TIBS 25 – MARCH 2000

990968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(99)01535-2

SEVERAL METHODS HAVE been devel-oped to address questions concerningthe in vivo regulation of the interactionsbetween cis elements and trans-actingfactors in the context of chromosomestructure and nuclear organization.These include in vivo footprinting, chem-ical and light-induced crosslinking andimmunocytochemistry. The ability toprovide direct evidence that given regu-latory proteins are associated ‘in timeand space’ with specific genomic regionsis a key determinant of the merits of thevarious techniques. In particular, thosemethods that use in vivo fixation com-bined with immunoprecipitation appearto have an advantage over other ap-proaches, such as in vivo footprinting,which provide indirect informationabout ‘occupancy’ of a given site by a nu-clear protein in chromatin. In addition,regulatory proteins that structurally re-semble histones are not detected by thelatter method.

The formaldehyde fixation and chro-matin immunoprecipitation approach(X-CHIP) offers the ability to detect anyprotein at its in vivo binding site di-rectly. In particular, proteins that are notbound directly to DNA or that dependon other proteins for binding activity invivo can be analysed with this method.

Macromolecular chromosomal struc-tures in living material, like tissue cul-ture cells or embryos, are fixed very efficiently, and the chromatin is used asa substrate for immunoprecipitation.Antibodies directed against the proteinof interest allow immunoselection of allgenomic binding sites. At the end, thecrosslinking can be fully reversed andthe DNA analysed.

The chemistry of formaldehyde crosslinking:the advantage of de-crosslinking

Formaldehyde is a tight (2 Å) crosslink-ing agent that efficiently produces bothprotein–nucleic acid and protein–proteincrosslinks in vivo. Formaldehyde is a veryreactive dipolar compound in which thecarbon atom acts as a nucleophilic cen-tre. Amino and imino groups of aminoacids (lysines, arginines and histidines)and of DNA (primarily adenines and cy-tosines) readily react with formaldehydeleading to the formation of a Schiff base.This intermediate can further react with asecond amino group and condense togive the final DNA–protein complex1,2.These reactions take place in vivo withinminutes after addition of formaldehyde toliving cells or embryos (Fig. 1). A key ad-vantage of the use of formaldehyde as acrosslinking agent is that the crosslinksare fully reversible. This is achieved pri-marily by protonation of imino groups atlow pH in aqueous solution.

For the identification and the charac-terization of the in vivo DNA targets of a

given protein, after immunoprecipi-tation of crosslinked chromatin, the DNAis purified and analysed by conventionalmethods like Southern-blot hybridi-zation and PCR analyses. Such analysisrequires the removal of all proteins fromthe immunopurified chromatin fraction.To achieve this, the DNA is released fromcrosslinked material by extensive diges-tion with proteinase K and mild heattreatment, and then purified by standardmethods3.

Specific crosslinking reversal condi-tions are also known for protein analysisin chromatin. It is often of interest to determine whether a given protein is present in the crosslinked chromatin. Inthis case, low pH and specific, highly de-naturing conditions (e.g. 8 M hydroxy-urea) are used4. A simpler alternative in-volves boiling crosslinked chromatin inconventional SDS-PAGE protein gel load-ing buffer for up to 1 hour5. Because theamount of the protein of interest is oftenbelow the limit of detection by simpleWestern blotting, total proteins can be labelled in vivo with 35S, which improvesthe sensitivity of detection of specificchromatin components after immuno-precipitation5.

It is technically difficult, however, todetermine the efficiency of immunopre-cipitation precisely based on the per-centage of precipitated protein of inter-est. Generally, it is hard to predict howmuch of this antigen will actually bechromatin-bound, how many sites willbe engaged and in which portion of thegenome. In addition, certain sites mightbe more available to the antibody thanothers. Hence, at the protein level, X-CHIP is a non-quantitative technique.

