Volume 12 Number 24 1984
Nucleic Acids Research
Complete concordance between glucose-6-phosphate dehydrogenase activity and hypomethyiation of
3' CpG dusters: implications for X chromosome dosage compensation
Stanley F.Wolf*, Suzanne Dintzis, Daniela Toniolo 1 , Grazidla PeTsico1, Keith D.Lunnen, Joyce
Axehnan and Barbara R.Migeon +
Depanment of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205,
USA, and 'Istituto Intemazionale di Genetic e Biofisica, Via G. Marconi, 10, Napoli 80125, Italy
Received 26 September 1984; Revised and Accepted 21 November 1984
ABSTRACT
To explore the m o l e c u l a r basis of X chromosome 1nact1vation,
we have examined the human locus for glucose-6-phosphate dehydrogenase (G6PD) 1n various human t i s s u e s . Studies of DNA from
males and females and from somatic cell hybrids with active
or Inactive X chromosomes, show that two remarkably dense clusters
of CpG d1nuc1eot1des 1n the 3* coding sequences are hypomethylated
1n active G6PD genes but e x t e n s i v e l y methylated 1n Inactive
ones.
Reacqu1s111 on of G6PD activity* either spontaneous or
Induced by 5 - a z a c y t 1 d 1 n e , Is accompanied by deraethy 1 at 1 on of
both clusters; however, the clusters remain methylated 1n reactivants
that express HPRT but not G6PD. Our observations Implicate these
3' CpG clusters 1n the transcription of G6PD and 1n maintenance
of dosage compensation for X linked housekeeping genes.
INTRODUCTION
It has been suggested that ONA methy1 at 1 on 1s responsible
for the Inactivity of X chromosomes In somatic cells of mammalian
females (reviewed 1n reference 1) and, therefore, 1s a key factor
1n achieving dosage compensation for X 1 Inked genes (2). Reactivation
of alleles on Inactive X chromosomes by 5-azacyt1d1ne (5-azaC),
an Inhibitor of methylation ( 3 ) , lends support to this hypothesis.
Ne have been examining DNA methylation of X chromosomal genes
to determine the role 1t plays 1n X dosage compensation. Studies
of X- linked genes provide o n e of the best means to examine
the relationship between methylation and expression of the locus.
Active and Inactive versions of the gene can be compared within
the same c e l l .
F u r t h e r m o r e , 1n this way one can observe the
Inactive version of genes that are const1tut1vely expressed,
not usually possible with autosotnal g e n e s . Our o b s e r v a t i o n s
of t h e X linked hypoxanthine phosphor 1bosy1 transferase (HPRT)
locus have revealed that active and Inactive genes differ 1n
© IRL Press Limited, Oxford, England.
9333
Nucleic Acids Research
their
patterns
difference
the
promoter
1n
active
ones
of
(4;
dense c l u s t e r
This
genes,
cluster
but
5).
of
The
most
1s
Invariably
extensively
5'
o f CpG d 1 n u c l e o t 1 d e s t h a t
HPRT
cluster,
have
g 1 ucose-6-phosphate
active.
of
Inactive
tissues
regulation
of
within
hypomethylated
methylated
been
1n
Inactive
are remarkably
found
dehydrogenase
1n
the
(G6PD)
suggest
of
the
X chromosome
and
reactivated
that
3
1
CpG
G6PD l o c u s
dosage
3'
clusters
(6)
1n
of
the
Our o b s e r v a t i o n s
G6PD a l l e l e s
are
and c o n s e q u e n t l y ,
similar
region
locus
X q 2 8 - q t e r s e g m e n t o f t h e human X c h r o m o s o m e ( 7 . 8 ) .
of
significant
CpG d 1 n u c l e o t 1 d e s
(4).
the
the
the
region;
HPRT
Two c l u s t e r s
to
DNA m e t h y l a t i o n
Involves
1n a
variety
Important
1n t h e
1n
the
maintenance
compensation.
MATERIAL AND METHODS
1.
Cel1s
and
Blood
females
from
had
Tisauas
and
been
3
dermal
males
stored
(10)
of
half
the
that
by
Hybrid
having
X)
24 and 34 e x p r e s s e d
treatment
11 and 20 had o n l y
(10)
the
the
GlxA9
the
with
5
and
late
x
both
10"
ExA9
X from
were
at
(3).
from
(but
no
Reactivant
human HPRT and G6P0 w h i l e
clones
human HPRT.
and Analysis of DNA
Methods
3•
on
approxi-
X
for purifying
DNA from
cells
and t i s s u e s ,
p u r i f i c a t i o n , n1ck-translat1on and Southern a n a l y s i s were
and
human
locus
Induced
Inactive
M 5-azaC
(human
previously
G6PD
1s e x p r e s s e d
1sod1centr1c
that
derived
(11).
hybrids
reactivants
6
and
The
and
lymphoblasts
were
replicating
X.
males
cultures
cells
(4,7)
7E1-4
hybrids
other
and
from
X karyotype,
active
1n t h e
The
clones
Isolation
nitrogen.
not
normal
Fibroblasts
derived
contain
activity.
from
were
6TGK 7 E 1 - 2
X chromosome
hybrids
(9).
46 X 1 s o d 1 c e n t r 1 c
clone81,but
Inactive
ExA9
liquid
active
2.
obtained
females
Clone81xA9
Included
fibroblast
the
1n
matings:
described
the
and
fibroblasts
Reactivants
mately
were
as p r e v i o u s l y d e s c r i b e d
normal
from
placenta
have been
Restriction
described
Enzyme
plasmid
standard
previously ( 1 7 ) .
