Methylation Patterns of the Human ApoA-I/C-III/A-IV Gene Cluster in ...

T H E.JOURNAL OF BIOLOCICAI. CHEMISTRY ‘c’1991 hy The American Society Sor Biochemistry and Molecular Biology, Inc

Vol. 266, No. 35, Issue of December 15, PP. 23676-23681,1991 Printed in U.S.A .

Methylation Patterns of the Human ApoA-I/C-III/A-IV Gene Clusterin Adult and Embryonic Tissues SuggestDynamic Changes in Methylation during Development* (Received for publication, April 22, 1991)

Ruth Shemer, Shlomo EisenbergS, Jan L. BreslowQ, and Aharon Razinll From the Departments of Cellular Biochemistry and SlnternalMedicine B, The Hebrew University, Hadassah Medical School, Jerusalem 91010, Israel and the §Departmentof Biochemical Genetics and Metabolism, The Rockefeller University, New York, New York 10021

We describe here a detailed analysis of the methylation patterns of the apoC-I11andapoA-IV genes in adult and embryonic tissues. Together with previously reported data on the human apoA-Igene (4), the results presented here constitute a comprehensive study on the methylation pattern of the apoA-I/C-III/A-IVgene cluster. The two genes (apoC-I11 and apoA-IV)display tissue-specific methylation patterns that correlate with their activity. This gene-specific methylation pattern indicates that the apoA-I/C-III/A-IVgene cluster is not one entity with respect to methylation. The cluster is almost entirely methylated in tissues that do not express any of the genes; however, individual gene regions are unmethylated in the tissue of expression. A comparison of the observed methylation patterns in adult tissues with those in embryonic tissues suggests that the mature tissue-specific methylation patterns are a result of an interplay between demethylation and de novo methylation events in the embryo. These changes inDNA methylation include demethylation in the early embryo followed by de novo methylation at later stages.A second round of tissue-specific demethylation and methylation de novo occurs in the late embryo as well.Evidence presented here supports the idea that CpG islands are protected in general from methylation de novo by a built-in signal and not by CpG density per se.

Although it is now quite clear that methylation patternsof specific mammalian genes reflect, in most cases, the state of activity of the genes, much has yet to be learned as to how these methylation patterns are formed and how they affect gene activity (for recent reviews, see Refs. 1-3). Wehave a previouslyshown that the human apoA-Igenedisplays tissue-specific methylation pattern. The 5’-endof the gene is methylated in sperm and all nonexpressing tissues and is unmethylated in tissuesthatexpressthe gene (liver and intestine) (4). This typeof correlation between the patternof methylation andgene activity holds true for many othergenes (5), including the apoB gene (6). Our studies with the apoA-I gene also revealed that in the embryo,tissue-specific expres-

* This work was supported by an Israel Ministry of Health grant and National Institutes of Health Grant GM 20483 (to A. R.) and a UnitedStates-IsraelBinationalFoundationgrant(to S. E.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticlemustthereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. ll T o whomcorrespondence and reprint requests should be addressed. Tel.: 972-2-428173; Fax: 972-2-415848.

sion precedes the formation of the adult pattern of methylation. In addition, the expression of a human apoA-I transgene correlated with its methylation pattern (4). The humanapoAI gene is a member of the apolipoprotein gene cluster on the long arm of chromosome 11 (7). In this gene cluster, apoA-I, apoC-111, and apoA-IV are juxtaposed to each other(see Fig. 1). This physical proximity of the three genes could have suggested a coordinated controlof their expression that could have been reflected in the overall methylation pattern of the cluster. This possibility prompted us to study in detail the methylation patterns of the apoC-I11 and apoA-IV genes. A comprehensive methylation patternof the entiregene cluster was expected to shedsome light on themode of control of the three members in this gene cluster. We demonstrate in this study that both apoC-I11 and apoA-IV have tissue-specific methylation patterns that correlate with their activity. Therefore, the cluster is not considered one entity with respect to methylation. Very littleis known concerningmethylation changes of individual genes during embryogenesis. A comparison of the methylation pattern of the cluster in embryonic and adult tissues suggests that the mature methylation pattern is a result of an interplay between demethylation and de nouo methylation during development. apoA-IV) Two of the genes inthecluster(apoA-Iand contain CpG islands in their lastexon. In general, these CpGrich regions are found at the 5’-ends of housekeeping genes and are invariably undermethylated (8).Although the island found in apoA-I is unmethylated in all tissues (4), surprisingly, the apoA-IV island is shown here to be methylated in alltissuesexceptintestine, where the gene is expressed. Experiments designed to elucidate the differencebetween these two islands reveal that the apoA-IV island is not protected from de nouo methylation. MATERIALS ANDMETHODS

