Proteins that bind to double-stranded regions of telomeric DNA

In eukaryotic organisms, the extremities of chromosomes, or telomeres, are essential to maintain genome integrity by enabling complete DNA replication and protecting chromosome ends against doublestrand break repair (see Ref. 1 for review). Telomeres also participate in various aspects of the functional organization of the nucleus: they interact with themselves and with other substructures such as the nuclear scaffold and nuclear envelope, they appear to play a role in homologous pairing by driving polarized chromosome movements during karyogamy and meiotic prophase I, and they exert position effects (telomeric position effect, or TPE) on transcription, the timing of DNA replication, transposition and recombination (see Refs 2 and 3 for reviews). In yeast, telomeres are also considered to be heterochromatinlike regions that serve as molecular sinks for factors involved in chromatin-mediated repression of gene expression (see Ref. 4 for review). Telomeric DNA comprises a tandem array of repeated sequences, which varies from species to species (see Ref. 1 for review). These repeats are extended by the ribonucleoprotein enzyme telomerase, which compensates for the loss of terminal sequences that occurs during DNA replication (see Ref. 5 for review). The number of repeats is tightly controlled during development in human cells. Telomerase is active in the germ line, where a constant average number of telomeric repeats is maintained. Conversely, telomerase is undetectable in most postnatal tissues, probably leading to a gradual decrease in telomere length with age. This reduction in telomeric repeats may limit the doubling capacity of normal mammalian cells, and its reversal may participate in carcinogenesis (see Ref. 5 for review). Telomeric DNA repeats are associated with various proteins, both at the overhang tail and along the duplex telomeric tract. These telomeric proteins were first described in ciliates, in which they cap the singlestranded DNA tail of the macronucleus telomeres (see Ref. 6 for review), and later in Saccharomyces cerevisiae, in which the major protein binding along the duplex part of telomeric DNA is the multifunctional repressor-activator protein 1 (Raplp; see Ref. 7 for review). In this article, we discuss the recent characterization of proteins that bind to doublestranded regions of telomeric DNA in yeasts and mammals and evidence for the universal role played by these proteins in telomere physiology. How to bind to double-stranded The answer is related to Myb

telomeric

DNA?

Raplp is an essentialprotein that binds specifically along the TG,-, telomeric repeats and many nontelomeric sitesin S.cerevisiaechromosomes(seeRef. 7 for review, Table 1 and Fig. la). Raplp contains two functionally important domains: a centrally located DNA-binding domain and a C-terminal 160-aminoacid domain required for TPE and telomere length regulation (Fig. 2a). The DNA-binding domain is organized into two regions (Rl and R2), each comprising a short N-terminal extension that binds in the minor groove of the DNA, and a bundle of three helice@. The third helix recognizes specific base trends

in CELL BIOLOGY

(Vol.

7) August

1997

Copyright

Proteins that bind to double-stranded regions of telomeric DNA In budding yeast, the DNA-binding protein negative feedback on regulation organization

Raplp orchestrates a

of telomere length and the

of a heterochromatin-like

telomeric compartment.

Recent studies have led to the identification

of /imctionally

telomeric proteins from fission yeast and mammals.

related

These

advances underline the key role played by the proteins that bind to the duplex part of telomeric DNA and reveal an important structural diversity among telomeric proteins.

pairs in the major groove. Remarkably, this structure can be superimposed,almost exactly, on the DNAbinding domain of the protooncogene c-MybsT9.A typical Myb repeat also contains three helices, and the third helix establishes specific contacts with basesof the cognate DNA sequence9.Despite their structural homology, the DNA-binding domains of Raplp and c-Myb are only distantly related in terms of primary sequenceconservation8. Another S.cerevisiae protein that binds to telomerictype DNA is the ‘ITAGGG-repeat binding factor 1 (Tbflp; Table 1). The TBFI gene is essential for mitotic growthlo. Tbflp binds specifically to TTAGGG DNA repeatsin vitr~*~~~~ and in vivo in a monohybrid assay(E. Binet-Brasseletand E. Gilson, unpublished). In yeast, TTAGGG repeatsor permutated versions of this sequence are found at a high density in the telomere-proximal portions of the X and Y’ middle repetitive sequences, which separate the simple telomeric DNA from other subtelomeric elementson many chromosomesw3 (Fig. 3a). These regions are bound by Tbflp in vitro l”,ll (C. Koering, E. Revardel and E. Gilson, unpublished), but whether Tbflp has a subtelomeric localization in vivo awaits further study. The sequence of Tbflp, in contrast to Raplp, can be aligned with that of c-Myb: it contains a single Myb repeat, which is essentialfor DNA binding14 (Fig. 2a). Interestingly, the DNA-binding domain of Tbflp is related to that of a human TTAGGG-binding protein: the TTAGGG repeat factor 1 or TRFl (Table 1 8 1997

PII: SO962.8924(97)01092-l

Elsevier

Science

Ltd. All rights

reserved.

0962-8924/97/$17.00

The authors are at the Laboratoire de Biologic Mokulaire et Cellulaire, UMR49, Centre National de la Recherche Scientifique, Ecole Normale Supkrieure de Lyon, 46 alke d’ltalie, 69364 Lyon Cedex 07, France. E-mail: egilson@ ens-lyon.fr

317

TABLE

1 - PROTEINS

Organism

Name Proteins RapI P

THAT

with

BIND

DOUBLE-STRANDED

TELOMERIC

DNA-binding properties

known telomeric functions 5. cerevisiae dsCTG,_,), ‘1 ss(TG,_,), >’ W,JQ, Induces DNA bending, untwisting and G-quartet formation

Tazl p

5. pombe

dG,_,~AWn

TRFl

Mammalian

dsCT,AC4, Induces DNA

Candidates Tbfl p

5. cerevisiae

ds(T+,), sequences the subtelomeric elements X and Y

5. cefevisiae 5. pombe S. pombe Trypanosoma

dsUG,-,I, n.d.

