Development 1(11 Supplement, 1(11 —KKS (I9K7)Printed in Great Britain © T h e Company of Biologists Limited 1987

101

Telomeres of the human X and Y chromosomes

HOWARD J. COOKE

MltC Clinical and Populations Cytogenetics Unit, Otttstation at King.s Buildings, Wesi Mains Road, Edinburgh EH9 3JT. UK

Summary

Analysis of the nature of the DNA sequences at thetelomeres of the short arms of the human sex chromo-somes suggests parallels with the structures found attelomeres of a number of lower eukaryotes. The exactnature of the end of the DNA has not yet been

established but it is clear that there are multiple levelsof variability both between and within individuals inthis region of the genome.

Key words: telomeres, X chromosome. Y chromosome,human.

Introduction

The human sex chromosomes have been the subjectof intensive genetic and molecular study. This hasbeen driven largely by the ease of both genetic andmolecular analysis afforded by their monozygosity inthe male and the absence of a Y chromosome in thefemale. In the case of both the X and the Ychromosomes, a large set of anonymous DNAmarkers have been assigned to these chromosomesand localized or mapped cytogenetically or by recom-bination. In the case of the Y chromosome, therationale for this work has been the search for thelocus responsible for male sex determination. Severalby-products have developed from this search, includ-ing the realization that the Y chromosome is a mosaicof sequences which are X related, autosome relatedand Y specific and the proof that a portion of thechromosome is genetically autosomal. The subject ofthis article is the nature of the telomeres of the X andY chromosomes. The finding of telomeric sequenceswas again a by-product of a search for genes presenton the Y chromosome. It is not surprising that, in thequest for understanding and control of gene ex-pression, differentiation, carcinogenesis and in-herited disease, genes are often regarded as the onlyfunctional entity that resides in the DNA. This is afailing of molecular biologists and especially of thoseof us who work on human systems. We have a goodexcuse; human genetics would not be regarded as'real' genetics by the standards of bacterial, yeast,Drosophila or even mouse genetics. The problems ofhuman molecular biology parallel those of humangenetics; manipulations possible in yeast are not

currently possible in mammalian systems. To find awider view of possible functions of DNA other thangenes one must borrow from other organisms anddisciplines. Cytology provides the concepts ofchromosomal elements such as centromeres and telo-meres which, although not genes, have importantfunctions. Organisms such as yeast and Tetrahymenahave been utilized to work out the molecular struc-ture of these noncoding but nevertheless functionalregions of DNA. In this article, I will review ourlimited understanding of human telomeres borrowingheavily from yeast and other lower eukaryotes toconstruct a model.

Telomeres are the natural end of a linear DNAmolecule. They have a number of functions to fulfil.All known DNA polymerases require a primer,usually a RNA. Excision of this primer would leaveone of the daughter DNA strands shorter than itsparent and over many rounds of replication thiswould lead to extensive deletions at the ends of thechromosomes. A number of replication schemes havebeen proposed which could avoid this problem (for areview see Blackburn & Szostak (1984)). Anotherfunction that the sequences at the telomeres mustdisplay is the protection against degradation or fusionof free ends of DNA in the cell. Fragments of DNAintroduced into mammalian cells are normally inte-grated into a chromosome whereas the chromosomesthemselves are stable. At the cellular level, telomeresare associated with the nuclear membrane and have aspecific distribution within the nucleus (Agard &Sedat, 1983). What features of the structure oftelomeres can be involved in these functions?