Cell Cultures-Mouse fibroblast L-cells were grown in Dulbecco’s modified Eagle’s medium supplementedwith 10% newborncalf serum, 2 mM glutamine, and 1000 units/ml penicillin plus 100 pg/ml streptomycin. Mouse teratocarcinoma F9 cells were grown in DulbecCO’S modified Eagle’s medium supplementedwith 10% fetal calf serum, glutamine, andpenicillin/streptomycin. DNA-mediated Gene Transfer-Transfections were carriedout using the following constructs. 1) The 2.2-kb’ PstI fragment of the apoA-I gene(see Fig. 5) was ligated into pUC19. Thisconstruct includes the apoA-I CpG island. 2) The 6-kb Hind111 fragment that includes the CpG islandof apoA-IV was ligated into pUC19. The two constructs were methylated in vitro with M. HhaI as described below. DNA-mediated gene transfer was performed with a Boehringer Mannheim lipofectin transfection kitaccording to the manufacturer’s

23676

The abbreviation is: kb, kilobase(s).

23677

ApoA-I/C-III/A-IV Gene Cluster: Methylation during Development

instructions.In alltransfections,pSV2Neo was includedas a selection highly demethylated in the liver, where the gene is predomimarker. 48 h after transfection,cells weretrypsinized and platedonto nately expressed (9). The gene in the intestine shows only medium containing 300 pg/ml G418. Cloneswerepooled after 2-3 partial methylation inaccord withits relatively lower activity. weeks of selection. Cells grownto mass culture were rinsed twice with However, in both the liver and intestine, the 1- and 0.7-kb phosphate-buffered saline andused to extract DNA. In Vitro Methylation of Plasmids-Approximately 20 pg of a given bands are visible, suggesting that the upstream M site and longer exposure, the 0.5plasmid was incubated with 5-10 units/pg M. HhaI methylase (New the S site are not methylated. Under England BioLabs,Inc.). Incubation was carried out overnight at 37 “C kb bandis also visible, indicating that the other M and S sites in the following reaction mixture: 50 mM Tris-HC1(pH 7.5), 160 p M are unmethylated. The 1.2- and 0.8-kb bands in the HhaI S-adenosylmethionine,10 mM EDTA, 5 mM 2-mercaptoethanol.The digest indicate that all HhaI sites indicated in the restriction extraction followed by a reaction was terminated byonephenol ch1oroform:isoamyl alcohol (24:l) extraction. To the extracted sam- map of Fig. 2 are unmethylated. This tissue specificity of the methylation pattern of the ples were added 0.4 M NaCl and 2 volumes of cold ethanol (-20 “C). apoC-I11gene inthe adult tissues is observed not in embryonic The DNA was dissolvedin50 mM Tris (pH 7.4) and used for transfection. The extent of methylation was determined by digesting tissues. Whereas thegene is completely methylated in embrythe DNA with a high excess (20 unitslpg) of the HhaI restriction onic leukocytes, it shows only little methylation in all other enzyme. Methylated DNAusedfor transfection wasalways com- tissues, including embryonic kidney. The complete methylapletely resistant to HhaI digestion. tion of the gene in the adultkidney is a good indication that RestrictionEnzyme Analysis and Southern Blotting-Genomic DNA was digested with the appropriate restriction enzymes, size- the gene undergoes tissue-specific de novo methylation later heavy methylation of fractionated by electrophoresis on 0.8% agarose gels, transferred to in development. On the other hand, the Zeta probe filters, and hybridized to the appropriate probes labeled the gene in sperm and the low methylation in mostembryonic by primer extension to a level of 2-3 X 10’ cpm/pg. Hybridization tissues suggest demethylation processes in the early embryo was performed at 65 “C for 15 h in a solution containing 10% poly- that are notassociated with gene expression. ethylene glycol, 7% sodiumdodecyl sulfate, 0.3 M NaCl, 15 mM Methylation Pattern of ApoA-IV Gene Is Tissue-Specific NaH2P04,1.5 mM Na,EDTA, and 250 pg of denatured salmon sperm DNA. Blots were then washed and exposed to x-ray film (Curix RP2, and Correlates with Its Activity in Adult-The methylation patterns of the 5’- and 3’-ends of the apoA-IV gene were Agfa) for autoradiography. analyzed separately. Four sites were analyzed at the 5’-end RESULTS three M sites, one of which is also an S site, and one H site To determine the methylation status of the human apoC- (see restrictionmap in Fig. 3). T o analyze thestatus of methylation of the S sites, DNA from various adult tissues I11 and apoA-IVgenes, we employed the methyl-sensitive restriction enzyme HpaII and its isoschizome (MspI) toprobe was digested with XbaI HpaII, XbaI SrnaI, and XbaI 1.8-kb XbaI fragment. As for methylation of CCGG sites (M sites). We also used the HhaI; blotted; and probed with the methyl-sensitive restriction enzyme HhaI to probe for meth- can be seen in Fig. 3, the two M sites, the S site, and the H site are completely methylated in sperm and leukocytes as ylation of GCGC sites (H sites)andSmaIto probefor judged by the 1.8-kb band. Also, other nonexpressing tissues CCCGGG sites (S sites). It should be kept in mind that these sites represent a subset of all CpGs in thisregion. Although such as kidney and liver are completely methylated in the H individual sites may differ in principal from other sites by site and the M site within the gene judged by the absence of their state of methylation, it is generally accepted that the the 0.7-kb band in thecase of the HpaIIdigest. Whereas it is especially in the HpaII and HhaI sites mayvery well represent the overall clear that theS site is almost unmethylated, pattern of methylation. In this study, it stands to reason that liver (judged by the 1.2- and 0.6-kb bands), the proximity of the methylation statusof >50 sites analyzed here represents the upstream M and S sites does not allow conclusive evaluthe overall methylation pattern of this gene cluster. ation of its methylation state. In contrast, all of the above Methylation Patternof ApoC-111Gene Is Tissue-specific and sites are unmethylated in the intestine, where the gene is Correlates with Its Activity in Adult-We have analyzed three expressed (lo), giving rise to 0.7-, 0.6-, and 0.5-kb fragments H sites and five M sites in thecoding region of the gene and by HpaII digestion; 0.8- and 1.0-kb bands by HhaI digestion; two H sites and oneM site in the 5”upstreamregion. Three and 1.2- and 0.6-kb bands by SrnaI digestion. Thus, the results two presentedin Fig. 3 show a clear correlation between the M sites were also S sites, which enabled us to distinguish adjacent M sites (see restriction map inFig. 2). DNA samples methylationpatternand expression of the gene inadult from various adult and embryonic tissues were digested with tissues (10). Also, two M sites in thebody of the gene (second HpaII,EcoRI HhaI, or EcoRI SmaI.The EcoRI intron) show similar methylation (see Table I). electrophoresed digests were blotted and hybridized with the In contrast, embryonic tissues show a similar pattern of 3.2-kb EcoRI fragment as described under “Materials and methylation in theliver, kidney, and intestine in spiteof the Methods.” The results presented in Fig. 2 and Table I dem- fact that theapoA-IV gene is known to be expressed only in onstrate that thegene is completely methylated in sperm and the embryonic intestine (10). This observation is similar to tissues that do not express the gene, such as kidney and what we have previously reported for the apoA-I gene (4). leukocytes. Both HpaII and HhaI digests reveal the entire Again, this result indicates thattissue-specific expression in 3.2-kb EcoRI fragment as the predominant band, indicating the embryo precedes the establishment of the tissue-specific that all M and H sites are methylated. As shown in Table I, methylation pattern of the gene. the S sites are methylated as well. In contrast, the gene is CpG Island in ApoA-IV Gene Is Fully Methylated in Em-

+

+

+

+

+

.P ”.s i I E c o R l

,P e t 1

II

I .

