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The cell has evolved many strategies to orches-trate gene activation or repression. Spilianakiset al.1 (page 637 of this issue) reveal a novelmechanism of gene regulation, throwing lighton how cells organize their genome to respondefficiently to stimuli. They show that genes ondifferent chromosomes that are destined to be expressed within a common cell lineage

are brought together in the nucleus. Suchinter-chromosomal communication has beensuspected for some time, but this is the firstevidence that it actually takes place.

Our understanding of gene regulation hasmoved from an initial notion of a one-dimen-sional array of regulatory elements next toeach other on the same thread of DNA as the

Figure 1 | Gene correction.Patients with severe combinedimmunodeficiency bear a particularmutation that influences thefunction of their T cells. Urnov et al.1 used the strategy shown hereto correct the fault in a significantpercentage of cultured cells carryingthe same mutation. a, Zinc-fingernucleases are designed so that their zinc-finger domains willrecognize specific sites near themutation. b, The nuclease domainsintroduce a double-strand breakinto the DNA. c, A wild-type(unmutated) sequence is introduced into the cells and used as the template for a cellularrepair process, homologousrecombination. d, The mutantsequence is corrected.

Zinc-finger domainsNuclease domains

Mutant sequence

Wild-type template

Corrected sequence

Homologous recombination

Double-strandedbreak

a

b

c

d

to different sequences. And this catalogue has been expanded using a technique calledphage display6.

The DNA-cleaving domain of ZFNs isderived from the enzyme FokI, and is not itselfspecific. As this domain must dimerize toachieve efficient DNA cleavage, the strategyrequires two ZFNs to bind at or near the site to be corrected. By linking four zinc fingers intandem for each of the two ZFNs, a combinedrecognition site of 24 base pairs is attained,specifying a unique address within thegenome (Fig. 1). So the first key reagent, allow-ing detection of the site to be corrected, isalready available.

This dimerized enzyme introduces adouble-strand break into DNA at or near the site of a mutation. The cell’s powerfulrepair mechanisms are then engaged. One ofthese, ‘homologous recombination’, repairs the damage by using similar sequences within thecell as a template, in a process that involvesswitching DNA strands between stretches of base-paired DNA. The similar sequencewould usually be found within the genome,but it could also be introduced into the cell.This solves the second problem — effecting a base change.

The most remarkable feature of this com-plex process is the frequency at which itoccurs, even in the absence of any selection forcorrected cells. Urnov et al.1 show correctionof 15–20% of mutated chromosomes in a cellline (that is, in serially cultured cells), allowingthe efficient introduction of two engineeredZFNs and the normal template to effect thechange. They even report around 5% correc-tion in human T cells taken from a subject,where introduction efficiencies are usuallyconsiderably lower. This contrasts starkly withthe rate of homologous recombination whenno double-strand breaks are introduced,which is closer to 0.001%.

What implications does this work have for treating human disease? The DNAsequence that Urnov et al. altered is one that is mutated in one form of severe combined

GENE REGULATION

Kissing chromosomesDimitris Kioussis

A three-dimensional examination of gene regulation suggests that portionsfrom different chromosomes ‘communicate’ with each other, and bringrelated genes together in the nucleus to coordinate their expression.

colleagues’ approach gets around this problem,offering the possibility of correcting the defectwithout serious side effects.

The same technique could be applied to thetreatment of other diseases in which bloodcells are affected, promising safer and morerealistic therapies for other forms of SCID andfor haemoglobin disorders such as sickle-celldisease. An attractive feature of this particulargene-correction strategy is that it does notrequire the long-term expression of either theZFNs or the normal template. So it can be usedas a ‘hit-and-run’ approach. To quote EdwardFitzGerald: “The moving finger writes; andhaving writ, moves on.”

Nevertheless, several hurdles remain beforethis technique can be successfully translated tohumans. For example, it is unclear whether theengineered ZFNs will provoke an immunereaction that rejects these enzymes. And fornow, the technique will almost certainly belimited to cell types that can be extracted fromthe patient, manipulated ex vivo and thenreturned, given that most of the strategies usedfor in vivo gene delivery are too inefficient.Finally, it will be important to determine just how specific the ZFNs are, and whetherDNA deletions or rearrangements occur at thetarget sites. Using Southern blotting, Urnov et al. detected neither mistargeting nor grossrearrangements or deletions, but moredetailed studies will be required. ■

Katherine A. High is at The Children’s Hospital ofPhiladelphia, 3615 Civic Center Boulevard,Philadelphia, Pennsylvania 19104, USA.e-mail: high@email.chop.edu

1. Urnov, F. D. et al. Nature 435, 646–651 (2005).2. Porteus, M. H. & Baltimore, D. Science 300, 763 (2003). 3. Bibikova, M. et al. Science 300, 764 (2003).4. Kim, Y. G., Cha, J. & Chandrasegaran, S. Proc. Natl Acad. Sci.

