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engaged in stable strand-exchange. Themost obvious possibility is that SC forma-tion actually disrupts unstable interho-molog DNA contacts. In fact, SC formationforces the chromatin loops of homologs toemanate outward in opposite directionsfrom their conjoined axes, which might tendto disrupt nascent contacts between homol-ogous loops. γ-H2AX–mediated delay of SCformation until commitment to strand-exchange would prevent this tendency.

Role of γ-H2AX in SC formation: aproposalMitotic and meiotic chromosomes undergoglobal cycles of chromatin expansion andcontraction, probably resulting from chro-matin fiber changes such as phosphoryla-tion and dephosphorylation. At meioticprophase, the resulting expansion and con-traction of chromatin loops will promote

extension and contraction along the chro-mosome axes (N.K. et al., unpublisheddata). Because leptotene is an expansionphase and zygotene is a contraction phase,the temporal program of SC formationwould be explained if SC formation wereprecluded by extension and permitted bycontraction, for example, through effectson molecular contacts along the axes.

H2AX phosphorylation and dephospho-rylation may represent a specialized cycle ofchromatin expansion and contraction,imposed on a local basis, in response to for-mation and repair of DSBs—a cycle thatpresumably influences DNA events in bothmitotic repair and meiotic recombination.However, recruiting this specialized cycle tothe meiotic process would also make SCformation specifically dependent on theDNA events of recombination. This special-ized coupling program would be superim-

posed on more basic global changes thatpreclude and permit SC formation, irre-spective of recombination. �1. Kleckner, N. Proc. Natl. Acad. Sci. USA 93,

8167–8174 (1996).2. Mahadevaiah, S.K. et al. Nature Genet. 27, 271–276

(2001).3. Zickler, D. & Kleckner, N. Annu. Rev. Genet. 33,

603–754 (1999).4. Keeney, S., Giroux, C.N. & Kleckner, N. Cell 88,

375–384 (1997).5. Bishop, D.K. Cell 79, 1081–1092 (1994).6. Schwacha, A. & Kleckner, N. Cell 83, 783–791 (1995).7. Padmore, R., Cao, L. & Kleckner, N. Cell 66,

1239–1256 (1991).8. Keeney, S. Curr. Topics Dev. Biol. (in press).9. Rogakou, E.P., Boon, C., Redon, C. & Bonner, W.M. J.

Cell Biol. 146, 905–916 (1999).10. Paull, T.T. et al. Curr. Biol. 10, 886–895 (2000).11. McKim, K.S. & Hayashi-Hagihara, A. Genes Dev. 12,

2932–2942 (1998).12. Dernburg, A.F. et al. Cell 94, 387–398 (1998).13. Downs, J.A., Lowndes, N.F. & Jackson, S.P. Nature

408, 1001–1004 (2000).14. Moens, P.B. et al. Chromosoma 106, 207–215

(1997).15. Plug, A.W. et al. J. Cell Sci. 111, 413–423 (1998).16. McClintock, B. Z. Zellforsch. Mikrosk. Anat. 19,

191–237 (1933).

Deconstructing DiGeorge syndromeMartina Schinke & Seigo Izumo

Cardiovascular Division, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USAe-mail: sizumo@caregroup.harvard.edu

DiGeorge syndrome is the most frequent contiguous-gene deletion syndrome in humans, occurring with an estimated frequencyof 1 in 4,000 live births. Extensive microdeletion mapping in a large number of affected individuals has failed to identify a singlegene or a combination of genes commonly deleted. Two new studies implicate the transcription factor TBX1 as a key candidategene for the aortic arch malformations seen in DGS, and are consistent with the concept that some congenital diseases are causedby a reduced dosage of genes that control development. However, a similar study focusing on the adaptor protein Crkol showsthat other genes within the deleted regions might affect the same developmental pathways.

Extensive microdeletion mapping has failedto identify a single gene or a combination ofgenes commonly deleted in people withDiGeorge syndrome (DGS), leading to thehypothesis that multiple genes contribute tothe complex phenotypic spectrum of thedisease. Three studies now shed light on thegenetics underlying the syndrome. On page286 of this issue, Loydie Jerome and VirginiaPapaioannou1 report that absence of thetranscription factor TBX1 may be critical tothe aortic arch malformations seen in DGS.A related study is reported in this week’sNature by Lindsay et al2. A third study byDeborah Guris et al.3 reports that mice witha homozygous deletion in Crkol show simi-lar defects in aortic arch developments.