Digestion
C o m p l e t e d i g e s t s were c a r r i e d o u t a c c o r d i n g t o m a n u f a c t u r e r ' s
Instructions
3-5
9334
using
10 u / u g D N A
for methyl
u / u g D N A f o r t h e rest, and o v e r n i g h t
sensitive
Incubations.
enzymes,
Nucleic Acids Research
Partial digests with Hpall were obtained b y adding different
a m o u n t s o f e n z y m e r a n g i n g from 0 - 5 u/ug D N A t o a series o f
s a m p l e s o f D N A that had been completely digested with a methyl
Insensitive restriction enzyme.
Specimens were Incubated f o r
30' at 37°C.
T h e reactions were stopped by adding EDTA to 10
raH.
RESULTS
G6PD Map a n d Prohes
F i g u r e 1 shows t h e 3 ' region o f t h e human G6PD locus near
the poly A addition site.
S e q u e n c e s 5 ' t o this region have
not y e t been cloned.
T h e m a p 1s based on restriction enzyme
and s e q u e n c e a n a l y s i s o f recorabinant p h a g e o b t a i n e d by s c r e e n i n g
a genonic library with a G6PD cDNA clone (6,12).
A notable
feature o f this region o f the gene are two clusters o f C p G d1nuc l e o t i d e s a t a p p r o x i m a t e l y 2.S k b (A-l c l u s t e r ) a n d 6.5 k b (A-2
c l u s t e r ) 5 1 f r o m t h e p o l y A a d d i t i o n s i t e . F o r e a s e 1n d e s c r i b i n g
the r e s u l t s , Hpall s i t e s a r e numbered beginning with t h e most
5 ' s i t e t h a t h a s b e e n I d e n t i f i e d 1n c l o n e d f r a g m e n t s .
T h e p r o b e s u s e d 1 n t h i s s t u d y a r e s h o w n 1n F i g u r e 1 . T h e
pGD1.4 probe hybridizes to mature G6P0 mRNA.
Both pGD3 and
pGD1.4 a r e highly conserved as they hybridize t o mouse genomic
DNA under s t r i n g e n t c o n d i t i o n s , and pGD3 has been used t o regionally
map t h e single locus on both human a n d murine X chromosomes
by l a situ h y b r i d i z a t i o n ( 8 ) .
CpG Clusters
Unmethvlated
In A c t i v e
G 6 P 0 „ .A 1 T a l e s
o f Mains
a r e Fxtnns1vaiv
M e t h y l a t i o n o f C p G d 1 n u c l e o t 1 d e s 1n D N A f r o m b l o o d , p l a c e n t a ,
lymphoblasts a n d fibrobiasts h a s been determined by cleavage
with the methyl sensitive enzyme, Hpall.
Less extensive analyses
w i t h H h a l a r e n o t d i s c u s s e d 1n d e t a i l , b u t p r o d u c e d
similar
results. The position o f unmethylated Hpall sites has been defined
by s u b s e q u e n t d i g e s t i o n s w i t h o t h e r r e s t r i c t i o n e n z y m e s a n d
Southern analysis using pGD3 and/or pGD1.4 probes.
T h e r e s u l t s s h o w n 1n F i g u r e 2 a n d s u m m a r i z e d 1n F i g u r e
IB c o n f i r m p r e v i o u s studies o f male leukocytes (6)and extend
o b s e r v a t i o n s t o D N A from other tissues.
T h e Hpall sites that
f l a n k b o t h c l u s t e r s 1n a c t i v e G 6 P D a l l e l e s , a r e c o n s i s t e n t l y
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Nucleic Acids Research
A-2
A
1
IM
i r
A-1
14 15
i ••) l
Smal
I
|
III II
Hpall
I
Aval
1 11
Hhal
in i R
I
I
i |
rr
p
S
, 11
i
I I
Hill
i n
inn
V
V
S
B
pGD3
P P
eg
poly A
pGD 14
x"
blood
placama
h/mphoblast
flbfOblBt
hybrid
H
It
0
—
/t
X1
pUOHltl*
tymphobUst
flbrobtat
hybrid
It—
if—
Figure 1. Map of the 3' coding region of hnnan G6P0 showing
location of A-1 and A-2 clusters and suanary of •ethylation
pattern.
A.
T h e p o s i t i o n of s i t e s f o r methyl s e n s i t i v e e n z y m e s
are I n d i c a t e d .
T h e relevant sites for other enzymes are also
shown (P, PstI) S, SstI; B g , Bgll; R I , E c o R I ) . The dark lines
represent the two probes used 1n this study. The poly A addition
site Is arrowed.
B. D i a g r a m o f representative fragments obtained 1n Hpall
digests of DNA from active X (X ) , Inactive X (X ) and reactivated
X. D o t s Indicate regions where variable methylation was found
(see t e x t ) .
S l a s h e s I n d i c a t e t h e b o u n d a r i e s of t h e regions
analysed.
(•) All I n a c t i v e g e n e s 1n p l a c e n t a a r e u n m e t h y 1 a t e d at
s i t e s H 1 4 o r H 1 5 so that p a t t e r n s of methy 1 at 1on 5 ' of those
sites can not be examined.