IBPl BPFl 67-kDae PPTe

Maize Parsley A. thaliana Physarum

ds(G,_,~AW, ds(subtelb) = ss(C- subtelb)

Related to Myb repeats and homeodomains

Telomere Telomere Telomeric

ligand” length regulation silencing

7,& 24,25, 29-32, 37,40

Telobox

Telomere Telomeric Telomere Telomere Telomere

length regulation silencing ligand” length regulation ligand”

18

Telobox

ds(T,AG,),

SPX TeRFl/lls ST-l e

Telomeric

Telobox

Mammalian

TBFP’

DNA-binding domain

bending

TRF2

DNA

functions

Refs

14-17 14,-h

of Telobox n.d. Telobox n.d. n.d.

10,11,14,-r 11 _i

Telobox Telobox n.d. nd.

14 14 22 21

20 23

>

W,AG,), = W,TA,), ds(Sh initiatof) ds(BoxPd) ds(T,AG,), = ss(TsAG,), ds(T,AG,),

“As determined by indirect immunofluorescence experiments on chromosomes. bSubtel, 29-mer subtelomeric sequence including TzAG, or T,AG,-like repeats. c The IBPl -DNA complex at the Shrunken promoter covers an exact plant teiomeric repeat (AC,T,). d BoxP sequences are rich in C,T, motifs. e Protein sequence has not yet been published. f This activity is distinct from Rap1 p both according to chromatographic behaviour and to binding specificity (TBFB does not recognize non-telomeric DNA-binding sites of Rap1 p). sThe sequences of TeRFl and TeRFll have not yet been published. Whether one of these two proteins corresponds to Tazl p is not known. hT. Bilaud, C. Brun, K. Ancelin, C. E. Koering, T. Laroche and E. Gilson, unpublished. i C. E. Koering, E. Binet-Brasselet and E. Gilson, unpublished. iSee text. Abbreviations: A. thaliana, Arabidopsis thaliana; ds, double-stranded; S. cerevisiae, Saccharomyces cerevisiae; S. pombe, Schizosaccharomyces pombe; ss, single-stranded; n.d., not determined.

and Fig. Za), which was identified in the course of two independent studies based either on its propensity to bind to duplex telomeric DNA sequence15 or as a protein related immunologically to Tbflp14. The C-terminal sequence of TRFl also exhibits a significant homology to a single Myb repeat14J5. This sequence is sufficient to bind to telomeric DNA in a southwestern assay14 but requires a dimerization domain to bind to DNA in solution16. Immunofluorescence experiments showed that TRFl is localized at telomeres in interphase nuclei and in metaphase chromosomesisJ7 (Fig. lb). Two other telomeric proteins containing a single Myb sequencehave been identified recently. The fission yeast Tazlp, found in a one-hybrid screenusing 318

S. pombe telomeric DNA as a target, is required for

telomeric functions in interphase cells and for meiosisor sporulationls (Table 1 and Fig. 2a). The human transcribed open reading frame TRFZ gene, first identified as an EST homologous to the Myb sequence of TRFl and TBFl (Ref. 14), encodes a protein localized at the tips of metaphasechromosomes(T. Bilaud, C. Brun, K. Ancelin, C. E. Koering, T. Laroche and E. Gilson, unpublished). The ‘telobox’ is characteristic telomeric proteins

of a large

class

of

An unrooted tree representing the sequencerelationships between representative members of the Mybrepeat family and the Myb sequencesof telomeric trends in CELL BIOLOGY

(Vol.

