Sequence of the EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region

G‘ene. 103 (IYYl)

169-176

c

Science

lYY1 Elsevier

GENE

Publishers

B.V. All rights reserved.

0378-l

i 19!91%03.50

16’)

05067

Sequence of the EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region (Genomic

DNA:

AP-I;

teratocarcinoma;

polyoma

enhancer;

intermediate

filament;

exon-intron)

Yoshitaka Tamai, Yoshihiro Takemoto *, Midori Matsumoto *, Takashi Morita, Aizo Matsushiro

and Masami Nozaki

of Microbial Genetics, Research Institute for Microbial Diseases, Osuka University>,3-l ( Yamada-oka, Suita, O.Paka 565 (Japan)

Department

Received by Y. Sakaki: 21 January 1991 Revised/Accepted: 8 April/15 April 1991 Received at publishers: 24 May 1991

SUMMARY

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5’ upstream, 3’ downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA.?) and AP-I elements. The 5’ regions upstream of the EtzdoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to bc hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylatcd in BALBiC 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.

INTRODUCTION

EndoA is a mouse type-11 cytokeratin that forms intermediate filaments together with type-1 cytokeratin, EndoB (Howe et al., 1986; Singer et al., 1986; Morita et al., 1988), in simple epithelium (Kemler et al., 1981a,b; Hashido et al., 1991). During mouse development, EndnA and EndoB gene expression is first detected at the four- to eight-cell stage, then both genes are expressed in the trophectoderm but not in the inner cell mass at the blastocyst stage (BrQlet et al.,

Corrr.~pon&wr Research

10: Dr. M. Nozaki,

Institute

for

Microbial

Department Diseases.

of Microbial Osaka

Genetics.

University,

3-1,

Yamada-oka, Suita, Osaka 565 (Japan) Tel. (81-6)X77-5121; Fax(81-6)876-2678. * Present ofPhysical (Japan) Faculty

addresses:

(Y.T.) Tsukuba

and Chemical

Research,

3-l-l

Meguroku,

Tokyo

I52 (Japan)

Abbreviations: pair(s): EndoA proteins

Life Science Center,

Tel. (81-29X)36-5266; (M.M.) of Life Science and Technology,

1980; Oshima et al., 1983; Duprey ct al., 1985; Nozaki et al., 1986; Chisholm and Houliston, 1987; Nozaki et al., 1988). Investigations into the mechanisms controlling EndoA and EndoB gene expression have led to an understanding of differentiation during early development. The structure of the EndoB gene has been previously analysed (Ichinose et al., 1988; Oshima et al., 1988). Moreover, one of the c®ulatory elements of the EndoB gent has been identified as an AP-1 -like motif in the first intron and is regulated by cTfis and cTjutT(Oshima ct al., 1990). In

Koyadai.Tsukuba.

The Institute Ibaraki

305

Department of Life Science, Tokyo Institute of Technology,

Tel. (81-33726-I

I I I.

and

respectively;

FY ceils;

AP-I.

activator

sulfate;

transcription

SSC,

protcu-1:

retinoic

endodermal

CK8; cytoskeletal

acid

and dibutyryl

or 1000 bp; nt, nucleotide(s);

PEA, polyoma

virus enhancer;

0.15 M NaCl.‘O.OIS M Na,

start point(s).

bp. base

CkX, gene encoding

EndoA and EndoB. genes encoding

F9RA,

kb, kilobase

oligodeoxyribonuclrotide; dodecyl

acid(s);

.4 and B, respectively;

EndoB.

treated

aa, amino

CAMP, cyclic AMP; CK, cytokeratin; and EndoB, mouse extra-embryonic

citrate

EndoA CAMPoligo,

SDS, sodium pH 7.h; 0p,

170 DNA is addition. the 5’ region of EndoB genomic methylated in non-epithelial cells. such as fibroblasts and myoblasts, in a nonpermissive transcriptional state. On the other hand, the EndoA gene has not been characterized and direct evidence of regulatory mechanisms for its expression has not been reported (BrOlet et al., 1985). In this study, we determined the sequence of the EndoA gene. Some possible regulatory elements and a CpG-rich region were identified from the sequence analyses. This CpG-rich highly methylated in fibroblast ceils.

