Methylation and expression of the human thyroglobulin gene

Vol.

134,

February

No.

3,

13,

1986

1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1109-1113

METHYLATION AND EXPRESSION OF THE HUMAN THYROGLOBULIN GENE F. LIBEKT, Institut *Service de Chimie, Campus Erasme, Received

December

G. VASSART* and D. CHRISTOPHE

de Recherche Interdisciplinaire (IRIBHN) Universite Libre de Bruxelles, Faculti de Medecine, Route de Lennik 808, B-1070 Bruxellrs, Belgique 16,

1985

The DNA methylation pattern at the 5'end of the human thyroglobulin gene has been determined in different tissues. Out of the four HpaII/MspI sites (5'-CCGG-3') present in this region, three were found to be non-methylated in thyroid DNA, while full metllylation was observed in liver-, salivary gland and sperm DNA. This demethylation therefore correlates with expression of the thyroglobulin gene. However, all four sites were found to be non-methylated in placental DNA, regardless of @ 1986 Academic Press, Inc. the activity of the gene.

In contrast systems

to its

of bacteria,

eukaryotes.

regulation

of

this

relationship

nied

tmy a

expressed to this

rerlain for

shown lower

rule,

these

studies,

recent

finding

extra-rsibrycnic intriging

data

suggest

but the

mechanisms

activity, unknown that

(see

data

transcriptional

Although

are

regardless

are

still

strongly

of their

new questions

about

1 for

it

a in It

is accompato the non-

seem to make exception used

incomplete.

expression,

in The

undennethylated

the role

plays

a review).

activity

genes

in

involved

due to the methods

to date

genes

role

that

as compared

other

ill r;,ind that

collected

numerous

an obscure

ref.

in 5-methylcytosines

tissues,

and raises

of experimental

largely

one must keep

that

plays

gene

of the gene.

all

in restriction-modification

still

many genes

content

form

function

DNA methylation number

in

has been

known

A large

role

the

well

in

is particularly

of DNA nethylation

(2). We have recently gene (3,4). Abbreviations M NaCl, 0.015

In

the

cloned frame

the 5'end of our

study

: kb : kilo base-pair; M Na citrate).

of the human thyroglobulin of the mechanism SSC : standard

(Tg)

by which

saline

citrate

Tg gene (0.15

0006-291X/86 1109

$l.SO

Copyright 0 I986 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

134,

No.

3, 1986

expression

BIOCHEMICAL

is controlled

we decided

DNA methylation

would

that

of methylation

the extent

reflect

the activity

seems to be very expression

AND

participate

RESEARCH

to investigate in

this

in adult

in extra-embryonic

of

COMMUNICATIONS

the possibility

regulation.

at the 5'end

of the gene low

BIOPHYSICAL

that here

We report

the Tg gene appears

somatic

tissue.

lineages

to

However,

, irrespective

it

of the

state.

MATERIALS

AND METHODS

DNA samples Human tissues taken post-mortem were kindly provided by Drs Kinnaert (liver and thyroid) and-Rocmans (salivary glands). Dr Simon supplied us with human placenta (from a normal delivery). Frozen human sperm (from the sperm bank of the H8pital Saint Pierre) was a gift of Dr Englert. DNA was prepared from fresh (salivary glands) or powdered frozen tissues (liver, thyroid, placenta) by lysis of the tissues in a 8 M urea - 2 % SDS solution (in 10 mM Tris-HCl pH 8.5, 10 mM EDTA and 300 mM NaCl), extensive phenol extractions and ethanol precipitation (5). Spermatozoa were pelleted from thawed total sperm and washed three times with a 10 mM TrisHCl pH 7.5, 10 mM EDTA and 150 mM NaCl solution before lysis. The DNA were digested with appropriate restriction enzymes (from Boehringer Mannheim or Amersham) using the three stock buffers system fragments were separated on 1 % agarose slab gels (6). The resulting and blotted on Hybond-N membranes (Amersham) following the recommendations of the manufacturer. Probe preparation The EcoRI 7.9 kb genomic fragment encompassing the 5'end of the human Tg gene (3) was excised from vector sequences and labelled by nicktranslation (6) using [ o-32,1 dATP (800 Ci/mnole, 10 mCi/ml aqueous solution from Amersham). Hybridizations DNA hybridizations were carried out in 6XSSC, 5X Denhardt's solution 0,5 % and 20pg/ml denatured sheared salmon sperm DNA at 65"C, overnight. Blots were further processed as reconrnended by the manufacturer of the wash being in 0.1 XSSC at 65°C. The membranes (Amershanm), the final membranes were then autoradiographed using Kodak XAR-5 films. RESULTS AND DISCUSSION We have recently region

