Analysis of a brain-specific isozyme. Expression and chromatin structure of the rat aldolase C gene and transgenes Iman Makeh, Muriel Thomas, Jean-Pierre Hardelin, Pascale Briand, Axel Kahn, Henriette Skala

To cite this version:

Iman Makeh, Muriel Thomas, Jean-Pierre Hardelin, Pascale Briand, Axel Kahn, et al.. Analysis of a brain-specific isozyme. Expression and chromatin structure of the rat aldolase C gene and transgenes. Journal of Biological Chemistry, American Society for and Molecular , 1994, 269 (6), pp.4194-4200. ￿hal-02704437￿

HAL Id: hal-02704437 https://hal.inrae.fr/hal-02704437 Submitted on 1 Jun 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THE JOURNALOF BIO~ICM. CHEMI~TRY Vol. 269, No. 6, Issue of February 11, pp. 4194-4200, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in USA Analysis of a Brain-specific Isozyme EXPRESSION AND CHROMATIN STRUCTURE OF THE RAT ALDOLASE C GENE AND TRANSGENES*

(Received for publication, July 12, 1993, and in revised form, October 15, 1993)

Iman MakehS§, Muriel Thomas§$ Jean-Pierre Hardelinnll, Pascale BriandS, Axel Kahnn**, and Henriette Skalan From the *boratoire de Recherche en Gkne'tique et Pathologie Expe'rimentales, Znstitut National de la Sante' et de la Recherche Me'dicale (ZNSERM) Unite' 380, Znstitut Cochin de Gkne'tique Mole'culaires, 22, rue Me'chain and the Xaboratoire de Recherche en Gkndtique et Pathologie Mole'culaires, INSERM Unite' 129, Znstitut Cochin de Gkndtique Mole'culaire, 24, rue du Faubourg St.-Jacques, 75014 ,France