RESULTS

region

was

AND DISCUSSION

(a) Isolation and nt sequence of the Endo,4 gene As described by Vasseur et al. (1985), EndoA is encoded by a gene containing seven introns in which a B-2 repetitive sequence and one pscudogene lacking any intron are comprised. We screened a genomic DNA library (Ichinose et al., 1988) with both 1.8-kb EndoA cDN.4 (Morita et al., 1988) and a synthetic oligo of B-2 repetitive sequence (Vasseur et al., 1985) and isolated positive clones. The sequence of the entire coding region of the EndoA gene along with 962 bp of the 5’ and 1778 bp of the 3’ flanking regions is shown in Fig. la. There is a TATAAA sequence 104 nt 5’ upstream from the putative start codon, ATG. A polyadenylylation signal AATAAA was found at the 3’ end of the gene. This site appeared 206 nt (nt 8442-8447) 3’ downstream from the translation stop codon. TGA (Fig. la). The exact polyadenylylation site for EndoA transcript was determined from a comparison of the cDNA and gene. It occurs at nt 8469 which is 22 nt 3’ downstream from the polyadenylylation signal. (h) Conservation of exon-intron pattern The coding sequence was divided into nine exons (Fig. lb), of which the nt sequence was precisely identified by alignment of the gene sequcncc with the E/ldoA cDNA sequence (Morita et al., 1988). Seven of eight introns within the ElzdoA gene were found at the region encoding sequcnccs predicted to form the coiled-coil rod domain; one is in domain IA, three in domain 1B and three in domain 2; the remaining intron was in the C-terminal domain (Fig. 2). The length of the cxons and introns is in agreement with the other type-11 Ck genes analysed previously except human Ck8, which lacks the fifth intron (Fig. 2). So far, the ErldoA gene has been reported to be composed of eight coding segments separated by seven intervening sequences (Vasseur ct al., 1985) using heteroduplex analysis between EtuloA gene a 1 and processed pseudogene x2. These inconsistent results are considered to be due to the deletion of scqucnces matched with some of the exons in pseudogene ~2. Each of the E~7hA spliccjunction scqucnccs conformed

to the expected consensus sequence, the donor AG/GT and acceptor AG/N sequences (Mount, 1982) except for the AA/GG donor site of exon 2 which is the same as that of exon 2 of the EndoB gene (Ichinose et al., 1988) and other type-II Ck genes (Johnson et al.. 1985; Tyner et al., 19X5: Glass and Fuchs, 1988; Krauss and Franke, 1990).

(c) Possible regulatory elements Homologous sequences of PEA motifs, which are crucial in the control of gene expression during differentiation of teratocarcinoma cells (Kryszke et al., 1987; Furukawa et al., 1990), were searched for possible regulatory sequences for the EndoA gene. As shown in Table I, several sequences identical with PEA1 and PEA3 elements were found 5’ upstream, 3’ downstream, and at the first and third introns. Of these sequences, six PEAS-motif repeats were clustered about 1 kb downstream from the EndoA gene (Fig. la). Recently, we identified that these repeats show enhancer activity in endodermal cells but not in F9 cells (Takemoto et al., 1991). The corresponding human gene (CkH) also contained PEAI and PEA3 at the 4th and 1st introns, respectively. EndoB gene expression is regulated by cTjutl and c+s expression through an AP-I -like sequence (EndoB AP-I ) in the first intron within the gene (Oshima et al., 1990). Since EndoA is also thought to be controlled by these oncogene products (Oshima et al., 1990), homologous sequences within the gene were examined. Three AP-I sequences were found in the EndoA gene, one of which is located in the first intron as in EndoB and human homologue Ck8. and the others in the first cxon and 3’ downstream (Table I).

(d) Comparison of the sequence of the EndoA gene with that of the human CkS gene and the EndoB gene The overall exon structure for EndoA is very similar to that of the human Ck8 gene. These genes are highly homologous at the nt and aa levels as described previously (Krauss and Franke, 1990). The sizes of cxons 2-8 of the EtldoA gene are identical with those of Ck8 (Table II), although the total size ofexons 5 and 6 is identical with exon 6 of Ck8, since the 5th intron is missing in the human Ck8 gene (Krauss and Franke, 1990). The modest difference in the size of the first and 8th exons, which code for the head and tail regions, reflects the variability of the size of noncoding sequences. Comparison of the flanking and intron sequences of the ErzdoA and the Ck8 genes revealed that sequences located 5’ upstream, surrounding the TATAAA box and 1st intron, were homologous (Fig. 3). For insight into the function of these homologous sequences, we searched genes that share nt sequence homology in computer databases. A 20-bp scquence, 5’-GGGCTGGCCTCTTCTTTTAT. upstream

subcloned

librar)

mto the vector pBluescript

(Stratagene)

and restriction

mapped.

Smaller fragments

for

which were

The EudoA genomic

b)

as described uas screened

into small fragments

to general methods.

