reviewarticle ocus peci c iochemical pigenetics chromatin...

Hindawi Publishing CorporationISRN BiochemistryVolume 2013, Article ID 913273, 8 pageshttp://dx.doi.org/10.1155/2013/913273

Review Article�ocus��peci�c �iochemical �pigenetics�Chromatin �iochemistryby Insertional Chromatin Immunoprecipitation

Toshitsugu Fujita and Hodaka Fujii

Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka,Suita, Osaka 565-0871, Japan

Correspondence should be addressed to Hodaka Fujii; [email protected]

Received 7 November 2012; Accepted 27 November 2012

Academic Editors: J. Bonnet, M. Frank-Kamenetskii, and J.-J. Lin

Copyright © 2013 T. Fujita and H. Fujii.is is an open access article distributed under theCreativeCommonsAttribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Comprehensive understanding of regulation mechanisms of biological phenomena mediated by functions of genomic DNAre�uires identi�cation of molecules bound to genomic regions of interest in vivo. However, nonbiased methods to identifymolecules bound to speci�c genomic loci in vivo are limited. To perform biochemical and molecular biological analysis of speci�cgenomic regions, we developed the insertional chromatin immunoprecipitation (iChIP) technology to purify the genomic regionsof interest. �e applied iChIP to direct identi�cation of components of insulator complexes, which function as boundaries ofchromatin domain, showing that it is feasible to directly identify proteins and RNA bound to a speci�c genomic region in vivoby using iChIP. In addition, recently, we succeeded in identifying proteins and genomic regions interacting with a single copyendogenous locus. In this paper, we will discuss the application of iChIP to epigenetics and chromatin research.

1. Introduction

Detailed biochemical and molecular biological analysis ofchromatin domains is critical for understandingmechanismsof genetic and epigenetic regulation of gene expression,hetero- and euchromatinization, X-chromosome inactiva-tion, genomic imprinting, and other important biologicalphenomena [1]. However, biochemical nature of chromatindomains is poorly understood. is is mainly because meth-ods for performing biochemical and molecular biologicalanalysis of chromatin structure are limited [2–8].

Identi�cation of regulatory regions of gene expressionhas been extensively attempted in the last several decades.Conventionally, these analyses have been performed byusing arti�cial methods such as reporter assay [9] and insilico identi�cation of genomic regions conserved amongspecies [10]. More recently, enhancer-speci�c modi�cationsare being used to identify enhancer regions in the genome(see review [11]). However, although these approaches havebeen successful for relatively easy targets such as immediateearly genes, it has been shown that they could produce arti-factual results inmany circumstances. In fact, deletion studies

of candidate regulatory endogenous genomic regions haveshown that the candidate regions identi�ed by using theseconventional methods could oen be dispensable for expres-sion of the genes of interest. Furthermore, these approachescannot be used when regulatory genomic regions are far fromregulated loci, for example, on other chromosomes. In fact,long-range interaction including interchromosomal interac-tion has been suggested to play important roles in regulationof gene expression and other biological phenomena [12]. Inthis regard, it has been shown that such regulatory regionshave physical contact with the regulated loci, forming a loop[13, 14]. is led to the idea of identi�cation of regulatorygenomic regions by detecting genomic regions interactingwith the genomic region of interest. us, development ofmethods to identify intra- and interchromosomal interactionis vital for the advancement of the �eld.

Identi�cation of molecules such as proteins and RNAsinteracting with speci�c genomic regions is also essentialfor understanding of epigenetic regulation and chromatinbiology. Conventionally, molecules interacting with a spe-ci�c genomic region have been identi�ed using arti�cialapproaches including a�nity puri�cation, yeast one-hybrid,

2 ISRN Biochemistry

electrophoretic mobility shi assay (EMSA), and others [15].Although these approaches are successful in some cases,especially for the analyses of easier targets such as immediateearly responses, they can be very problematic. For example,experimental conditions in these arti�cial approaches are farfrom physiological, causing artifactual or misleading results.erefore, researchers need to verify if the detected inter-action is physiological using other in vivo approaches. isrequires a lot of efforts and takes long time, oen more than10 years. ese problems have delayed the advancement ofthe �eld. erefore, development of technologies that detectmolecular interaction on the genome in vivo is absolutelyrequired.

In this paper, we will �rst discuss conventional tech-niques to analyze the molecular interaction on the genomein vivo. Subsequently, we will discuss insertional chro-matin immunoprecipitation (iChIP) we developed for thelocus-speci�c biochemical epigenetics/chromatin biochem-istry and its application.

