Loss of alpha I type I collagen gene expression in rat clonal bone cell lines is accompanied by DNA methylation

BIOCHEMICAL

Vol. 162, No. 3, 1989 August

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Loss of alpha I type I collagen gene ex ression in rat clonal bone cell lines is accompanied by ENA methylation Mary M.Y. Waye*, Ranga Robinson +, Anthony G. Orfanides and Jane E. Aubin Medical Research Council Group in Periodontal Physiology and + Department of Biochemistry University of Toronto Toronto, Ontario Canada Received

June

9, 1989

Four ctonal cell lines subcbned from a cbnal population of fetal rat calvaria cells show a loss of type I collagen synthesis. Northern blot analysis showed that the level of al (I) collagen mRNA expression in each of the clonal populations parallels the level of collagen protein expression in each of these cell lines. The methylation pattern of the collagen gene in these clonal cell lines was determined using the restriction endonucleases &@I and &I& It was found that the loss in collagen type I expression correlated positively with the degree of methylation of at (I) procollagen genes, indicating that methylation of CpG may be an important mechanism of collagen 0 1989 Academic Press, Inc. gene regulation.

DNA methylatbn has been implicated in regulatbn of gane expressbn in eukaptas

by,

for example,affectingDNA-protein interactions(for review, see ref. 1). Between 60?4, to 90% of the dinucleotide CpG’s are msthyktedat the 5th pasitbn of the cylosineringandanalysisof the frequency of CpG has lad to the suggestion that there ars clusters of non-methylated CpG in

eukyotes (l&a II Ilny Eragments, HTFsequences) [2]. Thereis evidencethat transcriptiinof genes with HTF islands k inhibited whenthe island k rnathyktsd p]. The chiian

a2(1) coflagen

gene k one of the genes that has HTF-likssequences [4], but DNA methylatbn as a mechanism of

*To whom correspondence Abbreviations

should

be addressed.

used:

Fl3S, fetal bovine saturn; HTF, J&all Tii

Fragmsnts; Kb, kilobase or 1000 basepairs;

minirmm assent&l medium; NaOAC, sodium acetate; PBS. phosphate-buffered violet. 0006-291x/89 Copyright All rights

$1.50

8 1989 by Academic Press, Inc. of reproduction in any form reserved.

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MEM,

salne; UV, ultra-

Vol. 162, No. 3, 1989

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regulation of the collagen gene is a contmversfat fssue. For exarnpte, the cq(l) collagen gene has heen reported to have unusual methytatbn patterns [5,6] and McKeon et at. [7,6] have shown that the DNA around the start site of transcrtptbn is not methylated whether or not the cells synthesize collagen. More recently, however, DNA methylatiin of the a2(1) promoter5

region was shown to

correlate with collagen production in rat liver epithetial cells [9]. Cbnal hone cell lines obtained from fetal rat cafvarfa have heen shown to he heterogeneous

in their synthesis of extracelblar

matrix components, including colagen [9]. Thus,

we were interested in examining the control of the type I collagen gene in several of the clonal cell lines which have heen characterized extensively in their collagen synthesis [l 11. Whereas both RCJ 3.2 and its suhcbne

RCJ 3.2.4 synthesize type I collagen, in two second step s&clones

(RCJ

3.2.4.1 and 3.2.4.4) derived from RCJ 3.2.4, collagen type I synthesis is not detectable. Since the loss in type I collagen synthesis was acccrrpanied

by other phenotypic changes (including

alterations in expression of types Ill, IV and V collagen, and morphological changes), there were SeWal

possible mechanisms that could have led to the bss in collagen synthesis. The studies

reported here indbate that the loss in collagen gene expression is due to a loss in collagen RNA expression and DNA methylatbn may play a role in the regulation