Aldolase C mRNA is detected by Northern blot in all (Schapira et al., 1970; Skala et al., 1987; Takashi et al., 1990). fetal tissues in rat; it is very abundant in theadult brain In contrast, thisisoenzyme is undetectable in normalnon-brain and undetectable in the other adult tissues. However, adult tissues. reverse transcriptase polymerase chain reaction ampli- In a previous work (Vibert et al., 1989) we have shown that fication indicates that this gene is not totally repressed although its promoter displays unusual housekeeping charac- in these tissues. A DNase-I hypersensitivity site located teristics for a tissue-specific gene, the aldolase C gene is regu- in a 116-basepair proximal promoter fragment is detect- lated at the transcriptional level. However, these characteris- able in the brain aswell as in other adult tissues. Two tics of the promoter are consistent with the low but ubiquitous MspVHpaII restriction sites located at -3800 and -460 expression of aldolase C in fetal tissues.Several questionsarise base pairs are demethylated in thebrain and totally or from these observations. partially methylated in othertissues. 1. Is theexpression of the aldolase C gene actually abolished In transgenic mice, a 12.6-kilobase genomicfragment in non-brain adult tissues, or is it slowed down to a very low is strongly and tissue specifically expressedin different lines, withconservation of a methylationpattern similar level that could be increased again in proliferating cells, espe- to that of the endogenous gene.A chloramphenicol acet- cially in cancer? yltransferase gene directed by either 800 or 116 base 2. It has been reported that contraryto specifically expressed pairs of aldolase C 6'-flanking sequences is tissue spe- genes, ubiquitous genes areassociated, in all tissues, withcon- cifically expressed in transgenic mice, but the level of stitutively open and hypomethylated chromatin (Bird, 1986, expression is very low. This level is greatly increased 1992). So, what is the chromatin structure and the methylation when the transgene consists of a chloramphenicolacet- pattern of aldolase C in tissues that expressaldolase C actively yltransferase hybrid gene directed by 6.6 kilobases of and in tissues thatdo not express it detectably? aldolase C 5'-flanking sequences. 3. What are the cis-acting regions important for a qualita- We propose therefore that the chromatin structure tively andquantitatively normal expression of aldolase C around the aldolase C promoter is accessible in fetal transgenes in vivo? tissues, then remains open in theadult brain, where the Here we report that thealdolase C gene behaves differently gene is very active, as well as in tissues in which it is from "classical" tissue-specific genes inthat its chromatin practically inactive. The specificityof expression in the structure is open even in tissues in which aldolase C is unde- brain is conferred by a short 116-basepair proximal pro- tectable. In particular, a DNase-I hypersensitivity site (HSS)' moter fragment that needs more upstream sequences to located in the vicinity of the start sites of transcription is as be fully active. strong in adult brainas in liver. "he persistenthypersensitivity at the promoter is inline with a persistent low level presence of aldolase C mRNA in adult non-brain tissues, detected only by reverse transcriptasePCR. However, a clear brainpreference of Fructose-l,6-bisphosphatealdolase is a glycolytic enzyme expression is conferred on a reporter gene by a short 115-bp that existsas three types: aldolase A (muscle type), aldolase B promoter fragmentin transgenic mice. More upstream se- (liver type), and aldolase C (brain type). Expression of these quencesincrease the level of transgene expression without isoenzymes in various tissues is modified during development modifying its specificity. and carcinogenesis (Lebherz and Rutter,1969; Numazaki et al., MATERIALS AND METHODS 1984). Aldolase C is strongly expressed in adult and fetal brain and at a lower level in all fetal tissues and proliferatingin cells Aldolase C Isoenzymes-Aldolase C isoenzymes were separated by cellulose-acetate electrophoresis with specific staining (Penhoetet al., 1966; Susor and Rutter, 1971). Northern blots and Reverse Dunscriptuse PCR Amplification"Tota1 * This investigationwas supported by grants from UniversityPans V cellular RNA from differenttissues was prepared as described (Kahnet Red Descartes, l'kssociation de Recherchesur le Cancer, and la Ligue al., 1981). The Northern blots were performed with10 pg of total RNAs Nationale de Recherche sur le Cancer. The costs of publication of this fractionated on formaldehyde(1.2% w/v) agarose gels andtransferred to article were defrayed in part by the payment of page charges. This Genescreen nylon membranes. The membranes were hybridized witha article must thereforebe hereby marked"advertisement" in accordance single-stranded probe complementary to the 3"noncoding extension of with 18 U.S.C. Section 1734 solely to indicate this fact. (Skala et al., Reverse transcriptase PCR $ The first two authors contributed equallyto this work. the aldolase C mRNA 1987). )I Present address: Unit4 de Gknktique Molkculaire Humaine, CNRS URA 1445, Institut Pasteur, 25, rue du Dr. Row, 75015 Paris, France. 1 The abbreviations used are: HSS, hypersensitive site; PCR, poly- ** To whomcorrespondence and reprintrequests shouldbe ad- merase chain reaction; bp, base paifis); kb, kilobaseb); CAT, chloram- dressed: ICGM, 24, rue du Fg St.-Jacques, 75014 Pans, France. phenicol acetyltransferase. 4194 Expression of the Rat Brain-specificAldolase C Gene 4195

12.5 kb aldolase C genomic clone A)

Brain Muscle Heart Lung Llver Gut 5' flanklng reglons 3' flanking region! EcoR 1 5.5kb 3.5kb 3.5kb EcoR 1 p- "

5.5/CAT (-5500/-79) B) EcoR 1 Xmal I I

0.8/CAT (-800/-79)

