Cell552

Figure 1. Model Of Transcription Factor Op-erating System in Early Mouse Developmentand ES Cells

Oct4 is crucial for the first embryonic lineagespecification, and Nanog is crucial for thesecond. Maintenance of the pluripotent epi-blast of postimplantation embryos requiresOct4, Sox2, and FoxD3. In the ES cell Oct4,Sox2, Stat3, and Nanog are essential for self-renewal: the pools of target genes controlledby each transcription factor or combinationof factors are shown in color.

Niwa, H., Burdon, T., Chambers, I., and Smith, A. (1998). Genes Dev.tribute to a better understanding of how stem cell re-12, 2048–2060.newal and differentiation are related to downstream tar-Smith, A.G., (2001). Annu. Rev. Cell Dev. Biol. 17, 435–462.get genes. At this stage, any new composition beyondStewart, C.L., Kaspar, P., Brunet, L.J., Bhatt, H., Gadi, I., Kontgen,the transcriptional tune we currently hum is bound toF., and Abbondanzo, S.J. (1992). Nature 359, 76–79.be instrumental.Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L.,Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A., and Gough,N.M. (1988). Nature 336, 684–687.Fatima Cavaleri and Hans R. Scholer

University of PennsylvaniaSchool of Veterinary MedicineCenter for Animal Transgenesis and Germ Cell

ResearchNew Bolton Center382 West Street Road

Fingering the Ends:Kennett Square, Pennsylvania 19348How to Make New Telomeres

Selected Reading

Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N., andLovell-Badge, R. (2003). Genes Dev. 17, 126–140. Telomerase-mediated healing of broken chromosomesBoiani, M., and Scholer, H. (2002). In Principles of Cloning, M.D. gives rise to terminal deletions and is repressed inWest, ed. (Academic Press).

most organisms. In ciliated protozoa, however, chromo-Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, some fragmentation and de novo telomere addition areS., and Smith, A. (2003). Cell 113, this issue, 643–655.

part of the developmental program. Work by Karamy-Hanna, L.A., Foreman, R.K., Tarasenko, I.A., Kessler, D.S., and La- sheva et al. (2003) in this issue of Cell indicates that inbosky, P.A. (2002). Genes Dev. 16, 2650–2661.

Euplotes crassus, this is mediated through switchingMatsuda, T., Nakamura, T., Nakao, K., Arai, T., Katsuki, M., Heike, between different telomerase reverse transcriptase iso-T., and Yokota, T. (1999). EMBO J. 18, 4261–4269.

forms.Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Taka-hashi, K., Maruyama, M., Maeda, M., and Yamanaka, S. (2003). Cell

Double-stranded DNA breaks can be repaired either by113, this issue, 631–642.homologous recombination or by non-homologous end-Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius,

D., Chambers, I., Scholer, H., and Smith, A. (1998). Cell 95, 379–391. joining. Alternatively, telomere sequences can be added

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to the broken ends by a process known as “healing.” (Est1p) enable the recruitment of telomerase to the telo-mere (Pennock et al., 2001).However, this leads to loss of the distal part of the

By analyzing the extension pattern of nontelomericbroken chromosome that lacks the centromere. Thus,DNA primers by telomerase in extracts from E. crassusit is not surprising that mechanisms evolved to disfavorcells in different developmental stages, the Shippende novo telomere formation relative to less mutagenicgroup showed that the Euplotes enzyme alters its bio-repair pathways (Schulz and Zakian, 1994).chemical properties from those of a canonical telomereIn ciliated protozoa, chromosome breakage and heal-elongation enzyme in vegetative cells to a de novo telo-ing by new telomere addition is not an accidental andmere elongation enzyme during macronuclear develop-rare event, but is part of the developmental programment. This switch in activity is accompanied by a drasticwhich gives rise to a new macronucleus. These unicellu-change in the molecular mass of the telomerase holoen-lar organisms contain at least one micronucleus andzyme from 280 kDa in vegetative cells to 1.6 MDa duringone macronucleus. The micronucleus is transcription-macronuclear development (Greene and Shippen, 1998).ally inactive during vegetative (asexual) growth. The