-

apoclll

apoAl

FIG.1. Physical map of apoA-I/CIII/A-IV gene cluster. This structural map is drawn to scale. Black boxes represent exons,and arrows represent start and direction of transcription. Restriction sites used for analysis are as presented in the map.

+

- I

apoAlV

Xbal

EcoRl I

I

In

I

I

I

Xbal I

I

I

EcoRl ”

Hindlll I

Ik b

ApoA-I/C-IIIIA-IV Gene Cluster: Methylation during Development

23678 ADULT

EMBRYO

various adult andembryonic tissues were digested with XbaI, HindIII, and HhaI; blotted; and hybridized with a 2.2-kb XbaI-EcoRI radioactive probe. As can be seen in Fig. 4, all tissues are methylated in all six HhaI sites as indicated by the intact 3.0-kb fragment. The only tissue where these sites are notmethylated is the adult intestine, in which the apoAIV gene is expressed, since the only visible band is the 0.7-kb : * fragment.Identicalresults were obtained when DNAwas 1 digested with XbaI, HindIII, and HpaII (data not shown). 1sl* I This is the firstexample of a non-X-linked CpG island that - 1.2 is methylated in theorganism. The extraordinary observation :. - 1 .o of a methylated CpG island prompted us to investigate the "r -0.8 origin of this phenomenon (see below). -0.7 .. i.., What Is the Mechanism Responsiblefor MaintainingApoA, :. . , -0.5 / ;I ' ". I V Island Methylated in Viuo?-CpG islands are especially common in 5'-ends of housekeeping genes and arecharacterEcoRl H S H M EcoRl izedby being unmethylatedin all tissues (8). Even CpG islands that have been found in tissue-specific genes thus far are all unmethylated (4,ll-13). Therefore, the observation of amethylated CpG island in the apoA-IV gene raises the .o question of whether this sequence is recognized by the orga1.2 nism as a CpG island. In a recent study in our laboratory (14), 0.8 we have found that the introduction of methylated island u. I sequences into mouse embryonic cells (F9) resultsin effective u . 5 demethylation of the island, whereas non-island flanking FIG. 2. Methylation status of human apoC-I11 gene. Adult sequences remain methylated. Somatic cells such as mouse and embryonic (12-week embryo) tissue DNA samples were digested fibroblast L-cells were not capable of this demethylation. A with EcoRI + HpaII (lanes M),EcoRI + HhaI (lanes H ) , or EcoRI + MspI (lanes M s p l ) . The digests were electrophoresed, blotted, and methylated island introduced into a transgenic mouse underhybridized as described under "Materials and Methods" to the3.2-kb went demethylation similarly to the transfected embryonic EcoRIfragment as a probe. K, kidney; Li, liver; In, intestine; Lu, F9 cells. These results suggested that island sequences are leukocytes. The lower part shows the sizes of the various expected recognized by the mouse embryo as an entity thatshould be restriction fragments. For EcoRI fragment location, see Fig. 1. maintained in an unmethylated state. To test thepossibility that the unprecedented in uivo methylated apoA-IV island TABLE I escapes this embryonic mechanism, we have prepared methSummary of the methylationpatterns ylated constructs of the apoA-I and apoA-IV islands by methof human apoA-I/C-IIIIA-IVgene cluster ylating them in uitro with the HhaI methylase. These conRelative extents of methylation of the individual sites (SmaI (S), HpaII (M), and HhaI (H)) are designated as follows: m, complete structs together with unmethylated controls were used to methylation; m, >75% methylation; 0,