2. Methods to Analyze Molecular InteractionIn Vivo

Several methods have been devised to analyze molecularinteraction with speci�c genomic regions in vivo.

2.1. Chromatin Immunoprecipitation (ChIP). ChIP wasdeveloped in 1988 [16] and has played instrumental rolesin detection of molecular interaction in the genome invivo. In ChIP, molecular interaction can be preservedby crosslinking with formaldehyde or other crosslinkers.Subsequently, chromatin is fragmented by sonicationor digestion with endonucleases. Immunoprecipitationwith antibodies against DNA-binding proteins of interestis performed to isolate genomic regions bound by theDNA-binding proteins (Figure 1). ChIP has been usedto identify in vivo binidng of transcription factors andother chromatin-associated factors. Recently, by combiningwith DNA microarray analysis (ChIP-on-chip) or next-generation sequencing (ChIP-Seq), ChIP has been used forgenome-wide search for target sequences bound by a givenDNA-binding protein [17].

Although ChIP is a very powerful technique and revo-lutionized epigenetics and chromatin research, it has somelimitations. For example, although ChIP is essential to iden-tify genomic loci to which a given protein binds, it cannot beused to identify unknown proteins binding to genomic lociof interest.

2.2. Imaging Analyses. Imaging techniques have been widelyused to examine molecular interaction with speci�c genomicregions [18, 19]. Fluorescent in situ hybridization (FISH) isused to visualize speci�c genomic loci. Proteins and RNAinteracting with a genomic locus of interest are detected byimmuno�uorescence and in situ hybridization, respectively.

ey have, however, certain limitations: (i) resolutionis low; that is, even if FISH and protein signals look co-localized, it does not necessarily mean the protein is in that

DNA-binding proteins

IP with Ab against the target protein

Genomic PCR (ChIP)

Microarray (ChIP-on-chip)

Next-generation sequencing (ChIP-Seq)

Reverse crosslink

Purification of DNA

CrosslinkFragmentation

F 1: Scheme of ChIP. In ChIP, molecular interaction can bepreserved by crosslinking with formaldehyde or other crosslinkers.Subsequently, chromatin is fragmented by sonication or digestionwith endonucleases. Immunoprecipitation with antibodies againstDNA-binding proteins of interest is performed to isolate genomicregions bound by the DNA-binding proteins.

locus. e protein can be localized far from that locus. Itcannot be judged by the imaging methods. (ii) Nonbiasedsearch for interacting proteins andRNA is not feasible. Beforecolocalization is examined by imaging techniques, candidateproteins and RNA should be identi�ed by other methods.

2.3. Chromosome Conformation Capture (3C) and Its Deriva-tives. 3Cwas developed in 2002 to examine genome-genomeinteraction [20]. In 3C, molecular interaction is maintainedby crosslinking with formaldehyde before digesting with arestriction enzyme(s). Aer ligation of DNA ends in thesame complex, proteins and RNA are removed by phe-nol/chloroform extraction to purify DNA. Interaction ofgenomic loci is detected by PCR using locus-speci�c primers(Figure 2). By using 3C, interaction of genomic loci includinginterferon-𝛾𝛾 and IL-4 loci [12] and odorant receptor loci andits regulatory locus [21] has been demonstrated. In addition,unknown interaction can be detected by PCR using primersannealing with both ends of target genomic fragments (4Cand 5C) [22] and HiC [23] (see the review [24] for detailson 3C derivatives). 3C has been widely used these days forthe genome-wide genome interaction analysis that is one ofimportant categories of epigenomics.

However, 3C-based approaches have some intrinsicdrawbacks. (i) 3C-based methods detect only genome-genome interaction. Information on neither interacting

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PCR


Target genomic region

Interacting genomic region

Crosslink

digestion

Sequencing

DNA microarray analysis

Purification of DNA

Reverse

crosslink

Ligation

F 2: Scheme of 3C-based identi�cation of interacting genomicregions. In 3C, molecular interaction is maintained by crosslinkingwith formaldehyde before digesting with a restriction enzyme(s).Aer ligation of DNA ends in the same complex, crosslink isreversed andDNA is puri�ed. Interaction of genomic loci is detectedby PCR using locus-speci�c primers or microarray�N�S.

proteins nor RNA can be obtained. (ii) 3C-based meth-ods require enzymatic reactions including digestion withrestriction enzymes and ligation of crosslinked chromatin.Especially, difficulty in complete digestion of crosslinkedchromatin can cause detection of artifactual interaction. Infact, it has been shown that interaction detected by 3Cdoes not necessarily correspond to that detected by imagingapproach [25]. (iii) Allele-speci�c analysis is very difficult,if not impossible; that is, 3C-based methods are not able todetect allele-speci�c interaction.is problem would make itdifficult to apply 3C-based methods to analysis of genomicimprinting for example.

2.4. Proteomics of Isolated Chromatin (PICh). PICh is anovel technique to isolate speci�c genomic regions retainingmolecular interaction [8]. PICh utilizes speci�c biotinylatednucleic acid probes such as locked nucleic acids (LNAs) thathybridize target genomic regions and isolates the regionsusing streptavidin beads to analyze interacting proteins(Figure 3). It has been shown that human telomeres canbe successfully isolated to identify interacting proteins [8].PICh would be especially useful to isolate genomic regionscontaining multiple repeats.

On the other hand, PICh also has its intrinsic problems.(i) It would be difficult to apply PICh to isolation of low-copynumber genomic loci. Since PICh requires partial denaturingof crosslinked target genomic loci, it would be very difficult


Denaturation

Hybridization with nucleic acid probes

Crosslink

Fragmentation

Reverse crosslink

LNA sequenceSpacerDesthiobiotin

Mass spectrometry

Affinity purification with avidin beads

F 3: Schemeof PICh. PIChutilizes speci�c biotinylated nucleicacid probes such as locked nucleic acids (LNAs) that hybridize targetgenomic regions and isolates the regions using streptavidin beads toanalyze interacting proteins.

to efficiently hybridize probes to low-copy number loci,making it very difficult to obtain sufficient amounts of thoseloci for biochemical analysis. (ii) When genomic regionscontaining repeated sequences are isolated by PICh, theisolated complexes are heterogeneous, that is, a mixture ofdifferent loci. For example, isolated telomeres by PICh aremixtures of telomeres of distinct chromosomes. It is notfeasible to isolate telomeres of a certain chromosome (e.g.,chromosome �). (iii) Allele-speci�c analysis is not feasible byPICh.

3. Insertional Chromatin Immunoprecipitation(iChIP)

3.1. Principle of iChIP. To perform biochemical analysesof speci�c genomic regions retaining molecular interaction,we developed insertional chromatin immunoprecipitation(iChIP) [26]. e scheme of iChIP is as follows (Figure 4).(i) A repeat of the recognition sequence of an exogenousDNA-binding protein such as LexA is inserted into thegenomic region of interest in the cell to be analyzed (Figure4(a)). (ii) e DNA-binding domain (DB) of the exogenousDNA-binding protein is fused with a tag(s) and a nuclearlocalization signal (NLS)(s) and expressed into the cell to beanalyzed (Figure 4(b)). (iii) e resultant cell is stimulatedand crosslinked with formaldehyde or other crosslinkers, ifnecessary. (iv) e cell is lysed, and DNA is fragmented bysonication or other methods. (v) e complexes includingthe exogenous DB are immunoprecipitated with an antibodyagainst the tag or isolated by other affinity puri�cationprocedures. (vi) e isolated complexes retain moleculesinteracting with the genomic region of interest. Subsequentpuri�cation of DNA, RNA, proteins, or othermolecules allowtheir identi�cation and characterization (Figure 4(c)).

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Genomic region of interest

(e.g., promoter/enhancer region)

Coding regionLexA-binding

elements

(a)

FLAG tagsNLSTagged LexA DB

(3xFNLDD) LexA DNA-binding domain + dimerization domain

(b)

DNA analysis (microarray, next-generation sequencing (iChIP-seq))

Protein analysis (mass spectrometry (iChIP-MS))


IP with Ab against the tag

Reverse crosslink

RNA

Interacting genomic regionfor example, distant enhancer

Crosslink

Fragmentation Irrelevant genomic DNA-proteincomplexes

RNA analysis (microarray, RNA-Seq)

(c)

F 4: Scheme of insertional chromatin immunoprecipitation (iChIP). e system consists of a locus of interest (e.g., a promoter, anenhancer, and an silencer of a gene) linked to LexA-binding elements (LexA BE) (a), and FLAG-tagged, nuclear localization signal (NLS)-fused LexADNA-binding domain (DB) (3xFNLDD) (b). LexA-binding sites are knocked-in in the genomic locus of interest in cells expressingthe tagged LexA DB. Alternatively, cells expressing the tagged LexA DB are transiently or stably transfected with the transgene tagged withLexA BE.ese cells are crosslinked with formaldehyde or other crosslinkers, if necessary, and lysed.en, DNA is fragmented by sonicationor other methods. Subsequently, the LexA-tagged genomic region is immunoprecipitated with an anti-FLAG antibody, and crosslink isreversed when a crosslinker is used. Molecules (DNA, RNA, proteins, and others) associated with the LexA-tagged genomic regions arecharacterized (c).

Knocking-in of LexA-binding elements (LexA BE) in theendogenous locus as well as transgene approach can be usedfor iChIP (Figure 5). Obviously, targeting an endogenouslocus would be more physiological (Figure 5(a)). In contrast,when the transgene is known to harbor critical regulatoryelements, random integration of transgenes retaining LexABE (Figure 5(b)) would be bene�cial because of potentialincrease in copy numbers, whichmakes biochemical analysesmuch easier. us, iChIP is a comprehensive approach topurify speci�c genomic regions of interest to identify inter-acting molecules including genomic DNA, proteins, RNAs,and others, with an emphasis on nonbiased search usingnext-generation sequencing (NGS), microarrays, and massspectrometry (MS).

iChIP has two precursory technologies as its origins.Obviously, one is ChIP as described above. e other islocus-tagging with recognition elements of DNA-bindingproteins.is technique has been widely used in live imagingof speci�c loci (reviewed in [27]). In addition, locus-tagginghas also been used for biochemical puri�cation of speci�cgenomic regions in yeast [6]. Since genomic DNA is toolarge to be isolated, some measures are needed to make thetarget regions short enough for puri�cation. �o this end,speci�c genomic regions were excised by using the Cre-loxP system. e use of Cre-loxP system circularizes the�oxed genomic regions suitable for biochemical puri�cation.However, Cre-mediated circularization may break interac-tion between the target loci and interacting genomic regions.

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Targeting vector

Homologous recombination

LexA-binding elements

Genomic DNA

Genomic DNA

iChIP

iChIP by using homologous recombination

A target region (Cre-LoxP)

Zinc-finger nuclease

(ZFN) or TALEN

Neor

-Neor

(a)

Reporter

A target region (e.g., promoter) Random integration

into the genome

Selection marker

iChIP

iChIP by using random integration

LexA-binding elements

Genomic DNA

(e.g., Neor)

(b)

F 5: Knock-in and transgenic approaches of iChIP. (a) Knocking-in of LexA BE into the endogenous locus or (b) random integration oftransgenes retaining LexA BE can be used for iChIP. �inc-�nger nucleases (�FN) and TALEN technology make gene targeting much easierin cultured cell lines.

In addition, Cre-mediated circularization could not be usedfor crosslinked chromatin. us, this approach cannot beused when endogenous conformation is important such asdetection of interchromosomal interaction.

3.2. Characteristics of iChIP. iChIP has many advantagesover other nonbiased search methods described above(Table 1). (i) iChIP enables us to perform nonbiased searchfor molecules interacting with speci�c genomic regions. (ii)Intergenomic interaction can be detected. It has not beenshown whether PICh can be used for these analyses. In addi-tion, “interaction” detected by 3C-based approaches doesnot necessarily mean physical interaction. In other words,since efficiency of enzymatic digestion is affected by locusaccessibility, signal derived from 3C-based approaches mayrepresent accessibility of the loci. In this regard, since iChIPcan be performed without any enzymatic processes, detectedsignals represent physical interactions. (iii) iChIP has beenused for detection of proteins and RNA interacting withthe genome. In contrast, detection of interacting proteinsand RNA is not feasible by 3C because they are removedin the procedure. It has not been shown whether PICh canbe used for detection of interacting RNA. (iv) iChIP can beperformed without any enzymatic reactions that may giverise to noise or artifactual signals. (v) Low-copy numberloci can be analyzed by iChIP. In fact, we succeeded inidentifying proteins interacting with a single endogenous

locus (manuscript in preparation. See below). In contrast,application of PICh to low-copy number loci may be difficultas described above. (vi) Allele-speci�c analysis is feasible withiChIP because a speci�c allele can be tagged.

Although iChIP has many advantages over other tech-niques as described above, it has some disadvantages. (i) Itrequires generation of cells for iChIP analysis, that is, inser-tion of LexA BE into the target loci and expression of a taggedLexA DB. In this regard, knocking-in into the genome of celllines has been more difficult than that of mouse embryonicstem cells. �owever, advent of zinc-�nger nucleases (�FN)[28] and TALEN technology [29] makes gene targetingmucheasier in cultured cell lines (Figure 5(a)). (ii) Insertion ofLexA BE may affect chromatin structure such as nucleosomepositioning and abrogate normal genome activities such asgene expression. Although the effects of insertion need tobe tested empirically for each locus, we have guidelines toavoid potential aberrant effects caused by insertion of LexABE. (a) For analysis of promoter regions near transcriptionstart sites (TSSs), the insertion site should be several hundredbase 5′ to the TSS so that the insertion would not inhibittranscription or disrupt nucleosome positioning. In contrast,for identi�cation of binding molecules of genomic regionswith distinct boundaries such as enhancers or silencers, theLexA BE can be directly juxtaposed to the regions because itis less probable that the insertion of LexA BE might inhibittheir function. (b)e insertion site should not be conserved

6 ISRN Biochemistry

T 1: Comparison chart of nonbiased methods to analyze speci�c genomic regions.

Method Non-biasedanalysis DNA analysis Protein

analysis RNA analysis Include enzymereactions

Low-copynumbergenes

Needtransgenic

Allele-speci�canalysis

iChIP Yes Feasible Feasible Feasible No Feasible Yes Feasible3C Yes Feasible Not feasible Not feasible Yes Feasible No Not feasible

PICh Yes Not reported Feasible(telomeres) Not reported No Not reported No Not feasible

among species because conserved regions are oen bindingsites of essential binding molecules.

4. Application of iChIP

4.�. I�enti�cation of Proteins an� �� Interactin� �it� Ins��a�tor. We applied iChIP to direct identi�cation of componentsof insulator complexes, which function as boundaries ofchromatin domains [30]. By combining iChIP with MS(iChIP-MS) and RT-PCR (iChIP-RT-PCR), we found that thechicken 𝛽𝛽-globin HS4 (cHS4) insulator complex contains anRNA helicase protein, p68/DDX5; an RNA species, steroidreceptor RNA activator 1 (SRA1); and a nuclear matrixprotein, Matrin-3, in vivo. Binding of p68 and Matrin-3 tothe cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). us, our results showed for the �rsttime that it is feasible to directly identify proteins and RNAbound to a speci�c genomic region in vivo by using iChIP.

e fact that p68/DDX5 was directly identi�ed as aninsulator component by iChIP clearly shows the powerof iChIP. It took only several months for us to identifyp68/DDX5 since the project was started. In contrast, it tookmore than ten years to identify p68/DDX5 as an insulatorcomponent by using conventional methods [31, 32] since theinsulator was �rst discovered [33].us, iChIP can acceleratethe process of identi�cation of components of chromatincomplexes by 10–100-fold.

We also successfully detected SRA1 RNA [32] in thepuri�ed cHS4 insulator complex. Combination of iChIP withmicroarray or RNA-seq would be promising for nonbiasedsearch for RNA associated with speci�c genomic regions,which cannot be achieved by other methods.

4.2. Detection of Genomic Interactions. It is of note that apioneering work used locus-tagging for detecting interactionof speci�c genomic loci by genomic PCR [34]. Aer theinitial publication of iChIP, iChIP has been used to detectgenomic interaction in budding yeast in a nonbiased man-ner. Genome-wide iChIP studies were performed to �ndthat pheromone-response genes regulated by a transcriptionfactor, Ste12, have increased interchromosomal interactionsin cells lacking Dig1 protein, a inhibitor of Ste12 [35]. eyfound that the increase in interchromosomal interactions isthe basis of increase in intrinsic and extrinsic noise in thetranscriptional outputs of the mating pathway. us, iChIPis a powerful technique to detect genomic interactions.

We are now attempting genome-wide search for genomicregions interacting with an endogenous locus by combiningiChIP andNGS (iChIP-Seq). Preliminary results showed thatiChIP-Seq is able to detect long-range genomic interactionssuch as interchromosomal interaction without ambiguity(manuscript in preparation), suggesting that it is useful forgenome-wide identi�cation of interacting genomic regions.

5. Future Application of iChIP

We have been optimizing experimental conditions of iChIPincluding development of a second generation 3xFLAG-tagged LexA DB, 3xFNLDD [36]. iChIP using 3xFNLDDwas able to consistently isolate more than 10% of inputgenomic DNA, several-fold more e�cient than the �rst-generation tagged LexA DB. In addition, elution conditionswith 3xFLAG peptide have been optimized.

To increase the utility of iChIP, we are attempting topurify molecular complexes associated with an endogenouslocus of higher eukaryotes. In this study, the LexA BE-inserted promoter region of the Pax5 gene, which encodesthemaster lineage commitment transcription factor for B celldevelopment, is puri�ed by iChIP and subjected to MS. ePax5 gene is on the Z chromosome in the chicken, and thechicken mature B-cell line, DT40, used in the study has oneZ chromosome. We identi�ed multiple proteins interactingwith the Pax5 promoter. e identi�ed proteins includedtranscription factors, DNA-binding proteins, histones, andother proteins potentially involved in transcriptional regula-tion (manuscript in preparation).

Combination of iChIP with SILAC (stable isotope label-ing using amino acids in cell culture) [37] or iTRAQ (isobarictag for relative and absolute quanti�cation) [38] wouldbe promising in comparing samples prepared in differentconditions, for example, different cell types, in the absence orpresence of stimulation, and so forth. In fact, we have beensuccessfully identifying proteins associated with the Pax5promoter region for the above-mentionedDT40-derived cellsusing iChIP-SILAC (manuscript in preparation). Recently,application of iChIP-SILAC to yeast cells was reported [39].

Another important direction of application of iChIP is todetect novel epigenetic marks such as histone modi�cations.It has been shown that various chemical modi�cations onhistones play crucial roles in genomic processes such as DNAreplication, DNA repair, transcription, heterochromatiniza-tion by binding speci�c factors that, in turn, serve to alter thestructural properties of chromatin [40]. Recent advancementof MS has enabled to identify novel histone modi�cations

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in a large scale [41]. In this regard, since distribution ofsome important epigenetic marks can be restricted in certaingenomic domains, these marks can be missed due to dilutionwhen whole genome is used as the source forMS. In contrast,since iChIP can purify speci�c genomic regions, it is possibleto identify such rare epigenetic marks concentrated in thosegenomic regions.

Application of iChIP is not restricted to cultured cell linesbut easily extended to organisms in vivo. In fact, iChIP wasrecently applied to cells of entire body of fruit �y [42].We andour collaborators are now applying iChIP to mice by usingknocking-in of LexA BE in ES cells and transgenic expressionof a tagged LexA in transgenic mice.

Taken together, iChIP will be a powerful and useful toolto dissect “interactome” of a given genomic loci.

Abbreviations

iChIP: Insertional chromatin immunoprecipitationChIP: Chromatin immunoprecipitation3C: Chromosome conformation capturePICh: Proteomics of isolated chromatinFISH: Fluorescent in situ hybridizationMS: Mass spectrometry.

Glossary

Epigenetics: e research �eld of heritable changesin gene expression or cellularphenotype caused by mechanisms otherthan changes in the DNA sequences

Chromatin: e complexes of DNA sequences andinteracting RNA and proteins in thenucleus.

Acknowledgments

is work was supported by Nakatani Foundation forAdvancement of Measuring Technologies in BiomedicalEngineering (T.F.), Japan Science and Technology Agency(JST) (H.F.) and Grant-in-Aid for Scienti�c Research onInnovative Areas (nos. 23118516 (T.F.) and 23114707(H.F.))from the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

References

[1] R. D. Kornberg and Y. Lorch, “Chromatin rules,” NatureStructural and Molecular Biology, vol. 14, no. 11, pp. 986–988,2007.

[2] �. Y. �hang and W. H�rz, “Analysis of highly puri�ed satelliteDNA containing chromatin from the mouse,” Nucleic AcidsResearch, vol. 10, no. 5, pp. 1481–1494, 1982.

[3] J. L. Workman and J. P. Langmore, “Nucleoprotein hybridiza-tion: a method for isolating speci�c genes as high molecularweight chromatin,” Biochemistry, vol. 24, no. 25, pp. 7486–7497,1985.

[4] L. C. Boffa, E. M. Carpaneto, and V. G. Allfrey, “Isolation ofactive genes containing CAG repeats by DNA strand invasion

by a peptide nucleic acid,” Proceedings of the National Academyof Sciences of the United States of America, vol. 92, no. 6, pp.1901–1905, 1995.

[5] A. Jasinskas and B. A. Hamkalo, “Puri�cation and initialcharacterization of primate satellite chromatin,” ChromosomeResearch, vol. 7, no. 5, pp. 341–354, 1999.

[6] J. Griesenbeck, H. Boeger, J. S. Strattan, and R. D. Kornberg,“A�nity puri�cation of speci�c chromatin segments fromchromosomal loci in yeast,”Molecular and Cellular Biology, vol.23, no. 24, pp. 9275–9282, 2003.

[7] R. Ghirlando and G. Felsenfeld, “Hydrodynamic studies onde�ned heterochromatin fragments support a 30-nm �berhaving six nucleosomes per turn,” Journal of Molecular Biology,vol. 376, no. 5, pp. 1417–1425, 2008.

[8] J. D�jardin and R. E. Kingston, “Puri�cation of proteins asso-ciated with speci�c genomic loci,” Cell, vol. 136, no. 1, pp.175–186, 2009.

[9] C. M. Gorman, L. F. Moffat, and B. H. Howard, “Recombinantgenomes which express chloramphenicol acetyltransferase inmammalian cells,”Molecular and Cellular Biology, vol. 2, no. 9,pp. 1044–1051, 1982.

[10] S. Aparicio, A. Morrison, A. Gould et al., “Detecting conservedregulatory elements with the model genome of the Japanesepuffer �sh, Fugu rubripes,” Proceedings of the National Academyof Sciences of the United States of America, vol. 92, no. 5, pp.1684–1688, 1995.

[11] G. A. Maston, S. G. Landt, M. Snyder, and M. R. Green,“Characterization of enhancer function from genome-wideanalyses,”Annual Review of Genomics andHumanGenetics, vol.13, pp. 29–57, 2012.

[12] C. G. Spilianakis, M. D. Lalioti, T. Town, G. R. Lee, and R. A.Flavell, “Interchromosomal associations between alternativelyexpressed loci,” Nature, vol. 435, no. 7042, pp. 637–645, 2005.

[13] S. A. Sajan and R. D. Hawkins, “Method for identifying higher-order chromatin structure,” Annual Review of Genomics andHuman Genetics, vol. 13, pp. 59–82, 2012.

[14] D. W. Heermann, “Physical nuclear organization: loops andentropy,” Current Opinion in Cell Biology, vol. 23, no. 3, pp.332–337, 2011.

[15] B. Dey, S. ukral, S. Krishman et al., “DNA-protein inter-actions: methods for detection and analysis,” Molecular andCellular Biochemistry, vol. 365, no. 1-2, pp. 279–299, 2012.

[16] M. J. Solomon, P. L. Larsen, and A. Varshavsky, “Mappingprotein-DNA interactions in vivo with formaldehyde: evidencethat histone H4 is retained on a highly transcribed gene,” Cell,vol. 53, no. 6, pp. 937–947, 1988.

[17] P. Collas, “e current state of chromatin immunoprecipita-tion,”Molecular Biotechnology, vol. 45, no. 1, pp. 87–100, 2010.

[18] A. G. Fisher and M. Merkenschlager, “Gene silencing, cell fateand nuclear organisation,” Current Opinion in Genetics andDevelopment, vol. 12, no. 2, pp. 193–197, 2002.

[19] P. Fraser andW.Bickmore, “Nuclear organization of the genomeand the potential for gene regulation,”Nature, vol. 447, no. 7143,pp. 413–417, 2007.

[20] J. Dekker, K. Rippe, M. Dekker, and N. Kleckner, “Capturingchromosome conformation,” Science, vol. 295, no. 5558, pp.1306–1311, 2002.

[21] S. Lomvardas, G. Barnea, D. J. Pisapia, M. Mendelsohn, J.Kirkland, and R. Axel, “Interchromosomal interactions andolfactory receptor choice,” Cell, vol. 126, no. 2, pp. 403–413,2006.

8 ISRN Biochemistry

[22] M. Simonis, P. Klous, E. Splinter et al., “Nuclear organizationof active and inactive chromatin domains uncovered by chro-mosome conformation capture-on-chip (4C),” Nature Genetics,vol. 38, no. 11, pp. 1348–1354, 2006.

[23] E. Lieberman-Aiden, N. L. van Berkum, L. Williams et al.,“Comprehensive mapping of long-range interactions revealsfolding principles of the human genome,” Science, vol. 326, no.5950, pp. 289–293, 2009.

[24] E. deWit andW. de Laat, “A decade of 3C technologies: insightsinto nuclear organization,” Genes and Development, vol. 26, no.1, pp. 11–24, 2012.

[25] J. Dosite and W. A. Bickmore, “Chromosome organization inthe nucleus—charting new territory across the Hi-Cs,” CurrentOpinion in Genetics and Development, vol. 22, no. 2, pp.125–131, 2012.

[26] A. Hoshino and H. Fujii, “Insertional chromatin immunopre-cipitation: a method for isolating speci�c genomic regions,”Journal of Bioscience and Bioengineering, vol. 108, no. 5, pp.446–449, 2009.

[27] A. S. Belmont, “Visualizing chromosome dynamics with GFP,”Trends in Cell Biology, vol. 11, no. 6, pp. 250–257, 2001.

[28] C. O. Pabo, E. Peisach, and R. A. Grant, “Design and selectionof novel Cys2His2 zinc �nger proteins,” Annual Review ofBiochemistry, vol. 70, pp. 313–340, 2001.

[29] A. J. Bogdanove and D. F. Voytas, “TAL effectors: customizableproteins for DNA targeting,” Science, vol. 333, no. 6051, pp.1843–1846, 2011.

[30] T. Fujita and H. Fujii, “Direct ideni�cation of insulator com-ponents by insertional chromatin immunoprecipitation,” PLoSOne, vol. 6, no. 10, article e26109, 2011.

[31] E. P. Lei and V. G. Corces, “RNA interference machineryin�uences the nuclear organization of a chromatin insulator,”Nature Genetics, vol. 38, no. 8, pp. 936–941, 2006.

[32] H. Yao, K. Brick, Y. Evrard, T. Xiao, R. D. Camerini-Otero, andG. Felsenfeld, “Mediation of CTCF transcriptional insulationby DEAD-box RNA-binding protein p68 and steroid receptorRNA activator SRA,” Genes and Development, vol. 24, no. 22,pp. 2543–2555, 2010.

[33] J. H. Chung, M. Whiteley, and G. Felsenfeld, “A 5′ elementof the chicken 𝛽𝛽-globin domain serves as an insulator inhuman erythroid cells and protects against position effect inDrosophila,” Cell, vol. 74, no. 3, pp. 505–514, 1993.

[34] A. Murrell, S. Heeson, and W. Reik, “Interaction betweendifferentially methylated regions partitions the imprinted genesIgf2 and H19 into parent-speci�c chromatin loops,” NatureGenetics, vol. 36, no. 8, pp. 889–893, 2004.

[35] E. McCullagh, A. Seshan, H. El-Samad, and H. D. Madhani,“Coordinate control of gene expression noise and interchro-mosomal interactions in a MAP kinase pathway,” Nature CellBiology, vol. 12, no. 10, pp. 954–962, 2010.

[36] T. Fujita and H. Fujii, “E�cient isolation of speci�c genomicregions by insertional chromatin immunoprecipitation (iChIP)with a second-generation tagged LexA DNA-binding domain,”Advances in Bioscience and Biotechnology, vol. 3, no. 5, pp.626–629, 2012.

[37] S. E. Ong, B. Blagoev, I. Kratchmarova et al., “Stable isotopelabeling by amino acids in cell culture, SILAC, as a simpleand accurate approach to expression proteomics,”Molecular &Cellular Proteomics, vol. 1, no. 5, pp. 376–386, 2002.

[38] P. L. Ross, Y. N. Huang, J. N. Marchese et al., “Multiplexedprotein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents,” Molecular and CellularProteomics, vol. 3, no. 12, pp. 1154–1169, 2004.

[39] S. D. Byrum, A. Raman, S. D. Taverna, and A. Tackett,“ChAP-MS: a method for identi�cation of proteins and histoneposttranslational modi�cations at a single genomic locus,” CellReports, vol. 2, no. 1, pp. 198–205, 2012.

[40] A. J. Ruthenburg, H. Li, D. J. Patel, and C. D. Allis, “Multivalentengagement of chromatin modi�cations by linked bindingmodules,” Nature Reviews Molecular Cell Biology, vol. 8, no. 12,pp. 983–994, 2007.

[41] M. Tan, H. Luo, S. D. Lee et al., “Identi�cation of 67 histonemarkes and histone lysine crotonylation as a new type of histonemodi�cation,” Cell, vol. 146, no. 6, pp. 1016–1028, 2011.

[42] M. Agelopoulos, D. J. McKay, and R. S. Mann, “Developmentalregulation of chromain conformation by Hox proteins inDrosophila,” Cell Reports, vol. 1, no. 4, pp. 350–359, 2012.

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