Extent of crosslinking: finding the rightbalance

Efficient fixation of a protein to chro-matin in vivo is crucial for the X-CHIPtechnique. The extent of crosslinking is probably the most important parame-ter. Two major problems concerning thesubsequent immunoprecipitation stepshould be taken into account: first, an ex-cess of crosslinking can result in the lossof material or reduced antigen availabil-ity in chromatin, or both, and second, the relative sensitivity of the antigen epitopes to formaldehyde.

Crosslinking times range between 10minutes and several hours. Nucleosomalproteins are normally analysed following acrosslinking time <10 minutes6–8. Longerexposure to formaldehyde leads to loss of immunoprecipitated material, althoughthe reason for this is not understood. It

TECHNIQUES

Mapping chromosomal proteinsin vivo by formaldehyde-

crosslinked-chromatinimmunoprecipitation

Valerio OrlandoGene regulation is a complex process. Numerous factors appear to be re-quired for the accurate temporal and spatial regulation of each gene. Oftenthese factors are assembled into multiprotein complexes, contributing tospecific gene regulation events. Understanding how all these factors areorganized in the chromosome and how their function is regulated in vivo isa challenging task. One of the most useful techniques for studying thislevel of gene regulation is the in vivo fixation by formaldehyde crosslinkingof proteins to proteins and proteins to DNA, followed by immunoprecipi-tation of the fixed material.

V. Orlando is at the DIBIT HSR BiomedicalScientific Park, Via Olgettina 58, 20132Milano, Italy.Email: v.orlando@hsr.it

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is possible that prolonged crosslinkingfavours the binding of nucleosome-associated proteins, thereby ‘masking’histone epitopes. In other cases, epitopesmight be lost because of the engagementof lysines and other preferred formalde-hyde-reactive sites. For proteins otherthan histones, longer crosslinking timesare required. Most proteins are readilycrosslinked following 30 minutes to onehour of crosslinking, although a time-course experiment is strongly recommended.

Another point of concern is the lossof antigen epitopes due to denaturation.Formaldehyde is a moderately denatur-ing agent for proteins and is known tointerfere with the secondary and, in par-ticular, tertiary structures, resulting inthe unfolding of the protein of interest.The general sensitivity of proteins toformaldehyde in vivo has to be deter-mined empirically. A good indicationcan come from immunolocalizationexperiments, whereby the formaldehyde

fixation time or concentration are var-ied. In these studies, if a loss of fluores-cence signal is observed upon the use ofstandard fixing conditions (indicatingprotein denaturation), shortening of theformaldehyde treatment might allow de-tection of the antigen.

Another parameter to be consideredwhen adjusting fixation conditions is themechanical shearability of the fixed ma-terial. Sonication is the only way to solu-bilize fixed material, in particular chromatin. Overfixed cells or embryosare refractory to sonication, resulting inthe loss of up to 90% of the starting material.

Antibodies and immunoprecipitationconditions

In the X-CHIP assay, the immunopre-cipitation step requires highly stringentconditions. A buffer, called RIPA, of intermediate ionic strength and contain-ing a combination of denaturing and non-denaturing detergents [Triton,

sodiumdeoxycholate and sodium dodecylsulphate (SDS)], is used to ensure solu-bility of chromatin6,9. The use of affinity-purified antibodies is highly recom-mended, and polyclonal antibodies arepreferred to monoclonals to avoid po-tential epitope masking problems incrosslinked material.

Prior to embarking on X-CHIP experi-ments, the antibody must be charac-terized with respect to immunopreci-pitation. Pilot immunoprecipitation ex-periments using simple nuclear extractsmust be designed to test each antibodywhether it is active in RIPA buffer.Obviously, the interaction of an anti-body with an antigen in solution mightdiffer from that in the crosslinked ma-terial. However, basic information ongeneral compatibility of immunopreci-pitation with detergents, in particularSDS, is essential. In addition, as men-tioned before, the ability of the antibodyto recognize its antigen in fixed materialcan be deduced from immunocytologicalexperiments.

