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could provide multiple therapeutic means tointerfere with leukocyte diapedesis withoutglobal blockade of leukocyte integrins, limit-ing side effects such as immunodeficiencyand dysregulated hematopoiesis.

1. Muller, W.A. Trends Immunol. 24, 326–334 (2003).2. Schenkel, A.R., Mamdouh, Z. & Muller, W.A. Nat.

Immunol. 5, 393–400 (2004).3. Springer, T.A. Cell 76, 301–314 (1994).4. Worthylake, R.A., Lemoine, S., Watson, J.M. &

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Rev. Immunol. 4, 1–14 (2004).6. Smith, A., Bracke, M., Leitinger, B., Porter, J.C. &

Hogg, N. J. Cell Sci. 116, 3123–3133 (2003).7. Mamdouh, Z., Chen, X., Pierini, L.M., Maxfield, F.R. &

Muller, W.A. Nature 421, 748–753 (2003).

8. Barreiro, O. et al. J. Cell. Biol. 157, 1233–1245(2002).

9. Cinamon, G., Shinder, V. & Alon, R. Nat. Immunol. 2,515–522 (2001).

10. Weber, C. & Springer, T.A. J. Immunol. 161,6825–6834 (1998).

11. Marschel, P. & Schmid-Schonbein, G.W. Ann.Biomed. Eng. 30, 333–343 (2002).

12. Gopalan, P.K. et al. J. Leukoc. Biol. 68, 47–57(2000).

NATURE IMMUNOLOGY VOLUME 5 NUMBER 4 APRIL 2004 353

Shh! Shh! It’s, oh, so quietDimitris Kioussis

The regulated expression of Rag genes in lymphocytes ensures the expression of a single antigen receptor on thelymphocyte surface. It now seems that a complex ‘ballet’ of activating and silencing elements controls the precisetiming of Rag expression in thymocytes.

Dimitris Kioussis is at the National Institute for

Medical Research, The Ridgeway, Mill Hill, London

NW7 1AA, UK.

e-mail: dkiouss@nimr.mrc.ac.uk

Silencers are DNA regulatory elements thathave attracted considerable attention in

recent years. The paper by Yannoutsos et al. inthis issue of Nature Immunology describessuch a silencing element that lies in the inter-genic region between recombination activa-tion gene 1 (Rag1) and Rag2 (ref. 1). Incommon with another T cell–associatedsilencer found in Cd4, this element also seemsto exert its function through binding of Runxtranscription factors2. One additional andvery interesting aspect of the paper is thedescription of another regulatory region 85kb away from the silencer upstream of Rag2,which the authors call the antisilencing ele-ment (ASE). This region seems to be neces-sary to allow expression of Rag genes at thedouble-positive stage of thymocyte develop-ment. Using transgenic mice bearing con-structs with different combinations ofregulatory elements from the Rag complex,they show that in the absence of ASE, thesilencer becomes dominant and expression ofRag genes ceases at the double-positive stage.Conversely, deletion of the intergenic silencerresults in expression of Rag genes within dou-ble-positive thymocytes, even in the absenceof the ASE distal element. ASE resemblesanother element similarly named that isfound in the human VIM locus, encodingvimentin, and that shares the properties ofthe Rag ASE in that it requires “a silencer ele-ment to be present in cis in order to exert apositive effect on gene expression”3.

It was once believed that the default state ofgenes is silence and repression, out of whichthey are rescued by the ‘shining knights’ ofenhancers and their binding factors. Yet thisidea sidelined the issue that some genes aretransiently and/or recurrently expressed dur-ing differentiation as a result of developmen-tal or environmental cues. The immunesystem offers several examples of this type ofgene regulation; for example, the regulationof Cd4 and Cd8 during thymocyte differentia-tion4. This impasse was broken in part whennonmammalian silencers in yeast anddrosophila were discovered. The mammalianequivalent in the immune system remainedelusive until elegant work from the labs ofHedrick and Littman demonstrated the Cd4silencer, a distinct DNA sequence found inthe first intron of Cd4 whose function is toprevent transcription of Cd4 at the double-negative and the CD8+ single-positivestages5,6. Since then, several other silencershave been identified; the Cd4 silencer and theRE-1 element found in several neuronal genesare the most extensively studied7.

However, the discovery of the silencers inmammalian genomes presented molecularbiologists with a new conundrum posed bythe coexistence within the same transcrip-tional unit of two ‘rival forces’: the enhancersthat orchestrate an activating process, whichresults in expression of the gene in question,and the silencers that impose a repressiveeffect aimed at halting the gene’s expression.When the Cd4 silencer was first described,Hedrick proposed a potential model for theinterplay of these two types of elements(enhancers and silencers) and postulatedcases in which the enhancer has the ‘upperhand’ and the gene is expressed and cases in

which this enhancer is overtaken by thesilencer and the gene’s expression is abo-lished. This model still dominates our exper-imental designs, and particular questionshave evolved as discussed below.

Why are silencers active only part of thetime? Silencers share many properties withenhancers. The activity of the latter is usuallymanifested when expression of the enhancer-binding factor begins. A similar hypothesiscould explain why silencing activity is presentat certain points in the cell’s life; that is, thefactors that bind on these DNA elements arepresent only temporarily. For example, Runxproteins that are important in the function ofthe Cd4 silencer are abundant in double-negative and single-positive thymocytes, pre-cisely where Cd4 needs to be silenced2.

How do silencers work? Mechanistically, sev-eral hypotheses seem to be possible given theavailable data. The prototype would indicatethat the repressive protein directly inhibitstranscription through a repressor domain (Fig. 1a). This may be true for bacterial geneexpression; in higher organisms, however, thepicture is not that simple. With the realizationthat chromatin structure and nuclear localiza-tion of the genes are central in the regulation oftheir expression, more possibilities haveemerged. Thus, it is possible that whatever fac-tor binds the silencer may have the ability totranslocate the complex (including the DNAand, by extension, the gene locus) to a positionin the nucleus where a repressive environmentexists (Fig. 1b). An alternative hypothesiswould postulate that the binding factor couldassociate with molecules with chromatin-remodeling activities (for example, histonedeacetylases, histone methyltransferases,ATPase remodelers and so on), resulting in the

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354 VOLUME 5 NUMBER 4 APRIL 2004 NATURE IMMUNOLOGY

local modification of chromatin and leading torepressive structures8–11 (Fig. 1c). Obviouslyone mechanism does not preclude the other;thus, sometimes the same silencer elementsmay be acting through any of these mecha-nisms, as demonstrated by the muscarinicreceptor gene Chrm4 (also known as M4)and the sodium type II channel gene Scn2a(also known as NaV1.2), both of which con-tain the RE-1 silencer but may be silenced indifferent ways12.

How do enhancers overcome silencers? It ispossible that the interplay between silencersand enhancers is one of competition. Thus,enhancer-binding complexes could recruit thegene to a transcriptionally active nuclear com-partment or alternatively could counteract therepressive activities of histone deacetylases orhistone methyltransferases recruited on site bythe silencer-binding complexes. Mass actionkinetics are important in such competitive sit-uations; thus, it is likely that the relative abun-dance of activating proteins over suppressiveones may lead to relief from silencing activitiesand vice versa. However, a more direct process

can also be envisaged: the enhancer-bindingand activating factor may associate directlywith the repressive silencer-binding factor,blocking its binding to the silencer and thusinhibiting the formation of repressive com-plexes on the locus (Fig. 1d). The reverse, ofcourse, is equally possible.

Can specific functions now be assigned topositive elements that antagonize silencers?Yannoutsos et al. propose that an indepen-dent ASE region upstream of the Rag2 locushas precisely and solely such a function. ForRag and also VIM expression, these ASE ele-ments seem to lack transcriptional-enhancingactivity and their function becomes apparentonly when a silencer is present and active incis. As assays become more sophisticated, thedifferences between a specific antisilencingactivity and an enhancing activity willbecome more apparent.

Is gene silencing always associated with thepresence of a silencer? In the two paradigms inlymphocyte differentiation, that is, the devel-opmental regulation of Cd4 and Cd8 expres-sion, diverse mechanisms seem to result in

their repression4. The Cd4 locus uses a silencerto prevent expression of the gene in the dou-ble-negative and CD8+ single-positive T cells.However, so far there is no evidence that theCd8 complex contains an element that can bedescribed definitely as a silencer. In conclusion,we can look forward to more experiments thatwill define the rules of the fascinating interplaybetween the forces of expression and those thatlead to the silence of the genes.

1. Yannoutsos et al. Nat. Immunol. 5, 443–450 (2004).2. Taniuchi, I. et al. Cell 111, 621–633 (2002).3. Wu, Y., Diab, I., Zhang, X., Izmailova, E.S. & Zehner,

Z.E. Oncogene 23, 168–178 (2004).4. Kioussis, D. & Ellmeier, W. Nat. Rev. Immunol. 2,

909–919 (2002).5. Siu, G., Wurster, A.L., Duncan, D.D., Soliman, T.M. &

Hedrick, S.M. EMBO J. 13, 3570–3579 (1994).6. Sawada, S., Scarborough, J.D., Killeen, N. &

Littman, D.R. Cell 77, 917–929 (1994).7. Griffith, E.C., Cowan, C.W. & Greenberg, M.E.

Neuron, 31, 339–340 (2001).8. Aalfs, J.D. & Kingston, R.E. Trends Biochem. Sci. 25,

548–555 (2000).9. Lusser, A. & Kadonaga, J.T. Bioessays 25,

1192–1200 (2003).10. Chi, T.H. et al. Nature 418, 195–199 (2002).11. Ballas, N. et al. Neuron 31, 353–365 (2001).12. Belyaev, N.D. et al. J. Biol. Chem. 279, 556–561

(2004).

Figure 1 How to silence a gene (or not). (a) A silencer-binding protein (SBP) with a repressor domain binds on the gene locus and silences it directly. (b) The SBP associates with heterochromatin-binding proteins (HBPs), which recruit the gene locus into repressive heterochromatic regions (blue box) in the nucleus. (c) The SBP recruits molecules with chromatin-remodeling activities (such as HDAC or Sin3), which generate heterochromatic structuresincompatible with expression. (d) The silencing activity can be antagonized by an enhancer or an ASE by removal of the SBP from its cognate site due to astrong interaction with the enhancer-binding protein (EBP) or ASE-binding protein.

HBPEBP

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SBP SBP

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