A.M. DiGeorge was the first to proposethat the concurrent absence of the thy-mus and parathyroid might be a result ofa perturbation in the development of the

third and fourth pharyngeal pouches4. Itwas soon realized that a spectrum ofother anomalies is also associated withthis syndrome, including cleft palate, cleftlip and neural tube defects. The majorityof people with DGS have heart defectsinvolving the cardiac outflow tract, withabnormal patterning of the aortic archarteries, improper alignment and septa-tion of the aortic and pulmonary outflowvessels, and defects in the septation of thetwo ventricular chambers. The unifyingfeature of the most common anomalieswas thought to be a distinct population ofneural crest cells that populate the aorticarch arteries and are essential for the nor-mal development of the thymus andparathyroid glands5. A subset of theseneural cells also migrates to the outflowtract of the heart, where they participatein outflow tract septation.

It was not until the 1980s thatresearchers linked DGS to chromosomaltranslocations and deletions of chromo-some 22 (ref. 6). Subsequent molecularcytogenetic studies have shown that 90%of patients have microdeletions of onecopy of the region 22q11.21–22q11.23,spanning approximately 2 Mb. Furtherdetailed mapping refined the minimalcritical region to 250 kb. Numerous can-didate genes have been proposed basedupon their location within the region oran expression pattern that is consistentwith the affected organs, or identifica-tion of patients with a DGS phenotypewho have a smaller deletion within thecandidate locus. But it has not been pos-sible to attribute an individual feature orthe entire phenotypic spectrum to aber-rations in a single gene or combinationof genes.

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Gene-targeting techniques are a powerfultool for to search for the gene(s) involved inDGS. Various models have been generatedwith attenuation or deletions of the mostpromising candidate genes, such as HIRA, atranscriptional corepressor of cell-cycle–dependent histone gene transcription7, andUFD1L, the homolog of a highly conservedyeast gene involved in the degradation ofubiquinated proteins8. In addition, Lindsayet al.9 deleted a 1.2-Mb fragment (Df1) ofmouse chromosome 16 (syntenic to humanchromosome 22q11), that covers manyorthologs of genes commonly deleted inDGS patients, such as UFD1L, but not oth-ers, such as HIRA (see figure).

However, all of these models fall shortin recapitulating the spectrum of featuresof DGS even when both chromosomalcopies are mutated. Furthermore, hap-loinsufficiency of Hira and Ufd1l results inmice with a normal phenotype. Only Df1-heterozygous mice exhibit cardiovasculardefects, including aortic arch abnormali-ties caused by defective development ofthe fourth pharyngeal arch arteries9, butthey do not manifest the entire pheno-typic spectrum of DGS. That said, thereare examples of mouse models of humanautosomal dominant disorders that dis-play more subtle and incomplete pheno-types10–12. Heterozygous mutations in themouse are not always sufficient to producea phenotype equivalent to humans.

Pieces of the puzzleThe studies by Jerome and Papaioannou2,and Guris et al.2 provide new pieces to thepuzzle that is DGS. Guris et al. engineeredmice deficient in Crkol, a gene thatencodes an adapter protein that has beenimplicated in response to growth factorsand focal adhesion signalling. It is highlyexpressed in tissues derived from theneural crest during development. It istherefore not suprising that Crkol–/– micedie during gestation and have multipledefects in neural crest derivatives—theaortic arch arteries, the cardiac outflowtract, the thymus and craniofacial struc-tures. This is similar to the DGS pheno-type. However, Crkol+/– mice do notdisplay this phenotype, which may beowing to different sensitivities to genedosage of mice and humans.

Jerome and Papaioannou1 providecompelling arguments for TBX1 being amajor genetic determinant in the etiologyof DGS. TBX1 belongs to a family of tran-scription factors that contain a conservedDNA-binding domain called the T-box.Other prominent members of this familyinclude TBX3 and TBX5, both of whichare responsible for human autosomaldominant developmental abnormalities,such as ulnar–mammary syndrome13 andHolt–Oram syndrome14, respectively.