(1.6 x 10h phage clones)

DNA library was constructed

by either EcuRI or Ba/nHI

et al.. 1988) according

13 kb, was digested

EudoA cDNA (Morita

“‘P-labeled

clone of approx.

et al., 1988). This genomic

(Ichinose

regions. A mouse genomic

previously

gene and 5’. and 3’-flanking

scqucnce

were subcloned

arc cxons.

EwRI

in the DDBJ. E, EcoRI;

I) is according

1; Takemoto

_I

1 he sequence

SR$P

signal are

to Vasseur et al. (1985). Start

Sequence

DataP, f.~fI; S. SocI.

and genomic maps ofthe EndoA gene. Open boxes

Nucleotide

et al., 199 1) is boldly underlined.

E

of

was by bent arrows

et al., 1977). Numbering

vector.

S E

TATA box and polyadenylylation EMBL and GenBank

(nt 9450-958

The putative

No. D90360. (b) Restriction B, BcrnzHI: D, DraI;

(Sanger

or IY phage

PP

site shown in (b). The exons are marked

chain-termination

method

DBB

start point ofexon sequence data will appear

enhancer bank under accession

The nt scqucncc

boxed. The Et&A

PESP

into the hl 13mpl8

are doubly underlined.

The r.7~(underlined,

and numbered. and stop codons

starts at the uppermost

by the dideoxynucleotide

analysis the scqucnce

determined

B

172 The expression of the EndoA and the E/r&B gents regulated coordinately during development. so that we tried to identify homology between these genes. We could not find novel homologous sequences except for two local

Human

homologies around the TATAAA box as described previously (Ichinose et al., 1988). These concentrated homologous sequences also located within a large homologous

Ck6

Fig. 2. Schematic diagrams CK-encoding Ck7 (Glass

showing the location of intl-ow

genes: CXI (Johnson et al.. 19X5), CM (Tyncr and Fuchs,

1988) and CkX (Krauss

111type-11

ct al., 19X5),

and Frankc,

1YYO). The

r-helical domains (coils 1.4, IB and 2) are shown by open boxes; head. tall and linker

regions by single lines. Upward

arrowhead\ mark the

locations of introns.

from the TATAAA box has been identified with that located in the intron Lvithin the human renin gene (Morris. 198h). A Wbp homologous stretch that is identical in the first intron of the _!Z’&L~and CL3 genes is embedded \+,ithin ill1r elements. Although repetitive scquenccs of the .4hr type have been found in the 5’ upstream region as well as in introns of some CK-encoding genes (Vnsseur et al., 1985; Ichinosc et al., 198X; Bader et al.. 1988: Kieger and Frankc. 198X), their functional

roles arc not clear.

Motif,’

Sequence”

PEA I (’

i-TGAC’TAA

PosItions

156X

region between EnhA and Ck8 (Fig. 3). Since upstream and downstream elements require efficient TATA-box binding of TFIID (Horikoshi et al., 1988a,b; Nakatani et al., 1990), endoderm and epithelial cell-specific factors may bind these homologous sequences surrounding the TATAA box in these genes and involve in efficient transcription. (e) Identification and methylation state of CpG cluster region of the EndoA gene The sequence of the E/U&~ gene shows that a region around the first exon contains a high frequency of C’pG dinucleotide (Fig. a). To investigate the methylation state cjt the CpC-rich region of the E&L~ gene in several ccl1 types. Southern analysis, utilizing the methylation-sensitive rcstriction enzyme HprrIl and its Incthyl3tion-insensiti\,c isoschizomer Msl)I, was performed. After digestion with

(nt)’

(1st intron)

5559 (4th mtron)

complement Y765 (3’ downstream) 5 -AGGAAG’l

PEA3’

Yqh5 9487 Y50Y Y53l

(3

199 I (1st mtron)

Y553 9575 downstream)

complement 73h (5’ upstream) 2382

5414 (4th WM)

(1st intron)

5246 (3rd introll) 5732 (4th exon) 5’-TGAGTCA

(1st intron)

2073 (1st intron)

X77X (3’ downstream)

1x45

5632 (4th intron)

c<>mplemrnt It157 (1st exan) (’ I’EA

motifs

in polyoma virus enhancer and .AP-I motif in OrcloB gene.

” Sequence of these motifs.

’

Left ends nt positions

” Gutmnn

and Wnsylyk

of these motifs (lY90).

’ Was\,lyk et al. (1990).

’

Oshimu

2 Krauas

et al. (1990). and Franke

(IYYO).

” Ichinose et al. (IYXX).

(in relation to nt = -+ I in Fig. la).

