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increased Rac1–GTP levels. And activating Rac1when Rap1 function was compromised restoredcell spreading.

Faced with several possibilities for how Rap1might induce cell spreading through Rac1, theauthors explored potential interactions of Rap1with Rac GEFs. Rap1 could bind to the centralDbl-homology and pleckstrin homology(DH–PH) domains of VAV2 and Tiam1, butnot to SWAP-70 or COOL-1. But although acti-vated versions of these GEFs enhanced spread-ing in control cells, they couldn’t do so in cells inwhich Rap1 activity was inhibited — unlike thecase mentioned above, in which activated Rac1could overcome impaired Rap1 activity.

Again, there were many possible reasons forthe requirement of Rap1 activity for VAV2 andTiam1 function. Active Rap1 didn’t seem neces-sary to stimulate the catalytic activities of theGEFs, so, instead, the authors studied the poten-tial influence of Rap1 on GEF localization. Instudies using extracts from fractionatedpseudopodia and by immunofluorescence, bothGDP- and GTP-bound Rap1 were found inmembrane protrusions that were associatedwith the substratum. So, too, was VAV2, but not

They belong to the same subfamily, but thesmall GTPases Rap1 and K-Ras show siblingrivalry when it comes to cell adhesion.Oncogenic mutations in K-Ras cause cells tolose adhesion, whereas Rap1 has a positiveinfluence on cell spreading and adhesion.William Arthur, Lawrence Quilliam andJonathan Cooper used the fact that the Rap1yeast orthologue activates a Rho protein bystimulating a guanine nucleotide-exchange fac-tor (GEF) to explore the possibility that, inmammalian cells, Rap1 might also mediate cellspreading by activating Rho-family GTPases.Their findings are reported in The Journal ofCell Biology.

When suspended cells were re-plated onto afibronectin surface, Rap1 and the Rho-familyGTPase Rac1 were both activated. And cellsexpressing active forms of Rap1, Rac1 or RacGEFs were all similarly spread. On furtherinvestigation of an interrelationship betweenRap1 and Rho-family GTPases, the authorsfound that inhibiting Rac1, but not Cdc42 orRhoA, antagonized Rap1-induced cell spread-ing, which implies that Rap1 requires Rac1 forspreading. In support of this, Rap1 activation

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R E S E A R C H H I G H L I G H T S

The regulation of the tumour suppressor andtranscription factor p53 is highly complex andinvolves different types of post-translationalmodification, including phosphorylation andubiquitylation. Recent findings reported inNature now add to the complexity, as DannyReinberg and colleagues have shown that p53can be specifically methylated, and that thisregulates its stability and the expression oftarget genes.

The histone lysine methyltransferase Set9 isknown to methylate Lys4 of histone H3 invitro.While searching for other substrates for

Set9, Reinberg and co-workers identified p53as an in vitro substrate — other proteins thatwere tested did not serve as Set9 substrates,and alternative protein methyltransferasescouldn’t methylate p53. Importantly, however,other polypeptides that are present in HeLa-cell-derived extracts also served as substratesfor Set9, and a recent report indicates that thetranscription-initiation factor TAF10 can alsobe methylated by Set9. The p53 methylationsite was mapped to a single Lys residue(Lys372) in the regulatory C-terminal regionof p53, which is subjected to multiple post-translational modifications.

The affinity of Set9 for a p53 peptide was, infact, higher than its affinity for an equivalenthistone-H3 peptide. This led the authors todetermine the structure of Set9 in complexwith a monomethylated p53 peptide, and

compare it with the previously solved complexof Set9 with a monomethylated histone-H3peptide. Overall, the structures are verysimilar, and the Set9 residues that interact withthe three amino-acid residues N-terminal tothe methylated Lys residue are the same for thep53 peptide and the H3 peptide, despite thedifference in peptide sequence. Reinberg andcolleagues speculate that more distant residuesin p53 might contribute to the substratespecificity.

To study the significance of Set9-methylationof p53 in vivo, the authors stably transfectedcells that expressed endogenous p53 with wild-type Set9 or with a methylation-defective Set9mutant. Using an antibody that is specific forLys372-methylated p53, methylated p53 couldbe detected in extracts from the former cellsbut not the latter.When untransfected cellswere treated with an anti-cancer drug thatinduces DNA damage and therefore a p53-responsive pathway, the amount of methylatedp53 was increased compared with untreatedcells. However, by introducing Set9 smallinterfering (si)RNA into these cells, the level ofmethylated p53 decreased after chemicaltreatment. So, Set9 methylates p53 in vivo andSet9-specific methylation of p53 occurs inresponse to DNA damage.

Next, the authors found that methylated p53is exclusively nuclear, which drew theirattention to the transcriptional activity of p53.Focusing on a well-characterized

Come closer to the edge

C E L L S P R E A D I N G

Complex control

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It’s so often the case — processes that were pre-viously considered separate and distinct turnout to be intimately related. Whereas post-tran-scriptional silencing occurs through RNA inter-ference (RNAi), transcriptional silencing isassociated with ‘silent’ heterochromatin, whichis mediated by histone methylation. The RNA-induced initiation of transcriptional genesilencing (RITS) complex is known to berequired for heterochromatic gene silencing,but it also contains short interfering (si)RNAsthat are generated by Dicer (Dcr), an enzymethat is involved in RNAi. In Nature Genetics,Noma et al. report the discovery of a self-enforcing loop mechanism through which RITSbinding to heterochromatic loci enables theRNAi machinery to function in cis to destroyaberrant RNAs and generate siRNAs for hete-rochromatin maintenance.