(f) Only one female 1ymphoblast line was analysed.
u n m e t h y i a t e d as shown by t h e f o l l o w i n g :
Hpall/PstI digests
probed with p G D 1 . 4 , show t h a t site H 3 6 at t h e 3' end of the
A-1 c l u s t e r Is open (0.9 kb f r a g m e n t . F i g u r e 2A lanes 1-4) j
H p a l l / S s t I d i g e s t s probed with pGD3 show t h a t t h e 5' and of
t h e c l u s t e r (site H 1 8 ) Is also unmethylated (data not s h o w n ) ;
and H p a l l / P s t I d i g e s t s p r o b e d with pGD3 Indicate that o n e or
the other of sites H 1 4 or H15 at the 3 ' end of the A - 2 cluster
9336
Nucleic Acids Research
I
II-
A
-2.8
-2.0
I 2 34 5 6 78
Figure 2. Southern blots showing differential aethylation of
clusters In Dales ( (fp ) and females ( Q )and tissue specific
differences.
H p a l l / P s t I d i g e s t e d D N A f r o m f i b r o b l a s t s (lanes 1 and 5 )
1 y m p h o b l a s t s (lanes 2 and 6 ) , blood (lanes 3 and 7 ) and p l a c e n t a
(lanes 4 and 8 ) probed with
A ) p G D 1 . 4 and B ) pGD3
N o t e : t h e 5 ' e n d o f t h e 0.9 kb f r a g m e n t 1s s i t e H 3 6 , as
shown by t h e 1.7 kb f r a g m e n t 1n H p a l l / B g l l d i g e s t s (See F i g u r e s
3C and 5 A ) . T h e relative weaker Intensity of t h e fragments
from t h e Inactive X m a y reflect h e t e r o g e n e i t y of Inactive X
m e t h y 1 at 1 on 1n f i b r o b l a s t s (See F i g u r e s 4,5,and t e x t ) , w h e r e a s
1n p l a c e n t a 1t 1s a t t r i b u t a b l e at least 1n p a r t , t o hyporoethy1 at1on
of t h e s e c l u s t e r s on t h e Inactive X (See t e x t ) .
1s a l w a y s u n n e t h y 1 a t e d
lanes 1 - 4 ) .
( 2 . 2 a n d 2.0 kb f r a g m e n t s . F i g u r e 2 B ,
Because t h e Hpall sites flanking both c l u s t e r s are n o t
m e t h y l a t e d , p a r t i a l M p a l l d i g e s t s w e r e u s e d t o d e t e r m i n e 1f
sites within t h e c l u s t e r s were also u n m e t h y 1 a t e d .
If m a n y Hpall
s i t e s were o p e n , then partial d i g e s t i o n with Hpall should p r o d u c e
m u l t i p l e f r a g m e n t s . T h e e x t e n t of m e t h y l a t i o n 1n t h e A-l c l u s t e r
w a s d e t e r m i n e d from O N A that had been c o m p l e t e l y d i g e s t e d with
P s t I or B g l l , b u t o n l y p a r t i a l l y d i g e s t e d w i t h H p a l l , using
pGD1.4 as the probe.
T h e A - 2 c l u s t e r w a s analyzed similarly
b u t w i t h c o m p l e t e P s t I d i g e s t i o n and t h e pGD3 p r o b e .
In t h e s e
p a r t i a l d i g e s t s (Figure 3 ) , we o b s e r v e d an array of f r a g m e n t s ,
r e f l e c t i n g m a n y u n m e t h y 1 a t e d H p a l l s i t e s 1n t h e A - l c l u s t e r
o f a c t i v e g e n e s from b l o o d , p l a c e n t a and 1 y m p h o b l a s t s , (Figure
3 A , lanes 3 and 9; F i g u r e 3 C , lanes 2 and 8 , r e s p e c t i v e l y ) .
A n a l y s i s of t h e A - 2 c l u s t e r also revealed m u l t i p l e b a n d s (Figure
9337
Nucleic Acids Research
blood
placenta
-1.95
-0.56
placenta
blood
-4.25
B
-2.25
- 1.95
O lymphoblast
9 lymphoblast
-4.25
I 2 3 4 5 6 7 8 9 10 II 12
FIgure 3.
Southern blots showing extent of •ethylation within
A-l and A-2 clusters.
L a n e s 1-6 and 7-12 contain aliquots of DNA Incubated with
I n c r e a s i n g a m o u n t s of H p a H (ranging from 0 - 5 u/ug DNA, s e e
Methods).
A . P a r t i a l H p a l l . c o m p l e t e PstI d i g e s t from b l o o d , probed
with pGD1.4j
B . Same digest, probed with pGD3j
C . Partial
H p a l l , c o m p l e t e Bgll digest of 1 y m p h o b i a s t s , probed with p G D 1 . 4 .
The size (kb) and position of X phage Hindlll f r a g m e n t s . Included
as m o l e c u l a r w e i g h t m a r k e r s , a r e Indicated on t h e right.
3B, lanes 3 and 1 0 ) .
Although
both
clusters
extensively
hypomethylated
ferences
patterns
9338
In
with
1n
active
there
respect
are
to
G6PD
some
the
alleles
tissue
extent
are
specific
of
always
dif-
m e t h y l a t 1 on
Nucleic Acids Research
or p o s i t i o n of methylated sites within each cluster.
Site H 1 5
w a s f r e q u e n t l y m e t h y l a t e d 1n b l o o d , b u t w a s o f t e n u n m e t h y l a t e d
1n p l a c e n t a a n d 1 n c u l t u r e d c e l l s ( C f . r e l a t i v e I n t e n s i t y o f
2.0 a n d 2 . 2 kb f r a g m e n t s . F i g u r e 2 B , lanes 1 - 4 ) . Furthermore,
d i f f e r e n c e s 1n t h e e x t e n t o f h y p o m e t h y 1 at 1 on w i t h i n t h e A - 2
cluster a r e also apparent as t h e multiple bands produced by
p a r t i a l H p a l l d i g e s t s a r e m o r e d i s t i n c t 1n b l o o d t h a n 1n p l a c e n t a
(Figure 3 B ) . On t h e other hand, tissue specific patterns did
not vary among I n d i v i d u a l s .