BamHlXmalll FIG.2. Panel A, Northernblot analysis of the aldolase C mRNA in different tissues during pre- and postnatal development.F18, 18 day- & old fetus; DO, newborn rats; 05 and D15, 5- and 15-day-old rats; A, adult rats. 10 pg of total RNA was electrophoresed on a 1.2% formal- dehyde (w/v) agarose gel, blotted, and the filters were hybridized with a 0.1 15lCAT (- 1991-84) single-stranded probe complementary to the 3'-untranslated region of the aldolaseC mRNA. Panels B and C, reversetranscriptase PCR analysis of the aldolase C, aldolase B, and cytochrome b5 reductase mRNAs in adult rat tissues.Amplification primers: for aldolase mRNA, -1 kb CAACTCC'ITC'ITCTGCTC (3' primer spanning from +31 to +48 bp) FIG.1. Scheme of the different analyzed transgenes. The 5' and TCTG'ITGCTAACCAGACC (5' primer, spanning from -75 to -57 EcoRI site of the 12.5-kb adolase C genomic fragment (12.5/aldC) is a bp).For aldolase B mRNA, TGTITG'ITCCTGCAAGCG (3' primer site. The 5"flanking sequencescloned in front of the CAT gene spanning from +372 to +391 bp) and CTGATACC'ITGGAGCACTC (5' (5.5/CAT, 0.8/CAT, and 0.115/CAT) are numbered with respect to the primer spanning from -44 to -25 bp). For cytochrome b5 reductase ATG translation initiator (position +l). The115-bp fragment obtained mRNA, GGGG'ITCTCGAGGGTGATGGC (3' primer spanningfrom +99 by PCR was cloned at the site SmaI. to +I20 bp) and GGTGTAGAACGGTGACACCACTG (5' primer span- ning from -37 to -16 bp). Each cDNA is numbered with respect to the amplification of fragments from aldolase C and aldolase B transcripts ATG translation initiator (+l); 1 pg adult RNAs was reversed tran- scribed, andcDNA was amplified for 20,25,30, or 40 cycles, as reported and from a ubiquitous reference transcript (cytochromeb, reductase) under "Materials and Methods." Amplified DNA fragments were elec- (Zenno etal., 1990) wasperformed as described (Noonanand Roninson, trophoresed on polyacrylamide gel, blotted, then hybridized with la- 1988). Reaction products were subjectedto polyacrylamide gel electro- beled oligonucleotides correspondingto internal sequences of the phoresis, transferred toa Genescreen membrane, and hybridized to a different fragments (AGTGAGCTGTGCCTGTG)for aldolase C, (TG- labeled internaloligoprobe (Chelly etal., 1989). Nucleotide sequence of GAAAAGGAATCACTCCG) for aldolase B, and (GC'ITCATGAAGAG- the primers complementary and identical to mRNA sequences are in- GCTGTAGACG) for cytochrome b, reductase mRNAs. dicated in the legend to Fig. 2. DNase-1Hypersensitivity and Methylation Analysis-Nuclei were respectively, which are cloned in front of the chloramphenicol acetyl- isolated essentially as described by Villeponteau et al. (1984). Tissues transferase (CAT) reporter gene. were removed, cut into pieces, washedin ice-cold nuclearisolation Production and Detection of ZFansgenic Mice-Inserts were isolated buffer (20 mM Hepes, pH 8, 50 mM NaCI, 1 mM EDTA, 0.25 m~ EGTA, from agarose gels and were further purified on Elutip-d columns ac- 0.15 mM spermine, 0.5 m~ spermidine, 0.5 M sucrose, 1 mM phenylmeth- cording to the instructions of the supplier (Schleicher& Schuell). The ylsulfonyl fluoride, 3.5 mM 2-mercaptoethanol) and then homogenized fragments diluted in 10 m~ Tris-HCI, pH 7.5, 0.1 mM EDTA were mi- in a Dounce homogenizer. The homogenate was filtered through gauze croinjected using established procedure into fertilized eggs resulting and centrifugedat 4 "C for 10 min. The nuclear pellet was resuspended from mating between B6D2F1 hybrids(Gordon and Ruddle, 1983). The in the nuclear isolation buffer in the presence of 0.5% (v/v) Nonidet animals were screenedfor the insertionsof the transgenesby Southern P-40, centrifuged, and then washed inthe samebuffer without Nonidet blot analysis of tail DNA preparations after hybridization with a ran- P-40. This step was repeated once, and then the nuclei were resus- dom primed CAT or aldolase C probe. Transgenic RNAs were detected pended in a small volume of nuclear isolation buffer containing 25% by SI nuclease protection analysis using radiolabeled single-stranded (v/v) glycerol. Nuclei samples, with 100 units of DNase-I added, were probes (Keyser et al., 1985). CAT activity generated by transgene ex- incubated at 25 "C from 0 to 20 min. Reactions were stopped by the pression in different tissues was measured on1-100 pg of proteins addition of 12 rn~EDTA and 0.5% (w/v) sodium dodecyl sulfate. Nuclei (Gorman et al., 1982). representing each point were digested overnight at 37 "C with 100 pg/ml (w/v) proteinase K. DNA from these nuclei was then phenol- RESULTS chloroform extracted andcleaved with the relevant restriction enzymes. Expression of the