dosage compensation of the active x chromosome in mammals

Dosage compensation of the active X chromosomein mammalsDi Kim Nguyen1 & Christine M Disteche1,2

Monosomy of the X chromosome owing to divergence between the sex chromosomes leads to dosage compensation mechanismsto restore balanced expression between the X and the autosomes. In Drosophila melanogaster, upregulation of the male X leadsto dosage compensation. It has been hypothesized that mammals likewise upregulate their active X chromosome. Together withX inactivation, this mechanism would maintain balanced expression between the X chromosome and autosomes and between thesexes. Here, we show that doubling of the global expression level of the X chromosome leads to dosage compensation in somatictissues from several mammalian species. X-linked genes are highly expressed in brain tissues, consistent with a role in cognitivefunctions. Furthermore, the X chromosome is expressed but not upregulated in spermatids and secondary oocytes, preservingbalanced expression of the genome in these haploid cells. Upon fertilization, upregulation of the active X must occur to achievethe observed dosage compensation in early embryos.

The X chromosome is unique in the mammalian genome because it ispresent in one copy in males and two copies in females, whereas eachautosome is present in two copies in diploid cells. Divergence betweenthe sex chromosomes and loss of genes from the Y chromosome leadto monosomy of the X chromosome in males1. X inactivation infemales is the classical form of dosage compensation that equalizesgene expression between the sexes2. A second form of dosage com-pensation must have evolved to protect mammals from deleteriouseffects due to functional monosomy (haploinsufficiency) of theX chromosome. Ohno proposed that ‘‘yduring the course of evolu-tion, an ancestor to placental mammals must have escaped a perilresulting from the hemizygous existence of all the X-linked genes in themale by doubling the rate of product output of each X-linked gene’’3.One example of such a functional compensation has been identified inD. melanogaster, in which upregulation of the X occurs in males only4,5.

A corresponding mammalian X chromosome upregulation processand its components have not been identified. We previously found thefirst indication of X upregulation in mice where the X-linked form ofthe Clcn4-2 gene from one species was expressed at twice the level ofthe autosomal copy from another species6. In this study, we usedmicroarray expression data to evaluate the global transcriptionaloutput from the mammalian X chromosome in comparison withthe rest of the genome. Array expression profiles can detect dosage-dependent changes in chromosome-specific gene expression in mono-somic or trisomic regions of the mouse and human genomes7–10. Ourprimary goal was to test Ohno’s hypothesis by calculating the ratio ofthe mean global expression of X-linked genes to that of autosomalgenes. This X:autosome expression ratio was predicted to be 1 if therewas a doubling of transcription from the X. If there were no such

doubling, the X:autosome expression ratio would be 0.5. Based on ouranalyses, the calculated ratio is indeed close to 1 in adult somatictissues, consistent with dosage compensation in all six mammalianspecies examined and in D. melanogaster, which was used as a control.

The mammalian X chromosome undergoes a cycle of inactivationand reactivation during germ cell development and early embryogen-esis11,12. In females, both X chromosomes are active in primaryoocytes13,14, whereas in males, the X chromosome is transientlysilenced at meiosis15,16. In female embryos, imprinted inactivationof the paternal X chromosome is established at the two- to four-cellstage17,18, followed by random X inactivation at the blastocyst stage19.We determined that the X chromosome was not upregulated inhaploid cells, thereby maintaining a balanced expression of thegenome. Thus, X upregulation must occur rapidly in early develop-ment to achieve the observed dosage compensation in embryos andadult somatic tissues.

RESULTSX chromosome is upregulated twofold in adult somatic tissuesWe compared the global transcriptional output from the X chromo-some with that of autosomes using microarray data from publicdatabases as well as data from our own arrays. We analyzed a total of1,554 microarrays, including cDNA and oligomer arrays (Affymetrixand 60-mer arrays) to demonstrate the consistency of our results(Supplementary Table 1 online). We calculated the mean fluorescenceintensity of X-linked versus autosomal genes for each set of arrayshybridized with labeled cDNA. The most complete sets of human andmouse arrays, designed to cover as much as possible of the protein-encoding transcriptome of each species in an unbiased manner, have

Received 20 June; accepted 11 October; published online 11 December 2005; doi:10.1038/ng1705

1Department of Pathology and 2Department of Medicine, University of Washington, Seattle, Washington 98195, USA. Correspondence should be addressed to C.D.([email protected]).

NATURE GENETICS VOLUME 38 [ NUMBER 1 [ JANUARY 2006 47

ART I C LES©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

been hybridized with cDNA from a total of 27 human tissues and 33mouse tissues (excluding brain and gonadal tissues, described below)from pooled males and females20,21. We calculated an overall averageX:autosome expression ratio of 0.94 in human and 1.01 in mouse(Fig. 1a; Supplementary Table 2 online). Normalized gene expressionvalues followed a normal distribution (Fig. 1b).

We conclude that the global transcriptional output from theX chromosome is doubled in both species to achieve dosage com-pensation. Despite an apparent tissue-to-tissue variation (Fig. 1a),X:autosome expression ratios were not significantly different betweentissues (by Student’s t-test). Analyses of a different type of arrayplatform containing cDNAs22 (P. Nelson, Fred Hutchinson CancerResearch Center, Seattle, personal communication) confirmed ourdata on the larger oligomer arrays, with X:autosome expression ratiosof 1.00 for human prostate and 0.91 and 0.98 for mouse liver andkidney, respectively. The rat X chromosome is also upregulated, withan average X:autosome expression ratio of 1.01 in 12 somatic tissues(excluding brain and gonadal tissues)23 (Fig. 2a; SupplementaryTable 2). Control analysis of array data sets from pooled male andfemale D. melanogaster yielded a ratio of 0.98, consistent with the well-known dosage compensation by upregulation of the male X in thisspecies (Fig. 2)24.