7) August

1997

proteins is shown in Fig. 4a. As expected, the classical Myb sequences fall into three groups, which include either the first (Rl), the second (R2) or the third (R3) repeat sequence9. The two Myb-related repeats of Raplp appear as distantly related members of the R3 family (Fig. 4a). The single Myb repeats from telomeric proteins are clustered in one separated group, which also includes the single Myb repeats of plant DNA-binding proteins (IBPl and BPFl) and uncharacterized open read- FIGURE 1 proteins, from yeast to human. ing frames14 (orfA, orfR1, orfR2 and Specific telomere of telomere proteins. SpX; Fig. 4a). A consensus sequence can Indirect immunofluorescence be derived from a multiple alignment of DNA is stained in blue by DAPI (4,6-diamidinoZ-phenylindole). Bar, 5 Pm. (a) Detection of sequences from the telomeric protein Rap1 p at the ends of all bivalents on a spread of group (Fig. 4b). Using this consensus Sacchoromyces cerevisioe pachytene nuclei (yellow). sequence as a probe for a BLAST search, RNA is stained with propidium iodide (red). the highest scores were found to correReproduced from Ref. 64, with permission from The Rockefeller University Press. (b) Detection spond to members of the telomeric tagged version of mouse TRFl expressed in human cells. Staining is visible at the ends of chromatids protein group but not to other Myb sea spread of metaphase chromosomes (green). Reproduced, with permission, from Ref. 15. quences, further supporting the grouping shown in Fig. 4a (E. Gilson, unpublished). These structural conservations, together screen for new telomeric protein candidates. Accordwith the specific binding to telomeric or telomericingly, IBPl, BPFl and the open reading frames from type DNA, led to the proposal that the telomeric plants and yeasts, indicated in Figure 4a and Table 1, protein group identifies a family of Myb-related can be predicted to be authentic telomeric proteins. telomeric DNA-binding proteins, christened ‘telobox’ On the basis of in vitro binding studies, additional proteinsl*. telomerlc candidates have been identified in yeast11,20, The DNA-binding specificity of telobox proteins, Physarum21, higher plants22 and 7’rypanosoma23 (Table 1). It is worth noting that the high affinity of corresponding to telomeric or telomeric-like DNA sequences, is highly conserved within the group and ST-l for subtelomeric DNA repeats in7’rypanosoma23 differs significantly from that of other Myb proteins is reminiscent of the subtelomeric binding of Tbflp (Fig. 4~). Yeast Tbflp and human TRFl and TRF2 proin yeast, and suggests that subtelomeric elements teins recognize the telomeric repeat ‘ITAGGG, and play a role in telomere physiology. Interestingly, the neither of them appears to bind to the c-Myb famcapacity to bind both to the duplex telomeric DNA ily consensus [C(C/A)GTT(G/A)]14. The portion of and to one strand of this sequence, first described the telobox consensus sequence that presumably, for Raplp24,25, is shared by two telomeric protein based on the c-Myb sequence, corresponds to the candidates, ST-l in Trypanosoma23 and a 67-kDa proDNA-recognition helix contains a highly conserved tein from Arabidopsis thalianaz2. This dual recognition VDLKDKWRT sequence (Fig. 4b) and shows only of DNA may be of functional importance since telolimited homology to the corresponding helix of the meres constitute one of the few instances in which other Myb repeats. Thus, this particular sequence single-stranded DNA is known to exist in vivo26,27. may account for the telomeric DNA-binding specificity of telobox proteins. Differences in the recogFunctional conservation among telomeric proteins nition helix correlating with distinct DNA-binding Several studies implicate Raplp in two functions at specificities have been reported for other groups of telomeres: transcriptional silencing (or TPE) and Myb proteinslg, as illustrated by MYB305 and telomere maintenance (seeRef. 7 for review). InterestMYBPl (Fig. 4a), which recognize a sequence motif ingly, these two functions are mediated by the same of GGTTGGA(T/G) rather than that recognized by C-terminal domain of the protein. This domain interother Myb proteins (Fig. 4~). The reason why teloacts either with the ‘silent-information regulatory’ proteins Sir3p and Sir4p to induce TPE2a34,or merit proteins have made such extensive use of Myb repeats may be related to common sequence features with the Raplp-interacting factor proteins Riflp35 between telomeric repeats and Myb-binding sites on and Rif2p3(j, to regulate telomere length (Fig. 2b). DNA. Indeed, a GTT sequence, found in various MybDeletion of the Raplp C-terminal domain or telobinding sites, is extended to GGG’IT in telobox bindmerit DNA alterations decreasingthe Raplp-binding affinity are not lethal, but greatly increasetelomere ing sites (Fig. 4~). Furthermore, it is worth noting that, in contrast to many protein-binding sites, the length, leading to heterogeneous telomeric tracts at Myb-binding sites and the telomeric repeats do not least ten times longer than wild type37-3g.A simiexhibit any dyad symmetry. In conclusion, the telolar phenotype is obtained in fission yeast by the box appears to be a signature motif for a large class disruption of tazl (Ref. 18). These experiments of proteins that bind to double-stranded regions of clearly show that yeasttelomeric proteins areinvolved telomeric DNA and that can be used as a probe to in TPE. It is not known whether human telomeres trends

in CELL

BIOLOGY

(Vol.