M a t h y l a t i o n o f C p G C l u s t e r ? ; In I n a c t i v e A T T a l e s 1s E x t e n s i v e
H p a l l d i g e s t e d DNA from f e m a l e s had all o f t h e f r a g m e n t s o b s e r v e d
1n m a l e s , b u t a d d i t i o n a l o n e s a s w e l l .
Based on studies o f
m o u s e - h u m a n hybrid c e l l s (see below), t h e extra fragments a r e
d e r i v e d f r o m I n a c t i v e a l l e l e s o n t h e s i l e n t X . In t h e s e I n a c t i v e
g e n e s t h e A-l and A - 2 c l u s t e r s a r e e x t e n s i v e l y m e t h y l a t e d ( F i g u r e s
2, 3 C , 4 a n d s u m m a r i z e d 1n I B ) . M e t h y l a t i o n w i t h i n t h e A - l
c l u s t e r 1 s e v i d e n t f r o m t h e f r a g m e n t s l a r g e r t h a n 0 . 9 k b 1n
Hpall/PstI digests o f blood a n d cultured cells (Figure 2A, lanes
5 - 7 ) , probed with pGD1.4.
However, Inactive genes o f placental
o r i g i n f r e q u e n t l y a p p e a r t o b e l e s s m e t h y l a t e d t h a n 1n o t h e r
tissues.
F o r e x a m p l e t h e b a n d g r e a t e r t h a n 0 . 9 k b In F i g u r e
2 A , l a n e 8 1s b a r e l y v i s i b l e (see b e l o w ) . T h a t t h e A - 2 c l u s t e r
1s m e t h y l a t e d w a s e v i d e n t f r o m t h e a r r a y o f b a n d s l a r g e r t h a n
2 . 0 k b 1n H p a l l / P s t I d i g e s t s p r o b e d w i t h p G D 3 ( F i g u r e 2 B , l a n e s
5-8).
frjfl-fcKyla'fcion o f I n a c t i v e Alifllfls I s HfltarooflnflQus
Although extensively methylated, t h e Inactive genes a r e
m e t h y l a t e d 1n a v a r i e t y o f w a y s .
F o r e x a m p l e 1n H p a l l / P s t I
digests probed with pGD3 o r p G 0 1 . 4 , there are multiple bands
d e r i v e d f r o m t h e I n a c t i v e G 6 P D g e n e (F1,gure 2 ) . B e c a u s e e a c h
gene should produce no more than one Hpall fragment (See F i g u r e
1), multiple bands Indicate that all Inactive genes a r e not
methylated t h e same way.
The relative Intensity o f bands from the Inactive X d i f f e r s
c o n s i d e r a b l y from o n e tissue t o another, s o that Inactive g e n e s ,
like active ones show tissue specific methylation o f t h e 3 '
c l u s t e r r e g i o n ( F i g u r e s 2 - 4 ) . M o r e o v e r In s o m e t i s s u e s t h e
relative Intensity o f fragments derived from active and Inactive
9339
Nucleic Acids Research
0.56 —
Figure 4. Southern blots showing variable nethylation of Inactive
G6PD tlleles.
H p a l l / E c o R I d i g e s t s of ( A ) b l o o d and (B) p l a c e n t a , and
H h a l d i g e s t of ( C ) blood and ( D ) p l a c e n t a , prohed with p G 0 3 .
T h e size and position of x phage Hindlll fragments are Indicated.
X chromosomes 1s unequal; too few Inactive X fragments are detected
(Figures 2A and B, lanes 5 and 8 ) . This nay reflect heterogeneous
methylation of Inactive chromosomes, I.e., the signal 1s distributed
among many fragments and therefore can not be detected. A l t e r n a t i v e l y , some Inactive genes may mimic active o n e s , I.e., having
an open site at the end of a c l u s t e r , but extensive tnethylation
within the cluster. Or In some cells the clusters may» In factf
be relatively hypomethylated. T h e latter 1s the case for placenta,
9340
Nucleic Acids Research
i n u t i ive A
mutnve A
A
Reactivated X
B
-2.8
-2.0
I 2 3 4 5 6 7 8 9 10 II 12 13 1415 16 17 18 19 202122
Figure 5. Partial Hpall digests of Inactive, active and reactivated
G6PD alleles, showing extent of aethylation within clusters.
G l x A 9 hybrid with Inactive X (lanes 1 - 5 ) , E x A 9 hybrid with
1 s o d 1 c e n t r 1 c I n a c t i v e X ( l a n e s 6 - 1 1 ) , C l o n e 8 1 x A 9 hybrid with
a c t i v e X ( l a n e s 1 2 - 1 7 ) , and C l o n e 8 1 x A 9 hybrid, (6TG 7 E 1 - 4 ) ,
with r e a c t i v a t e d X (lanes 1 8 - 2 2 ) . Size ( k b ) of g e n o m i c f r a g m e n t s
1s I n d i c a t e d on t h e r i g h t .
(Conditions for partial digests
are t h e same a s 1n F i g u r e 3 L e g e n d , e x c e p t t h a t L a n e 18 1n F i g u r e
5A c o n t a i n s D N A d i g e s t e d with 0.01 unit H p a l l / u g D N A ) .