102 H. J. Cooke

Table 1. Terminal repeals

TetrahvmenaS ceri'vixicie

TrypaiwsomaOxvmcia

(CCCCAA),,(C, ,A)n

(CCCTAA),,(CCCCAAAA),,

Terminal repeats

In those organisms in which the chromosome terminihave been studied at the molecular level, a variablenumber of short terminal repeats have been found.These sequences are presented in Table 1. Within acloned population of cells, the number of repeatsfound at the end of a particular chromosome variesfrom cell to cell. One result of this is that restrictionfragments that terminate at telomeres are hetero-geneous in size. In yeast, the average number ofrepeats is under genetic control. CDC17, a tempera-ture-sensitive lethal mutation which causes a block inthe cell cycle, increases the length of terminal restric-tion fragments by increasing the number of repeats(Carson & Hartwell, 1985). Two other genes areknown in S. cerevisiae which are also lethal and giverise to telomeres which have fewer terminal repeatsthan wild-type cells. These genes may be involved inturnover processes which subtract and add repeatsfrom the telomeres (Lustig & Petes. 1986). Only onesuch process has been directly demonstrated. InTetrahvmena an activity has been isolated which canadd the Tetrahymena macronuclear terminal repeats,in a terminal transferase-like manner, to either Tetra-hymena or yeast terminal repeat sequences acting asprimers (Greidler & Blackburn. J985).

The first clues suggesting that we were observinghuman telomeres came from observations (Cooke etal. 1985) that a probe isolated from a Y-chromosome-derived cosmid detected heterogeneously sized re-striction fragments in DNA samples from singleindividuals when digested with some restriction en-zymes whilst detecting homogeneous fragments whenthe DNAs were digested with other enzymes. Thiswas directly comparable with the observations de-scribed above on the telomeres of yeast and otherorganisms. Other criteria that any naturally occurringend of a DNA molecule must satisfy are that this endshould be nuclease sensitive in intact DNA andshould appear as a restriction site for all restrictionenzymes when a restriction map is generated. Oneobvious additional criterion that a telomere shoulddemonstrate is that sequences adjacent to it shouldmap to the ends of a chromosome cytologically. Thesequences detected by this probe fulfilled all thesecriteria.

The observation of heterogeneity in DNA frag-ments that originate from a single individual is

consistent with the idea that human telomeres are likethe telomeres of other organisms which have beenstudied at the molecular level in that there is avariation from cell to cell in the number of shortrepeats found at the end of the chromosome. Therepeat length is such that the quantal nature of thisvariation cannot be seen. In this case, our probe ismore than 15 kb from the chromosome end and wewould not expect that a repeat unit of less than l(X)bpwould be resolved. The repeat unit may, of course, bevery much smaller than this and its existence mustremain a hypothesis until it has been cloned andsequenced. It remains, however, the most likelyexplanation for the observation of heterogeneity.

The extent of the heterogeneity is the same withindifferent tissues of a single individual with twoexceptions. When sperm and blood DNAs from asingle individual are compared the distance betweenthe terminal BamH\ site and the telomere is greaterby about 5 kb in the germ line DNA and the hetero-geneity is either reduced or absent (Cooke & Smith.1986). This situation is reminiscent of the phenom-enon of germ-line diminution found in the nematodeAscaris. In this organism, up to 25 % of the germ-lineDNA is present as heterochromatic blocks of tan-demly repeated satellite DNA which is lost during thedevelopment of the somatic tissues (Blackburn &Szostak, 1984). A loosely comparable situation existsin the holotrichous ciliates such as Tetrahymena andO.xytricha in which the micronucleus constitutes thegerm line. After conjugation and fusion of the micro-nuclei, the DNA is fragmented and in this processspecific DNA sequences are lost. It appears that arelated process is taking place on a smaller scale athuman telomeres. I stress that this is a loose analogy,the bulk of the complex changes that occur in thedevelopment of the ciliate nuclei are not reflected inthe human system. The second exception is that alymphoblastoid line, although heterogeneous, showsa much reduced range of terminal fragment lengths incomparison to blood DNA from the same individual.The simplest explanation is that the number ofterminal repeats is loosely fixed in cell lineages butvaries considerably between lineages.

Changes in the number of terminal repeats havebeen observed in trypanosomes which suggest thatone repeat is added per cell division in a processwhich can continue through many cell cycles and isterminated by a sudden loss of repeats (Bernards et al.1983). Human chromosomes, at least in culturedcells, do not seem to be subject to such a process sincea comparison of early and late passages of a lympho-blastoid line reveals no differences in terminal restric-tion fragment length. Perhaps, after the loss of avariable amount of DNA from the chromosome endsat some stage during development, a steady state is

Telomeres of the human X and Y 103

reached in which processes of addition and deletionof repeats are balanced giving rise to the small degreeof heterogeneity seen in transformed cell lines(Fig. 2).