7) August

1997

of a on

319

(4

Silencing Length-sensing

DNA-binding Rl R2

that a sensing mechanism can discriminate the precise number of Raplp molRap1 p II ecules bound to a chromosome end. A threshold number of Raplp molecules Telobox Dimerization at a telomere could therefore be the sig66 263 376 431 m 439 TRFI II nal that prevents elongation (Fig. 2b). Inhibition of elongation does not seem 559 613 to activate a recombinase or a nuclease Tazl p II I663 since, at most abnormally long telomeres, the amount of DNA lost at each 406 467 m Tbfl p 11 1562 cell division corresponds to a few nucleotides41, compatible with incomplete DNA replication. Rather, these results sug(b) gest that an excess of Raplp or RaplptCentromere binding factors at a telomere inhibits the access and/or the activity of telomerase38,40. This inhibition could result from a direct interaction between Raplp and the catalytic part of the telomerase or a putative telomerase regulatory complex formed by Estlp4Z, Cdc13p43,44 and 1 I Stnlp, a Cdcl3p-interacting protein45 (Fig. 2b). It could also result from a 000 Elongation Shortening Tbfl p higher-order structure transition of the telomeric chromatin that prevents the action of telomerase. An excess of Raplp might also induce its own binding to the single-stranded DNA overhang24,25, producing an inappropriate substrate for the telomerase. Raplp molecules interacting with Sir3p and Sir4p seem to be excluded from the length-sensing mechanism40, suggesting the existence I I of two separate regions within a telomere: one involved in silencing and asso000 Tbfl p ciated with Sir3p and Sir4p, and a Sir-free region that is kept constant in size and FIGURE 2 requires Riflp and Rif2p (Fig. 2b). In adDomains and functions of Rap1 p and telobox proteins. (a) Structure of Rap1 p, TRFl, Tazl p and dition to this negative regulation, telomTbfl p. The DNA-binding domain of Rap1 p is formed by two Myb-related regions, Rl and R2 (blue). erase may need to be activated when a The C-terminal domain (purple) is necessary and sufficient for both telomere-length sensing and telomeric tract becomes shorter than transcriptional silencing by interacting with Rifl p and Rif2p or with Sir3p and Sir4p, respectively (see normal or when the pool of telomeretext). TRFl, Tazl p and Tbfl p share at their C-terminal domain a single Myb-related sequence specifically bound proteins is below the regulated contacting telomeric DNA bases, named the telobox (orange). DNA binding by TRFl requires its threshold number. However, telomerdimerization domain (black). Whether such a domain is present in Tbfl p or Tazl p is unknown. (b) A ase does not require Raplp to act since model for Rap1 p functioning at yeast telomeres. A telomere is bound by a regulated number of Rap1 p telomeric DNA mutations preventing molecules. Some of these (at the internal end of the TC,_, repeats) initiate the loading of SirZp, Sir3p Rap1 binding do not impair telomerand Sir4p to the adjacent nucleosomes 34,65 (bent arrows). When a specific threshold number (n) of ase activity38,39. Rap1 p molecules (not interacting with Sir3p and Sir4p, and presumably interacting with Rifl p and Rif2p) is reached, a signal is produced (grey rectangle) to inhibit the telomerase or putative regulatory A second pathway involved in telofactors (e.g. Estl p, Cdcl3p or Stnl p). When the loss or degradation of telomere repeats results in the mere length regulation was revealed in a loss of Rap1 p-binding sites (n - l), the telomere switches to a new state that allows its elongation by context where telomeres are abnormally telomerase. Tbfl p-binding sites are present in most of the subtelomeric regions of long4l. This process, termed rapid deSocchoromyces cerevisiae chromosomes, and a putative binding of Tbfl p at these sites is indicated. letion, appears to be capable of efficiently returning long telomeres to near wildtype size in a single-step recombination process that is independent of the C-terminal porcan mediate TPE, but, in human cells, overextion of Raplp41. pression of a dominant-negative TRFl protein also leads to elongated telomeres”. Thus, all proteins In immortalized human cells, TRFl overexpression tested so far that bind along the duplex portion of leads to a decrease in telomere length and to an intelomeric DNA appear to be involved in the control crease in the amount of TRFl signal at telomeresl’. of telomere length. Overexpression of a dominant-negative TRFl mutant protein also leads to elongated telomeres17. These reA recent study showed that the number of Raplp sults suggest that TRFl is involved in a telomereC-terminal domains assembled at a telomere is maintained at a constant mean value40. This indicates shortening pathway. At least two non-exclusive 361 410 446

320

552

667 -1827

trends in CELL BIOLOGY

(Vol.

7) August

1997

(a)

hypotheses could account for this activity of TRFl. An excess of TRFl may inhibit telomerase, as suggested for Raplp, and/or activate a telomere-degradation pathway. Alternatively, excess TRFl could inhibit TRF2 by competing for telomeric tract binding and/ or by forming putative TRFl-TRF2 heterodimers. In this case, telomere shortening might result from the loss of a positive regulatory effect of TRF2 on telomere elongation, as already proposed for mammalian telomeric DNA-binding proteins46, and not from a direct action of TRFl. This raises the exciting possibility that the activities of TRFl and TRF2 are in opposition to each other and that telomere dynamics result from the ratio between each protein. It is not yet known whether TRFl and TRF2 activities are regulated by a feedback mechanism sensing telomere length. One general activity of telomeric proteins may be to organize higher-order chromatin structures. These domains could regulate the activity of telomeric effectors such as telomerase. In agreement with this hypothesis, the double-stranded part of telomeric DNA is bound to the nuclear scaffold in human cells47,48 and exhibits a non-nucleosomal pattern after nuclease digestion in nuclei prepared from ciliates, yeasts and human cells4g-52. A role for telomerit proteins in this association is suggested by the fractionation of Raplp and TRFl with the nuclear scaffold48J3 and the disruption of telomeric chromatin in yeast cells lacking Tazlpis. Interestingly, the assembly of a telomere-specific chromatin domain could involve DNA-binding properties shared by telomeric proteins, such as the ability to distort DNA structures, demonstrated in vitro for Raplp (see Ref. 7 for review) and TRFl (Ref. 16). Evolution

of telomeric

proteins

The proteins that bind to the duplex part of telomerit DNA exhibit only a low degreeof sequenceconservation outside their DNA-binding domain14J8,54 (Fig. 2a), but their functions appear to be similar (see above). Especially striking is the fact that the Raplp C-terminal domain is sufficient to mediate several Raplp functions but does not share any significant sequence homology with known telobox proteins. Furthermore, homologues of the Raplp-associated proteins, Sir3p, Sir4p, Riflp and Rif2p, have not yet been identified outside budding yeasts, suggesting that they may be specific for Raplp. Thus, telomeres from different organismsappear to usedifferent sets of proteins to achieve common functions. Several mechanismscan account for the diversity of telomeric proteins observed at telomeres of different organisms. First, the telomeric DNA-binding ancestor protein, perhaps a telobox sequence,could associatewith a large number of different domains. Second, assupported by the differences observedbetween the mouseand the human TRFl (Ref. 54), the sequencesof certain domains of telomeric proteins could have diverged rapidly. Third, telomeres can undergo massive modifications in their nucleotide sequencecomposition, as illustrated by Drosophila, in which the telomeric repeats are replaced by a mosaic of retroposonss5,which are replicated by a reversetranscriptaserelated to the catalytic subunit of trends

in CELL BIOLOGY

(Vol.