A. Partial HpalI/complete Bgll, pGD 1.4, probing A-l cluster.
B. Partial Hpall/complete P s t I , pGD3, probing A - 2 cluster.
as c e l l s e x p r e s s i n g b o t h G 6 P D a l l e l e s h a v e b e e n o b s e r v e d 1n
c h o r i o n i c v 1111 ( H i g e o n , B . R . , W o l f , S . F . , A x e l m a n , J.<
K a s l o w , D . C . and S c h m i d t , M . , m a n u s c r i p t s u b m i t t e d ) .
F u r t h e r m o r e , I n d i v i d u a l s vary c o n s i d e r a b l y 1n m e t h y l a t i o n
o f I n a c t i v e g e n e s w i t h i n t h e same t i s s u e .
This heterogeneity
1s r e a d i l y a p p a r e n t 1n H p a l l / E c o R I d i g e s t s o f b l o o d ( F i g u r e
4 A ) a n d 1n H h a l d i g e s t s of p l a c e n t a ( F i g u r e 4 D ) . V a r i a b i l i t y ,
a l t h o u g h m o r e s u b t l e , 1s also found 1n o t h e r d i g e s t s of blood
and p l a c e n t a ( F i g u r e s 4 B and C ) . T h e m u l t i p l e b a n d s m i g h t r e f l e c t
the h e t e r o g e n e i t y o f cell p o p u l a t i o n s w i t h i n t h e t i s s u e ; h o w e v e r ,
as t i s s u e c o m p o s i t i o n among I n d i v i d u a l s 1s r e l a t i v e l y s i m i l a r , t h e
9341
Nucleic Acids Research
12
3
4 5 6 7
8 9 ION
12 13 14
Figure 6 . C e l l u l o s e acetate electrophoresis showing G6PD phenotype
of r e a c t i v a t e s .
B, G6PD B; A, 66PD A; M, G6PD Mouse ( A 9 ) .
Unlabeled I n t e r m e d i a t e bands are heterod 1mers.
Raactivants:
Clone81 f i b r o b l a s t s
Clone81xA9
hybrid
5)}
Clone81xA9
hybrid
(lane 2 ) ;
HAT 7 E 1 » w i t h
(6TG 7 E 1 - 4 ) .
both
with
human
only
X chromosomes
the
(lane
reactivated
locus
(lane 6 ) .
ExA9
c l o n e 24 ( l a n e 1 0 ) a n d c l o n e 34 ( l a n e 1 3 ) r e e x p r e s s i n g
G6PD B .
ExA9 c l o n e 20 e x p r e s s i n g human PGK and HPRT, b u t no human G6PD
(laae 9 ) .
Controls:
G6PD B ( l a n e 3 ) , G6PD A ( l a n e 7 ) , G6PD AB ( l a n e s 4 . 1 1 , 14)
G6PD m o u s e ( l a n e s 8 , 1 2 ) .
G6PD AB 6 9 XXY f i b r o b l a s t s
(lane
1 ) . s h o w i n g t h e AB h e t e r o d 1 m e r .
extent
of
variability
Individuals
not
makes
Identically
1n t h e s i z e
1t
likely
methylatedf
and I n t e n s i t y
that
even
Inactive
within
of
bands
among
X chromosomes
t h e same
M a t h y l a t I q n o f A c t i v e and I n a r t i v a A l l a l a . s Snpa r a t e d
type
of
1n
are
cell.
frlousa-Hiunan
Hybrid. C e l l s
Mouse-human
and
Inactive
the
Inferences
The
array
12-17)
females
retaining
9342
bands
seen
active
that
from
Hpall
other
t o those
t h e means t o s e p a r a t e
female
cells
on s e x d i f f e r e n c e s
In
partial
X from
digests
active
of
Hpall
a female
(Figure
as t h o s e
also
confirm
patterns.
of
1n a c t i v e
an a d d i t i o n a l
X chromosomes,
1n m a l e s .
and t h u s
digests
cell
hyporaethyl a t e d
DNA f r o m
the active
1n m e t h y l a t i o n
t h e A - l and A - 2 c l u s t e r s
a r e as e x t e n s i v e l y
Complete
similar
based
the
shows
provide
chromosomes
of
containing
hybrids
hybrids
5,
lanes
genes
from
three
produced
from
males.
hybrids
fragments
Nucleic Acids Research
B
-4.4(M)
-3.5
-3.2
N
3.0
- 1.4
r-(M)
12 3 4 5 6
12
3 45
F1gu re 7. Southern blot of reactivant C1one81 and derivative
hybrids showing deeethylation of Hpall sites
A.
Hpall digests probed with pGD3.
Fibrobiast clone
with a c t i v e G6PD B a l l e l e , b u t I n a c t i v e G6PD A (lane 1 ) . C l o n e 8 1
of s a m e o r i g i n a s c l o n e 1n lane 1» b u t with both a l l e l e s a c t i v e
(lane 2 ) . Clone81xA9 hybrid. (HAT 7E1) retaining both active
and I n a c t i v e l o d (lane 3 ) , t w o d e r i v a t i v e 6 T G h y b r i d s ( 7 E 1 - 2 ,
7 E 1 - 4 ) with only t h e r e a c t i v a t e d l o c u s (lanes 4 . 5 ) and a n o t h e r
d e r i v a t i v e 6 T G hybrid ( 7 E 1 - 3 ) w i t h n o human X c h r o m o s o m e ( l a a e
6).
N o t e : F r o m H p a l l / S s t I d i g e s t s 1t 1s c l e a r t h a t t h e 3 . 2
and 3.0 kb f r a g m e n t s In l a n e s 1-5 result from u n m e t h y 1 a t e d s i t e s
at H 1 4 o r H 1 5 a n d H 1 8 a n d t h a t the 3.5 kb f r a g m e n t 1n lane 1
r e s u l t s from m e t h y l a t i o n of H15 t o H35. Thus the reactivated
a l l e l e 1n c l o n e B l d i f f e r s f r o m t h e s a m e a l l e l e when I n a c t i v e
(lane 1 ) and r e s e m b l e s a c t i v e g e n e s .