Aldolase Gene in Fetal and Adult Southern blots were hybridized with genomic fragments labeled by C Tis- random priming. The level of methylation of MspYHpaII sites was sues-Fig. 2A confirms (Mukai et al., 1991; Skala et al., 1987) investigated by cutting 10 pgof genomic DNA with a 10-fold excess of the dual specificity of aldolase C expression; mRNAs were de- the correspondingrestriction enzymes. Fragments were electropho- tected by Northern blot in all fetal tissues tested (brain,muscle, resed ina 0.8% (w/v) agarosegel, in thepresence of T3radioactive size heart, lung,liver, gut) butnot in adult tissues,except in brain markers provided by Amersham Corp. were then blottedon hybond N+ where its level was much higher than in fetaltissues. However, membrane. The 1.3-kbgenomic BamHI fragment described previously a minute amountof aldolase C mRNA was detectable after 40 (Vibert et al., 1989), encompassing 5"flanking sequences, first exon, and part of the first intron, was digested withSmaI, generating three cycles of reverse transcriptase PCR amplification in adult liver subprobes that where separated and isolatedfrom a low melting point and lung, whereas it wasdetected in brain after25 cycles (Fig. agarose gel. 2B ). This low expression of the aldolase Cgene in the lung and DNA Constructs-The different DNA constructs used as transgenes liver cannot be considered equivalent to the illegitimate tran- are described in the Thomas et al. (1993) and Vibert al.et (1989) and scription phenomenon, that is to say to verythe low level tran- schematized in Fig. 1. The 12.51aldC transgene consistsof an unmodi- scription of any tissue-specific gene outside its cognate expres- fied rat aldolase C gene with about5.5 kb of 5'- and 3.5 kbof 3"flanking sequences. The 5.5/CAT, 0.8/CAT, and O.l15/CAT transgenes consist in sion sites (Chelly et al., 1989); indeed, under the samereverse upstream sequences spanning, with respect to the ATG translation transcriptase PCR conditions, the liver-specific aldolase B initiator, from -5500 to -64 bp, -800 to -64 bp, and -199 to -84 bp, mRNA (Tsutsumi et al., 1985)was not detected in the lung and 4196 Expression of the Rat Brail %-specific AldolaseC Gene Probe A Probe 6 sively with three types of probes: probe 1was the BamHllSmaI fragment spanning from -800 to -450 bp; probe 2, the Small SmaI fragmentlocated between -450 and +70 bp; and probe 3, the SmallBamHI fragment spanningfrom +70 to +400 bp. To standardize the results and facilitate the positioning on the genomic fragment of the different described sites, we arbi- trarily attributed theposition +1 at the beginning of the cDNA described by Mukai et al. (1991), which is located in themajor start sites of transcription (Vibert et al., 1989). . Fig. 4A shows that probe 1revealed different DNAfragments when digestion was performed with HpaII or MspI, using ei- ther liver or brain DNA, indicating that a CCGG site located at position -3.8 kb was methylated in theliver and demethylated EcoR I HSS EcoRV HSS EcoRl in thebrain, whereas thesite at -1.7 kb was methylatedin both gene I tissues. Probe 2 revealed that theCCGG site located at position 13kb n I -450 bp was partially methylated in the liver and demethyl- Probe A 3.2kb - ated in the brain, whereas theat site +70 bp remained demeth- ylated in both tissues. Finally, probe 3 detected two additional FIG.3. DNase4 hypersensitivity analysis of the aldolase C downstream MspllHpaII sites, inside (+2.7 kb) and outside gene. Nuclei from adult rat liver and brain were incubated with 100 (+4.6 kb) the aldolase C gene; the former was always methyl- units of DNase-I for 1-20 min at 25 "C. DNA was then purified, digested ated and the latter always demethylated. It is worth noting with EcoRI and EcoRV enzymes, electrophoresed, and then blotted. The that this downstream demethylated site is in the immediate blots were hybridized with either probe A or probe B located here and there on the EcoRV site. Southern blot analysis and scheme of the HSSs vicinity of the downstream DNase-I HSS (located at +5.1 kb) with respect to the gene (open box): the length of the generated frag- and conserved region described above and mightcorrespond to ments is summarized in the lower part of the figure. a downstream gene, as already discussed. In Fig. 4B we took advantage of the presence of two PstI brain, whereas, as expected, it was very abundant in theliver restriction sites upstream and downstream of the +1 site to (Fig. 2C). In contrast, the messenger for cytochrome b5 reduc- locate more accurately some of the MspllHpaII sites and to tase, a ubiquitous enzyme(Zenno et al.,1990), was amplified in demonstrate that the site -450 at bp was