ANDRCJ 3.2, RCJ 3.2.4, RCJ 3.2.4.1 and RCJ 3.2.4.4 were maintained in a mintmal essential medium (a MEM) containing 15% v/v fetal hovtne serum (FBS) and antihbtbs as descrthed In ref. 11. For fsofatiin of DNA or RNA, cells were pfated Into 4xT-150 flasks at a density of 12x1 O4 cells/cm2 and grown in the same mediim. For the studies reported here, cells were recovered from frozen stocks and were suhcuftured for only about 3-4 weeks after each thawing to avoid any changes in phenotype. DNAFINA m The ce44swere washed w4th icedci phosphate buffered satins (PSS) and then trypsbtzed (0.01% in citrate saline). The trypstn was neutralized by a MEM containing 10% FBS. DNA was prepared as described in ref. 12. RNA was prepared as in ref. 13 except for the folbwing rnodiibatbns: monolayer cultures of 8x107 cells were washed with PBS, trypeinized and pelleted. 1 ml of 3M LCI-GM urea was added and the cells were passed through a 18.5 gauge needle ten times, and precipitated overnight on be. The RNA was centrifuged in a microfuge at 4’C for 40 min and resuspended in a buffer containtng 0.1 M sodium acetate (NaOAC) pH 5.5, 0.2% SDS and 1000 U/ml hepartn. The RNA was extracted twice with phenolchloroform and once with chloroform. The aqueous phase was adjusted to 0.3M NaOAc (pH 5.5) and precipitated with 2.5 vol of absolute ethanol overnight. 1447

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RNA w

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RNA was denatured and transferred to Biiyne

0.2 micron

membrane filters (Pall Ltd.) essentially as described by Thomas [14] , except that the RNA was cross-linked by ultra-violet (UV) light, by placing the damp filter with transferred RNA 8 inches away from a germicidal lamp and exposing to UV light for 20 minutes. DNA was digested by restriction endonucleases (BRL, Bethesda or New England Biilabs) according to the manufacturer’s specifications. Digested DNA was fractionated on a 1% or 1.5% agarose gel, transferred to Biodyne filters as described by Southern [14,15] and UV-cross-linked on the filters as described above. m The rat collagen probe al R2 1161was kindly provided by Dr. David Rowe. al R2 contains the cDNA which codes for the entire 3’ non-coding region and one-half of the Cterminal of the propeptide of the al chain of collagen type I. The probes used for Southern blot or Northern bott analysis were made by the random primer method [17]. Filters were prehybridized with diethylpyrocarbonate (DEPC)-treated Bbtto in 50% formamide with or without 100 mg/ml single-stranded, sheared salmon testis DNA for Northern blots [18] or 50% formamide with 5xSSC (750 mM NaCl and 75 mM sodium citrate), 10x Denhardt’s solution (0.2% each of Fiill, polyvinyl pyrrolidone and BSA), 0.2% SDS, and 100 rngIml single-stranded, sheared salmon testis DNA for Southern blots. The fitters were hybridized wlth the same solution containing the DNA probe for 24 hr at 42’C. The filters were washed at 50°C three times for 30 min each, once in 2xSSC with 0.1% SDS and twice in 0.1x SSD with 0.1% SDS. The fitters were then autoradiiraphed. For analysis of DNA digestion by f&g1 and &all, cellular DNA. Electrophoresis,

blotting and hybriiizations

0.02 pg of X DNA was mixed with 20 ug of were done as above. After

autoradiography, the collagen probe was removed from the filters by heating the membrane filter at 65’C in 10 mM Na Phosphate pH 6.5 with 50% fomtamide. The filters were washed once in 250 ml of 2xSSC with 0.1% SDS per 100 cm2 of membrane for 15 min at room temperature with vigorous agitation. Fitters were prehybridlled and then rehybridized with 32P X DNA.

RESULTS We have shown previously that RCJ 3.2 and its subclone RCJ 3.2.4 synthesized predominantly type I collagen with appreciable amounts of type Ill collagen (5% and 22% respectively) and small amounts of type V collagen. In contrast, second step subclone RCJ 3.2.4.1 synthesized prima@ type Ill collagen with small amounts of type V collagen and subcfone RCJ 3.2.4.4 did not synthesize any interstitial coftagen but synthesized type V collagen 1111. NoWem

Blot Anafvats of Coflaaen r&f) RNA In Rat Bone Cell Lines To quantitate ths amount of

collagen at (I) RNA in the rat bone cell lines, denatured total RNA was run in a 1% gel, transferred to nitrocellubse

and hybridized with a radiolabelled rat collagen cDNA clone-al R2 [16] (figure 1).

One prominent band of molecular weight 4.3 Kb was seen in RCJ 3.2 and RCJ 3.2.4 RNA, but no 1448

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MSPI

HP8

-+

-+

11

1234

.