B . H p a l l / P s t I d i g e s t s p r o b e d with pGD1.4. GlxA9 hybrid
w i t h h u m a n a c t i v e X ( l a n e 1 ) , E x A 9 hybrid with human I n a c t i v e
X (lane 2 ) , C l o n e 8 1 x A 9 hybrid w i t h a c t i v e X (lane 3 ) , C l o n e 8 1 x A 9 hybrid 6 T G 7 E 1 - 4 w i t h r e a c t i v a t e d X (lane 4 ) , a n d m o u s e
A9 (lane 5 ) .
T h e faint b a n d s at 4.4 and 0.9 kb a r e from h o m o l o g o u s m o u s e
sequences ( H ) .
T h e Inactive X w a s a l s o e x a m i n e d 1n t w o h y b r i d s , e a c h w i t h
a d i f f e r e n t I n a c t i v e human X c h r o m o s o m e . O n e ( E x A 9 ) c o n t a i n e d
an 1 s o d 1 c e n t r 1 c X c h r o m o s o m e with t w o I n a c t i v e l o d . A l l t h r e e
I n a c t i v e a l l e l e s w e r e e x t e n s i v e l y m e t h y l a t e d 1n both c l u s t e r s
(Figure 5. lanes 1 - 1 1 ) .
Some of t h e Inactive X chromosomes
w e r e m e t h y l a t e d d i f f e r e n t l y t h a n o t h e r s ( F i g u r e 5 . lane 1 1 ) ,
so t h a t t h e v a r i a b i l i t y o b s e r v e d 1n f e m a l e c e l l s 1s also p r e s e n t
1n h y b r i d s .
9343
Nucleic Acids Research
B
.
•NMM» - 1.4
'
2 6
P«
<
-0.9(M)
- 1.7
12 3 4 5
Figure 8 . A n a l y s i s of the A - l c l u s t e r using partial Hpall digests
o f O N A f r o n 5 - a z a C Induced H P R T r e a c t i v a n t s .
L a n e s 1-5 c o n t a i n D N A w i t h I n c r e a s i n g a m o u n t s of H p a l l .
(see F i g u r e 3 ) . M , h o m o l o g o u s m o u s e s e q u e n c e s .
A. E x A 9 C l o n e 2 4 also e x p r e s s i n g G6PD B , H p a l l / B g l l .
B. E x A 9 C l o n e 2 0 , not e x p r e s s i n g human G 6 P D , H p a l l / P s t l .
Mathy.lAt.1on P a t t a r a s . a f Ra.nr.t.1 vatnri C6PJ3. AT Ifll as
T o a s s e s s t h e functional I m p o r t a n c e of m e t h y l a t i o n 1n t h e
A - l a n d A - 2 c l u s t e r s , we a n a l y z e d G 6 P D a l l e l e s t h a t had been
reactivated.
In o n e case, t h e r e a c t i v a t i o n e v e n t o c c u r r e d s p o n t a n e o u s l y In a n o t h e r w i s e n o r m a l c l o n e o f h u m a n f i b r o b l a s t s
( C l o n e 8 1 , 1 0 ) . In t h e s e c e l l s t h e G6PD A a l l e l e on t h e I n a c t i v e
X had a p p r o x i m a t e l y half w i l d t y p e a c t i v i t y ( F i g u r e 6, lane
2 ) , b u t n e i t h e r t h e PgK l o c u s ( 1 0 ) n o r t h e HPRT locus (4) 1s
expressed. The mouse-human hybrids retaining the relevant X
c h r o m o s o m e ( F i g u r e 6, lanes 4 and 5 ) have been d e s c r i b e d ( 1 0 ) .
F i g u r e 7 s h o w s t h a t 1n r e a c t i v a n t C l o n e S l (Figure 7 A , lane 2 )
a n d d e r i v a t i v e h y b r i d s , ( F i g u r e 7 A , l a n e s 5 a n d 6; 7 B , lane
4 ) t h e H p a l l s i t e s flanking t h e A - l c l u s t e r had been d e m e t h y l a t e d .
P a r t i a l H p a l l d i g e s t s of DNA from o n e o f t h e s e hybrids (6TG
7 E 1 - 4 ) , revealed that both c l u s t e r s were extensively unmethyiated ( F i g u r e 5 , lanes 1 8 - 2 2 ) .
G 6 P D r e a c t i v a n t s Induced by 5 - a z a C w e r e also e x a m i n e d . T h e
t w o r e a c t i v a n t s (ExA9 clones 2 4 and 3 4 ) w e r e derived from hybrid
c e l l s r e t a i n i n g o n l y t h e I n a c t i v e X c h r o m o s o m e , by t r e a t m e n t
w i t h 5 - a z a C and s e l e c t i o n 1n H A T m e d i u m .
They were Identified
b y s c r e e n i n g f o r t h e p r e s e n c e o f h u m a n G 6 P D ( F i g u r e 6, l a n e s
10 a n d 1 3 ) .