Karamysheva et al. (2003) show now that Euplotesmacronucleus is generated from the micronucleus aftercrassus contains three different TERT genes, with dis-mating (Jahn and Klobutcher, 2002). Macronuclear de-tinct expression patterns during macronuclear develop-velopment involves (1) chromosome polytenization, (2)ment. Strikingly, EcTERT-2 expression is limited to theDNA fragmentation (to gene-sized chromosomes in Eu-time of de novo telomere formation, whereas EcTERT-1plotes), (3) telomere addition to each DNA fragment,and -3 are mainly expressed when telomere mainte-and (4) DNA amplification. This process results in annance is required. EcTERT-2 mRNA peaks just prior toenormous number of telomere-containing minichromo-the period in which telomerase acquires relaxed speci-somes. In Euplotes, every macronucleus contains ap-ficity. It is restricted to macronuclear developmentproximately 2 � 107 minichromosomes. Therefore, cili-through two successive and very unusual events. Toated protozoa have been exploited as biochemicalactivate the gene, noncoding DNA that disrupts the opensources for the study of telomeres and telomerase. Forreading frame is excised from the micronuclear versionexample, the first telomere end binding proteins wereof EcTERT-2 via a kind of DNA splicing event. To inacti-identified in Oxytricha nova (Gray et al., 1991), telom-vate the gene upon completion of macronuclear devel-erase activity was discovered in Tetrahymena ther-opment, the EcTERT-2 gene is eliminated from the ma-mophila (Greider and Blackburn, 1985), and the catalyticture macronucleus. This study provides the first examplesubunit of telomerase was first isolated from Euplotesof a role for different TERT isoforms in telomere biology.aediculatus (Lingner et al., 1997). In ciliates, telomeraseWhile the correlation of isoform expression with differenthas two modi operandi. In vegetative cells, telomerasetelomerase activities strongly supports a model in whichcompensates for sequence loss at the telomere, whereastelomerase catalytic subunit exchange leads to a speci-during development of the macronucleus, telomeraseficity switch, the availability of isoform-specific antibod-adds telomeric repeats to DNA fragments which lackies should provide more direct evidence in support oftelomeric sequences. The molecular events enablingthis model in the future. It is as yet unclear whetherthis switch have so far remained mysterious.expression of other end replication factors may also beTelomerase is a large ribonucleoprotein complex thatregulated.contains, among others, a catalytic subunit (the telom-

What gives EcTERT-2 its “healing” properties? Inter-erase reverse transcriptase, TERT) and an RNA moietyestingly, the sequences of all three TERT proteins are(TR) (Kelleher et al., 2002). The telomerase RNA providesvery closely related. As for other eukaryotic TERT sub-the template for the synthesis of telomeric repeats andunits, the conserved motifs defining the reverse tran-it serves as an assembly platform for the telomerasescriptase family are found in the central domain of allholoenzyme. Telomerase re-extends shortened telo-three proteins. This indicates that the folding of the ac-

meres that arise from the inability of the canonical DNAtive site is similar to retroviral RTs. The RT structure is

replication machinery to replicate DNA ends. Therefore,usually described as a semi-closed right hand. The

it is required for unlimited proliferation of unicellular or- thumb contacts and stabilizes the primer-template com-ganisms such as yeast or protozoa, as well as for immor- plex, the palm contains the active site with its two metaltal cell lines in multicellular organisms, such as germ ions and the fingers enclose with the palm the nucleotidecells and many cancer cells. Telomerase specificity is that will be incorporated into the nascent DNA (see Fig-guided in part by base pairing between its RNA template ure 1). Amino acids that are unique to EcTERT-2 andand the telomere 3� end. However, telomerases from that could account for its relaxed specificity are verymost organisms can act in vitro on primers that show limited in number. All but two of them cluster in a regionvery little or no complementarity between their 3� ends localized at one of the finger tips (see yellow region inand the RNA template. A telomeric sequence in the 5� Figure 1). This region is seven amino acids long in HIV-1region of such primers greatly enhances the efficiency RT but approximately 100 amino acids long in EcTERTsof elongation. Explanations for these and other observa- (and in other telomerases). Several hypotheses can betions have invoked the existence of a so-called anchor raised to explain how this region may determine sub-site in telomerase that binds to primer 5� ends and helps strate specificity. It may bind directly to nontelomerictelomerase to recognize and elongate telomeric sub- chromosome ends at the site of polymerization or it maystrates. Telomerase specificity is also mediated through bind to a more 5� region defining a modified anchorprotein-protein interactions. This is well documented in site with relaxed specificity. Alternatively, the modifiedyeast, where the specific interactions between a telo- finger-domain may bind to proteins that confer the ability

of telomerase to be recruited to nontelomeric DNA ends.mere binding protein (Cdc13p) and a telomerase subunit