As with most other candidate genes,deletion of Tbx1 results in embryonic

lethality. Jerome and Papaioannou1 havesucceeded, however, in showing thatTbx1–/– embryos display virtually all(testable) features of DGS, includinghypoplasia of the thymus and parathyroidglands, cardiac outflow abnormalities,abnormal facial features and cleft palate.This detailed analysis was facilitated by thelate gestational death of Tbx1–/– embryosand reflects the authors’ exceptional abili-ties to detect subtle anatomical defects inthe embryo.

Importantly, embryos haploinsufficientfor Tbx1 have a less penetrant phenotypeand do not reflect the entire phenotypicspectrum of DGS. Depending on theirgenetic background, they show a varyingdegree of absent or reduced fourth aorticarches. This phenotype nicely matchesthe malformations seen in mice lackingDf1 (ref. 9), suggesting that Tbx1 is thedisease-causing gene within the Df1-deleted region.

Lindsay et al.2 come to the same con-clusion using a more complex but ele-gant transgenic approach. They firstnarrowed down the Df1 critical regionfrom 1.2 Mb (Df1) to 200 Kb (Df3/Df4;see figure) using chromosomal engineer-ing and rescue transgenesis, and then,using gene targeting, confirmed Tbx1 ascritical for the formation of the fourthpharyngeal arch. Complete loss of func-tion of Tbx1, as tested in Tbx1/Df1

Details of a common deletion. Schematic representation of the human chromosomal region 22q11 associated with DGS and the syntenic region of mouse chromo-some 16. The gene order is inverted between human and mouse in a segment of this region, as indicated by crossed arrows. The table below summarizes the pheno-types of mice homozygous or heterozygous mutant for chromosomal deletions (horizontal arrows) or gene mutations (X, and highlighted by vertical bars). In contrast,HIRA expression was attenuated by antisense oligonucleotides in chick embryos (marked by ↓). The distance between genes is not drawn to scale. (Image provided byIuan-Bor Chen.) Df-deletions and gene mutations as cited in the text, except for deletion 1 (ref. 16).

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embryos, revealed severe disruption ofthe pharyngeal arch artery system at anearly developmental stage (embryonicday 10.5). Because Lindsay et al.2 did notanalyze later developmental stages, it isyet to be determined whether other DGSfeatures are related to the loss of functionof Tbx1 in their model.

Putting it all togetherThat TBX1 haploinsufficiency is likely tobe the major determinant of aortic archabnormalities in people with DGS comesas welcome news. It should be noted, how-ever, that point mutations in the codingsequence of TBX1 have not been found inpeople who present with DGS but lack a22q11 deletion2,15. The new studies ofTbx1 will undoubtedly reopen the search

for such point mutations. As the TBX1locus is not always included in the 22q11deletion associated with DGS, and thephenotypic spectrum of DF1+/– andTbx+/– mice is limited to aortic arch mal-formations, it seems likely that there are atleast two DGS-related loci within the22q11 region. Other genes, such as CRKL,might act independently or in conjunc-tion with TBX1 to affect the same develop-mental pathways. The analysis ofheterozygous mice that lack two or morecandidate genes, and people with featuresof DGS but no 22q11 deletion may help toaddress these issues. �

Note added in proof: An independent report byMerscher et al.17on the involvement of TBX1 inDGS is in press at Cell.

Chipping away at chromatinRobert Martienssen

Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.e-mail: martiens@cshl.org

When DNA-binding proteins are tethered to dam methylase from Escherichia coli, adenine methylation is directed to eukaryotictarget sites in vivo. Hybridization of methylated DNA to microarrays allows binding sites to be displayed genome-wide, provid-ing a versatile alternative to chromatin immunoprecipitation.

Methylation of adenine, forming N6-methyl adenine (m6A), is widespread inprokaryotic DNA but occurs at very lowlevels, or not at all, in the nuclear DNA ofeukaryotes. Nonetheless, the cells of mam-mals, Drosophila and yeast can toleratemethylation by the prokaryotic DNA m6Amethyl-transferase, dam. On page 304, Basvan Steensel and colleagues1 describe amethod that takes advantage of this differ-ence between prokaryotic and eukaryoticDNA to profile chromatin-binding sites incomplex genomes.