Noma et al. began by showing, using chro-matin immunoprecipitation (ChIP) analysisand immunofluorescence, that all three knownsubunits of the RITS complex — Argonaute,Chp1 and Tas3 — stably associate with allknown heterochromatic domains in theSchizosaccharomyces pombe genome, such ascentromeres, telomeres and the mat locus.

RITS is known to be essential for hete-rochromatin assembly at centromeres, so theauthors investigated its possible role in hete-rochromatin assembly at the mat locus. Theyfound that RITS cooperated with Atf1 and Pcr1,which belong to the ATF-CREB (cAMP-respon-sive-element-binding protein) family, to nucle-ate heterochromatin assembly at the mat locus.Mapping of RITS at the mat locus showed that,surprisingly, RITS could spread from the initialnucleation site, a centromere-homologous(cenH) repeat, to the neighbouring sequences.This presumably allows RITS to exert controlover sequences that are incapable of initiatingan RNAi response. For RITS to spread fromcenH throughout the mat locus, though, Swi6— a protein that mediates spreading of Lys9-methylated histone H3 — was required. Dcr-generated siRNAs, though, were not requiredfor this spreading, but were needed for the ini-tial targeting of RITS to cenH. The authorsfound that cenH was transcribed bidirection-ally, and that the level of cenH transcript was

regulated by both transcriptional and post-transcriptional silencing mechanisms: a lack ofSwi6 (which is dispensable for RITS targeting atcenH but is required for transcriptional silenc-ing) or a lack of Dcr (which isn’t needed for het-erochromatin maintenance at the mat regionbut does process dsRNAs into siRNAs) causedcenH transcripts to accumulate — substantiallyso in double mutants.

Noma et al. also investigated whether thestable association of RITS with heterochromaticloci was essential for the processing of rarerepeat transcripts that escape transcriptionalsilencing. Indeed, they found that a point muta-tion in the H3-Lys9-binding domain of Chp1,or deletion of the Clr4 methyltransferase, notonly prevented RITS from binding to chro-matin but also abolished RITS-associatedsiRNAs. Moreover, loss of Clr4 caused cen-tromeric-repeat transcripts to accumulate. SoRITS needs to be associated with chromatin toprocess transcripts into siRNAs and to gener-ate heterochromatin.

The model proposed by the authors estab-lishes a mechanism through which RITS canfunction in cis to bring about effective genesilencing. Heterochromatic marks are initiallyestablished in a Dcr-dependent manner, andtherefore presumably through the RNAi path-way. Such siRNAs somehow guide Clr4 and fac-tors that mediate heterochromatin assembly tohomologous target sequences. RITS then local-izes at these specific loci and mediates the pro-cessing of nascent RNA transcripts, probably byrecruiting Dcr or RNA-dependent RNA poly-merase. This thereby generates more siRNAs —so the mechanism continues…

Katrin BussellReferences and links

ORIGINAL RESEARCH PAPER Noma, K. et al. RITS acts in cisto promote RNA interference-mediated transcriptional and post-transcriptional silencing. Nature Genet. 36, 1174–1180 (2004)WEB SITEShiv Grewal’s laboratory:http://ccr.cancer.gov/staff/staff.asp?profileid=7275

Let us jointogether insilence

E P I G E N E T I C Swhen Rap1 activity was inhibited — indeed,this caused displacement of VAV2 from the celledge.

It follows from these results, then, that artifi-cially targeting Rap1-dependent Rac GEFs suchas VAV2 and Tiam1 to the edge of the cell,where Rap1 is normally active, could compen-sate for a lack of Rap1 activity. By fusing themembrane-localization signals that are presentin the Rap1 C-terminal ‘CAAX’ box and itsadjacent N-terminal hypervariable domain toVAV2, the requirement for GTP-bound Rap1 inRac1 activation could be bypassed.

The resulting Rac1-induced formation ofproductive membrane protrusions associatedwith the adjacent extracellular matrix therebyinduces cell spreading. Such protrusive activitywould, in turn, create new sites for Rap1 local-ization, and so the cycle should continue.

Katrin Bussell

References and linksORIGINAL RESEARCH PAPER Arthur, W. T., Quilliam, L. A. &Cooper, J. A. Rap1 promotes cell spreading by localizing Racguanine nucleotide exchange factors. J. Cell Biol. 167, 111–122(2004) FURTHER READING Kinbara, K. et al. Ras GTPases: integrins’friends or foes? Nature Rev. Mol. Cell Biol. 4, 767–776 (2003)

transcriptional target of p53 — p21 — theyshowed that the increased expression of p21correlated with elevated levels of methylatedp53. In addition, the induction of DNAdamage further increased the level of p21expression. By contrast, overexpression ofcatalytically inactive mutant Set9, or thesiRNA-mediated depletion of Set9, impairedp21 expression and decreased the total levelof p53.

This latter finding suggested to Reinbergand colleagues that methylation of p53might affect its stability. Indeed, the stabilityof p53 increased in cells that expressed wild-type Set9, but not in those that expressedthe methylation-defective mutant.Consistent with this ‘hyper-stabilization’ ofp53, cells that overexpressed Set9 alsoshowed higher apoptotic staining, which isindicative of a stronger p53-dependentapoptotic response.

The in vivo role of methylation in p53regulation implies complex regulatorymechanisms, the coordination of whichremains unclear. In addition, it raises thequestion of how the methylation of a singleresidue might interfere with theubiquitylation and subsequent degradationof p53.

Arianne HeinrichsReferences and links

ORIGINAL RESEARCH PAPER Chuikov, S. et al.Regulation of p53 activity through lysine methylation. Nature18 Nov 2004 (doi:10.1038/nature03117)