In S. cerevisiae the only structures necessary forstabilization and replication of artificial chromosomeends are the terminal repeats. When terminal repeatsfrom Tetrahvmena rDNA are used as the ends of a

linear plasmid, yeast terminal repeats are added tothem (Shampay et al. 1984). The general structure ofthese repeats in single-cell eukaryotes is conserved.The probable reason for this is that these sequencesare involved in interactions with specific proteins. InOxytricha, two different proteins have been isolatedtightly bound to the macronucleus telomeres. One ofthese proteins will bind in vitro to a synthetic telomere

Bam Bgl Hin EcoRI Pst Sfi Sst Taq

j ^ ^ ^

B

Fig. 1. (A) Restriction ofheterogeneous ends. Digestionwith a restriction enzyme thatcuts at a site such as a gives apattern of heterogeneousfragments when the probe (thethickened line in this drawing)is located between therestriction site and the end ofthe molecule. An enzyme thatcuts at sites b gives rise tohomogeneously sized fragmentsdetected by the same probe.(B) In this blot Bam, EcoR\and Sfi give a type a pattern andthe other enzymes type bpatterns.

104 H. J. Cooke

LWWWWWWWN] \:X::r\::l.::::]:y:-:-: G e r m lino

I^\\\\\\\\\\\\\N1

k\\\\\\\\\\\\\\>3 t-:-:-:-:::-:::-::::t:::::-:-.:::::::-:-:'MWiwiw5gH

k \ \ \ \ \ \ \ \ \ \ \ \ \ \ N J I- •:':'•:••:-:--:-:-:-i: iimim

— Somatic dimnuitiun

k\\\\\N\NM Minisatellite

Terminal turnover J Invariant

P^\\\\\\\\\\\\VJt:: : : ::::::-::i MiniMitcllitc

Terminalrepeats

t\\\\\\\\\\\\\\^] l::>

Fig. 2. Variations in the numbers of terminal repeats. Deletion of sequences from the ends of the chromosomes occursduring development. This process is arrested at different stages in different cell lineages. A turnover of repeat units maythen occur giving rise to microheterogeneity seen in cell lines. Integral regions are unaffected by these processes.

5'

1'

J

EXO III SENSITIVE

EXO III SENSITIVE

M U N G B E A N + E X OSENSITIVE

SNAP BACK

SI OR BAL 31SENSITIVE

Fig. 3. Possible structures for the end of a chromosome.

in a manner that suggests that the presence of asingle-strand tail and duplex region, which must havethe natural DNA sequence, are both recognized bythe protein (Gottschling & Zakian, 1986). In S.cerevisiae telomere binding activity has also beendetected but here the sequence specificity is simpleras both internal and external copies of the yeastterminal repeat C ^ A are recognized (Berman et al.1986). Do human telomeres fall into this generalpattern? The indirect evidence strongly suggests thepresence of terminal repeats but their structure andthe presence of bound proteins awaits direct proof.

What is the end?

The nature of the DNA terminus has been investi-gated in a number of ciliated protozoa using /// vitrolabelling techniques. These methods are impracticalfor the study of human telomeres because there areonly 92 ends per nucleus. Oxytricha for example hasapprox. 1()7 ends per macronuclcus. Instead nucleaseswith different specificities can be used as probes forthe nature of the terminus. Exonuclease III willdegrade molecules with blunt ends or 5' extensionsbut will not degrade molecules with 3' extensions.Treatment of high molecular weight human DNAwith exonuclease III leaves telomeric restriction frag-ments intact. If the terminus was a 3' extension itshould be possible to remove this with an enzymesuch as mung bean nuclease to give molecules whichare then sensitive to digestion with exonuclease III.This is not the case for the X/Y telomeres. Theremaining possibility is that there is a loop structurepresent. If this was a covalcnt joining of the twostrands of the DNA then these molecules would'snap-back' into a duplex when renatured at lowconcentration. Again this does not happen leavingthe most probable structure as that of a hairpin whichis not covalently closed. Such a structure would bepredicted to be BalB\ sensitive, as is observed to bethe case. This is consistent with one model fortelomere replication and with the results from label-ling methods in a number of ciliated protozoa.