which

position

This

contain tion

the

cloning

of the human genome encompassing

gene from (4).

reported

-530

sequence

exhibited

any HpaII/MspI sites

were

contains

found 5.5

to position

in the

the 5'end

+ 151 (referring

a strong

restriction

and sequencing

CC suppression

site.

7.9 kb EcoRI

kb of upstream

sequences 1110

Only

of a small

of the

thyroglobulin

to +l as Cap site) (7)

and did

two HpaII/MspI

fragment

cloned

and 2.5 kb of

not

restric-

previously the

transcri-

(3)

BIOCHEMICAL AND BIOPHYSICAL

Vol. 134, No. 3, 1986 bed region Luckily,

including this

therefore three

sequence

be used

HpaII/MspI

restriction

the two first lacked

to probe

highly

(0.95;

involved

blots,

Probing

(3)

blots

(see

of HpaII

the 7.9 kb fragnent

to the

(i)

- no band was detected

(ii>

- two bands

(iii>-

all

three

in placental In order amount

(2.9

(225

and could

the detection

kb).

The four

of these containing

of

HpaII/MspI

fragments the 5'end

(plus

of DNA from various following

in liver,

kb and 0.95

bands

elements

3).

were of the

2). digests

led

phage

2 and ref.

allowing

in the generation

fig.

fig.

2.9 and 4.9

mapped on A hTg1 DNA, a recombinant human Tg gene

(see

repetitive

Southern

fragments

sites

exons

RESEARCH COMMUNICATIONS

kb)

some extra

results

humn

(see

tissues

fig.

salivary

gland

appeared

in thyroid

ones;

see below)

with

1) :

and sperm DNA. DNA. were

revealed

DNA. to monitor pg,

to the various

equivalent samples

the activity

of the restriction

to a single in parallel

gene dose)

experiments.

A B C D E F

enzyme, of AhTgl

A canplete

a small

DNA was added restriction

kb

- 7.8 -49 -38 -29

FIGURE 1. Autorediograph tissues after lane a : DNA lane b : DNA lane c : DNA lane d : DNA lane e : DNA lane f : ibid.

of a Southern blot of HpaII digested DNA from various hybridization with the EcoRI 7.9 kb Tg genomic prohe. from human thyroid from human liver fran human salivary glands from human sperm fom human placenta with 225 pg of AhTgl DNA aided as restriction control.

1111

Vol.

134.

No. 3, 1986

of the

test

BIOCHEMICAL

DNA ws

observed

in all

kb and 7.8 kb extra

bands

when

co-restricted

XhTgl

conclude

DNA ws that

these

the sample. all

(not

We also tide

found

for

were

found

to be fully

From

1) : the 3.8

more

intense

Therefore

from incanplete

the expected

AvaI

enzymes

sequence

which

we

digestion

three

and HlaI

enzymes

mrthylated

was still

the results

summarized

residue.

of

band pattern

in

the CG dinucleo-

(see

fig.

shown).

2).

DNA when this

However liver

The control

completely

the

Restriction

in the DNA from

(not

experiment

contain

and does not cleave

on the cytosine

no band was detected

parallel

arrose

fig.

DNA were

the human DNA.

MspI gave

is methylated

were

since

with

restriction

other

recognition

dinucleotide

one (see

COMMUNICATIONS

shown).

used

in their

but

RESEARCH

in placental

bands

with

BIOPHYSICAL

cases

detected

additional

Digestion

tissues

AND

sites these

sites

and thyroid A hTg1

DNA added

in

digested.

in fig.