To determine whether X chromosome expression differed betweenthe sexes, we analyzed our own set of eight Affymetrix arrayshybridized with cDNA from human female and male heart, femalespleen and male liver, together with deposited array data on heart andmuscle for which the sex was specified25,26. We calculated similar

X:autosome expression ratios for male(XY:AA) and female (XX:AA) tissues fromhuman and mouse (Fig. 3). Thus, dosagecompensation was consistently achieved, pre-sumably by a combination of overall doublingof transcriptional output from the active X inboth sexes and X inactivation in females.

A number of genes (about 15%) escapeX inactivation in human (that is, they areexpressed from both X chromosomes),whereas few genes escape in mouse. ‘Escapegenes’ could potentially increase the X:auto-some expression ratio in females. However, asdescribed above, we did not observe signifi-cant differences between the sexes. To address

this paradox, we used microarray data to determine the sex-specificexpression of a subset of 27 human genes expressed in at least five ofnine rodent hybrid cell lines containing an inactive X (ref. 27) andfour mouse escape genes28. The average female-to-male expressionratio of these genes was 1.11 in humans and 1.38 in mouse. Thefemale-to-male ratio varied from 0.10 to 2.94 for 27 individual escapegenes examined in three human tissues, indicating that only a fewescape genes have a significant increase in expression in females,

0.4

0.6

0.8

1.0

1.2

1.4

0.4

0.6

0.8

1.0

1.2

1.4

Adr

enal

gla

ndB

one

mar

row

Hea

rtK

idne

yLu

ngLy

mph

nod

e P

ancr

eas

Pla

cent

aS

aliv

ary

glan

dS

kele

tal m

uscl

e S

plee

nT

hym

usT

hyro

idTr

ache

aLi

ver A

dren

al c

orte

x M

yelo

id c

ells

Epi

thel

ial c

ells

Car

diac

myo

cyte

sF

etal

live

rF

etal

lung

F

etal

thyr

oid

Den

driti

c ce

llsS

kin

Sm

ooth

mus

cle

Tong

ueW

hole

blo

od

Oth

er ti

ssue

s av

erag

e

Am

ygda

la Cer

ebel

lum

Cin

gula

r co

rtex

Dor

sal r

oot g

angl

ion

Pre

fron

tal c

orte

xH

ypot

hala

mus

Olfa

ctor

y bu

lb Pitu

itary

Spi

nal c

ord

Cau

date

nuc

leus

Cor

pus

callo

sum

C

iliar

y ga

nglio

nF

etal

bra

in

Glo

bus

palli

dus

Med

ulla

obl

onga

taO

ccip

ital l

obe

Par

ieta

l lob

eP

ons

Sub

thal

amic

nuc

leus

S. c

ervi

cal g

angl

ion

Tem

pora

l lob

eT

hala

mus

Trig

emin

al g

angl

ion

Who

le b

rain

Bra

in ti

ssue

s av

erag

e

Adr

enal

gla

ndB

one

mar

row

Kid

ney

Lym

ph n

ode

Pan

crea

s Pla

cent

aS

aliv

ary

glan

dS

kele

tal m

uscl

eT

hym

usT

hyro

idTr

ache

aLi

ver

Adi

pose

B c

ells

Bon

eB

row

n fa

tC

D4+

T c

ells

CD

8+T

cel

ls Dig

itsE

pide

rmis

Epi

thel

ium

Larg

e in

test

ine

Ret

ina

Sm

all i

ntes

tine

Sno

ut e

pide

rmis

Sto

mac

hTo

ngue

epi

derm

isU

mbi

lical

cor

dV

omer

onas

al

Oth

er ti

ssue

s av

erag

e

Bla

dder

Spl

een

Hea

rt

Lung

Am

ygda

laC

ereb

ellu

mC

ingu

lar

cort

exD

orsa

l roo

tFr

onta

l cor

tex

Hyp

otha

lam

usO

lfact

ory

bulb

Pitu

itary

gla

ndS

pina

l cor

d lo

wer

Spi

nal c

ord

uppe

rD

orsa

l str

iatu

mH

ippo

cam

pus

Pre

optic

ner

veS

ubni

gra

Trig

emin

al

Bra

in ti

ssue

s av

erag

e

a

b

Expression bin

Freq

uenc

y

HumanOther tissues Brain tissues

Other tissues Brain tissuesMouse

HumanOther tissues

0.00

0.05

0.10

0.15

0.20

–5.0

–4.2

–3.3

–2.5

–1.6

–0.8 0.1

0.9

1.8

2.6

3.5

4.3

Human Brain tissues

–5.0

–4.2

–3.3

–2.5

–1.6

–0.8 0.1

0.9

1.8

2.6

3.5

4.3

Mouse Other tissues

–5.0

–4.2

–3.3

–2.5

–1.6

–0.8 0.1

0.9

1.8

2.6

3.5

Mouse Brain tissues

–5.0

–4.2

–3.3

–2.5

–1.6

–0.8 0.1

0.9

1.8

2.6

3.5

X:A

exp

ress

ion

ratio

X

:A e

xpre

ssio

n ra

tio

Figure 1 Twofold upregulation of the X

chromosome in human and mouse somatic

tissues. (a) Human (diamonds, in all figures) and

mouse (squares, in all figures) adult somatic

tissues (excluding brain and sex-specific tissues)

and brain tissues. Each point represents the

X:autosome expression ratio (mean ± s.e.m.)

for a given tissue listed at the bottom of each

panel. Filled diamonds and squares represent

overall averages ± s.d. (b) Distribution histograms

of the expression of X-linked (filled bars) and

autosomal (open bars) genes in adult somatic

tissues and brain tissues. Gene expression levels

were transformed into log2 and binned before

graphing the data in arbitrary units. Thenormalized distributions of expression of 523

X-linked and 12,511 autosomal human genes

and 602 X-linked and 18,816 autosomal mouse

genes are shown.

0.4

0.6

0.8

1.0

1.2

1.4

0.4

0.6

0.8

1.0

1.2

1.4

Hum

an

Chi

mpa

nzee

Gor

illa

Mac

aque

Mou

se Rat

D. m

elan

ogas

ter

Hum

an

Mou

se Rat

a b

X:A

exp

ress

ion

ratio

X:A

exp

ress

ion

ratio

Figure 2 Twofold upregulation of the X chromosome in mammalian species

and D. melanogaster. (a) Somatic tissues (excluding brain and sex-specific

tissues). Each data point is the X:autosome expression ratio (mean ± s.d.)for all tissues examined in human, mouse, rat and whole D. melanogaster.

(b) Brain tissues. Each data point is the X:autosome expression ratio (mean

± s.d.) for brain tissues examined in human, chimpanzee, gorilla, macaque,

mouse and rat. The primate cDNA had been hybridized to human arrays.

48 VOLUME 38 [ NUMBER 1 [ JANUARY 2006 NATURE GENETICS

ART I C LES©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

whereas most show a modest increase, no increase or even a decreasein expression. This is due to low expression from the inactive X andto additional sex-specific effects (such as hormonal effects) ongene expression.

X-linked gene expression higher in brain than in other tissuesThe mean X:autosome expression ratios in adult brain tissues were1.08 in human (24 tissues) and 1.13 in mouse (17 tissues), based on acombination of our own arrays and on deposited data20,21,25,29

(Fig. 1a; Supplementary Table 2). In spite of tissue-to-tissue varia-tion, the differences in X:autosome expression ratios between all braintissues as a group and all other tissues as another group were significant(Student’s t-tests: human P ¼ 1.03 � 10–10; mouse P ¼ 7.22 � 10–7).Scatter plots also demonstrated an excess of X-linked genes with highexpression in brain tissues (Fig. 4). Indeed, the proportion of geneswith a twofold higher expression in the brain tissue group than in the‘other tissue’ group was 2.8 and 2.5 times greater for the X chromo-some than for the autosomes in human and mouse, respectively. Whenconsidering genes selected as brain-specific genes (defined by at least atwofold higher expression in brain than in other tissues), the averageX:autosome expression ratios were 1.43 in human and 1.18 in mouse,suggesting a greater proportion of highly expressed X-linked genes inhuman. The distribution of normalized gene expression in brain tissuesalso showed a shift toward higher values for X-linked versus autosomalgenes in human but not in mouse (Fig. 1b).

The global transcriptional output from the X was also high in braintissues of other mammalian species, including chimpanzee, gorilla,

macaque, and rat (Fig. 2b; Supplementary Table 2)23,29. The higherexpression of X-linked genes in brain versus other tissues was inde-pendent of gender (Fig. 3)25. X: autosome expression ratios were 1.12and 1.16 in human male and female brain tissues, respectively, and1.19 and 1.19 in mouse male and female brain tissues, respectively(Fig. 3; Supplementary Table 2). Taken together, our data indicate ahigh expression of X-linked genes in mammalian brain tissues.

X chromosome reactivated but not upregulated in spermatidsThe mammalian X chromosome is silenced in male germ cells,specifically in spermatocytes at meiosis I (refs. 15,16). Some X-linkedgenes are reactivated in spermatids30–33, but the global transcriptionaloutput from the X chromosome was unknown in these haploid cells(X:A or Y:A). We observed low X:autosome expression ratios in wholeadult testis from mouse, rat, and human, which represent a mixtureof supporting cells and germ cells (Fig. 5a; SupplementaryTable 2)21,34–36. The adult testis contains 75% haploid cells (sperma-tids and sperm), 9% diploid cells (somatic cells, spermatogonia) and12% tetraploid cells (meiotic primary spermatocytes)34,37. In post-partum mouse testes, the global transcriptional output from theX chromosome selectively decreased with age from postnatal day 11onward, coincident with the progressive onset of spermatogenesis, aspreviously reported (Supplementary Fig. 1 online)38.

To sort out the X chromosome transcriptional output in differentcell types, we examined data from purified rat spermatocytes36, whichhad a very low X:autosome expression ratio (0.22), consistent withrepression of the X chromosome in late pachytene. In contrast,spermatogonia and somatic tissues (Sertoli, Leydig and otherinterstitial cells) had ratios of 1.00 and 1.22, respectively (Fig. 5a;

0.4

0.6

0.8

1.0

1.2

1.4

0.4

0.6

0.8

1.0

1.2

1.4

a

bH

ypot

hala

mus

Occ

ipita

l cor

tex

Str

iatu

m

Who

le b

rain

Who

le b

rain

Mus

cle

Live

r

Hea

rt

Mus

cle

Spl

een

Hea

rt

Hyp

otha

lam

us

Hyp

otha

lam

us

Who

le b

rain

Live

r

Live

r

Hyp

otha

lam

us

Kid

ney

Kid

ney

X:A

exp

ress

ion

ratio

X:A

exp

ress

ion

ratio

Figure 3 X chromosome upregulation in males and females. (a) Human

tissues. Each diamond represents the X:autosome expression ratio (mean ±

s.d.) for a given tissue. Each data point represents 15 males and 15 females

for muscle; seven males and four females for heart; seven males and five

females for hypothalamus; and one individual each for occipital cortex,

striatum, whole brain, liver and spleen. (b) Mouse tissues. Each square

represents the X:autosome expression ratio (mean ± s.d.) for a given tissue.

These ratios were calculated from three individual mice for each tissue type.

Symbols with black outline represent males; symbols with gray outline

represent females. Filled symbols, brain tissues; open symbols, other tissues.

0.01 0.1 1 10 100

0.01

0.1

1

10

100

0.01 0.1 1 10 100

Other tissues expression Other tissues expression

Human Mouse

Autosomes

X chromosome

Autosomes

X chromosome

0.01 0.1 1 10 100

0.01 0.1 1 10 1000.01

0.1

1

10

100

0.01

0.1

1

10

100

0.01

0.1

1

10

100

Bra

in ti

ssue

s ex

pres

sion

Bra

in ti

ssue

s ex

pres

sion

a b

Figure 4 X-linked genes are highly expressed in brain. Scatter plots of

normalized expression values (in log2 based arbitrary units) for genes on

autosomes (top) and on the X (bottom) in brain tissues versus other tissues,

based on a large set of array data21. Red symbols represent genes withtwofold higher expression in brain tissues compared with other tissues;

green symbols represent genes expressed with twofold higher expression

in other tissues compared with brain tissues. Gray symbols represent genes

with similar expression in both tissue types. (a) Human tissues (24 brain

tissues versus 27 other tissues). (b) Mouse tissues (17 brain tissues versus

33 other tissues).


ART I C LES©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

Supplementary Table 2). Early spermatidspurified to 90% (ref. 36) and presumablyrepresenting equal numbers of X chromo-some– and Y chromosome–bearing cells hada ratio of 0.46. Thus, X chromosome–bearingspermatids would have a ratio close to 0.9,the ratio being theoretically 0 in Y chromo-some–bearing spermatids. This implies thatthe X chromosome is active and not upregu-lated in X chromosome–bearing early sper-matids (Fig. 5a,b; Supplementary Table 2).We found a marked shift towards X-linkedgenes with low expression in spermatocytesand early spermatids (Fig. 5b), in agreementwith overall decreased expression from theX chromosome and depletion for genesexpressed in late spermatogenesis38. Similarresults were obtained in mouse, albeit with ahigher ratio in spermatocytes purified byattachment in culture37 than in rat cellspurified by centrifugation and thus morerepresentative of the in vivo state36.

X chromosome not upregulated inprimary and secondary oocytesBoth X chromosomes are reactivated in pri-mary oocytes, which arrest at prophase I untilpuberty, when they complete meiosis I toform secondary oocytes13,14. Meiosis II iscompleted only upon fertilization. Based onarray data21,39–41, the X:autosome expressionratio was 0.67 in primary mouse oocytes(diploid, XX:AA) and 0.87 in secondarymouse oocytes (haploid, X:A; Fig. 5a; Sup-plementary Table 2). Notably, a large num-ber of X-linked genes showed low expressionin primary oocytes, as indicated by a shift inthe distribution of expression values com-pared with autosomes (Fig. 5b). Thus,neither the two active X chromosomes inprimary diploid oocytes nor the singleX chromosome in secondary haploid oocyteswas upregulated. The absence of doubling of transcriptional outputfrom the X chromosome in primary and secondary oocytes wouldmaintain balanced expression with the autosomes. There may even bepartial repression of the X chromosome in these cells, which will needto be confirmed by additional analyses.

X:autosome expression ratios were high in human and mouse ovary(1.18 and 1.21) and in human and mouse uterus (1.13 and 1.09),indicating a possible role for the X chromosome in these female-specific organs20,21,25 (Supplementary Table 2).

X chromosome upregulation during early mouse embryogenesisAs the X chromosome in haploid spermatids (X:A or Y:A) andsecondary oocytes (X:A) was found to be active but not upregulated,the observed X upregulation in adult somatic tissues must be initiatedin embryos (XY:AA or XX:AA). To follow the expression of theX chromosome during development, we examined mouse array datafor these stages21,39–41 (Supplementary Table 1). The gender of thefertilized eggs and embryos was not known for any of these sets ofarray data. However, 30 to 500 zygotes or embryos, presumably

representing equal number of males and females, had been pooledfor each stage.

The X:autosome expression ratio in mouse fertilized eggs andembryos up to blastocysts ranged from 0.87 to 1.02 (Fig. 5c; Supple-mentary Table 2). From 6.5 days post coitum (d.p.c.) embryosonward, the mean X:autosome ratio remained fairly constant andwas slightly but not significantly higher (1.09 to 1.12) than in earlierstages (Fig. 5c). Our results indicate that dosage compensation of theX chromosome to maintain balanced expression of the genome isachieved immediately at zygote formation. Potential differences inX-linked gene expression between male and female zygotes and/orembryos would not be detected in our analyses of pooled male andfemale embryos. To resolve this issue, sexed zygotes and embryos willneed to be purified.

DISCUSSIONGene expression from the mammalian X chromosome is upregulatedin somatic tissues of males and females, a process that achieves dosagecompensation by a doubling of the X transcriptional output, as shown

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4a c

Zyg

otes

Two-

cell

Fou

r-ce

ll

Eig

ht-c

ell

Bla

stoc

ysts

6.5

dpc

7.5

dpc

8.5

dpc

9.5

dpc

10.5

dpc

16-c

ell

Test

is

Spe

rmat

ogon

ia

Spe

rmat

o-cy

tes

Spe

rmat

ids

Mal

e-sp

ecifi

cso

mat

ic t

issu

es

Ova

ry

Prim

ary

oocy

tes

Fem

ale-

spec

ific

som

atic

tiss

ues

Sec

onda

ry o

ocyt

es

b

X:A

exp

ress

ion

ratio

Freq

uenc

yFr

eque

ncy

Mor

e

Expression bin

Spermatocytes

0.00

0.05

0.10

0.15

0.20

0.25Primary oocytes

Spermatids

0.00

0.05

0.10

0.15

0.20

–8.8

–6.2

–4.2

–2.2

–0.2 1.8

3.8

Mor

e

Expression bin

–8.8

–6.2

–4.2

–2.2

–0.2 1.8

3.8

Secondary oocytes

aXPrimary oocytes Spermatocytes

Y : A A

Secondary oocytes Spermatids

Zygotes

X:A = 0.7

X:A = 0.9

X:A = 0.2

X:A = 0.5

X:A = 0.9

Soma X:A = 1.0

aX

Female Male

iX

aX

: A A

: A aX : A and Y : A

aXaX : A A Y : A AaX

Y : A AaXaX iX : A A

d

0.25

Figure 5 X chromosome expression in male and female sex-specific tissues and germ cells and in

embryos. (a) X:autosome expression ratios (mean ± s.d.) are shown for mouse (square), rat (circle) and

human (diamond). Male-specific somatic tissues are Leydig and Sertoli cells, testicular somatic cells

and prostate. Female-specific somatic tissues are uterus and mammary gland. (b) Distribution

histograms of normalized expression values for the X chromosomes (filled bars) and autosomes (open

bars) for various tissues. Gene expression levels were transformed into log2 and binned before graphing

the data in arbitrary units. (c) X:autosome expression ratios (mean ± s.d.) are shown for mouse

embryonic tissues at specific stages. (d) Schematic of X chromosome upregulation in relation to the

number of autosomal sets (AA, diploid; A, haploid). The schematic summarizes hypothetical changes

in the X chromosome(s) in female and male germ cells, zygotes and soma. In female primary oocytes,

secondary oocytes, and zygotes, the X chromosome(s) are active (Xa) but partially repressed or not

upregulated (one downward black arrow), whereas in female soma, one X is active and upregulated

(large X) and the other is inactivated (Xi, two downward black arrows). In male spermatocytes, the

X chromosome is inactive; in spermatids, it is active but partially repressed or not upregulated; inzygotes and soma, it is upregulated. The X:autosome expression ratios (X:A) for each stage are shown.


ART I C LES©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

by our analyses of microarray data. In haploid germ cells, the balanceof expression between the X chromosome and autosomes is main-tained by a lack of X upregulation. These findings indicate a need toachieve dosage compensation for X-linked genes in most cells.A notable exception includes spermatocytes, in which global expres-sion of the X chromosome is very low owing to silencing at meiosis I.Our findings in mammals mirror those in D. melanogaster in whichmicroarray analyses have also shown that dosage compensation isachieved both in somatic and germ cells42.

An obvious question is how X upregulation is established inmammals. One possibility is that the basal expression level of eachX-linked gene may have become upregulated via modifications ofthe DNA sequence to compensate for the loss of the correspondingY-linked gene during evolution. Promoter or enhancer functionsmay have changed to increase gene expression. The microarrayexpression data analyzed in our study represent steady-state RNA,which could also be increased by an enhancement of mRNA stabilitydue to changes at the 3¢ end or another region of the gene. Althoughour study indicates that dosage compensation is established at thetranscriptional level, further regulation may be at the translational level.A second possibility is that upregulation is an active processthat involves epigenetic changes, similar to those mediated bythe MSL complex in D. melanogaster43. Mouse orthologs of theD. melanogaster msl-1, msl-2 and msl-3 genes showed high expressionin zygotes and early two-cell embryos, followed by a decrease in latetwo-cell and four-cell embryos, based on our analysis of array data(data not shown)21,39–41. This transient burst of expression of msl-related genes coincides with the overall high expression of chromatinremodeling genes in early embryos39,40,44. Whether upregulation of themammalian X involves the formation of a protein complex remains tobe determined. At present, there is no evidence that the chromatinstructure of the active X chromosomes in mammals differs in any wayfrom that of autosomes, which argues in favor of a gene-by-gene evolutionary process resulting in enhanced basal expression ormRNA stability.

If X upregulation results from permanent DNA sequence modifica-tions, the low transcriptional output we observed in spermatids andoocytes would imply an X-specific partial repression process in thesecells. In embryos, removal of these repressive marks would benecessary to initiate X upregulation. On the other hand, ifX upregulation results from epigenetic modifications and/or theformation of a protein complex on the active X, this complexwould presumably be removed in germ cells to account for theobserved lower expression, which would then represent the basalexpression. Our study suggests that there must be epigenetic mechan-isms either to decrease specifically expression of the X chromosome(s)in diploid oocytes with two active X chromosomes (XX:AA) andhaploid germ cells (X:A or Y:A) or to increase specifically expression ofthe single active X chromosome in diploid somatic cells (XY:AA orXX:AA). Both scenarios imply dynamic changes in the global tran-scriptional output of the X chromosome or possibly the autosomes attransitions between germ cells and somatic cells and vice versa(Fig. 5d). Changes in specific sets of expressed X-linked genes mayalso have a role in the observed modulations of global expression inthese cell types.

Regulation of the active X chromosome probably differs dependingon the sex of the embryo. At conception, X upregulation might takeplace immediately in males, whereas both X chromosomes might bepartially repressed before X inactivation in females (Fig. 5d).X inactivation provides a mechanism to protect the organism fromfunctional ‘tetrasomy’ of upregulated X-linked genes. If X upregulation

were an early event in female embryos, silencing should be as well, asrecently reported for early imprinted paternal X inactivation inmouse17,18. However, as imprinted X inactivation is apparently incom-plete and may not occur in other mammalian species17, X upregulationcould be progressive in female embryos. At the blastocyst stage inmouse, the paternal X becomes reactivated and random X inactivationtakes place in the inner cell mass19; we did not detect a significantchange in X:autosome expression ratios at this stage, perhaps owing tothe small number of cells involved, the timing of this event and/or otherregulatory mechanisms. It will be interesting to determine expressionfrom the X chromosome in sexed zygotes and early embryos.

The global transcriptional output from the X chromosome wassimilar in adult male and female tissues, although larger sets of dataultimately may demonstrate sex-specific differences. A significantcontribution from genes that escape X inactivation in females wasnot detected in our study. This could be explained by the modestincrease in expression of individual escape genes in females, consistentwith low expression from the inactive X chromosome27,45. As deter-mined by allele-specific RT-PCR, only one-fifth of human escapegenes show expression from the inactive X chromosome that reaches50% of that of the active X chromosome27. Factors that influenceindividual gene expression such as tissue-specific, hormonal andmetabolic differences between the sexes must also be considered.

We found that X-linked genes were highly expressed in brain tissuesof several mammalian species. Our results are consistent with previousestimates of the proportion of X-linked genes involved in humanbrain function, based on the frequency of X-linked mental retarda-tion46,47. The data also suggest a greater proportion of highlyexpressed X-linked genes in human versus mouse brain. Duringevolution, the X chromosome seems to have become a repositoryfor genes specifically and highly expressed in brain. Such genes mayhave a role in enhancing cognitive functions, thereby providing aselective advantage to males in sexual reproduction46.

Our findings of X upregulation in mammals unify the concept ofbalanced expression in a given genome. Haploinsufficiency owing tomonosomy of a whole chromosome is not well tolerated in mostorganisms48. We report on the balanced expression of X-linked genesin diploid somatic tissues and haploid germ cells from severalmammalian species and in somatic tissues from D. melanogaster.Similar results have been obtained in somatic tissues and germ cellsfrom D. melanogaster and in somatic tissues from C. elegans andmouse42. Hence, the evolution of mechanisms to protect fromdeleterious effects of haploinsufficiency are found in several organismsin conjunction with sex chromosome differentiation. Whether thesemechanisms evolved piecemeal, on a gene-by-gene basis or for anentire chromosome at a given time remains to be determined49.

METHODSMicroarrays. Array characteristics are listed in Supplementary Table 1. Data

were obtained in part from public array databases. We analyzed the following

Affymetrix arrays: human HG-U95 set A, B, C, D, E; human HG-U133A;

human HG-U133 set; human HG-U133 2.0 plus; mouse MG-U74 set A, B, C;

mouse 430 set A, B; rat 230 set A, B; rat RG-U34 set A,B; and Drosophila

melanogaster genome array. Other types of arrays were 60-oligomer arrays

custom-made at the US National Institutes of Health and GNF-1H and GNF-

1M custom-made 25-oligomer arrays21. In addition, we acquired eight HG-

U133 2.0 plus arrays from Affymetrix for hybridization to gender-specific total

RNA (Stratagene). Probe labeling, array hybridization and scanning were done

by the University of Washington Microarray Center. Mouse and human cDNA

array data were provided by P. Nelson (Fred Hutchinson Cancer Research

Center, Seattle). Mouse and human cDNA arrays contained cDNAs from the

Research Genetics sequence-verified set of IMAGE clones.


ART I C LES©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

Microarray data analysis. Data downloaded from public databases were not

uniformly deposited in terms of format. It was critical for our purpose and for

consistency in the analysis that the chromosome location and raw fluorescent

intensity for each spot on the array be available for each set of data. In the case

of Affymetrix arrays, we reanalyzed the raw data files using the Affymetrix

software (GCOS 1.1) based on the annotation files available at the Affymetrix

website. In the case of cDNA arrays or other types of 60-oligomer arrays, the

authors provided annotation files as well as their preferred cut-off point for

genes with low expression; without this information, the spots could not be

normalized and sorted. Each set of array data was initially extracted and

analyzed differently depending on the format in which they were originally

deposited. For example, some data were extracted in .CEL file format

(reanalyzed using GCOS 1.1, Affymetrix), whereas others were in .XLS format

(Microsoft Excel) or .TXT format. After data extraction, all arrays were analyzed

in a similar manner. After elimination of background and genes with a low level

of expression, the mean fluorescence intensity of duplicated spots representing

the same gene was calculated and normalized to the mean fluorescence

intensity of the whole array. Genes were then sorted by chromosome location.

The ratio of the mean expression of X-linked genes to that of autosomal genes

was calculated, as well as the average of the above ratios for all arrays hybridized

with cDNA prepared from the same tissue type.

Statistical analyses. From each set of arrays extracted from the databases, a

gene expression distribution histogram (Microsoft Excel) was created to

determine whether expression values (log2 based and binned) for all genes

surveyed followed a normal distribution. The percentage of X-linked spots on

the arrays ranged between 3.8–4.0% of the total numbers of spots, consistent

with the percentage of X-linked genes in the mammalian genome, 3.8–4.4%,

depending on the species. For example, using the GNF-1H and GH-U133A

arrays, we compared 743 X-linked genes to 18,193 autosomal genes.

X:autosome expression ratios remained similar when we compared genes on

the X chromosome and on a single autosome of similar size, chromosome 3,

which had 1,143 genes available for analysis.

Accession codes. Gene Expression Omnibus: GSE3413

URLs. Cardiogenomics database, http://www.cardiogenomics.org; National

Center for Biotechnology Information (NCBI) Gene Expression Omnibus,

http://www.ncbi.nlm.nih.gov/geo/; European Bioinformatics Institute Array

Express, http://www.ebi.ac.uk/arrayexpress/; Stanford MicroArray Database,

http://genome-www5.stanford.edu/; National Institute of Aging Laboratory

of Genetics microarray data, http://lgsun.grc.nia.nih.gov/microarray/data.

html; Gene Expression Atlas, http://expression.gnf.org/; Biozentrum Swiss

Institute of Bioinformatics, http://www.biozentrum.unibas.ch/personal/primig/

rat_spermatogenesis/; Prostate Expression Database, http://www.pedb.org/;

University of Washington Microarray Center, http://ra.microslu.washington.

edu/; Affymetrix, http://www.affymetrix.com; Array Expression Database, http://

www.ebi.ac.uk/arrayexpress/.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSThis work was supported by grants from the US National Institutes of Health(NIH). We thank J. Birchler (University of Missouri), B. Oliver (National Instituteof Diabetes & Digestive & Kidney Diseases, NIH) and M. Cheng (University ofWashington), for helpful discussions. We thank C. Pritchard, I. Coleman andP. Nelson (Fred Hutchinson Cancer Research Center); J. Hogenesch, T. Wilshireand J. Walker (Genomics Institute of the Novartis Research Foundation);G. Martin and B. Cool (Department of Pathology, University of Washington);C. Bondy (National Institute of Child Health, NIH); M. Ko (National Institute ofAging, NIH); P. Khaitovich (Max-Planck-Institute for Evolutionary Anthropology,Leipzig); R. Bumgarner and the staff at Array Expression Database(http://www.ebi.ac.uk/arrayexpress/) for providing data for these analyses.We thank the Locke Computer Center and the Department of Biostatistics(University of Washington) for help with the statistical analysis, L. McKitrickand H. Vendettuoli, for technical assistance.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

1. Charlesworth, B. & Charlesworth, D. The degeneration of Y chromosomes. Phil. Trans.R. Soc. Lond. B 355, 1563–1572 (2000).

2. Lyon, M. Gene action in the X-chromosome of the mouse (Mus musculus L). Nature190, 372–373 (1961).

3. Ohno, S. Sex Chromosomes and Sex Linked Genes (Springer Verlag, Berlin, 1967).4. Birchler, J.A., Pal-Bhadra, M. & Bhadra, U. Dosage dependent gene regulation and the

compensation of the X chromosome in Drosophila males. Genetica 117, 179–190(2003).

5. Straub, T., Dahlsveen, I.K. & Becker, P.B. Dosage compensation in flies: mechanism,models, mystery. FEBS Lett. 579, 3258–3263 (2005).

6. Adler, D.A. et al. Evidence of evolutionary up-regulation of the single activeX chromosome in mammals based on Clc4 expression levels in Mus spretus andMus musculus. Proc. Natl. Acad. Sci. USA 94, 9244–9248 (1997).

7. FitzPatrick, D.R. et al. Transcriptome analysis of human autosomal trisomy. Hum. Mol.Genet. 11, 3249–3256 (2002).

8. Saran, N.G., Pletcher, M.T., Natale, J.E., Cheng, Y. & Reeves, R.H. Global disruption ofthe cerebellar transcriptome in a Down syndrome mouse model. Hum. Mol. Genet. 12,2013–2019 (2003).

9. Amano, K. et al. Dosage-dependent over-expression of genes in the trisomic region ofTs1Cje mouse model for Down syndrome. Hum. Mol. Genet. 13, 1333–1340 (2004).

10. Zhou, X. et al. Identification of discrete chromosomal deletion by binary recursivepartitioning of microarray differential expression data. J. Med. Genet. 42, 416–419(2005).

11. Gartler, S.M. & Andina, R.J. Mammalian X-chromosome inactivation. Adv. Hum.Genet. 7, 99–140 (1976).

12. Cheng, M.K. & Disteche, C.M. Silence of the fathers: early X inactivation. Bioessays26, 821–824 (2004).

13. Gartler, S.M., Liskay, R.M., Campbell, B.K., Sparkes, R. & Gant, N. Evidence for twofunctional X chromosomes in human oocytes. Cell Differ. 1, 215–218 (1972).

14. McMahon, A., Fosten, M. & Monk, M. Random X-chromosome inactivation in femaleprimordial germ cells in the mouse. J. Embryol. Exp. Morphol. 64, 251–258 (1981).

15. Monesi, V. Differential rate of ribonucleic acid synthesis in the autosomes and sexchromosomes during male meiosis in the mouse. Chromosoma 17, 11–21 (1965).

16. Turner, J.M. et al. Silencing of unsynapsed meiotic chromosomes in the mouse. Nat.Genet. 37, 41–47 (2005).

17. Huynh, K.D. & Lee, J.T. Inheritance of a pre-inactivated paternal X chromosome inearly mouse embryos. Nature 426, 857–862 (2003).

18. Okamoto, I., Otte, A.P., Allis, C.D., Reinberg, D. & Heard, E. Epigenetic dynamics ofimprinted X inactivation during early mouse development. Science 303, 644–649(2004).

19. Mak, W. et al. Reactivation of the paternal X chromosome in early mouse embryos.Science 303, 666–669 (2004).

20. Su, A.I. et al. Large-scale analysis of the human and mouse transcriptomes. Proc. Natl.Acad. Sci. USA 99, 4465–4470 (2002).

21. Su, A.I. et al. A gene atlas of the mouse and human protein-encoding transcriptomes.Proc. Natl. Acad. Sci. USA 101, 6062–6067 (2004).

22. Pritchard, C.C., Hsu, L., Delrow, J. & Nelson, P.S. Project normal: defining normalvariance in mouse gene expression. Proc. Natl. Acad. Sci. USA 98, 13266–13271(2001).

23. Walker, J.R. et al. Applications of a rat multiple tissue gene expression data set.Genome Res. 14, 742–749 (2004).

24. Wang, J. et al. Function-informed transcriptome analysis of Drosophila renal tubule.Genome Biol. 5, R69 (2004).

25. Rinn, J.L. et al. Major molecular differences between mammalian sexes are involved indrug metabolism and renal function. Dev. Cell 6, 791–800 (2004).

26. Welle, S., Brooks, A.I., Delehanty, J.M., Needler, N. & Thornton, C.A. Gene expressionprofile of aging in human muscle. Physiol. Genomics 14, 149–159 (2003).

27. Carrel, L. & Willard, H.F. X-inactivation profile reveals extensive variability in X-linkedgene expression in females. Nature 434, 400–404 (2005).

28. Disteche, C.M., Filippova, G.N. & Tsuchiya, K.D. Escape from X inactivation. Cyto-genet. Genome Res. 99, 36–43 (2002).

29. Uddin, M. et al. Sister grouping of chimpanzees and humans as revealed by genome-wide phylogenetic analysis of brain gene expression profiles. Proc. Natl. Acad. Sci.USA 101, 2957–2962 (2004).

30. McCarrey, J.R., Dilworth, D.D. & Sharp, R.M. Semiquantitative analysis of X-linkedgene expression during spermatogenesis in the mouse: ethidium-bromide staining ofRT-PCR products. Genet. Anal. Tech. Appl. 9, 117–123 (1992).

31. Hendriksen, P.J. et al. Postmeiotic transcription of X and Y chromosomal genes duringspermatogenesis in the mouse. Dev. Biol. 170, 730–733 (1995).

32. Odorisio, T., Mahadevaiah, S.K., McCarrey, J.R. & Burgoyne, P.S. Transcriptionalanalysis of the candidate spermatogenesis gene Ube1y and of the closely relatedUbe1x shows that they are coexpressed in spermatogonia and spermatids but arerepressed in pachytene spermatocytes. Dev. Biol. 180, 336–343 (1996).

33. Khalil, A.M., Boyar, F.Z. & Driscoll, D.J. Dynamic histone modifications mark sexchromosome inactivation and reactivation during mammalian spermatogenesis. Proc.Natl. Acad. Sci. USA 101, 16583–16587 (2004).

34. Malkov, M., Fisher, Y. & Don, J. Developmental schedule of the postnatal rat testisdetermined by flow cytometry. Biol. Reprod. 59, 84–92 (1998).


ART I C LES©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

35. Schultz, N., Hamra, F.K. & Garbers, D.L. A multitude of genes expressed solely inmeiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets.Proc. Natl. Acad. Sci. USA 100, 12201–12206 (2003).

36. Schlecht, U. et al. Expression profiling of mammalian male meiosis and gametogenesisidentifies novel candidate genes for roles in the regulation of fertility. Mol. Biol. Cell15, 1031–1043 (2004).

37. Hamra, F.K. et al. Defining the spermatogonial stem cell. Dev. Biol. 269, 393–410(2004).

38. Khil, P.P., Smirnova, N.A., Romanienko, P.J. & Camerini-Otero, R.D. The mouseX chromosome is enriched for sex-biased genes not subject to selection by meioticsex chromosome inactivation. Nat. Genet. 36, 642–646 (2004).

39. Hamatani, T. et al. Age-associated alteration of gene expression patterns in mouseoocytes. Hum. Mol. Genet. 13, 2263–2278 (2004).

40. Wang, Q.T. et al. A genome-wide study of gene activity reveals developmental signalingpathways in the preimplantation mouse embryo. Dev. Cell 6, 133–144 (2004).

41. Zeng, F., Baldwin, D.A. & Schultz, R.M. Transcript profiling during preimplantationmouse development. Dev. Biol. 272, 483–496 (2004).

42. Gupta, V. et al. Global analysis of X-chromosome dosage compensation. J. Biol.(in the press).

43. Akhtar, A. Dosage compensation: an intertwined world of RNA and chromatinremodelling. Curr. Opin. Genet. Dev. 13, 161–169 (2003).

44. Zheng, P. et al. Expression of genes encoding chromatin regulatory factors in deve-loping rhesus monkey oocytes and preimplantation stage embryos: possible roles ingenome activation. Biol. Reprod. 70, 1419–1427 (2004).

45. Migeon, B.R. et al. Differential expression of steroid sulphatase locus on active andinactive human X chromosome. Nature 299, 838–840 (1982).

46. Zechner, U. et al. A high density of X-linked genes for general cognitive ability: a run-away process shaping human evolution? Trends Genet. 17, 697–701 (2001).

47. Ropers, H.H. & Hamel, B.C. X-linked mental retardation. Nat. Rev. Genet. 6, 46–57(2005).

48. Birchler, J.A., Riddle, N.C., Auger, D.L. & Veitia, R.A. Dosage balance in generegulation: biological implications. Trends Genet. 21, 219–226 (2005).

49. Charlesworth, B. The evolution of chromosomal sex determination and dosage com-pensation. Curr. Biol. 6, 149–162 (1996).


ART I C LES©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

dosage compensation of the active x chromosome in mammals

Documents