6X04 (6th mtron)

31 I-l

(151mtron)

173 TABLE

II

Comparison

of exon and intron sizes between

cellulose spanning

mouse OrdoA and human

Ck8

bes A-D). Using the 2.6-kb EcoRI fragment containing a CpG cluster as probe A, largely HpaII-resistant fragments were detected in 3T3 cell DNA. On the other hand. these sites - except a small percentage - were HprrIIsensitive in PYS-2, F9 and F9RA cells (Fig. 5A). Probes B. C and D hybridized to fragments that were highly resistant to the enzyme HpaII in all cells. Since the control M.s/)I cleaved HpaII sites smaller fragments were detected (Fig. 5,

_ Exon

EndoA

Ck8

Intron

EndoA

Ck8

NO.

size (bp)

size (bp)

No.

size (bp)

size (bp) 2535

I

421

407

2184

2 3

209 61

209 61

776

628

1046

414

4 5

96 165

96 -

487

518 -

6

126

291

221

221

316 135

864

7

519

829

8

59

59

9

427

436

257

B-D). These data indicate that the CpG cluster region is highly methylated in 3T3 cells but not in the other cell types and the other areas were almost all methylated in all cells examined. The EndoA gene is first expressed in trophectodcrmal cells during mouse development. In the adult, its expression is restricted to simple and transitional epithelial cells (Kemler et al., 198 la,b; Oshima et al., 1983; Hashido et al., 1991). In this study, the CpG-rich 5’ region of the EndoA gene was highly methylatcd in non-epithelial 3T3 cells which do not express the EndoA gene, but nonmethylated

158

_ Intron

5 of the human

Ck8 gene is deleted

(Krauss

and Franke,

1990).

EcoRI, fragments from F9 and 3T3, which do not express EndoA, as well as from PYS-2 and F9RA, which do express EndoA, were treated with either HpuII or MspI, clectrophoresed through agarose gel, and transferred onto nitro-

(a) 5' upstream

EndoA Ck8

EndoB

EndoA Ck8

EndoB

EndoA Ck8

(b)

Ck8

1st EndoA Ck8

Fig. 3. A comparison

around

the TATA

box

-80 -140 -130 -120 -110 -100 -90 TCTCTGCTGCCCTCACCTGAGTCCCGTCTGGACGTTGCAGACGTTGGGTT-AGGCCCTGCCCTCTAGTGTCTAGC :::::::::::: :::::::::: : : ::::: :::: : ::::: : :: ::: ::: : : ::::: TCTCTGCTGCCCCCACCTGAGTCT-GCCCCGAGGTGGCAGGTGACGGGTTCACGCGACGCC-TCT-G-GCCTAGC -60 CTGGGGCGTGGCCT ::::::: :::::: -10 -70 -60 -50 -40 -30 -20 CACTCAGGTAAGAGGCCCTTTTCCCTC-CCCTGGGGGC-TGGCCTCTTCTTT WGGGCC-TTCGCTGGGCGC :::::::::: :::::: :: ::: : ::: ::::: ::: ::: :::::::::: :::: ::: ::: : :: CACTCAGGTACGAGGCCTTTCCCCCACTCCCCGGGGC-TGGGATCT-CTTTTATAAAAGGCCATTC-CTGAGAGC t40 +50 GCTTCGCTCTCCTCTC :::: :::: : :::: 1 +10 +20 +50 t30 +40 TGTCC-CACCGTCTAGAAGCAGCTGCTTAGCTCSC-TCTCTCG~CCTCCGTCT ::::: :::: :::::::::: :: :::: ::: : : ::::: :: TCTCCTCACC---AAGAAGCAGCTTCTCCGCTC~T-TCTAGGATCTCCGCCT

distal

EndoA

region

filters, which were hybridized with four probes the 5’- to 3’-flanking regions (Fig. 5, pro-

5' upstream

region

120 130 TTCATTCATTCCTCAAATATCTACC :::::::::::: :::::::::::: TTCATTCATTCCACAAATATCTACC 430 440

intron 2250 2260 2270 2280 2290 GCTGACTGGCTCCTGAGAGGCGGTGTGGGAACG(;GAGGAGGGACACGGGGCTGAG ::::::::::::::: ::::: :::::::::::::::::::::: : ::::: ::: GCTGACTGGCTCCTGGGAGGCATTGTGGGAACG(;GAGGAGGG~TCCTGGGGCAGAG 2520 2570 2530 2540 2550 2560

of nt sequences

located

5’.upstream

and at the 1st intron

of the EndoA, EndoB and human

Ck8 genes.