7) August

1997

0

TTGGGA GGGATT AGGGTT TTAGGG

l

l l l

---

0 l

o

l

00 l

l

em* l oomeea

l

l l

m’

n

core X

X element TTGGGA GGGATT AGGGTT TTAGGG--

0

00

em

l e l o

a a

0

00

Y’ gene

0

l l *

0

--

l l a

I

ra

TGA

ARS

m a

--

!TGj -3)1,

Y’ element 1

++ FIGURE 3 Do subtelomeric regions of Soccharomyces cerevisiae correspond to ancient telomeres? (a) The structure of 5. cerevisiae chromosome ends. In the vast majority of chromosomes, the telomeric repeats are juxtaposed to the subtelomeric elements, X or Y’. The X sequence can be divided into two parts: core X, containing an autonomously replicating (ARS) sequence, and the subtelomeric repeats (STR A, 6, C and D)rz. The Y’ sequences are formed by a long open reading frame (Y’) and, at the 3’ of this gene, an ARS sequence, located roughly 200 base pairs from the beginning of the telomeric repeats13. The occurrences of the TTACCC sequence and of its permuted version AGCCTT are indicated in orange. Each dot represents the presence of one of these sequences or of a variant with one mismatch (orange). For comparison, occurrences of the mirror symmetry of the two versions of the TTACGG repeat are also shown (green). The regions of X and Y’ that contain a high density of telomeric sequences are indicated in orange. It is remarkable that both subtelomeric elements, although they do not share any sequence homology, contain a high density of TTAGGC repeats between their junction with telomeric repeats and the first ARS encountered from the telomere. Note that a gradient of such sequences is visualized from the telomeric repeats to the ARS sequence in the X element. Tbfl p binds to all of these elements in vitro (see text). (b) Speculative model for coevolution between Rap1 p, Tbfl p and telomeric DNA. A Tbfl p ancestor (orange box) was present at telomeric DNA (orange arrow) early in evolution. During budding yeast evolution, telomeric DNA evolved rapidly, probably through changes in genes encoding telomerase, leading to unusual repeats (blue arrow). This new telomeric sequence was then stabilized through its interaction with a Rap1 p ancestor (blue triangle). This hypothesis implies that the former telomeric repeat became subtelomeric, which is in agreement with the presence of traces of TTACGC sequences in the subtelomeric regions juxtaposed to telomeric (Km3), sequences (see above).

S.cerevisiaeand Euplotestelomerases6.Such telomeric modifications may have occurred during evolution through telomerase mutations, as shown by the changes in the repeat DNA that can be elicited by mutation of the RNA moiety of the telomerases7,58. In this case,additional proteins could have been recruited to the newly formed telomeric sequences. This may be the casefor Raplp, which is not found outside budding yeasts. This, together with the fact 321

(a)

Telobox FIGURE

4

The telobox A multiple used

family. sequence

to build

methodI

(a) Dendrogram of Myb and telobox alignment generated by CLUSTALW

an unrooted

(W.

Saurin

representative

and

examples

groups

corresponding

c-Myb,

A-Myb

indicates

by the unpublished).

of vertebrate to the

and

B-Myb

Rl,

and

representative

including Antirrhinum

phylogeny E. Gilson,

Myb RZ and

neighbour-joining Grey

repeats

Xmybl

of plant

three

of human

(Ref.

Myb

indicates

showing

R3 repeats

Xenopus

examples

repeats. was

9). Green

proteins,

the two repeats (a and b) of the maize MYBPl MYB305 (Ref. 19). Orange indicates the telobox

which

includes

orfR2,

IBPl,

recently

previously BPFl,

orfl

described

, orf2/TRF2)14,

telomere

proteins

characterized

(mTRF1)54,

sequences

/TRFITRFl

Tazl pJ7 and

open

reading

frame

(SpXa,

amino

acids

52-108;

and

number

910274).

SwissProt

accession

(TM1 p, orfA, together

and

TRFl

an uncharacterized

pombe

containing

and of group, orfR1,

with

from

two

mouse

Scbizosaccboromyces two

SpXb,

telobox

amino Blue

sequences

acids

137-l

indicates

92;

the

two

Myb-related sequences of Saccharomyces cerevisiae Rap1 p (Rap1 and Rap1 R2)*. (b) From multiple alignment of the 12 available telobox

sequences,

conserved

a consensus

residue

for each

at each

position

are plotted

4, 33 and 41, where frequencies,

the

the alterations changes rather position

can be derived

position. above

the consensus.

two residues are present

sum of the frequencies from than

30 where

by taking

The frequencies

the consensus deletion/addition

in the c-Myb diagram.

For positions

has been

sequence

0.6-

homologous

in the text. 6

(blue) telobox telomeric

plotted.

‘1 II III II IllI III

are different

from

RKRRKWTVEEEEALVEGVEKHGTGNWRKILRANYNLKNRTSVDLKDKWRTLKHTA F PO 1 IO 20 30

Telobox

consensus

Y

40

50

sequence

03 Recognized Budding

DNA yeast

telomeric

Saccharomyces Ktuyveromyces

sequences

Rap1 family mag

la&

:;;; Telobox

sequences

sequence

BOXP S. cerevisiae S. pombe Vertebrate

Promoter Vertebrates Plants

322

proteins

cerevisiae

Telomeric and telomeric-type Higher plant telomeric repeat Sh initiator

DNA-binding

sequence

subtelomeric telomeric telomeric

repeat repeat

TA

?