Cell554

Figure 1. The Crystal Structure of HIV-1Reverse Transcriptase

Telomerases and retroviral reverse tran-scriptases share several specific sequencemotifs, which are expected to confer similarthree-dimensional geometries. The reversetranscriptase domain of HIV-1 RT can be sub-divided into three subdomains named thepalm, fingers, and thumb, for their analogy toa right hand. This hand encloses the primer-template complex. One of the fingers con-tacts the dNTP that is incorporated into thenascent DNA chain. In combination with thepalm domain, it defines the so-called nucleo-tide binding pocket (Huang et al., 1998). Strik-ingly, telomerases contain a large insert pro-truding from the top of one finger (yellow loopwith question mark). Most amino acid differ-ences between EcTERT-2 and its isoformsEcTERT-1 and EcTERT-3, are clustered inthis region. This domain might determineDNA substrate specificity (Karamysheva etal., 2003) either by contacting the DNA primerdirectly, or by interacting with proteins thattarget telomerase to telomeres or brokenchromosome ends.

Lingner, J., Hughes, T.R., Shevchenko, A., Mann, M., Lundblad, V.,Such proteins may function in a manner analogous toand Cech, T.R. (1997). Science 276, 561–567.that of Cdc13p and Est1p, the partners that mediatePennock, E., Buckley, K., and Lundblad, V. (2001). Cell 104, 387–396.access of telomerase to telomeric chromosome ends.Schulz, V.P., and Zakian, V.A. (1994). Cell 76, 145–155.The increase in native molecular mass of telomerase

during macronuclear development is consistent with analtered subunit composition.

In summary, the paper by Karamysheva et al. (2003)describes unanticipated mechanisms for gene regula-tion, which, though fascinating, may be a peculiar quirkof ciliated protozoa. The sequence alterations in the

CUE’d up for Monoubiquitinfinger domain of the natural TERT-variant EcTERT-2,which may transform a telomere-extension enzyme intoa chromosome-healing enzyme, point to a region thatdistinguishes the telomerase reverse transcriptase fam-

The first structures have been obtained for complexesily from reverse transcriptases encoded by retroele-between CUE domains and monoubiquitin, one byments. Further structural and mechanistic analysis ofNMR (Kang et al., this issue of Cell) and one by X-raythis region promises to provide new insights in thecrystallography (Prag et al., this issue of Cell), thusmechanism and specificity of telomerases.providing insights into ubiquitin recognition by CUEdomains. Structural comparisons suggest that differ-

Gael Cristofari and Joachim Lingner ent CUE surfaces can interact with ubiquitin, indicat-Swiss Institute for Experimental Cancer ing that not all CUE domains are created equal.

Research (ISREC)CH-1066 Epalinges Many important cellular processes are regulatedSwitzerland through posttranslational modification by ubiquitin, a 76

amino acid protein that is conjugated via it C terminusSelected Reading to lysine residues within a target protein (Hochstrasser,

1996; Hershko et al., 2000; Pickart, 2001). Several E2Gray, J.T., Celander, D.W., Price, C.M., and Cech, T.R. (1991). Cellconjugating enzymes and numerous E3 ligase cofactors67, 807–814.work in unique combinations to recognize and conjugateGreene, E.C., and Shippen, D.E. (1998). Genes Dev. 12, 2921–2931.ubiquitin to target proteins. It has become increasinglyGreider, C.W., and Blackburn, E.H. (1985). Cell 43, 405–413.clear that proteins can be initially conjugated to a singleHuang, H., Chopra, R., Verdine, G.L., and Harrison, S.C. (1998).ubiquitin moiety, and the monoubiquitinated target canScience 282, 1669–1675.serve either as an active signal or as a substrate forJahn, C.L., and Klobutcher, L.A. (2002). Annu. Rev. Microbiol. 56,polyubiquitin conjugation (Pickart, 2000).489–520.

Monoubiquitin conjugation plays an important role inKaramysheva, Z., Wang, L., Shrode, T., Janna Bednenko, J., Hurley,modulating numerous cellular pathways, including tran-L.A., and Shippen, D.E. (2003). Cell 113, this issue, 565–576.scription regulation, cellular localization, vesicle bud-Kelleher, C., Teixeira, M.T., Forstemann, K., and Lingner, J. (2002).

Trends Biochem. Sci. 27, 572–579. ding, histone modification, and protein trafficking in the