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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
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS Pages
15, 1989
1446-1452
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.
1446
MEM,
salne; UV, ultra-
Vol. 162, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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
Vol. 162, No. 3, 1969
RNA w
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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
Vol.
162,
No.
3, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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
BIOCHEMICAL
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.
REFERENCES
1. Doerfler, W. (1983) Ann. Rev. Bbchem. 52,93-124 2. Bird, A., Taggart, M., Ftommer, M., Miller, 0. J. and Mackod, D. (1965) Cell 4891-99. 3. Keshet, I., Yisraeli, J. and Cedar, H. (1985) Pmt. Netl. Acad. Scf. USA 82, 2560-2564 4. Bird, A. P. (1986) Nature. 321,209-213 5. Chandler, L. A., De&r& Y. A., Bogenmann,
E. and Jones, P. A. (1988) Cancer Res. 46,2944-2949
6. Parker, M. I. and Gevers, W. (1984) Bbchem. Bbphys. Res. Comm. 124, 236-243 7. McKeon, C., Ohkuho, H. and Pastan, I., ds Crombrugghe. B. (1962) Cell, 29, 203-210 8. McKeon, C. and Pastan, I. de Cromtwugghe, B. (1964) Nucleic A&s 9. Smith, B. and Marsffb, E. (1968) Bbchem. J. 253,269-273
Res.. 12, 34913502
10. Auhln, J. E., Heersche, J. N. M., Merrflees, M. J. and Sodek, J. (1962) J. Cell. Bbl. 92,452-461 11. Bellows, C. G.. Sodek, J., Yao, K. -. L. and AuMn, J. E. (1986) J. Cell Bbchem. 31,153-169 12. Kunkel, L. M., Smith, K. D., Bayer. S. H., Borgaonkar, D. S., Wachtel, S. S., Miller, 0. J., Breg, W. 13. 14. 15. 16. 17.
R.,Jones, H. W. and Rary, J. M. (1977) Pmt. Natf. Acad. Scf. 74,1245-1249 Mohun. T. J., Bmnnan, S., Dathan, N., Fairman, S. and G&on, J. B. (1964) Nature 311.716721 Thomas, P. S. (1960) Proc. Natl. Acad. Sci. USA 77,5201-5205 Southern, E. M. (1975) J. Mol. Bill. 98,503-517 Genovese. C.. Rowe, D. and Kream. B. (1964) Bfcchemfstry 23.62106216 \ Feinherg, A. and Vogelstein, B. (1982) Anal. Bbchem. 132.613
18. Siegel, L. I. and Bresnbk, E. (1966) Anal. Bbchem 159.82-87 19. Parker, M. I., Judge, K. and Gevers, W. (1982) Nucleic Acfds Res. 10, 58796697
1452
Vol. 162, No. 3, 1989 August
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS Pages
15, 1989
1446-1452
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.
1446
MEM,
salne; UV, ultra-
Vol. 162, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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
Vol. 162, No. 3, 1969
RNA w
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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
Vol.
162,
No.
3, 1989
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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
BIOCHEMICAL
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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