TCTC

Maize

TGTTGGTG

Parsley

GGGTA

Tbfl p

CA

Tazl p

repeat Go A

1

regions CMSTP

TRFl,

family IBPl BPFI

TRF2

Myb proteins HUG, HuA, HUB, Xl

GmGAK

Maize

GmGGAK

Antirrhinum

MYBPl MYE305

shared

by many

DNA

of

and

and

is shown

Rap1 p-binding Huyveromyces

the telomeric of the telobox DNA sequences

recognized

in the position

above

the

discussed sites are /acti~~~

repeats

recognized

by

family recognize containing a GGGlT

some Myb-binding which may correspond sites

residues

helix

by the proteins

of telomeric

sequence (orange). Interestingly, a ClT sequence (underlined), motif

sequences

5. cerevisioe

proteins. Members or telomeric-like

Most

of up to seven

recognized

The sequences between

but

R3 repeat

sequences

2,

with similar

are the result of amino acid events, except after

an additional

R2 and

(c) DNA

the most

of occurrence

can be present. The position of the DNA-recognition telobox consensus is extrapolated from its corresponding 0.8 -

Rl

sites include to a core

by Myb

proteins.

that the telomeric Raplp-binding site is different from classicaltelomeric repeats recognized by telobox proteins (Fig. 4c), suggeststhat Raplp represents an exception among telomeric proteins. It is worth noting that unusual telomeric repeats are found in all budding yeasts testeds9,suggestingthat a rapid divergence in telomeric repeat sequencesoccurred early in the evolution of this group of yeasts. Interestingly, in S. cerevisiae,TTAGGG-like subtelomeric sequencesexhibit many features of authentic telomerit repeats. Indeed, they are oriented towards the ends like canonical telomeric repeats and, in the caseof the STRelements of X, exhibit a gradient of conservation from the telomere to the centromere that is reminiscent of the degeneration of TTAGGG repeats observed in human telomeric sequences (Fig. 3a). The juxtaposition at S. cerevisiaetelomeres of authentic telomeric repeatsnext to telomere-like repeats suggeststhat, during the evolution of budding yeasts,a new telomeric repeat sequence(TGlm3) has been added to the existing one (TTAGGG-like) (Fig. 3b). The direct connection between these two sequencessuggeststhat the passagefrom one type of telomere to another occurred suddenly rather trendsin

CELL BIOLOGY

(Vol.

7) August

1997

than by a progressive drift of telomeric sequences. We suggest that a Raplp ancestor was recruited to (TG1-3)n for efficient telomere maintenance, whereas the ancestral telomeric proteins became subtelomerit. Tbflp, which binds to the ‘ITAGGG-like subtelomeric repeats, may well derive from the ancestral telomeric protein. This suggestion is further supported by the high sequence homology between the teloboxes of Tbflp and Tazlp, a genuine telomeric protein in the fission yeastI (Fig. Za). The conservation of Tbflp in yeast is probably due to nontelomeric functions, which remain unknown but may be linked to transcriptional regulation. Towards

an integrated

view of telomeres

Although proteins that bind to the double-stranded part of telomeric DNA repeats play essentialroles at chromosome ends both asstructural components of telomeric chromatin and asregulatory factors, they are only limited pieces of the telomeric puzzle. A wide variety of other factors are also involved in telomeric function, including polymerases (telomerase,DNA polymerases),DNA-metabolism enzymes (helicase, exonuclease, recombinase, etc.) and cellcycle-checkpoint proteins (seeRefs1 and 3 for reviews). Recently, yeast genetics has identified several proteins that are involved in the maintenance of genome integrity and that also play a role at telomeres. In contrast to the telomeric DNA-binding factors, these proteins display a high sequence homology from yeast to human. They include the Sidp family involved in TPE, DNA repair, anti-recombination, chromosome stability and cell-cycle progressiorP, the ATM/Tellp family implicated in telomere length regulation, cell-cycle checkpoint and DNA repair (seeRef. 61 for review), and the DNA end-binding Ku complex, HdflpHdf2p, which isinvolved in telomere length regulation and double-strand break repair62,63. These findings raise the exciting possibility that telomeresassociatewith proteins that have a dual role - at telomeres and in the generalcontrol of cell-cycle and DNA metabolism. Future studieson these highly conserved factors and their putative interactions with the basic structural components of telomeric chromatin will take biologists onto new ground in terms of relationships between nuclear architecture, DNA metabolism and cell-cycle regulation. References ZAKIAN, V. A. (1995) Science 270, 1601-l 607 GILSON, E., LAROCHE, T. and CASSER, 5. (1993) Trends Cell Biol. 3, 128-l 34 ZAKIAN, V. A. (1996) Annu. Rev. Genet. 30, 141-l 72 MARCAND, S., GASSER, S. M. and GILSON, E. (1996) Curr. Biol. 6, 1222-l 225 AUTEXIER, C. and CREIDER, C. W. (1996) Trends Biochem. Sci. 21, 387-391 FANG, G. and CECtl, T. R. (1995) in Jelomeres (Blackbum, E. H. and Creider, C. W., eds), pp. 69-l 05, Cold Spring Harbor Laboratory Press GILSON, E. and GASSER, S. M. (1995) Nucleic Acids Mol. Biol. 9, 308-327 KONIG, P., GIRALDO, R., CHAPMAN, L. and RHODES, D. (1996) Cell 85, 125-l 36 trends

in CELL BIOLOGY

(Vol.

