Controlling the X chromosome

X-INACTIVATION

MARY F. LYON

Controlling

the X chromosome

Inactivation of the mammalian X chromosome begins at the X-inactivation centre. The gene Xist has a unique expression pattern, suggesting that it may be a functional part of the centre. There is anticipation in the air that the mammalian X chromosome may soon yield some long-held secrets concern ing the control mechanism of its activity. Some animals that use differences in the ratio of ‘sex chromosomes’ to determine sex, compensate for the resulting difference in the proportions of some genes in males and females. Mammals are unique: they achieve dosage compensation for the different doses of X-linked genes in chromosomally XX females and XY males by inactivating one of the two X chromosomes of females in every cell early in development. Thus males and females both effectively have a single dose of genes that occur only on the X chromosome. In female germ cells, the inactive X chromosome becomes reactivated, and the cycle begins again for the new generation. Although the phenomenon of X-chromosome inactivation has been known for over 30 years, the mechanism remains an enigma [l-3]. One fact that has been clear from the early days, however, is that the process involves an ‘Xinactivation centre’ on the X chromosome, from which an unknown signal spreads to cause inactivation. It is the increasing understanding of this X-inactivation centre that is now generating considerable excitement. The first evidence for the existence of the X-inactivation centre came from observations of the effects of translocations between the X chromosome and an autosome in mice. In females heterozygous for these translocations, only one of the two segments into which the X is broken undergoes inactivation. In addition, X chromosomes in which a segment is deleted may fail to undergo inactivation. These oservations are interpreted as showing that the segments of X chromosomes that remain active in all cells lack an X-inactivation centre. By comparing the behaviour of different translocations and deletions, it has been possible to map the position of the inactivation centre to a specific stained band on the X chromosome: XD in mouse and Xq13 in human. A gene has been cloned from this rem gion [46] - named XlST in man and Xist in mouse which has the unique property of expression from the inactive but not from the active X chromosome. This gene is therefore a strong candidate for a component of the X-inactivation centre. At the earliest stages of female embryonic development, both X chromosomes are active and the X-inactivation centre is thought to provide the earliest signal for inactivation of one of the pair. One essential requirement in a candidate gene for the inactivation centre is, therefore, that it must be expressed before the first appearance of X-inactivation. Kay ct al. (71 studied the expression of X%t in mouse early embryos. They show that X&t fPJA 242

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is not present in Z-cell embryos but it can be detected by the 8-cell stage (Fig. 1). This is well before the appearance of any other signs of inactivation: the first detectable phenomenon is asynchronous replication of the X chromosome in the trophectoderm lineage of late blastocyst stages (64 cells or more). Thus, as expression of Xist OCcurs before inactivation, it is potentially a cause rather than a consequence of inactivation. Other characteristics of X&t expression also favour a causal role. For example, although the earliest embryos studied could not be sexed, Kay et al. [7] were able to show at the blastocyst stage that only female embryos expressed X&t. It had previously been established that X-inactivation occurs earlier in the trophectoderm and primitive endoderm lineages of the embryo, progenitors of some of the extraembryonic membranes, than in the primitive ectoderm that will give rise to the embryo itself. In addition, in the cells in which inactivation occurs early, the paternally derived X chromosome is preferentially inactivated, in contrast to the random inactivation seen later in the development of the embryo proper (Fig. 1). Kay et al. used mice with genetically distinguishable X chromosomes to show that when expression of Xist first appeared, RNA could only be detected from the paternal allele, in line with the pattern of X-inactivation in different parts of the developing embryo. In slightly later embryos, both paternal and maternal alleles were active, consistent with the change from paternal to random X-chromosome inactivation later in development. In the male, the single X chromosome becomes inactive in the germ cells of the testis. Kay et al. [7] examined expression of Xist in the testis, and here their conclusions are at variance with those of three other recent studies [8-101. All four groups of authors agree that, whereas Xst is expressed in all tissues in the adult female, in the male its activity is seen only in the testis. Kay et al. reported that the detected activity was not in the germ cells, as it was present in mice believed to lack germ cells. But, by contrast, the other authors concluded that Xist is active in germ cells of the testis. Salido et al. [ 101 found no Xist activity in cultured Sertoli cells, the somatic cells of the testis; there was also no Xist activity in testes of mouse fetuses before spermatogenesis had begun (15.5-21.5 days after fertilization) [ 81. But Xist RNA could be detected at early postnatal stages, when spermatogonia were the only spermatogenic cells present [9,10]. In particularly compelling evidence, after separating cells from the adult testis into different fractions, Xist RNA was found in a fraction that contained only germ cells, the type A and type B spermatogonia and spermatocytes [S]. 1993, Vol 3 No 4