B e c a u s e t h e human I n a c t i v e X c h r o m o s o m e In t h e s e
h y b r i d s w a s an 1 s o d 1 c e n t r 1 c X w i t h t w o c o p i e s of t h e G 6 P D l o c u s ,
1t Is not c e r t a i n 1f one or b o t h o f t h e loci had been r e a c t i v a 9344
Nucleic Acids Research
ted.
had
N e v e r t h e l e s s , t h e A-l c l u s t e r
been
extensively
1n both G 6 P D
demethylated
(one
reactivants
1s shown
1n F i g u r e
8A)
and at least several sites 1n the A - 2 c l u s t e r had been d e m e t h y l a t e d
(data not s h o w n ) .
(Clones
GlxA9
(one
11
and
hybrids
As c o n t r o l s , we e x a m i n e d 2 s i b l i n g
2 0 ) , and
1n w h i c h
Is shown
4
Independent
5-azaC had
1n F i g u r e
8B).
reactivants-
reactivants
reactivated
In each of t h e m
derived
H P R T but not
G6PD
the
G6P0
Inactive
alleles were methylated throughout the c l u s t e r s , although
demethylat1on
had
occurred
at t h e H P R T
locus
from
(4 and
relevant
unpublished
results).
DISCUSSION
Hvpomathylatioji of. C l u s t e r s is C o r r e l a t e d
Although hypomethylat1on
has been a s s o c i a t e d
of m a n y g e n e s , t h e c o r r e l a t i o n
relationship
14).
The
heterogeneous
examined.
and
are
active
of
a gene
II.
housekeeping
X chromosome,
that
established
observations
of the
lod
has
that
The
(reviewed
may
and
be
expression
1n
reference
attributable
tissues
1n w h i c h
I.e.
revealed
Inactive
with
genes
1s c o n s t 1 t u t 1 v e l y
ability
gene.
associated
and
variation.
g e n e on
the silent
activity
within
presumed
Important
Studies
X,
to e x a m i n e
Inactive
features
f e r e n c e s have been d i s c r i m i n a t e d
specific
with
1s not a b s o l u t e , nor has a causal
to
they
the
were
He have studied t h e r e l a t i o n s h i p b e t w e e n DNA m e t h y l a t i o n
polymerase
this
clearly
nature
expression
by RNA
of
been
discordant
w1th.Expression
Inactive
alleles
of t h e
the
locus.
same c e l l ,
reactivants
Indicate that t h e
for e x p r e s s i o n
versions
on t h e
of t h e m e t h y l a t i o n
from t h o s e t h a t
of
transcribed
of t h e
By
silent
pattern
comparing
functional
reflect
that
dif-
tissue
reexpress
f e a t u r e s of t h e
locus, are
the
pattern
Indeed
func-
t i o n a l , as t h e y d i r e c t l y c o r r e l a t e with r e a c q u 1 s 1 1 1 on of a c t i v i t y .
B e c a u s e t h e two CpG c l u s t e r s
1n t h e 3 ' c o d i n g
region
are
always
h y p o m e t h y l a t e d 1n a c t i v e G6PD g e n e s , l a r g e l y m e t h y l a t e d 1n I n a c t i v e
genes,
of
and
these
demethylated
clusters
In
reactivated
1s c l e a r l y
a
genes,
significant
m e c h a n i s m s that m a i n t a i n the a c t i v i t y of t h e G 6 P D
T h e Role af
h y p o m e t h y l at 1on
component
the
locus.
CpG Clusters.1JI Transcription
S e q u e n c i n g of t h e h o u s e k e e p i n g g e n e s , d 1 h y d r o f o l a t e
(DHFR)
of
(15) and H P R T
(16) has revealed
unusual
reductase
f e a t u r e s of
their
9345
Nucleic Acids Research
promoter
regions;
whereas,
dense
Comparison
TATA
of
5 'dusters
locus
(4).
of this
observations
of
location
these
of
the
function,
clusters
may
HPRT
be
and
digestion,
Tracts
3'
but
of
In
of
alleles
G6PD
an
ectopic,
Indicate
expression
alleles,
1n t h e 3 1
Important
we
that
have
region
role
that
of
1n
shown
upstream
expression
lod
clearly
of
housekeeping
these
gene.
several
demonstrate
clusters
The
nature
hypothesis
3f
In p r o p e r t e r m i n a t i o n .
(2) CpG
clusters
only
1f
of
the
are
they
region.
In
hypersensitive
are
purines
clusters,
the
CpGs
and
regarding
(1) The
be f u r t h e r
architecture
of H P R T
and
explored:
could
hypomethy1ated
and
but
fact,
to
1n
precise
the
suggests
that
cooperatively.
Clustering
m e t h y l at 1 on
has
little
sites
the
cluster
of
effect
are
5'
1s
present
clusters
between
not
these
known,
the
existence
dInucleot1des
may
CpGs
might
that
chance
1s u n l i k e l y
not
1n
clusters
the
these
as
1n
sequences.
(15).
relationship
machinery
both
nuclease
(manuscript
pyr1ra1d1nes
also
(26) and D H F R
transcr1pt1onal
within
G6PD
suggests
clusters
G6PD
clustered
and
hypomethy1 at1on
G6PD
regions
the
HPRT
relevant
alternating
Although
and
HPRT
or
present.
(3) T h e s e c l u s t e r s may serve as e n h a n c e r
the
promoter
of
Involved
the
preparation).
only
absent
are
regulating
of
site, play
clusters
that
Influence
51
1n
studies
between
expression
may
role
of CpG d 1nucleot1des
A addition
concordance
their
are
locus.
Our
the
sequences
Inactive
a
similar
that dense clusters
poly
and
have
From
CAT
of C p G d 1 n u c l e o t 1 d e s
active
these
of the
and
clusters
ensure
function
methylation
to
silence
the
at
Interphase
d_a
of
novo
a
active
few
G6PD
gene.