7) August

1997

9 LIPSICK, J. S. (1996) Oncogene 13, 223-235 10 BRICATI, C., KURTZ, S., BALDERES, D., VIDALI, C. and SHORE, D. (1993) Mol. Cell. Biol. 13, 1306-l 314 11 LIU, Z. and TYE, B. K. (1991) Genes Dev. 5, 49-59 12 LOUIS, E. J., NAUMOVA, E. S., LEE, A., NAUMOV, G. and HABER, J. E. (1994) Genetics 136, 789-802 13 LOUIS, E. 1. and HABER, J. E. (1992) Genetics 133, 559-574 14 BILAUD, T. et al. (1996) Nucleic Acids Res. 24, 1294-l 303 15 CHONG, L. et al. (1995) Science 270, 1663-l 667 16 BIANCHI, A., SMITH, S., CHONG, L., ELIAS, P. and de LANGE, T. (1997) EMBO ].16,1785-1794 17 van STEENSEL, B. and de LANGE, T. (1997) Nature 385, 740-743 18 PROMISEL COOPER, J., NIMMO, E., ALLSHIRE, R. and CECH, T. (1997) Nature 385, 744-747 19 MARTIN, C. and PAZ-AREZ, 1. (1997) Trends Genet. 13, 67-73 20 DUFFY, M. and CHAMBERS, A. (1996) Nucleic Acids Res. 24, 1412-1419 21 COREN, J. S., EPSTEIN, E. and VOGT, V. M. (1991) Mol. Cell. Biol. 11,2282-2290 22 ZENTGRAF, U. (1995) Plant Mol. Biol. 27, 467-475 23 EID, 1. E. and SOLLNER-WEBB, B. (1995) Mol. Cell. Biol. 15, 389-397 24 CILSON, E., MULLER, T., SOGO, J., LAROCHE, T. and CASSER, 5. M. (1994) Nucleic Acids Res. 22, 531 O-5320 25 GIRALDO, R. and RHODES, D. (1994) EMBO /. 13, 241 l-2420 26 WELLINGER, R., WOLFF, A. 1. and ZAKIAN, V. A. (1993) Cell 72, 51-60 27 MAKAROV, V., HIROSE, Y. and LANGMORE, J. P. (1997) Cell 88, 657-666 28 APARICIO, 0. M., BILLINGTON, B. L. and GOTTSCHLING, D. E. (1991) Cell 66, 1279-l 287 29 MORElTl, P., FREEMAN, K., COODLY, L. and SHORE, D. (1994) Genes Dev. 8, 2257-2269 30 BUCK, S. W. and SHORE, D. (1995) Genes Dev. 9, 370-384 31 COCKELL, M. et 01. (1995) /. Cell Biol. 129, 909-924 32 LUSTIC, A. 1. C., LIU, C., ZHANG, C. and HANISH, J. P. (1996) Mol. Cell. Biol. 16, 2483-2495 33 MARCAND, S., BUCK, S. W., MORElTl, P., GILSON, E. and SHORE, D. (1996) Genes Dev. 10,1297-l 309 34 HECHT, A., LAROCHE, T., STRAHL-BOLSINCER, S., GASSER, 5. M. and GRUNSTEIN, M. (1995) Cell 80,583-592 35 HARDY, C. F. J., SUSSEL, L. and SHORE, D. (1992) Genes Dev. 6, 801-814 36 WOTTON, D. and SHORE, D. (1997) Genes Dev. 11, 748-760 37 KYRION, C., LIU, K. and LUSTIG, A. J. (1993) Genes Dev. 7, 1146-1159 38 McEACHERN, M. J. and BIACKBURN, E. H. (1995) Nature 376, 403409 39 KRAUSKOPF, A. and BIACKBURN, E. H. (1996) Nature 383, 354-357 40 MARCAND, S., CILSON, E. and SHORE, D. (1997) Science 275, 986-990 41 LI, 8. and LUSTIG, A. J. (1996) Genes Dev. 10, 131 O-l 326 42 VIRTA-PEARLMAN, V., MORRIS, D. K. and LUNDBIAD, V. (1996) Genes Dev. 10, 3094-3104 43 NUGENT, C. I., HUGHES, T. R., LUE, N. F. and LUNDBLAD, V. (1996) Science 274, 249-252 44 LIN, 1. J. and ZAKIAN, V. A. (1996) Proc. Not/. Acad. Sci. U. 5. A. 93,13760-l 3765 45 CRANDIN, N., REED, 5. I. and CHARBONNEAU, M. (1997) Genes Dev. 11,512-527 4s HANISH, J. P., YANOWITZ, J. and de LANGE, T. (1994) Proc. Natl. Acad. Sci. U. 5. A. 91, 8861-8865 47 de LANGE, T. (1992) EMBO /. 11, 717-724

Acknowledgements This article is dedicated to the memory of Thomas Bilaud. We are grateful to 5. Gasser and C. Fourel for critical reading, and to W. Saurin for his help during sequence analysis. Work in our laboratory is supported by Association de la Recherche contre le Cancer, Ligue Nationale contre le Cancer, Region RhBne-Alpes and Association Frangaise de Lutte contre la Mucovisidose. C. B. thanks Ligue Nationale contre le Cancer for her fellowship.

