference or naturaldevelopment during aging, occurs prior to the develop-ment of visibly altered phenotypes. The affected genesthat underlie the changed phenotypes may not be known.Therefore, unbiased genome-screening may be a helpfultool to monitor and pin down specific epigenetic path-ways involved in diet-related diseases that are changedby nutritional and environmental modifications/interventions.

Acknowledgments

We thank Benjamin Rodriguez and Daniel E Deatheragefor helpful discussions and feedback on this manuscript.We apologize to colleagues whose work could not be citedbecause of space limitations.

Declaration of interest. The authors have no relevantinterests to declare.

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