Telomeres of the human X and Y 105

Subterminal variability

Within 30 kb of the X/Y short-arm telomeres, there isa great deal of variability in the population (Cooke &Smith, 1986). The basis of this variability is a numberof blocks of minisatellite sequences. The probe thatdefines DXYS14 contains over I kb of a minisatellitecomposed of multiple imperfect copies of a 31-basepair repeat. This minisatellite lies within a block ofsequences, which may be duplicated or not, ondifferent chromosomes. The size of the minisatelliteblock is itself variable. In combination, these twolevels of variability result in at least 150 differentalleles at this locus. Separated from this region byabout 5 kb of invariant DNA is a block of a differentminisatellite, about 20kb long, which shows internalrestriction site polymorphisms. Neither of these mini-satellites are present elsewhere in the human genomenor are they converved outside the higher primates.This distribution contrasts sharply with those mini-satellites described by Jeffries el al. which are presentat multiple locations (Jeffreys et al. 1985). Because ofthis distinction, these telomere-associated minisatel-lites cannot be involved in any function that involvesall telomeres such as replication or chromosome"capping". Perhaps they are involved in positioning oridentifying telomeres of chromosomes that pair. If so,one would predict that this class of sequence wouldexist at all telomeres but that the repeat on differentchromosomes would have a different sequence. Theonly human telomere-associated sequences knownare at the X/Y pairing region and so this hypothesisremains untested.

S. cerevisiae also has telomere-associated repeatedsequences which are more complex in structure thanthe terminal repeats. All yeast chromosomes have acopy of a sequence designated as X with between 0and 4 copies of a sequence Y'. This last sequence wasoriginally isolated as containing a yeast origin ofreplication. Stretches of C|_3rA repeats separatethese elements and Y' elements when these aretandemly repeated. Like the human telomere-associ-ated repeats these sequences are highly variable andhave been shown to be involved in recombination(both meiotic and mitotic) (Horowitz el al. 1984) andto exist as free circular forms. Although the simi-larities in position of these yeast and human telo-mere-associated repeats invite the conclusion thatthey may have some similar role which is perhapsmore subtle than either replication of the ends or'capping' of the DNA termini, the distribution ofthese sequences is different.

Many questions remain to be answered abouttelomeres in general and human telomeres in particu-lar. 1 have posed some here already. The majorproblem is that we have no sequence information

about the end of the chromosome. The most centro-mere-distal sequences that we have so far been ableto clone are a minimum of 2 kb from the end of theshortest X/Y chromosome in the cell populations thatwe use. The most reasonable model would suggestthat terminal repeats on all chromosomes would becapable of being replicated by the same enzymaticmachinery and that this constraint would result inrelated, if not identical, sequences at the chromo-some ends. Cloning of such sequences from the X/Ychromosome short arms should then allow the analy-sis of the telomeric region of a number of differentchromosomes. From this data, it will be possible todraw some conclusions about conserved featureswhich may be significant for telomere function.

A knowledge of the terminal sequences will allow asearch for proteins that recognize these sequencesand either bind to them or are involved in theirreplication. It will be possible to look for the presenceof cryptic telomeres in the human genome, i.e.sequences that are normally present internally in achromosome but that are potentially capable oftelomere function perhaps as a result of breakage.The most interesting prospect is that of the construc-tion of synthetic chromosomes for mammalian cells.Functional telomeres are only one requirement forthis. Centromeres will also be necessary and theremay be other, as yet undefined, essential elements.We are close to at least one telomere. Intriguing datafrom injection of viral constructs into mouse eggssuggest that centromere function may be exercised byrelatively small DNA elements (Rassoulzadegan et al.1986). I do not think it is too far-fetched to suggestthat constructs that mimic at least some aspects ofmammalian chromosome behaviour will be availablein the near future.

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106 H. J. Cooke

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