323

48

49 50 51 52 53 54 55 56 57

LUDERUS, M. E. E., van STEENSEL, B., CHONG, L., SIBON, 0. C. M., CREMERS, F. F. M. and de LANGE, T. (1996) /. Cell ho/. 135, 867-881 GOTTSCHLINC, D. E. and CECH, T. R. (1984) Cell 38,501-510 PRICE, C. M. (1990) Mol. Cell. Viol. 10, 3421-3431 WRIGHT, J. H., COTTSCHLINC, D. E. and ZAKIAN, V. A. (1992) Genes Dev. 6,197-210 TOMMERUP, H., DOUSMANIS, A. and de LANGE, T. (1994) Mol. Cell. Biol. 14, 5777-5785 HOFMANN, J. F-X., LAROCHE, T., BRAND, A. H. and CASSER, 5. M. (1989) Cell 57, 725-737 BROCCOLI, D. et al. (1997) Hum. Mol. Cenet. 6, 69-76 PARDUE, M. L., DANILEVSKAYA, 0. N., LOWENHAUPT, K., SLOT, F. and TRAVERSE, K. L. (1996) Trends Genet. 12, 48-52 LINGNER, J., HUGHES, T. R., SHEVCHENKO, A., MANN, M., LUNDBLAT, V. and CECH, T. R. (1997) Science 276,561-567 YU, C. L., BRADLEY, 1. D., AllARDI, L. D. and

Mammalian cell mutants of membrane phospholipid biogenesis Cultured mammalian

ceil mutants defective in the biosynthesis of

membrane phospholipids, although limited in number, are increasing our understanding

of the molecular

mechanisms

underlying the

biogenesis and the biological significance of membrane phospholipids in higher eukaryotes. This review summarizes isolation and characterization

of such mutants,

the progress in the focusing on those

isolated from cultured Chinese hamster ovary (CHO) cells.

Yhospholipids are major components of biological membranes and show great heterogeneity in their specific fatty acid and polar head group compositions. Indeed, there may be approximately 100 or 1000 distinct molecular species of phospholipids in prokaryotes and eukaryotes, respectively. Although light is now being shed on the roles of certain eukaryotic phospholipids, such as inositol and choline phospholipids, and sphingolipids, in signal transduction’, numerous issues regarding the metabolic regulation and function of these and many other

324

Copyright

Q 1997 Elsevier Science Ltd. All rights reserved.

PII: SO962-8924(97)01084-2

58 59 60

61 62 63 64

65

BLACKBURN, E. H. (1990) Nature 344, 126-132 SINGER, M. S. and GOTTSCHLING, D. E. (1994) Science 266, 404409 McEACHERN, M. 1. and BLACKBURN, E. H. (1994) Proc. Not/. Acad. Sci. U. S. A. 91, 3453-3457 BRACHMANN, C. B., SHERMAN, J. M., DEVINE, 5. E., CAMERON, E. E., PILLUS, L. and BOEKE, 1. D. (1995) Genes Dev. 9,2888-2902 ZAKIAN, V. A. (1995) Cell 82, 685-687 PORTER, 5. E., GREENWELL, P. W., RITCHIE, K. B. and PETES, T. D. (1996) Nucleic Acids Res. 24, 582-585 BOULTON, S. I. and JACKSON, 5. P. (1996) Nucleic Acids Res. 24,4639-4648 KLEIN, F., IAROCHE, T., CARDENAS, M. E., HOFMANN, 1. F-X., SCHWEIZER, D. and GASSER, 5. M. (1992) /. Cell Biol. 117, 935-948 MOAZED, D. and IOHNSON, A. D. (1996) Cell 86,667677

phospholipid species remain to be resolved. A powerful approach towards understanding the metabolic control and biological significance of individual phospholipids is the isolation and biochemical characterization of cell mutants with specific defects in phospholipid metabolism. This approach has been productive in studies on Escherichia cob2 and the yeast Saccharomyces cerevisiaP. Various types of mammalian cell mutants with altered phospholipid metabolism have now been isolated too. These mutants have been used to elucidate the physiological roles of the metabolic pathways deduced from in vitro studies, to isolate the genes and cDNAs for the enzymes involved in phospholipid metabolism and to modify the phospholipid composition in viva, thereby allowing studies of phospholipid functions in intact cells. In this review, we describe somatic cell genetic approaches used to study the biogenesis and functions of membrane phospholipids in mammalian cells, particularly Chinese hamster ovary (CHO) cells4. Why choose phospholipid

CHO cells for genetic metabolism?

studies

of

CHO cells possessseveral ideal characteristics for somatic genetic studies. Most importantly, a large number of recessive CHO mutants have been isolated despite their being somatic cells. The most likely reason for this successis that CHO cells are probably functionally hemizygous, displaying monoallelic expression of various biallelic genes. Other characteristics of CHO cells are also advantageous for genetic studies,namely their stablepseudo-diploid karyotype, their high efficiency of colony formation and the way they are amenable to genetic manipulations such as cell fusion and DNA-mediated gene transfer. Since CHO cells can grow in a wide range of temperatures (33-40°C) and can utilize exogenously added phospholipids for membrane biogenesis,conditional mutants defective in the metabolic pathways for synthesis of essential phospholipids can be isolated (e.g. temperature-sensitive and lipidauxotrophic mutants). In addition, although a major difficulty in the genetic study of mammalian

0962-8924/97/$17.00

trends

in CELL BIOLOGY

(Vol.

7) August

1997