02

VJ Figure 1

A. Northern

hybrfdiiation

were denatured transfened collagen

3

‘2

analysis of type I collagen

by glyoxal.

to Biine

fractionated

membrane

4

RNAs. Total cellular

by electrophoresis

RNAs (20 pg)

on a 1% agarose

filters. RNAs were hybridized

cDNA. Clones RCJ 3.2 (lane 1) and RCJ 3.2.4 (lane 2) hybridize

mRNA, whereas

gel, and

with al R2, the al (I) to a 5.5 Kb

RCJ 3.2.4.1 (lane 3) and RCJ 3.2.4.4 (lane 4) show no collagen

mRNA. Figure

2

Southern

hybridization

digested

with fykgl (lanes 1 and 2) or bll

electrophoresis hybridiied

analysis of type I collagen

on a 1.5% agarose

with alR2.

similar manner,

indicates

band

at the same

whereas

&all

approxlmateiy collagen

Bbtto

weight

digested

pfus kImon

testis

typa

I colfagen

(RCJ

collagen

was much

smaller

32.4.1

and 3.2.4.4)

I (RCJ wera

RNA.

metbyfatbn

3.2 and RCJ 3.2.4) anafyzeci

1449

then

I-”

A minor

(data

of

from the same

not &own).

in the rat bone calf Unes,

and two cbnes

and the DNAs

band

band was not

and it was abaenf

DNA was used in the hybridiiation

type

filters and

is synthesized;

and RCJ 3.2.4.4

To study synthesizing

membrane

(lane 1) and RCJ 3.2 (lane 2) DNA in a

In all 4 call Ilnes. This minor

-11 two dories

to Biiyne

by

is not synthesized.

in RCJ 3.2.4.1

weighf

DNAs (2Opg) were

RCJ 3.2.4.4 (lane 3) much less extensively

type I collagen

was seen

since its mgfearfar

RCJ 3.2.4.4

cell lines in which type I collagen

was detectable

1.5 Kb molea~far

al (I) mRNA.

blots when

cell lines in whit

positions

gel, transferred

Mspl digested

RCJ 3.2 (lane 4). “+” indiites

DNA. Total cellular

(lanes 3 and 4). fractionated

were

not synthesizing

digested

with -11

BIOCHEMICAL

Vol. 162, No. 3, 1969

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Mspl. both of whfch mcognke 5 CCGG. Whenever the internal C-&due mcdlfied to 5’C%GG-3’

of the sequence is

by methytatbn, the endonuctease J&all cannot cut whereas Mspl does

art at such a site. In the &gl

digest, one band of 1 Kb was observed in both RCJ 3.2 and RCJ

3,2,4,4 (ffgure2). However, the RCJ 3.2.4.4 DNA digested with JjgM had several higher molecular weight bands (of more than 3 Kb) compamd to RCJ 3.2 DNA. Likewise, one band of 1 Kb was observed in both RCJ 3.2.4 and RCJ 3.2.4.1 in the &@I digest. However, RCJ 3.2.4 diisted &all

with

showed a smaller molecular weight band (of less than 2 Kb), compared to RCJ 3.2.4.1 (see

Figure 3, lanes 1 to 4). To rule out the possibilii

that the difference in digestion pattern was due to

incomplete digestion of DNA, we mixed 0.02mg of lambda (k) DNA with the total cellular DNA and analyzed the DNA with the collagen and ths X probe consecutively after Mspl or J&all digestion and blotting. After analysis with the collagen probe and autoradiiraphy, removed, the membrane was prehybriiied,

the collagen probe was

and then rehybrtdiied wlth 32P 1 DNA . The 1 DNA

mixed with cellular DNA was digested to compietion (data not shown), thus we conclude that the

1

2

3

6.7’ 4.4.

Figure 3

Southern digested

. . . .

hybridiiation with m

4

56 i .>

70

.,

analysis of type I collagen

DNA. Total cellular

DNAs (3opg) were

(lanes 1 and 2) or L@all (lanes 3 and 4), fractiotWed

by

fitters and fvtxfdked wttf! alA2. Mspl digested the RCJ 32.4 (lute 1) and RCJ 3.2.4.1 (Lane 2) coltaoen al(l) gene In a sknilar manner. In contrast. l&3 II dIgested RCJ 3.2.4 (lane 3) iessextemJvelytfianRCJRCJ3.2.4.1 (tane4).A6aamtd.~(tanes5andG)and &&RI (lanes 7 and 6) were Shown to di@%sIthe RCJ 3.2.4 (lanes 6 and 6) and RCJ 3.2.4.1 (tams 5 and 7) collagen al (I) gene ln a sbnilar manner. electrophoresis

on a 1.5% agarose

gel, transferred

1450

to Bibdyne

membrane

Vol. 162, No. 3, 1969

difference in restrbtiin

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pattern of the WI1 and j&l

digest was due to rnethylatbn of the DNA and

not due to a difference in the degree of digestion. To show that there is no major rearrangement

of

the al(I) collagen gene, the DNAs of two clones (RCJ 3.24 and RCJ 3.2.4..1) were chosen for further analysis with 9 different restriction endonucleases. two clones with ml,

Bl,

hll,

ml,

ml.

Mhpl, &$3A

Southern blot analysts of DNAs of the I, ljigdlll and &RI

showed no

detectable difference between the collagen at(l) gene of the two rat cbnal cell lines. Only one representative Southern blot showing the Mbpl and -3&U

digest ls shown (Figure 3, lanes 5 to

8).

DISCUSSION

Our previous studies have shown marked hetemgene.ky in collagen synthesis in clones and suhdones of fetal rat calvarta osteohtast-ttke cells [lO,ll].

In particular, two cell lines (RCJ 3.2

and its subclone RCJ 3.2.4) synthesize type I collagen. whereas two second step suhcbnes (RCJ 3.2.4.1 and RCJ 3.2.4.4, derived from RCJ 3.2.4 do not synthesiie

collagen type I [ll].

In this

study, we have shown that collagen type I mRNA is expressed in RCJ 3.2 and RCJ 3.2.4, hut not in RCJ 3.2.4.1 and RCJ 3.2.4.4 and that the bss of type 1 collagen expression in RCJ 3.2.4.1 and RCJ 3.2.4.4 rntght he explained by the hypemtethytatbn

of the collagen at(l) gene. Thtt result

contrasts with observations in chbken ernhryo fbrohlasts [8], human forskin fihrobtasts and human tumor cell lines [5] in which the level of expression of the a 2(l) collagen gene was reported to he independent methylatbn

of methylat’bn. However, the presence of HTF islands rich in CpG suggests that could be a method of regulation of the collagen gene [4]. In support of the latter

pcssbillly and consistent with our results, Parker 6tal(19] collagen synthesis in SV40-transformed

have reported that decreased type I

human fibroblasts was accompanied by hypermethylatbn

of type I collagen genes. Furthermore, Smith and Marsib [9] have shown that DNA methylat.bn of the a2(l) promoter-S

region could contribute to the altered collagen production in chemically

transformed rat liver epithelial cells [9]. Our results extend their findings regarding DNA methylatbn of the promoter-6

region, by shoWrg that methylatbn of the 3’ non-coding region of the collagen

at (I) gene also regulates cotlagen expressian at least in these cbnal rat calvarb cell lines. The observed difference in digestion pattern is unlikely to he the result of gross chromosomal

1451

Vol. 162, No. 3, 1989

rearrangement

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as we have shown that 9 different restriction endonucleases

cut the collagen al(I)

gene of the two cbnal cell lines in a similar manner. In concluskn, we have demonstrated that In these clonal osteoblast-like cell lines derived from fetal rat calvaria, loss of collagen synthesii ls regulated at the transcrtptbnal

level and that

methylation of the al (I) procollagen gene can contribute to the loss of type I collagen gene expression. These cell lines am thus unique in that their complete blockage of collagen at(l) synthesis is probably due to DNA methyfation.

B

This study was supported hy a group grant from the Medical Research Council

of Canada. We thank Dr. Jam Sodek for advice, Ms. Rebecca Ber, Ms. Eda Lui and Ms. Susan Eng for technical help, Dr. D. Rowe for providing the type I collagen prohe and Ms. Mbhelina Viinti and Mrs. Elba Kdssflas for preparing the manuscrtpt.

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