2, the

following

conclusions

can

be drawn. There

(i)

is

a tissue-specific

demethylation

5'region

of the human Tg gene

with

the

expression

both

the

transcribed

thV& liyer.

salivary

*perm placenta

gland.

in the

of the gene. portion

thyroid

This

of HpaII DNA which

demethylation

and the proximal

upstream

n

0

0

0

n

n

n

n

0

0

00

sites

in the

can be correlated event

occurs

region

in

of the

n =methylated 0 =non

methyl.ted

FIGURE 2. Map of the 5'end of thr human thyroglobulin gexle showing the positions and methylation states of the CG dinucleotides considered this study. E, = exon n; X means that the relative positions of thta two sites not been detenined.

1112

in has

Vol.

134,

No. 3, 1986

BIOCHEMICAL

The HpaII

gene.

site

site)

is not affected.

(ii)

A general

placental

observed Itmust in their

This

be noted

study

(AvaI

thylation

puristic

states

and HhaI)

only

point

methylation (8)

over

were

task

presently.

is still

found

of all

the

has also

caution

of

sites

been

a variation

the Tg gene in

examined

in these in

in this

tissues.

the methylation/deme-

in the

"methylable"

(9).

showed

the 5'end

investigating

gene

which

(CCGG) sites

interpretation

positions.

of Fran

the relationship

of the Tg gene would

200 kb-long

in

know11 to express

to be methylated

a subset

6 kb from cap

unknown.

CG-containing

great

COMMUNICATIONS

is observed

phenonenon,

of a specificity

however,

(about

is not

when conparing

and imposes

of view

this

of this

RESEARCH

sites

tissue

The other

and expression

cing

HpaII

the HpaII

the existence

process

involving

(Z), only

DNA.

emphasizes

data

that

methylation and thyroid

This

genes

BIOPHYSICAL

upstream

of all

The meaning

other

liver

further

extra-embryonic

gene. with

is

demethylation

DNA.

thyroglobulin

which

AND

This

require is clearly

such a

between

genanic

sequen-

an unrealistic

ACKNOWLED@tk:NTS The continuous support and interest of Dr J.E. Dumont is greatly acknowledged. The authors are grateful to Huguette Brocas for the preparation of salivary gland and placental DNA and to Mrs D. Leemans for the preparation of the manuscript. This study was supported by grants frun the Ministkre de la Politique Scientifique (Action Concert&e), frun NIH (n" AM 21732) and FRSM, and from the asbl hsociation Recherche Bianddicale et Diagnostic. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9.

RAZIN, A. and SZYF, M. (1984) Biochim. Biophys. Acta 782, 331-342. SANFORD, J.P., CHAPMAN, V.M. and ROSSANT, J. (1983) Trends in Genetics 1, 89-93. TARGOVNIK, HM., POHL, V., CHRISTOPHE, D., CABRER, B., BROCAS, H. and VASSART, G. (1984) Eur. J. Biochem. 141, 271-277. CHRISTOPHE, D., CABREK, B., BACOLLA, A., TARGOVNIK, H., POHL, V. and VASSART, G. (1985) Nucl. Acid. Res. 13, 5127-5144. GOOSSENS, M., DUMEZ, Y., KAPLAN, L., LUPKER, M., CHABRET, C., HENRION, R. and ROSA, J. (1983) New Engl. J.Med. 309, 831-833. MANIATIS, T., FRITSCH, E.F. and SAMBROOK, J. Molecular Cloning, a laboratory manual (1982) Cold Spring Harbor Laboratory. EHRLICH, M. and MNG, R. (1981) Science 212, 1350-1357. CHURCH, G.M. and GILBERT W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995. VAN OMMEN, G.-J-B., ARNBERG, A-C., BAAS, F., EROCAS, H., STERK, A., TEGELAERS, W.H.H., VASSART, G. and de VIJLDER, J.J.M. (1983) Nucl. Acid Res. 11, 2273-2285. 1113