DISPATCH

1 )

X chromosomes

] 1

Active

Xisf

h

I’ (6)

Germ

ceils

Female Xist activity, No methylation

No Xist activity

13.5

12.5 days

days

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Extra-embryonic ectoderm

1

or

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Male Spermatocytes Y both inactive

( and

Inactive X chromosome

Spermatogonia Xist activity

appears

Primitive ectoderm or

I Y chromosome

I

Methylated

inactive

;( chromosome

Fig. 1. Cycle of changes in activity of the X chromosome and the Xist gene through the life cycle of the female mouse and the male germ cell. Each panel shows the stage of the embryo (above) and the state of the X chromosomes within the cells (below); the paternal X chromosome is blue and the maternal X chromosome is red. In the male the single maternally derived X chromosome is active and the Xist gene inactive, except in the germ cells, in which both X and Y chromosomes become inactive.

It is not clear why the findings of Kay et al. dilfer, but the results of the other authors indicate that the onset of xist expression occurs during spermatogonial stages. The exact time at which X-inactivation begins in male germ cells is not known, and hence it is not clear whether X&t expression precedes inactivation as in the female embryo. However, expression of Xist in the testis does suggest that the mechanisms of X-inactivation in male germ cells and in female somatic cells may have features in common, and that male germ cells may provide a suitable model system for the study of X-inactivation. Systems for studying X-inactivation are in fact few, as cultured adult somatic cells seem refractory to changes in X-chromosome activity. Ilowever, changes in activity can be induced by appropriate treatment in cultured embry onic stem (ES ) cells. In particular, in some lines of female ES cells, both X chromosomes are active but inactivation can be induced by allowing the cells to differentiate. Kay et al. [7] showed that induction of differentiation in such ES cells was accompanied by the onset of expression of X&t. Hence, these ES cells may also prove valuable

in elucidating the role of X&t. What is the nature of the Xist gene product? Detailed analyses of both human and mouse x&t genes have revealed that neither appears to encode a protein. The complete transcripts have been sequenced~-15kbinmouse 1111 and17kbinhuman [12] - and in neither species is there any substantial open reading frame. The general structure of the gene is conserved between the two species, with a number of repeats in each, but conservation of the detail of the sequence is not strong. Furthermore, the transcript is located almost entirely within the nucleus, rather than the cytoplasm, and thus is not ,associated with the translation machinery. The evidence therefore suggests that the functional gene product may be RNA and that details of its sequence may not be important to its function. Knowledge of the exact function of the X-inactivation centre is not adequate to suggest how ‘an RNA gene product might work. There is some mechanism that counts chromosomes, so that however many X chromosomes are present only a single one remains active per two autosome sets - one X is active in diploids and two in tetraploids.

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1993, Vol 3 No 4

This suggests that either the quantity of some substance is limiting, or that some agent must bind cooperatively in order to repress only one X-inactivation centre [l-s]. The primary function of the inactivation centre appears to be the initiation of a spreading signal that is in turn responsible for inactivating the chromosome, but at present the nature of the spreading signal is not known. In eutherian mammals, the 5’ promoter regions of genes on the inactive X chromosome are heavily methylated. But this differential methylation is not thought to be the primary spreading signal, as it is not found in marsupials. which also have inactivation of one X chromosome in females. nor in the extraemblyonic cell lineages of mice, which undergo selective irjstivation of the paternal X chromosome. Rather, methylation is thought to be involved in stabilizing inactivation once it has occurred. Other prop erties of the inactive X chromosome include replication of the LIPJAlater than other chromsomes, and condensation of the chromatin throughout the cell cycle. These arc common characteristics of ‘heterochromatin’ and are thus mther non-specific and unlikely to be the primary signals for inactivation. Current opinion favours some change in the i,hronlatin fibre itself, or the association of DNA with proteins, ;IS the primary spreading signal. A point of doubt concerning the role of Xst is that there have, as yet, been no reports of an Xist homologue in mar supials. This is important as studies of the kangaroo have brought down trt%ured hypotheses before - including the suggestion tha( methylation is the prirnaI); signal in the spreading effect. If no marsupial Xist homologue can be found, then perhaps AYistonly functions in those aspects of ?<-inactivation in which eutherians differ from m;u-supials. These include random inactivation in eutherians. rather than preferentially paternal X-inactivation in tnarsupials, and a more stable type of inactivation in eutherians. associated with differential tnethylation. However, Kdy et a[ 171 showed that 2Yist expression apparently owut-s in the ~nouse extraembrl\ronic cell lineages that have paternal inactivation, and it also occurs in male germ cell X-inactivation. neither of which involves differential methylation. Thus, ;II present, there is nothing to suggest that .\;i.
in the adult - perhaps including methylation and late replication - which ensures the stability of the inactive state and protects against the effects of accidental loss of Xist expression. Mouse fetal ooqtes are accordingly an additional source of material that is likely to be valuable in investigating function. In view of the apparently complex nature of X-inactivation, and as one can only Speculate on the role of a gene expressed only as RNA, it seems unlikely that Xi.st will prove to encode the whole of the X-inactivation centre function. However, it will be ;I sad disappointment if it does not reveal some new insights into the mechanisms of X-chromosome inactivation. References I.