totally demethylated all tested tissues. Therefore, the aldolase C geneis transcribed in the brain only, whereas it was partially methylated in the in fetal tissues and is strongly repressed, but not totally extin- adult liver, spleen (data not shown) and fetal liverthat express guished, in the corresponding non-brain adult tissues. In con- the aldolase C gene minimally or weakly. Therefore, although trast, the gene is strongly activated during pre- and postnatal the chromatinconformation of the aldolase C gene appears to development of the brain. This raises the question of whether be open in all tissues,a clear difference in methylation of up- these differences between aldolase C gene expression in the stream CG dinucleotides exists between the brain, where the brain and other tissuesoccurs in parallel with changes in the gene is very actively transcribed, and the othertissues. To gain chromatin structure of the gene. insight into the function of the flanking regions possessing DNase-I Hypersensitivity Analysis of the Aldolase C Gene in either the DNase-I HSS or differentially methylated CG di- Expressing and Nonexpressing Tissues-Nuclei from adult nucleotides, we subsequently created transgenic mice carrying brain and liver were purified and digested by DNase-I for dif- either the aldolase C gene, or constructs in which a reporter ferent times, and then DNA was purified and digested with gene was directed by aldolase C 5"flanking sequences (Fig. 1). EcoRI and EcoRV enzymes. The digestion products were ana- Expression of the Rat Aldolase C Gene in DansgenicMice- lyzed by Southern blotting (Fig. 3) using probes recognizing We introduced into fertilized mouse eggs the entirerat aldolase either the endsof a 13-kb 5' fragment (probe A) or of a 3.7-kb C gene, an EcoRllEcoRI genomic fragment (Vibert et al., 1989) 3' fragment (probe B). Fig. 3 shows that two strong HSSs were including 5.5 kb of 5'-flanking region, the gene (3.5kb), and 3.5 similarly detected in the liver and in thebrain. The upstream kb of the 3'-flanking region (Fig. 1). We produced four male HSS, detected with probe A, is located nearby the major start founder lines(37,39,41, and42) carryingfrom 3 to 30 copies of sites of transcription (Vibert et al., 1989). The downstream the transgeneper haploid genome. The founders were outbred HSS, detected with probe B, is located downstream from the to generate independent mouse transgenic lines. The expres- gene, at about 1.5 kb from the polyadenylation site. In fact, this sion of the transgene indifferent tissues was determined by S1 downstream HSSis located in a DNAfragment that appearsby nuclease mapping. Fig. 5 shows that the expression of the al- zooblotting to be a conserved region in different mammals and dolase C gene was very high in the brainof F1 animals, much birds (not shown) and could, therefore, correspond to the pres- higher than theexpression of the endogenous gene in ratbrain. ence of another gene unrelated to aldolase C. In anycase, these A considerably lower expression was detected in some non- results indicate that a strong HSS is detected in thepromoter brain tissues, e.g. liver, gut (especially in line 371, and lung of the aldolase C gene, in tissues that transcribe this gene (investigated in line 42). The high expression of the transgene strongly as well as in tissues that transcribeit only minimally. in the brain was confirmed by detection of the activity of the Since the difference of gene expression in these two tissues different isozymic forms after cellulose acetate electrophoresis could not be ascribed to differences in chromatin accessibility, (Fig. 6); the brainaldolase activity is generated by the various we addressed the question of a possible relationship between tetramers resultingfrom the combination between subunits A gene activity and methylation of CG dinucleotides. (predominant form in muscle) and C. In line 39, harboring Methylation Analysis-Methylation of CG dinucleotides be- three copies of the transgene, C4 homotetramers were rein- longing to MspllHpaII restriction sites (CCGG) in and around forced; they were largely predominant in line 41 harboring 30 the aldolase C gene was analyzedby comparison of the restric- copies. From these results, we can suppose that the enzyme tion patterns obtained withMspI that is not inhibitedby meth- activity generated by the 12.5/aldC transgenes is more or less ylation and with HpaII that isinhibited. Digests were analyzed correlated with the number of integrated copies. by Southern blotting, and hybridization was performed succes- Expression of AldolaselCAT Chimeric Genes in nansgenic Expression of theBrain-specific Rat Aldolase C Gene 4197 A