USA 93, 1156–1160 (1996).5. Tupler, R. et al. Nature 409, 832–833 (2001).6. Klug, A. FEBS Lett. 579, 892–894 (2005).7. Cavazzana-Calvo, M. et al. Science 288, 669–672 (2000).8. Hacein-Bey-Abina, S. et al. N. Engl. J. Med. 348, 255–256

(2003).

immunodeficiency (SCID) — a disease that,without bone-marrow transplantation or genetherapy, is fatal early in childhood. Severalyears ago, Cavazzana-Calvo et al.7 reportedsuccessful gene-transfer studies of SCID. Theyused a retrovirus to add a normal gene to tar-get cells (blood cells that express the CD34marker) taken from patients. After growth inculture, the cells were re-infused into thepatients. About 20–40% of the re-infused cellshad taken up the normal gene. In the patients,this level of gene transfer was adequate,because the treated cells had a robust survivaladvantage over the mutant cells in vivo. So ifthe rates of cell correction achieved by Urnov et al. in human T cells can be achieved inCD34-expressing cells, they will probably beadequate to restore immune function.

Moreover, this approach has the advantageof avoiding unwanted ‘integration events’, asthe strategy is to correct the gene rather thanto insert a normal copy. In the earlier study7,problems occurred as a consequence of gene integration at an untoward site (next to a growth-promoting gene)8. Urnov and

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gene (Fig. 1a) to an appreciation that genes areassociated with groups of proteins, formingmultimolecular complexes that are arrangedin structures generically known as chromatin2.The subsequent discovery that distant, con-tiguous sequences can have a profound effecton gene expression introduced a seconddimension onto the scene, with ‘looping’ and‘scanning’ (probably mediated by the attachedproteins) invoked to explain these long-rangeinteractions (Fig. 1b)3.

But to explain how genes that are farremoved from each other in the genome, andeven on different chromosomes, can be coor-dinated to be expressed together, or to pre-clude the expression of one another, required aleap into a third dimension. The spatial loca-tion of a gene within a cell nucleus can deter-mine whether it is expressed or not: genesresiding in areas of chromatin that containrepressive factors (heterochromatin) are silent;conversely, genes in nuclear regions full ofactivating proteins (euchromatin) are usuallyswitched on (Fig. 1c)4,5. How do genes findtheir appropriate location in the nucleus of acell, and how are genes that must be expressedherded into active neighbourhoods?

To address these questions, Spilianakis andcolleagues1 used immune cells called T cells.As they mature, T cells organize themselvesinto subsets that are assigned specific duties.Thus, T helper (TH) cells produce factors thathelp other cells of the immune system to func-tion optimally. After antigen stimulation,naive (undecided) TH cells develop into either

TH1 cells, which produce one set of effectormolecules (for example interferon (IFN)-γ), orTH2 cells, which produce a different set (forexample interleukin (IL)-4 and IL-5)6. Theauthors explored the organization of twogenomic regions within the TH subsets: thegene encoding IFN-γ (called Ifng), which ismainly active in TH1 cells, and a multi-genecomplex including the genes encoding IL-4and IL-5 (Il4 and Il5), which is mainly active inTH2 cells.

Viewed through two-dimensional analyses,Ifng seems to be regulated by elements foundnear it on chromosome 10, whereas expressionof Il4 and Il5 on chromosome 11 seems to beregulated by a ‘locus control region’ (LCR) onthe same chromosome, which directs theentire TH2 gene complex. Spilianakis et al.asked whether these two genetic regions are inclose proximity or interact in the nuclei ofnaive TH, TH1 or TH2 cells.

Biochemical and imaging experimentsshowed that the two regions (on two differentchromosomes) are in close proximity in thenucleus of naive TH cells, but after stimulationsthat induce a TH1 or a TH2 state they seem tomove away from each other (Fig. 1c). Theauthors’ interpretation of this is that in thenaive TH cell, the two gene complexes are closetogether in a region of the nucleus that ispoised for gene expression. Upon receiving aspecific stimulus, the gene to be activated (forexample Ifng after a TH1 stimulus) is allowed tobegin expression, whereas its counterpart thatis to remain silent (in this case the TH2 genes)

is moved, presumably to a more repressedregion of the nucleus (Fig. 1c).

Spilianakis et al. provide some evidence thatfollowing stimulation, the inter-chromosomalinteractions in the naive cell are replaced byintra-chromosomal ones between regulatoryelements within the same gene region. More-over, the inter-chromosomal association doesseem to regulate to some extent the expressionof the genes involved, as deletion of a regulatory element (HSS7) from the TH2 LCR on chromosome 11 disturbs the inter-chromosomal associations, and this is coupledwith delayed expression kinetics of Ifng fromchromosome 10.

These remarkable findings will puzzle us forsome time to come. Are inter- and intra-chro-mosomal associations a general phenomenonoccurring in all types of cell7 or not8? What arethe mechanisms that bring two functionallyrelated genes on two different chromosomestogether? Between cell divisions, chromosomesexpand to occupy ‘chromosomal territories’ inthe nucleus9,10. To allow genes from differentchromosomes to come into close proximity,therefore, perhaps chromatin strands mightextend from the main body of a chromosome’sterritory to an area of the nucleus where tran-scription is possible (Fig. 1d)7,11.

Do both copies of the Ifng gene find theirfunctional counterparts of the TH2 gene complex at some point in the cell’s develop-ment programme? The results presented bySpilianakis et al. are snapshots and indicatethat at any particular time the majority of thecells show only one copy of Ifng in close prox-imity to one TH2 complex. The remaining Ifngand TH2 complex are found separately. Doesthis mean that there is fluidity in this inter-action, allowing genes to come together andthen drift apart again after some time? Or doesit imply that these genes are expressed fromonly one of their copies? To start addressingthese questions, and others that similar sys-tems may bring up, we would have to developtechnologies that allow the detection of inter-chromosomal interactions not as a snapshot,but as a movie. Is it time to go 4D? ■

Dimitris Kioussis is in the Division of MolecularImmunology, National Institute for MedicalResearch, The Ridgeway, London NW7 1AA, UK.e-mail: dkioussis@nimr.mrc.ac.uk

1. Spilianakis, C. G., Lalioti, M. D., Town, T., Lee, G. R. & Flavell, R. A. Nature 435, 637–645 (2005).

2. Felsenfeld, G. & Groudine, M. Nature 421, 448–453(2003).

3. De Laat, W. & Grosveld, F. Chromosome Res. 11, 447–459(2003).

4. Brown, K. E. et al. Cell 3, 207–217 (1999).5. Kioussis, D. & Festenstein, R. Curr. Opin. Genet. Dev. 7,

614–617 (1997).6. O’Garra, A. Immunity 8, 275–283 (1998).7. Osborne, C. S. et al. Nature Genet. 36, 1065–1071 (2004).8. Kim, S. H. et al. Cytogenet. Genome Res. 105, 292–301

(2004).9. Parada, L. A., Sotiriou, S. & Misteli, T. Exp. Cell Res. 296,

64–70 (2004).10. Cremer, T. & Cremer, C. Nature Rev. Genet 2, 292–301 (2001).11. Kosak, S. T. & Groudine, M. Genes Dev. 18, 1372–1384

(2004).

Figure 1 | The three dimensions ofgene regulation. a, Linear view ofgene regulation. The promoter (P)near the start of a gene provides theminimal information needed forgene expression. The function of the promoter is supplemented byenhancers or silencers (E), fartheraway, where regulatory proteinsbind to activate or represstranscription of the gene (arrow). b, The ‘looping–scanning’ model ofgene regulation. The locus controlregion (L) regulates several genes.Proteins binding here scan throughlarge portions of DNA, looping theintervening region out, until theyfind the relevant gene. c, Generegulation in 3D. Spilianakis et al.1

find that genes from differentchromosomes (A and B) are in closeproximity until a developmentalsignal stimulates the cells, when the genes split apart. One moves to a region that represses geneexpression (heterochromatin, red)and the other relocates to an area with many active genes(euchromatin, green). d, Genesfrom different chromosomes might come into contact when the chromatin containing themloops out from their chromosome‘territory’.

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