The use of a caesium chloride gradientIn the original protocol by Varshavsky

and co-workers designed for Drosophilatissue culture cells, the purification ofcrosslinked chromatin using caesiumcloride (CsCl) gradients was described6.This procedure is rather time-consum-ing as it involves 72 hours of ultracen-trifugation6,10. An alternative method is the sonication of fixed material inhigh-detergent buffer and adjustment toimmunoprecipitation-compatible condi-tions by simple dilution11. This pro-cedure has been successfully applied inthe study of Saccharomyces cerevisiae12.In fact, none of the published applica-tions of the X-CHIP technique in yeast in-clude the isopycnic centrifugationstep12,13 (see also Table 1 and referencestherein).

However, in some biological systems,it appears that CsCl gradients cannot beavoided. In Drosophila tissue culturecells and, in particular, in embryos, theomission of the gradient step results insevere reduction of specific enrichmentand the presence of significant amountsof background DNA. The same appearsto hold for mammalian cells, bothmurine and human (Refs 5,14,15). One ofthe reasons for this might lie in the com-position of cell membranes, which af-fects the solubility of lysed materialsafter sonication. CsCl gradients can beuseful because the density of lipid–pro-tein aggregates allows their easy elimi-nation by isopycnic centrifugation.

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living cells

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(a) (b)

Figure 1Chromatin fixation and immunoprecipitation. (a) Living cells are fixed with formaldehyde(HCHO) and chromatin is first solubilized by sonication and then purified over caesium chlo-ride (CsCl) gradients. Immunoprecipitation is performed with antibodies directed againstspecific chromosomal proteins and the corresponding, enriched DNA. Two main types ofanalyses are used to identify the in vivo binding sites. The first is a quantitative PCR analysisof genomic fragments using specific pairs of primers12,13. A second one uses the bulk ofimmunoprecipitated DNA as a probe to scan specific genomic regions by Southern analysis9,10.(b) The cartoon shows the basic concept of in vivo fixation (FIX), chromatin immunoprecipitation(CHIP) with antibodies (forked symbols) and detection of binding site either by the Southernor quantitative PCR approaches (MAP). Nucleosomes and associated proteins (orange circles) and nucleosomes containing the protein of interest (blue circles).

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The original papers describe ultra-centrifugation conditions with centrifu-gation times of up to 72 hours6,10.Shorter centrifugation times can beused, and a 36-hour spin gives morethan satisfactory results. Recently, stepgradients have also been successfullyexploited with centrifugation times <24hours (Ref. 14; B.S. Parekh and T.Maniatis, pers. commun.).

Analysis by PCR of X-CHIP DNAMichael Grunstein’s team has de-

scribed a method for the analysis of X-CHIP products based on a quantitativePCR approach12,13. After DNA purifi-cation from immunoprecipitated chro-matin, the enrichment of specific ge-nomic fragments corresponding tospecific protein-binding sites are scoredby quantitative PCR.

Specific pairs of primers coveringcontiguous fragments of the genomic re-gion of interest are designed. An empiri-cally defined number of amplificationcycles is used and the amplified frag-ments monitored by densitometry.Because of intrinsic differences in theGC content of the genomic fragmentsand length of the corresponding PCRproducts to be analysed, optimal ampli-fication conditions have to be deter-mined for each combination of templateand primers. The same PCRamplification reactions arecarried out with control DNAresulting from a mock X-CHIPexperiment. The appearanceof specific fragments withinthe immunoprecipitated frac-tion scores the binding sites(Fig. 2). In some cases, a mul-tiplex PCR reaction includingall primers at the same timeis set up, a condition clearlydiffering from the single-primer-pair reaction and re-quiring specific adjustments(Fig. 2; for multiplex PCRanalysis, see Ref. 16).

The quantitative PCR ap-proach is rapid and sensitiveand allows fine mapping ofchromosomal proteins in re-gions as small as 300 bp.Recently, spectacular datahave been obtained by thisapproach, showing sequentialrecruitment at specific sites of multiprotein complexes involved in replication and activated transcription15,17–21.Important results have alsobeen obtained in humancells, showing dynamicchanges of acetylation stateof histones in response toviral infection and hormone-induced activation (Refs14,15; for more examples andreferences, see Table 1). Inthe future, the availability ofcomplete sequence informa-tion of several genomes, in-cluding Drosophila, mouse

and human, will markedly increase thepotential power of this type of analysis.