GAKT.I-K SPJ,I)\hlR MA. GOIDMILV some inactivation. In .~Ioleczrlrr~ Frictlmann

2. .3 t.

7’. Nev~ York:

,Mh: Mammalian X-chroml,. Genetic Press

Academic

.Iledicine Editd 1992, Vol .Z. 121

I.\‘o~ MF:

Some milestonesin the history of X-chromosome inactivation. ,+z~zI( KW (;cwPt 1992, 26:15-L’ f&c& AI). fWlt:I’K GP: X-chromosome inactivation and ccl1 memory. 7jptz~L~ C;PM 1992, 8: l(i9-174. BKOWN CJ. ~~ALIMIO A, ‘~‘ONLOKJiNLI I<. v[‘II.lAIU?

~~U’EK’I’ jI..

b’KI:NII:W

I<(;.

~KOW’1;

HF: A gene from the region human X-inactivation centre is expressed exclusively the inactive X chromosome. Ric~ttf~ 1991, 349:3X-+-i.

5

7

expressed

from

the

inac-

from Ku

the inactive X chromosome. Mr/rf~ 1991, 351:3?&~31. GF. I%NNY GD, f-‘PirEI. D, AV{M’ORI’II A. B~~ocltmo~c~~~ N. KkSr4N S: Expression of Xist during mouse development suggests a role in the initiation of X-chromosome inactivation. 1993. 72:171-182. JR. L~ll.sw~nl

MC~AIW~.Y

germ

cells

IIITP c;wc~t 9.

gene

1~991, S51~3.Z 329. ~~ROCIU~~I~I+ N, ASInvoHr~i A, UY GF. Coo~vx P. Shlli’ll S. MCCAI~ VU. NORRIS UP. PUNY GD, PATPI l), E&TAN 5: Conservation of position and exclusive expression of mouse .Yist

Cdl 8.

hi.

of thf. fron;

fklRS:Wl c;. ‘I‘ONI.OKI?$Y R. SIM!vlIIR MC. I~hSIY~I.0 1.. AKNAl’lr I) Chl’RA ‘v’. GKOh~ll’li $1. f’V%IITI A. iVNl%N‘I D. tAWRI:N~.l: (; /:I .I/..

Characterization of a murine tive X chromosome. Nattrlr 6.

Is 1hl I

~ICHl.I:R

testis tuw

correlates 1992.

DD: Expression with X-chromosome

Gcvret

1992.

Xist in inactivation.

mouse %?I~

2:2on-203.

(1. SOREQ 11, WAHRMAN

is correlated

of

with

inactive

j.

X-inaCtiVation

X-specific

in

mammakIn

transcription.

IO.

%.IIIO EC. YEN I’H, MOHANDAS TK. S~IAPIRO IJ: Expression the X-inactivation-associated gene Xisf during spermatogenesis. %r/tr~ @)Ic’t 1992, 2: 196~ 199.

II

I~KO~:KIXXW~ N. ASH\X’OKTH A, Ic\\ GI:. !&x:AHE VII. COOI~I:K 1’1. Sw~‘i 5. &.s’r;ll! 5: The product of Xist gene is a 15 kb inactive X-specific transcript

12

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2.192-195.

NOKKl\

of

IX’,

the

mouse containc:cll 1992.

ing no conserved ORF and located in the nucleus. 71:515~ 526. BIKI\V~ CJ. I~I~NIXI(:H UD, Rllt’tiKT JL, II\FI*NII:KI; RG. XIN(; I’. I.4w~rx:c J, WIUAKE IlF: The human Xist gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. G/l 1992. 71 52’ $42

Mary F. Lyon, Genetics Division, MRC Radiobiology Unit, Chilton, Oxon OX11 ORD, LlK,