Pstl genomic clone Fet. Hpall racognltlon sltm rle2.8kb

0.5kb = Braln = Probe 2 0.5kb I adult and 2.8kb I1fetal llver b0.5kb

Pstl genomic clone PStl Hpall recognltlon shes d Braln methylatlonBraln pattern psm Llver methylatlon pmern p*m FIG.4. Methylation analysis of the endogenous gene. Panel A, digestion by HpaII or MspI. DNA from rat liver (Li)and brain (Br)was digested with HpaII or MspI, then analyzed by Southern blot and hybridized with probes1.2. or 3. defined above. The length and the position of the fragments obtained after hybridization with each probe are presented near the corresponding Southern blots. The lotuer part of the fimre summarizes the methylation patternof the aldolaseC gene: the methylated MspYHpaIl sites are represented by hlack squaws, the demethylnted sites by open squares, and the partially methylated sitesby hatched squares. Lad. molecular weight markers.The questton mark ( !) signifies that a upstream site cannot he studied in brain because of the downstream complete cleavage.Panel R,digestion by Hpall and I'stl. To focus on the region surrounding the promoter, double digestionsof DNA from rat liver (Li), brain (Rr),and fetal liver (Fet. Li) wcre performed. Thc Southern blot was hybridized with probe 2. 4198 Expression of the Rat Brain-specificAldolase C Gene tionis sufficient to confer a brainspecificity of expression. However, CAT activityin the brain ranged from 62 to 436 arbitrary units for five lines harboring the5.5lCAT transgene, AGT C this activity being especially low only in line 32 harboring a single copy of the transgene. In contrast, theCAT activity was considerably lower in all lines with the0.RlCAT and the 0.1151 CAT transgenes, ranging from0.04 to 2.4 arbitrary units, even "- in animals harboring a relatively high copy number (lines 13 ~ll>C'b'* 5 10," .. , ,., >1,..>,," and 9; Table I). In addition, no clear correlation was observed FIG.5. Nuclease S1 protection assay of the transgenic rat al- dolase C mRNA in mice. 10 pgof total RNA (brain, liver, gut, kidney, for either typeof transgene between the numberof integrated and lung) from adult transgenic mice were hybridized with a radio- copies and the CAT activity. These results suggested that ele- labeled single-stranded 160-bp aldolase C cDNA probe and then di- mentslocated upstream of the tissue-specificproximal pro- gested with 200 units of S1 nuclease for 30 min at 37 "C. The protected moter of the aldolase C gene are necessary for stimulating its cDNA fragment (120 bp) was analyzedon a sequencing polyacrylamide gel. The positions of the undigested probe and a rat brain control are activity in the chromosomal context. indicated. The different linesof transgenic mice harboring the12.5/aldC Since the upstream 5"flanking sequence that seems to be transgene and the numberof integrated copies estimated with respect important for a quantitatively correct expression of the trans- to the signal generated on Southern blots by the endogenous gene are genes contains the differentially methylated MspVHpaIIsites, precised. we investigated the methylation of these sites in the trans-

QJ genes.

(D Transgenlcbraln MethylationAnalysis of the7kansgmes-The methylation status of the transgenes was analysed as reported for the en- dogenousgene. The methylationpattern of the MspVHpaII C4" sites was identical in the endogenous gene and in eachcopy of the 12.5laldC transgene (Fig. 7A shows the methylation pat- tern of the line 41 bearing 30 copies), indicating that this pat- A4* I tern depends on information intrinsic to the aldolase C frag- I I mentsand not on the transgene chromosomal location. FIG.6. Isozymic pattern of aldolase C in transgenic and non- Moreover, we can conclude from this analysis that these copies transgenic tissues. Tissue extracts wrre electrophoresedon cellulose acetate strips that were then stained for aldolase activity. Nontrans- are integrated headto tail. The DNA methylation patternof the genic animals: A4, homotetramer present in muscle; A4, A3C, A2C2, other lines (42,37, and 39) gave similar results. In contrast,all AC3. and C4, tetramers present in brain. 'Ransgenicmice: brain extract MspVHpaII sites of the 5.5lCAT transgene were similarly par- from line 41 F1 (30 integrated copies) and from line 39 F1 transgenic tially methylated in both brain and liverof the different trans- mouse (3 integrated copies). genic mouse lines. Fig. 7R shows the resulh obtained for the line 29 after hybridization with probe 1. A similar pattern was TARLEI obtained with probe 2 with, as expected, an additional frag- CAT activity in different tissuesof aldolase1CAT transgenic mice ment of about 550 bp correspondingto the partial digestion of CAT actlvlty, cpm/mln/pg of protein the HpaII sites located at -450 bp and in the CAT gene (data Mouse Coov not shown). - - EralnLlver heart kldney sDleen lung Intestm DISCUSSION 5 5/CAT 32 I 1.3 (0.01 (0.01 (0.01 (0.01 0 I (0.01 Aldolase C is a brain-specific isoenzyme that is synthesized 5.5/CAT 9 2 436 (0.01 0.2 (0.01 0.08 (0.01 0 2 with aldolase A throughout the brain, although some brain regions appearto be richer in this enzyme than others(Popovici 5 5/CAT 29 3 203 (0.01 (0.01 ~0.01(0.01 0 4 (0.01 et al., 1990; Thompson et al., 1982). In addition to this absence