Southern-blot analysis of X-CHIP DNAA different approach utilizes the X-

CHIP DNA as a probe in Southern-blotanalysis9,10. The advantage of this ‘one-step’ analysis is the rapid identificationof binding sites within large genomic re-gions without relying on multiple PCRreactions. The resolution with thismethod is also ~300 bp. In this type ofanalysis, the genomic fraction obtainedby immunoprecipitation, containing vir-tually all the in vivo binding sites of agiven chromatin protein, is radio-labelled and used as a probe againstDNA fragments encompassing large ge-nomic regions (Fig. 3). The amount ofDNA obtained by X-CHIP is usually in therange of a few nanograms, so theamount of specific DNA target present in

TECHNIQUESTable 1. Proteins mapped with the

formaldehyde fixation and chromatinimmunoprecipitation (X-CHIP) method in

various biological systems

Protein Organism Refs

Polycomb Drosophila 10,22,23Polyhomeotic Drosophila 29Posterior sex combs Drosophila 29Trithorax Drosophila 23Trithorax-like (GAGA) Drosophila 22Mcm7 Yeast 17Orc2 Yeast, 17,30

DrosophilaCdc6 Yeast 17TBP Yeast 21TFIIB Yeast 21TFIIH Yeast 21RNA polymerase II Yeast, human 15,21Sir2 Yeast 12,13Sir3 Yeast 12,13Sir4 Yeast 12,13Rap1 Yeast 12,13Swi2 Yeast 19Swi5 Yeast 19Swi4 Yeast 19Ada2 Yeast 19Ash1 Yeast 19Histone H1 Tetrahymena 11Histone H3 (6 acetyl) Yeast 8,31

Drosophila 6Human 14,15

Histone H4 (6 acetyl) Yeast 7,8,18,31Drosophila 6Human 14,15

HMG B Tetrahymena 11HMG C Tetrahymena 11HoxC8 Mouse 25Oct4 Mouse 5Beaf-32 Drosophila 32ER-a Human 15p300 Human 15CBP Human 15ACTR Human 15Brg1 Human 15Smc1 Yeast 26Smc3 Yeast 26Scc1p Yeast 26Scc3p Yeast 26

Abbreviations: ACTR, activator thyroid receptor;Ada, adenosine deaminase; Ash1, asymmetricsynthesis of HO; Beaf-32, boundary-element-associated factor 32; CBP, REB-binding protein;Cdc, cell division clone, ER, estrogen receptor;HMG, high mobility group; HxC8, homeoboxprotein C8; Mcm, minichromosome main-tenance; Oct4, octamer-binding protein 4; Orc2,origin recognition complex 2; Rap1, repressoractivator protein 1; Scc, sister chromatid co-hesion; Sir, silence information repressor; Smc,structural maintenance chromosome; TBP, TATA-binding protein; TFIIB/H, transcription factor (RNApolymerase) II B/H.

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Figure 2In vivo binding-site detection by quantitative PCR of X-CHIP DNA. Formaldehyde-crosslinked chromatinfrom wild-type and mutant yeast S. cerevisiae strainswas immunoprecipitated with antibodies against atelomere component, the SIR4 (for ‘silence informa-tion repressor’) protein. Quantitative PCR analysis ofimmunoprecipitated chromatin was performed usingthree sets of gene-specific primers situated in sub-telomeric regions. The first set is used to amplify thesilent mating type loci HMRa and HMLa and the ex-pressed MATa locus and the euchromatic GAL1 gene.The second set includes a telomeric copy of URA3next to the telomere of the left arm of chromosome VIIand URA3 at its normal locus on the left arm of chro-mosome V. The third set contains a 0.77-kb regiondistal from the telomere on the right arm of chromo-some VI and the ACT1 gene, located at 52 kb on thesame chromosome. Lanes 1–4: PCR products of DNAprecipitates from yeast strains wild type or mutant forspecific SIR genes; lanes 5–8: PCR products from therespective input extracts; lanes 9–10: 2.5-fold serialdilutions of wild-type input extracts. By comparing therelative enrichments of specific fragments found in X-CHIP fractions (lanes 1–4) with the control PCRs(lanes 5–10), it is possible to identify the presence ofthe SIR proteins at specific sites. The anti-SIR4 anti-bodies immunoprecipitate sequences from the silentloci HMRa and HMLa, but not the expressed MATalocus and the GAL1 gene (lanes 2 and 6). Similar anal-ysis was performed on SIR2 and SIR3HA (not shown;Ref. 13; courtesy of Andreas Hecht and MichaelGrunstein; reproduced, with permission, from Ref. 13).