(a) 196-m sequences

arc

aligned for the EndoA and Ck8 genes and numbered starting at the putative fs~~(nt position 1). TATAAA boxes are underlined. The upper sequences are homologous elements of the EndcoB gene. (b) The upper sequence is EndoA and the lower is Ck8. Numbering of the Ck8 sequence is that of Krauss and Frankc

(1990). Colons

indicate

identical

nt.

173

Fig. 4.

CpG or GpC

dinuclcotide posItiona in the Et~loA gent. lxch vertuxl

along the gent structure

refer to the nt positions.

line represents

around the lirst exon and undermeth~lation of thcsc regions is correlated with expression. These data indicate that differential methylation is one of the general mechanisms for transcriptional repression of tissue-specific genes in nonexpressing somatic cells (Cedar, 1988). Consistent with the possible importance of methylation in regulation of the

in other cells. Although most housekeeping genes have CpG islands in their 5’ regions, dc novo methylation was not seen at the CpG islands. However, such CpG cluster region contains many potential targets for DNA methylation (Bird. 1986; 1987). EudoA (in this study) and i+doB (Oshima ct al.. 1988) genes contain a CpG-rich region

[

a CpG or GpC dinucleotlde on the gene sequence. I’he numbcrb

Open boxes are axons.

prcbcA

12345678

12345678

12345678

12345678 kt,

kb

kb

kb

2.62 l-

C

A Fig. 5. Mcthylation

states ofthe Ort/oA gene. DNA

cells (Inne!, 7 and 8) were digested with EwRI

isolated from PYS-2

D

cells (lanes I and Z), 3T3 cells (lanes 3 and 4), F9 cc& (lanes 5 and 6). or t9RA

+ Nl~rrll (lanes 1,3, 5 and 7) or A4spI (laws fragments acre transferred to nitrocelluloae

2, 4, 6 and 8). The digested DNAs ( 10 pg per Imc) were loaded filters (Nitroplus 2000. M.S.1) as described by Southern ( 1975 ). onto I.()“,, agarose gels. After clectrophoresis, Hybridiratlon was performed with labeled probe A, B. C or D (panels A, B, C, and D, respectively) and the filters were washed for I h in 0.1 x SSC/O.S”,, SDS at 68°C. The filters were then cxposcd to x-ray film (AR-5 Kodak) at -7O’C with an intensifier screen. Size markers arc indicated on the lcli (111 kb). B, RwIHI; L, EcoRI (downward arroahcads). Short \crtlcal lines under the map rrfcr to Il~~rll,‘MsyI restriction situ.

175 EndoA expression tocarcinoma potent

is the observation

derived

inhibitor

that treatment

of tera-

fibroblast

cells with 5-azacytidine,

a

of methylation,

results in the induction

of

Endo. expression (Darmon, 1985; Semat et al., 1986). Recently, however, methylation at CpG islands ofgenes has been found to be widespread in cultured cell lines such as 3T3 or L cells (Antequera et al., 1990). The islands affected were those present at genes whose expression is probably not required CpG islands

in culture. Methylation was not seen at the of housekeeping genes. In their study, Ante-

quera et al. (1990) found that some CpG islands which are methylated in cultured cell lines are nonmethylated in some tissues in adult mouse. Accordingly, the possibility that the CpG-rich region of the EndoA gene is methylated in nonepithelial tissues must be further examined.

adult tissues Horikoshi,

mammalian Horikoshi,

TFIID-promoter factor

establishment keratins

methylation

J. and

and altered

Bird,

A.P.:

chromatin

High

structure

levels

of de nova

at CpG islands

in cell

B.L., John,

L. and Franke,

8. 18 and

umbilical

cord

W.W.: Low level expression

19 in vascular

and

in cultured

analysis ofthe chromosomal

smooth

muscle

cells derived

locus containing

of cyto-

cells of human

therefrom,

with

the cytokeratin

an

19 gene.

A.P.: CpG-rich

islands

and the function

A.P.: CpG Genct.

islands

sequence

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nucleus.

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M., Kaghad,

M.. Morello.

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assembly

in the

101 (1987) 565-582.

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M., Babinet,

Brulet,

P. and Jacob,

early mouse embryogenesis.

C.. Condamine,

H.,

of the cytokeratin

endoA gene

Proc. Natl. Acad.

Sci. USA 82

F.: Expression

A.: Nucleotide

endoB gene. Gene

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67.kDa

P.M.:

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M.-T., Gaillard,

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Y., Ogawa, repressor

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reveals polypeptide

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Gene 68 (1988) 109-l 17.

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of the human

renin and inactive

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T., Besnard, initiation

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