Implications
f o r X Inact_1 v a t Ion
Because the
ctive,
most
X
special
has
(reviewed
pattern
silence
Our
1n
of
of
differences
9346
for
DNA
the
dosage
features
reference
previous
chromosomes
Inactive X chromosome
models
that
1).
studies
using
X
of
random
1n m e t h y l a t i o n
are
assume
responsible
We h a v e
been
that
might
raethylation
Inactive
compensation
for
Its
searching
for
serve
chromosome,
but
have
methylation
of
active
DNA
probes
between
did
1s so
that
not
to
distin-
the
silent
Inactivity
a
specific
maintain
Identified
and
reveal
the two chromosomes
the
none.
Inactive
X
significant
(17,18).
Nucleic Acids Research
F o r b o t h c h r o m o s o m e s , t h e p a t t e r n o f m e t h y l a t i o n 1n a 2 2 kb
r e g i o n o f D N A w a s v a r i a b l e , even w i t h i n clonal p o p u l a t i o n s o f
cells.
A l t h o u g h , w e h a v e I d e n t i f i e d Important d i f f e r e n c e s 1n
m e t h y l a t i o n of t h e h o m o l o g o u s HPRT loci on a c t i v e and I n a c t i v e
X c h r o m o s o m e s ( 4 ) , 1t w a s t h e a c t i v e genes that had t h e c o n s e n s u s
p a t t e r n , n a m e l y h y p o m e t h y l a t 1 o n 1n t h e 5 ' C p G c l u s t e r and e x t e n s i v e
m e t h y l a t i o n distal to t h e p r o m o t e r ; t h e I n a c t i v e genes had c l u s t e r s
that were m e t h y l a t e d , but o t h e r w i s e showed t h e same v a r i a b i l i t y
we had o b s e r v e d with t h e random p r o b e s .
B e c a u s e Inactive H P R T
genes lack a c o n s e n s u s p a t t e r n , we suggested that X d o s a g e c o m p e n sation w a s m a i n t a i n e d by p r o p a g a t i n g t h e activity of t h e a c t i v e
X, rather than s i l e n c i n g t h e I n a c t i v e o n e . F u r t h e r m o r e , b e c a u s e
t h e c o n s e n s u s p a t t e r n of m e t h y l a t i o n t h a t c h a r a c t e r i z e d a c t i v e
MPRT genes resembled t h o s e o b s e r v e d for t h e t w o autosomal h o u s e k e e p i n g g e n e s t h a t h a v e b e e n a n a l y z e d ( 1 9 ) , 1t seemed l i k e l y
t h a t e x p r e s s i o n o f t h e l o c u s on t h e a c t i v e X w a s m a i n t a i n e d
1n t h e s a m e w a y as t h a t o f h o u s e k e e p i n g g e n e s on a u t o s o m e s .
The 3 1 C p G c l u s t e r s from a c t i v e G6PD g e n e s a r e also c o n s i s t e n t ! y
h y p o m e t h y l a t e d , F u r t h e r m o r e , none of the f e a t u r e s of G6PD m e t h y l a t i o n
t h a t w e h a v e I d e n t i f i e d c a n b e a t t r i b u t e d a s p e c i a l role 1n
maintaining X chromosome 1nactivat1on.
Although the 3' clusters
of C p G s a r e u n u s u a l , s t u d i e s to date do not p r e c l u d e t h e i r p r e s e n c e
1n o t h e r h o u s e k e e p i n g g e n e s .
Moreover, our studies of clone81
r e a c t i v a n t s show that h y p o m e t h y l a t 1on o f t h e s e c l u s t e r s 1s s p e c i f i c
for e x p r e s s i o n o f t h e G 6 P D l o c u s ; o t h e r loci on t h e I n a c t i v e
c h r o m o s o m e remain I n a c t i v e and that c h r o m o s o m e remains late
r e p l i c a t i n g ( 1 0 , 2 0 ) . In a l l t h e 5 - a z a C Induced r e a c t i v a n t s
that were selected for HPRT a c t i v i t y , t h e HPRT c l u s t e r w a s h y p o m e t h y l a t e d ; h o w e v e r , t h e 3 1 G 6 P D c l u s t e r s were h y p o m e t h y l a t e d
o n l y 1n t h o s e t h a t a l s o e x p r e s s e d G 6 P 0 .
T h e r e f o r e , It s e e m s
t h a t r e e x p r e s s i o n 1s c o n t r o l l e d locus by locus rather than by
some m a s t e r s w i t c h . T h e r e a c t i v a t i o n event 1s Invariably a s s o c i a t e d
w i t h a c q u i s i t i o n of t h e m e t h y l a t i o n p a t t e r n that c h a r a c t e r i z e s
t h e g e n e on t h e a c t 1 v e c h r o m o s o m e .
These observations enhance
the l i k e l i h o o d that X c h r o m o s o m e d o s a g e c o m p e n s a t i o n 1s t r a n s m i t t e d
from o n e cell to Its p r o g e n y by m a i n t a i n i n g t r a n s c r 1 p t 1 o n a 1
a c t i v i t y of t h e Individual g e n e on t h e a c t i v e X.
9347
Nucleic Acids Research
ACKMOWLEDGMEHTS
We
1n
this
This
are
grateful
to
Dr.
work
and
for
project
was
supported
financial
support
from
making
CNR,
Lucio
a
by
Luzzatto
fruitful
NIH
for
his
collaboration
grant
HD
05465
and
Interest
possible.
through
Italy.
•Present address: Genetics Institute, 225 Longwood Avenue, Boston, MA 02115, USA
+
To whom correspondence should be addressed
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