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each immunoprecipitation is normallybelow the detection level for this type ofSouthern-blot analysis. To circumventthis problem, before radiolabelling, theimmunopurified chromatin DNA is lig-ated to a synthetic linker and PCR-ampli-fied22. Direct comparison of the hy-bridization profile obtained by X-CHIPwith that of a control probe (obtainedby mock X-CHIP) identifies the bindingsites (Fig. 3). In all cases, the exact evalu-ation of the significance of the enrich-ments has to be determined by phosphorimager analysis9,10,22,23.

When the genomic target of a proteinis known, the enrichment of that partic-ular sequence can be tested directly byslot-blot analysis of the immunoprecipi-tated chromatin DNA9,10. In this tech-nique, the bulk of the DNA obtained byX-CHIP, without PCR amplification, is im-mobilized on a hybridization filter andprobed with a specific genomic frag-ment. Comparison with the signal in theslot containing ‘mock’ X-CHIP DNA pro-vides the enrichment factor. The inten-sity of the hybridization signal can becompared with a series of standards de-fined as genomic equivalents, represent-ing the amount of DNA of a defined sizepresent in a given amount of genomicDNA. This allows the calculation of the amount of target sequence presentin the immunoprecipitated chromatin.This value, compared with the input(amount of chromatin DNA used for theimmunoprecipitation), indicates the effi-ciency of immunoprecipitation of thatparticular target9,10. Slot-blot analysis isalso used to evaluate the overall effi-ciency of immunoprecipitation.

It must be emphasized again that therelative enrichment is defined as theratio of genomic fragments representinga bound fraction over a background(non-bound) present in the control X-CHIP (e.g. without antibody). In the original version, X-CHIP-ed DNA was digested with restriction enzymes andPCR-amplified after ligation to a sticky-ends linker9,10. The fidelity of amplifi-cation of genomic fragments enrichedfrom chromatin relied on a ‘fair’ distri-bution of particular restriction sites in agiven genomic region. The risk in thiscase is the amplification of backgroundfragments just because of ‘unfair’ distri-bution of restriction enzyme sites andthe loss of enriched fragments due tothe lack of appropriate restriction siteswithin a particular genomic region.

The introduction of direct ligation of a blunt-ended linker to undigested im-munoprecipitated chromatin DNA22 thus

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5 iab46 abd-A promoter7 abd-A promoter8 iab2

9 bxd10 ubx promoter11 bx

Figure 3Southern analysis of X-CHIP DNA. This figure shows an example of binding-site detectionby hybridization of total DNA obtained by immunoprecipitation of in vivo fixed Drosophilaembryo chromatin. The factors analysed are the Polycomb (Pc) and Trithorax (Trx) proteins,members of chromatin remodelling multiprotein complexes involved in the process ofmaintenance of cellular memory in Drosophila. In vivo fixed chromatin was prepared fromstaged Drosophila embryos collected 5–8 h after egg lay and immunoprecipitated with anti-Pc and anti-Trx polyclonal antibodies23. Pc- and Trx-enriched chromatin DNA from a specificdevelopmental time window was PCR-amplified. A small aliquot was then radiolabelled andhybridized with various regulatory regions of the locus bithorax complex (BX-C) listed in thefigure. The same filters were stripped and rehybridized with control DNA obtained frommock chromatin immunoprecipitation. The latter pattern provides the base line for back-ground estimation. Several specific restriction fragments are detected. The Trx protein isbound to unique sites corresponding to core promoters and other Pc/Trx cis-specific elements in early embryonic stages. The Pc protein clearly differs from Trx showing in itscharacteristic ‘spread’ binding profile around the same regulatory regions (adapted, withpermission, from Ref. 23).

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represented a significant improvement.This allowed a substantial refinement ofprevious mapping results, allowing ahigher resolution of the binding sites ofPolycomb, Trithorax and GAGA-factorproteins in the bithorax complex ofDrosophila22,23.

So far, the Southern-blot approach hasbeen applied only to Drosophila cellsand embryos10,22–24. In genomes of highercomplexity, the initial unfavourable ratiobetween single-copy sequences and therest of the genome can create problemsin Southern-blot analysis, even after PCRamplification. Here, competition with un-labelled, non-specific DNA (e.g. repeti-tive DNA) appears to improve the ratiobetween enriched and background sequences. Also, pre-blocking of the protein-A sepharose beads with nonspe-cific DNA appears to enhance the signal-to-noise ratio significantly. In general, ifthe sequence of the genomic target re-gion is known, the quantitative PCR approach remains the best choice. Inthis case, enrichments of a particular sequence obtained by X-CHIP are wellmaintained, thanks to the use of specificprimers, and easily detected comparedwith control X-CHIP DNA.

Future applications and conclusions:identification of target genes

The ability to determine the in vivobinding sites of chromosomal proteinsby X-CHIP could be of interest for theidentification of target genes of specifictranscription factors. X-CHIP has beenused successfully in mammals for theidentification of target genes of theHoxC8 and Oct4 proteins5,25. In the caseof HoxC8, fixed chromatin from whole-mouse spinal cord was used for X-CHIPand the specifically enriched DNA sub-cloned in l-vectors. Each subclone wasthen used as a probe to screen a ge-nomic library. In this way, the l(2)gl tu-mour suppressor gene was identified asa target of HoxC8. This approach re-quires a substantial amount of work be-cause each individual product of the im-munoprecipitation has to be cloned,purified, radiolabelled and hybridized toa genomic library. In addition, severalclones will contain either non-specificDNA or repetitive elements. Therefore,each positive result will require furtherhybridization analysis in order to iden-tify specific genomic fragments.

In a second application, targets of theOct4 protein in mouse enterochromaffin(EC) cells were found5. In this case, acombination of X-CHIP with quantitativePCR of empirically determined targets

was used. Genomic sequence analysis ofgenes active in mouse early embryogen-esis and containing the Oct4 DNA con-sensus was performed. On the basis ofphenotypic criteria of EC cells ectopi-cally expressing Oct4, the osteopontin-specific (Osn) gene was chosen amongseveral computer-scored candidates.Quantitative PCR analysis of X-CHIP-edDNA with primers specific for the regionof osteopontin containing the Oct4-binding site revealed a high enrichmentof the Osn gene in the immunoprecipi-tated fraction.

A future application for the identifi-cation of in vivo targets of transcriptionfactors is the combined use of chro-matin immunoprecipitation with DNAmicroarrays (genome chip) technology(X-CHIP-CHIP). Access to the completesequence of the genomes of yeast,Drosophila and human offers the possi-bility to create bioinformatic libraries ofputative targets of DNA-binding pro-teins. These libraries can be PCR-ampli-fied and become available for analysiswith the products of chromatin im-munoprecipitation.

In conclusion, the X-CHIP techniqueprovides a powerful approach to thestudy of chromosome structure andfunction. Thanks to its widespread application in the analysis of many chromosomal proteins, considerableprogress has been made in our under-standing of gene regulation and chromo-some structure (Table 1). Major break-throughs have been achieved, forexample, in the sequence of events thatfollow mitosis and lead to resetting of transcriptional competence by chromatin remodelling complexes19.Analogous results were obtained withregard to the cell replication machin-ery17,26 and the hierarchy of events in-volving acetylation of chromatin compo-nents and other regulatory factors uponhormone-induced transcriptional acti-vation15. Another important advance inthe understanding of gene regulation,obtained with X-CHIP, was the descrip-tion of the dynamics of recruitment ofTATA-binding protein and associatedfactors at promoters21. The structure ofchromosomal domains such as telo-meres and centromeres have also beenextensively studied by the X-CHIPmethod12,13,26.

Further results are anticipated withrespect to the distribution of proteins innuclei and at single-gene loci. The eu-karyotic nucleus could in fact provideseveral ‘opportunities’ for nuclear fac-tors to be stored, compartmentalized,

mobilized, etc. The occupancy by a pro-tein of given genomic sites does not al-ways allow inference of the functionalrole of those binding sites. Othercrosslinking approaches like UV-crosslinking have revealed associationof transcription factors with genes phenotypically unrelated to the activityof those factors, as well as portions ofgenes that do not include defined cis-regulatory regions24,27,28.

Finally, we still know little about thechromatin composition of genes, espe-cially at high resolution. Many pro-tein–DNA interactions remain to bemapped and there will be much to learnby studying proteins in their chromoso-mal context in vivo.

AcknowledgementsI would like to thank Andreas Hecht

and Michael Grunstein for providingtheir original data, Bhavin Parekh andTom Maniatis for sharing technical infor-mation prior to publication, Renato Paroand members of his laboratory for having shared the navigation in the X-CHIP hyperspace, and Paul Orban forproofreading the manuscript. This workis supported by the AssociazioneItaliana Ricerca sul Cancro and Telethon.

References1 McGhee, J.D. and von Hippel, P.H. (1975)

Formaldehyde as a probe of DNA structure. I. Reactionwith exocyclic amino groups of DNA bases.Biochemistry 14, 1281–1296

2 McGhee, J.D. and von Hippel, P.H. (1975)Formaldehyde as a probe of DNA structure. II. Reactionwith endocyclic imino groups of DNA bases.Biochemistry 14, 1297–1303

3 Solomon, M.J. and Varshavsky, A. (1985).Formaldehyde-mediated DNA-protein crosslinking: aprobe for in vivo chromatin structures. Proc. Natl. Acad.Sci. U. S. A. 82, 6470–6474

4 Jackson, V. (1978) Studies on histone organization inthe nucleosome using formaldehyde as a reversiblecrosslinking agent. Cell 15, 945–954

5 Botquin, V. et al. (1998) New POU dimer configurationmediates antagonistic control of an osteopontinpreimplantaion enhancer by Oct-4 and Sox-2. GenesDev. 12, 2073–2090

6 Solomon, M.J. et al. (1988) Mapping protein–DNAinteractions in vivo with formaldehyde: evidence thathistone H4 is retained on a highly transcribed gene.Cell 53, 937–947

7 Braunstein, M. et al. (1993) Transcriptional silencing inyeast is associated with reduced nucleosomeacetylation. Genes Dev. 7, 592–604

8 Kuo M.H. et al. (1996) Transcription linked acetylationby Gcn5 of histones H3 and H4 of specific lysines.Nature 383, 269–272

9 Orlando, V. et al. (1997) Analysis of chromatinstructure by in vivo formaldehyde crosslinking.Methods 11, 205–214

10 Orlando, V. and Paro, R. (1993) Mapping Polycomb-repressed domains in the BX-C using in vivoformaldehyde crosslinked chromatin Cell 75,1187–1198

11 Dedon, P.C. et al. (1991) A simplified formaldehyde-fixation and immunoprecipitation technique for studyingprotein-DN interactions. Anal. Biochem. 197, 83–90

12 Hecht, A. et al. (1996) Spreading of transcriptionalrepressor SIR3 from telomeric heterochromatin. Nature383, 92–96

13 Strahl-Bolsinger et al. (1997) SIR2 and SIR4interactions differ in core and extended telomericheterochromatin in yeast. Genes Dev. 11, 83–93

TECHNIQUES

Dennis Chapman (Fig. 1) passed away at his home in Beaconsfield,Buckinghamshire, UK on 28 October1999. He will be best remembered for hiscontributions in the field of membranestructure and dynamics and, towardsthe later stages of his career, for takingmembrane science to the market place.

Dennis was born on 27 May 1927 inSunderland, County Durham, UK. Hecompleted his undergraduate studies inphysics at the University of London andwent on to complete his PhD in electrical engineering at the Universityof Liverpool. During his scientific careerhe published more than 450 scientificpapers and some 16 books, and was oneof the top 300 most cited scientists inthe world between 1965 and 1978. Hestarted his working life as a research sci-entist at Unilever where he employed di-verse physical techniques to character-ize the phase properties of triglyceridesand cocoa butter. Between 1960 and1963, Dennis enjoyed a brief interlude inCambridge as a Comyn Berkeley Fellowat Gonville and Caius College, but re-turned to industry after the 3 years.With his academic background inphysics and engineering, Dennisbrought a fresh angle to the study ofbiomembranes. He realized that a betterunderstanding of biochemical pro-cesses required a multidisciplinary ap-proach and he was one of the first to

apply many of the physical techniquesthat are now routinely used in structuralbiochemistry. For example, he was oneof the pioneers in the use of both protonand deuterium NMR spectroscopy tostudy biomembrane structures. In the1960s, Dennis’s research focused oncharacterizing the fluidity of phospho-lipid membranes. For example, heshowed that above the lipid-phase tran-sition temperature, addition of choles-terol reduces the mobility of hydro-carbon chains of fluid phospholipidmembranes, and he was brave enoughto tackle complex systems such as ery-throcyte membranes using NMR spec-troscopy. While at Unilever, Dennis usedinfrared spectroscopy (IR) to study thestructure and phase properties of fattyacids and phospholipids. Due to limi-tations of IR at the time, these studieswere restricted to non-aqueous media.However, many years later, followingtechnological developments that al-lowed the use of IR for analysis of aque-ous systems, Dennis embraced thismethodology, realizing its potential forstudying not only lipids but also mem-brane-bound proteins. It is now one ofthe popular techniques for characteriz-ing lipids and membrane proteins. Fromhis work we have obtained a wealth ofinformation regarding the structure anddynamics of membranes, and we nowhave a picture of biomembrane struc-

ture that is not static but a highly dy-namic system where lipids and proteinsdisplay diverse motions within thephospholipid membrane.

Dennis’s first academic post was atSheffield University, where his inaugurallecture in 1968 was entitled ‘Industry andthe role of universities – collision or coop-eration’. In Sheffield, Dennis had a highlyproductive research group, and many ofhis former students and postdocs arenow leading membrane scientists. In1976, Dennis moved to London as aSenior Wellcome Trust Research Fellow atChelsea College, University of London. In1977, he became Professor of BiophysicalChemistry at the Royal Free HospitalSchool of Medicine, University of London.In 1988, Dennis founded a new depart-ment, which he called the Department ofProtein and Molecular Biology. Thechoice of wording is significant and re-flects his awareness that the study of pro-teins has become more fashionable thanlipids. It is therefore not surprising thatmuch of his work at the Royal Free fo-cused on using biophysical techniques tocharacterize membrane proteins andlipid–protein interactions.

Dennis was a dynamic man with an abil-ity to adapt to changes in time and the sci-entific climate. It was at the Royal FreeHospital that Dennis initiated anotherarea of research for which he will be re-membered for many years to come. Thiswas his pioneering work on the use ofbiomembrane components to developnovel biomedical materials, initiating anew field of research, which he coined as‘biomembrane mimicry’. This novel classof biomaterials was based on mimickingthe outer leaflet of the lipid bilayer of red blood cells. Dennis was aware of a previous study, which demonstrated that

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Dennis Chapman(1927–1999)