5 5/CAT I 5 62 (0.01 (0 01 0.6 a01 (0.01 (0.01 of a narrow specificity for a particular neuronal population, aldolase C is also characterized by its low expression in any 5 5/CAT 23 7 393

Brain Uver Probe 2

MsplESkb IBraln Liver

B 5.5/CAT transgene methylation

Hpall recognition sites Lad BrLi Mkb 13kb fl 9.6kb 7.7hb 2 6.2kb - 4.Zkb -. ?! 1.3kb n *..I 3.5hb r2kb -e .. L) 3.4kb n 2.Yb Probe 1 -. ue r3.8kb 1.8kb - Brain 1.4kb ** .) Liver 0.9kb -

kbL=== 24 kbL=== 0.6 kb -0

0.4 kb a Brain and Liver methylation pattern e Probe 1

FIG.7. Methylation analysis of the transgenes. DNA was processed as inFig. 4. The lengthof the fragments and the position and methylation pattern of the sites were mapped, as indicated in Fig. 4. Panel A, 12.5/aldC transgene. Liver (Li)and brain (B)DNA from line 41 (30integrated copies) was cut by HpaII or MspI. The Southern blots were hybridized with the three probes dencribed above. Only the hybridization pattern obtained with probe 2 is presented. On the scheme. two transgenes integrated headto tail were represented. Panel B,5.WCAT transgene. Liver (Li)and brain (Br)DNAfrom line 29(3 copies) wascut by HpaII. The Southernblot was hybridized withproh 1. The methylation pattern analysis shows that all sites are partially methylated in the brain aswell as in theliver. tively transcribed in fetal liver and repressed in adult liver examples discussed above. In the case of the aldolase C gene, (Tilghman and Belayew, 1982). However, DNase-I HSSs are the DNase-I HSS located in the immediatevicinity of the major still present upstreamof the a-fetoprotein gene in adult liver, start sites of transcription is as strong in the liver, where the although they are absent in spleen where the gene is never gene ispractically inactive, as in the brain,which suggests that expressed (Godbout and Tilghman,1988). Similar results have in non-brain tissues the promoter remains accessible to tran- recently been reported in our laboratory, by in vivo footprinting, scriptionfactors but either lacks an essential brain-specific for the L-pyruvate kinase gene that isactive in fetal intestine positive factor or is blocked by a negative factor. and repressed in the exocrine pancreas derived from an intes- The CAT transgenes directed by either 115 or 800 bp of tinal evagination while its chromatin remains accessible (Mi- 5"flanking sequences are tissue specifically but very weakly querol et al., 1994). Importantly, a common characteristic of expressed in the brain. Transgeneexpression is strongly stimu- this type of genes extinguished in differentiated tissues after lated by more upstream sequences located between -5500 and they havebeen active during thefetal period is that they canbe -800 bp. This 5"flanking region does not contain any addi- reactivated, for instance in cancer or in cultured cells in the tional DNase-I HSSs, often associated with remote enhancers 4200 Expression of the Rat Brain-specific Aldolase C Gene

(Elgin, 1988; Gross and Garrard, 1988) but includes the MspV Acknowledgment-We are grateful to Allan Strickland for careful HpaII site at -3800 bp which, in the endogenous aldolase C revision of the manuscript. gene, is differentially methylated in the brain and liver. REFERENCES Although the association between gene expression and de- methylation has been described (Meehan et al., 1992; Peek et Bird, A. (1986)Nature 321,209-213 Bird, A. (1992)Cell 70,W al., 1991; