DNAVolume I, Number 3, 1982Mary Ann Lieben, Inc., Publishers

Methylation and a Polymorphic Restriction Site Adjacentto Human ß-Interferon Gene

CHARLES H. RIGGIN, JR., and PAULA M. PITHA

ABSTRACT

High-molecular-weight DNA from cultured human fibroblast cells and tissues was analyzed with the restric-tion endonucleases Msp I and Hpa II for the presence of methylated sites in the sequences flanking the/3-interferon gene. The majority of the DNAs analyzed were methylated in the restriction site (CCGG) forthese two enzymes and thus were sensitive to cleavage by Msp I, but resistant to Hpa II. A polymorphicMsp I restriction site was identified approximately 1000 bp upstream from the /3-interferon gene. The resultsshow an association of the /3-interferon gene with Msp I fragments of either 2.7 kb or 4.2 kb, which are in-herited as Mendelian alíeles. An unusual Msp I site upstream from the /3-interferon gene was present in 22%of the DNA from peripheral leukocytes of healthy individuals, and in 36% of the DNA from leukocytes of in-dividuals with different forms of leukemia. Induction of /3-interferon with poly(rI • rC) did not alter themethylation pattern in the sequences flanking the /3-interferon gene, and the levels of /3-interferon induced incells by poly(rI • rC) could not be directly related to the presence or absence of a polymorphic Msp I restric-tion site in the 5-flanking region.

INTRODUCTION

Human a -and /3-interferons are inducible cellular gly-coproteins. Exposure of sensitive cells to interferon

confers resistance to viral infection and induces alterationsin a number of cellular functions. Genes for human a- and/3-interferons were recently cloned and shown to be locatedon human chromosome 9 (Meager et al., 1979; Owerbach etal., 1981; Pitha et al., 1982). Unlike the majority ofcellular genes, they contain no introns (Derynck et al.,1980; Lawn et al., 1981; Houghton et al., 1981; Taniguchiet al., 1980; Tavernier et al., 1981). Several differenta-interferon genes were identified (10-15 copies) in humanDNA (Nagata et al., 1980), all with a high degree of DNAhomológy (90%), while the total sequence homology be-tween ß- and the a-interferons is only about 45 °?o(Taniguchi et al., 1980). The (3-interferon gene is present inthe human haploid genome in a single copy (Houghton etal., 1981).The synthesis of a- and /3-interferons in cells can be in-

duced by viral infection and dsRNA [e.g., poly(rI • rC)].Induction seems to be inducer- and tissue-specific; for ex-ample, infection of peripheral human leukocytes with virus(Newcastle disease or Sendai virus) induces synthesis ofa-interferon preferentially, while treatment of fibroblastswith poly(rI • rQ induces synthesis of /3-interferon only.Sequence analysis of the 5'-flanking region of severala- and the /3-interferon gene shows a similarity between the

DNA sequences in the upstream region of the ß- anda-interferon genes (DeGrave et al., 1981); it was suggestedthat this sequence may play a role in the control of the ex-

pression of interferon genes.Recent data from several laboratories indicate that the

methylation of DNA may be one of the determinants ofeukaryotic gene expression control (reviewed by Doerfler,1981). Therefore, the distribution of the restriction sites forthe isoschizomers Msp I and Hpa II was mapped in the se-

quences flanking the /3-interferon gene in DNA isolatedfrom induced and uninduced cells. It has been shown thatMsp I restricts CCGG and CMeCGG sites while Hpa II isactive only at the unmethylated site (Bird and Southern,1978; Waalwijk and Flavell, 1978). Our results indicate thatalmost all Hpa II sites near the /3-interferon gene are

methylated and that induction of interferon with poly(rl • rQ does not alter the methylation pattern of CCGGsequences flanking the /3-interferon gene. However, a com-mon polymorphic Msp I site was located approximately1000 bp upstream from the /3-interferon gene.

MATERIALS AND METHODSi

DNA extraction and blottingHigh-molecular-weight DNA was extracted from cells as

described (Riggin and Pitha, 1982). DNA (10 pg per sam-ple) was restricted with 2 units of restriction endonuclease

The Biochemical Virology Lab, The Johns Hopkins University, Baltimore, MD 21205.

267

268 RIGGIN AND PITHA

per pg of DNA in the buffer recommended by the vendor(Bethesda Research Laboratories). Restriction was carriedovernight at 37°C, and stopped by heating the samples to65 °C for 5 min and extracting with phenol and chloroform-¡soamylalcohol. DNA was precipitated with ethanol andelectrophoresed on horizontal 0.8% (w/v) agarose gels in Ebuffer (90 mMTris-borate pH 8.2, and 2.5 mAfEDTA) for24 hr at 25 volts. DNA was denatured and transferred ac-

cording to the modified (Riggin and Pitha, 1982) Southernprocedure (Southern, 1975) to a cellulose nitrate filter andhybridized with nick-translated pHF/3cDNA probe whosespecific activity was — 1 x 108 cpm/^g (Rigby et al., 1977).

Interferon induction and assay

Human fibroblast cells at confluence were superinducedwith poly(rI • rQ as described before (Raj and Pitha,1981). The levels of interferon in the medium were deter-mined 24 hr after induction by cytopathic assay (Finter,1979) on human cells trisomie for chromosome 21 (GM2504)using vesicular stomatitis virus (VSV) as the challenge virus.The titers are given in international research referenceunits.

Cell lines

Human fibroblast cells trisomie for chromosome 21(GM2504) were obtained from Human Genetic Mutant CellRespository. Normal human fibroblasts and a chemicallytransformed subclone were obtained from Dr. B. Vogel-stein (The Johns Hopkins University). The human fibro-blast MDL(P") line which has translocations on chromo-some 5 and 15 and was selected as a high interferonproducer was obtained from Dr. J. O'Malley (Roswell ParkInstitute, Buffalo, NY). Namalva cells which are lym-phoblastoid, positive for EBV, and high interferon pro-ducers were supplied by Dr. K. Osther. The placental cellline was obtained from Dr. S. Panem (University ofChicago, Chicago, IL).

RESULTS

Using the pHF/3cDNA as a probe, we have investigatedthe methylated sites surrounding the human /3-interferongene. Fig. 1 shows an autoradiogram of restriction en-donuclease patterns produced by Msp I and Hpa II diges-tion of human DNA isolated from poly(rI • rC)-treated and-untreated cells. The /3-interferon gene has no internalrestriction site for Msp I or Hpa II (Houghton et al., 1981);therefore, it was expected that only a single band would bedetected. However, when genomic DNA from two differ-ent cultures of diploid human fibroblast cells, HF andMDL(P-), was digested with Msp I and hybridized to/3-interferon probe, we observed /3-interferon sequences in4.2- and 2.7-kb fragments. Transformed and subclonedfibroblasts (tHF) derived from the same human fibroblastcells (HF) gave with Msp I restriction only a 4.2-kb frag-ment.After restriction of these DNAs with Hpa II, the

/3-interferon gene was identified predominantly inunrestricted sequences greater than 23 kb, indicating that

the CCGG sites in the DNA flanking /3-interferon gene are

probably methylated. Identical patterns were observedwhen the DNA isolated from induced cells was analyzed,indicating that induction appears to have no effect on themethylation of Hpa II sites in this region of DNA.DNA from parents and their offspring (Fig. 2) was

analyzed. Based on Msp I restriction, two types of in-dividuals were identified: common homozygotes for a4.2-kb fragment, and hétérozygotes with equal amounts of4.2- and 2.7-kb fragments. From 18 normal individuals (3families), 14 individuals were homozygous for a 4.2-kbfragment and 4 were heterozygous having both 4.2-kb andthe 2.7-kb fragment. When both parents were homozygousfor the 4.2-kb fragment, their children were homozygous;when one parent was heterozygous for the Msp I site, atleast one child was heterozygous. From these families, wedid not identify a rare homozygous individual with only the2.7-kb fragment. Therefore, we analyzed, a number ofother DNAs (Fig. 3) isolated from different tissues or celllines inducible for interferon. From all the DNAs tested (45samples), we found that only Namalva cells had DNAwhich has the rare homozygous pattern with only a 2.7-kbfragment. Namalva cells are lymphoblastoid cells derivedfrom a Burkitt lymphoma; we analyzed the genomic DNAfrom leukocytes of individuals with various forms of

HF_rHF IDL (P~)123 4 5 6789 10 11 12M H M H M H M H M H M H

KB

I I I It -23.7;* -9.5

¡¡fe.-

- —6,7

I —1.3

—2,3—2.0

FIG. 1. Cultured human fibroblast (HF), the same cellstransformed (tHF), and MDL(P") cells (human fibroblastswith translocations from chromosomes 5 and 15) were in-duced for interferon production for 5 hr with 100/ig/mlpoly(rI-rQ. The probe used was a Bgl 11/Pst I fragment ofcloned /3-IFN DNA, nick-translated to a specific activity of- 1 x 10* cpm//ig. DNA in slots 1,2,5,6,9, and 10 were allfrom cells induced for interferon. DNA in slots 3,4,7,8,11,and 12 was from uninduced controls.

METHYLATON AND POLYMORPHISM NEAR B-IFN GENE 269

12 3 4 5 6 7 8 9 10 1112 13 14 15 16 HF Jt£_ Pu

m » m - .-m «i m> ilfi df

KB

4.33.32.8

—

0.55-0.45

P P E EM M H

P P E P P E Em n n

'

FIG. 2. Human /3-interferon genes from the peripheralleukocytes of four pairs of parents (slots 1,2; 4,5; 9,10; 13and 14) and their offspring (slots 3; 6,7,8; 11,12; and15,16), respectively. DNA was restricted with Mspl, blot-ted, and annealed to pHF/3cDNA probe.

FIG. 4. DNAs from human fibroblasts (HF), transformedhuman fibroblasts (tHF), and human placenta were re-stricted with Msp I (M), Pvu II (P), and Eco RI (E). Doubledigests were done with Pvu II and Msp I, and Eco RI andMsp I. Molecular-weight markers were Hind Ill-digested Xand Hae III ¿X174 RF DNA fragments.

leukemia and lymphoma to determine if this rare homozy-gous form can be found with higher frequency in the in-dividuals which have disorders in leukocyte maturation.From 22 individuals analyzed, 14 individuals were

ta_ Leu (D211)n .H

Leu (D?15)M H

ILA.M H

FIG. 3. DNA from Namalva (Nam), human leukocyte(Leu D211 and Leu D213) and placenta (pía) were restrictedwith Mspl (M) and Hpa 11 (H). Conditions for elec-trophoresis and blotting are in the text.

homozygous for the 4.2-kb Msp I fragment and 8 in-dividuals were heterozygous (data not shown). None of theleukemic individuals tested was homozygous for the 2.7-kbband. Several lines of evidence indicate that the differentpatterns observed with Msp I restriction resulted from analteration of a specific Msp I restriction site: (i) identicalpatterns were observed in DNA digested with a great excessof restriction enzymes; (ii) cleavage patterns with other en-donucleases excludes large deletions near the /3-interferongene; and (iii) segregation patterns in several families exam-

ined are consistent with Mendelian inheritance.DNA from both homozygous and heterozygous in-

dividuals was restricted with Eco RI, Pvu II, and Msp I insingle and double digests to locate methylated sites ofMsp I relative to other known sites in and flanking the/3-interferon gene (Fig. 4). The restriction patterns of Pvu IIand Eco RI are similar to patterns published by others(Houghton et al., 1981). In the 5' flanking regions, there are

E Pi iM'1111-

M

-*-3'

I M"

5"-

0 300 IOOO bp

FIG. 5. Modifications by methylation are shown in a

physical map of the human /3-interferon gene and its flank-ing sequences. Restriction site were deduced from the blotshown in Fig. 3. The cross-hatched region represents the/3-interferon structural gene. The direction of transcriptionis from left to right. M,E, and P are Msp I, Eco RI, andPvu II, respectively. M* represents the polymorphic Msp Isite.

270 RIGGIN AND PITHA

Table 1. DNA Restriction Pattern and Interferon Expression

Cell Lineor

Tissue

Restriction Pattern

Units of ß-InterferonInducible per ml

Msp/(Band(s) kbp)

Hpa//(Band(s) kbp)

HFtHFMDL (P")Leukocyte (D211)Leukocyte (D213)PlacentalNamalva

<10-300"2500°<10"<10*> 160">1000»

4.2 and 2.74.2

4.2 and 2.74.2 and 2.74.2 and 2.7

4.22.7

>23>23>23>23>23>23>23

Methods for induction of /3-IFN with poly rl-rC and its assay on human fibroblasts were bypublished procedures (Raj et al., 1981)."Induced by poly(rI-rQ.'Induced by Sendai virus.

two Msp I sites. In the 3' flanking region there is oneMsp Isite about 900 bp downstream from the /3-interferon gene,about 200 nucleotides upstream from the Eco RI restrictionsite. The polymorphic Msp I site was located in the 5' flank-ing region of the /3-interferon gene 800 bp upstream fromthe flanking Eco RI site and is marked M* on Fig. 5.While normal leukocytes induced with virus produce

a-interferon predominantly (or exclusively), virus-inducedNamalva cells produce small amounts of /3-interferon(1-10% of total interferon). To test whether there is a

quantitative difference in interferon production in cellswhich are homozygous for the 4.2-kb fragment and cellswhich are heterozygous for the 4.2- and 2.7-kb fragments,we measured the levels of interferon in the media of thesecells induced with virus or poly(rI*rQ (Table 1). Theresults do not indicate any simple correlation between thepolymorphic Msp I site and the levels of /3-interferon syn-thesized. Fibroblast cells homozygous for the 2.7-kb frag-ment may be better producers than those heterozygous forhomozygous for the 4.2-kb fragment, but this had not beentested because such a line has not been found.

DISCUSSION

There are many examples from a diverse selection ofeukaryotic genes that show that actively transcribed genesare hypomethylated in their flanking sequences (Doerfler,1981). Whether the extent of methylation itself is sufficientto determine gene activity is not clear. Our data indicatethat there are methylated CCGG sites present in the vicinityof the /3-interferon gene. Induction of /3-interferon expres-sion in human fibroblast cells by poIy(rI*rQ was not cou-pled to altered levels of methylation in Hpa II sites. Theseresults, however, are not inconsistent with models in whichabsence of methylation at specific residues is necessary forgene expression, since a methylated site crucial for the con-trol process may represent only a small fraction ofmethylated cytosines in the DNA region and may or maynot be in Hpa II sites. With a restriction enzyme thatrecognizes only one of many methylated sites, we haveidentified a site which has a unusually high frequency ofpolymorphism.In the course of this work, we did not find unusual

restriction pattern around the /3-interferon gene with any ofthe following restriction endonucleases: Hind III, Bgl II,Eco RI, Hinz II, Tag 1, and Xba I. Thus, we found one

polymorphic site out of six restriction sites examined. Itwould be interesting to determine if the 5'-flanking region isa high-incidence locus for DNA polymorphism, such as oneidentified near the /3-globin gene cluster (Antonarakis et al.,1982). It remains to be determined if polymorphism in the5'-flanking region has an effect on /3-interferon expression.

ACKNOWLEDGEMENT

We wish to thank Dr. N. Babu K. Raj for advice andassistance, Mr. M. Kellum for technical assistance, and Ms.E. Siegler for DNA samples from leukemic patients.P.M.P. is a Leukemia Society Scholar.

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METHYLATON AND POLYMORPHISM NEAR B-IFN

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Address reprint requests to:Charles H. Riggin, Jr.

The Johns Hopkins UniversityThe Biochemical Virology Lab

600 North Wolfe St., Room 1-109Baltimore, MD 21205

Received for publication May 24, 1982.