In mammalian cells2, yeast3 andDrosophila4, dam methylase of Escherichiacoli has been used to probe chromatin inmuch the same way as nucleases andrestriction enzymes have been used in thepast. In each case, restriction or methyla-tion of a given DNA sequence is inter-preted to mean that the DNA is relativelyfree of chromatin and other hindrances toenzyme access. Using Drosophila DNA as atemplate, van Steensel and Henikoff5

modified this strategy by fusing the dammethylase gene with the DNA-bindingdomain of chromatin proteins and tran-

scription factors such as Gal4. They foundthat, on exposure to these constructs, thebinding sites of Gal4 and HP-1, in addi-tion to a few kilobases of surroundingDNA, were methylated—as revealed byrestriction digests and Southern blotting.They called the technique ‘DamID’.

Now, these same authors describe amethod to map dam-methylated sites usingmicroarrays1. DNA methylated by damfusion proteins becomes sensitive to diges-tion with DpnI (see figure). Small genomicDpnI fragments were isolated from cellsexpressing the fusion protein and allowed tohybridize (or not) with microarrays. Probesrepresenting target genes bound fragmentsmethylated by dam fusion proteins, but notcontrol fragments of genomes methylatedby dam alone. Specificity of binding wasassessed in replicate experiments. Whereasthe relative affinities of different sites werealso estimated, their accuracy relies on lim-iting amounts of both tethered and unteth-ered proteins relative to their binding sites,which was not tested directly.

Although the arrays were comprised of acomparatively small set of probes repre-

senting genes and transposons, the resultsindicate the feasibility of the technique forgenome-wide mapping. The authors pro-vide several demonstrations. In one, HP1-dam fusions were shown to methylate 12of 13 transposons on the array. This isconsistent with the location of trans-posons in heterochromatin, which isknown to bind HP-1. It also gives credibil-ity to the idea that heterochromatin func-tions as a genome defense mechanism toprevent transposon spread, although itcould also simply reflect its evolutionaryorigin6,7. In another demonstration, theGAGA-binding factor (GAF) was shownto bind a variety of euchromatic targetgenes which had significantly elevated lev-els of GA dinucleotides. The HP1- andGAF-binding sites were non-overlapping,a result confirmed by immuno-localiza-tion using tagged proteins in culturedDrosophila cells.

In a third test, the authors fused one offour Drosophila homologs of the yeastsilencing gene SIR2 to dam and expressedthe fusion product in cultured Drosophilacells. They found that Sir2-binding sites in

1. Jerome, L.A. & Papaioannou, V.E. Nature Genet. 27,286–291 (2001).

2. Lindsay, E.A. et al. Nature 409, (in press) (2001).3. Guris, D.L., Fantes, J., Tara, D., Druker, B.J. &

Imamoto, A. Nature Genet. 27, 293–298 (2001).4. DiGeorge, A.M. Birth Defects Orig. Art. Ser. IV,

116–121 (1968).5. Emanuel, B.S., Budarf, M.L. & Scambler P.J. Heart

Development (eds. Harvey, R.P. & Rosenthal, N.)463–478 (Academic, New York, 1998).

6. Moerman, P., Goddeeris, P., Lauwerijns, J. & Van derHauwaert, L.G. Br. Heart J. 44, 452–459 (1980).

7. Farrell, M.J. et al. Circ. Res. 84, 127–35 (1999).8. Yamagishi, H., Garg, V., Matsuoka, R., Thomas, T. &

Srivastava, D. Science 283, 1158–1161 (1999).9. Lindsay, E.A. et al. Nature 401, 379–383 (1999).10. Wilkie, A.O.M. et al. Nature Genet. 24, 391–395

(2000).11. Biben, C. et al. Circ. Res. 87, 888–895 (2000).12. Peters, H., Neubuser, A., Kratochwil, K. & Balling, R.

Genes Dev. 12, 2735–2747 (1998).13. Bamshad, M. et al. Nature Genet. 16, 311–315

(1997).14. Basson, C.T. et al. Nature Genet. 15, 30–35 (1997).15. Chieffo, C. et al. Genomics 43, 267–277 (1997).16. Kimber, W.L. et al. Hum. Mol. Genet. 8, 2229–2237

(1999).17. Merscher, S. et al. Cell 104, 1–20 (2001).

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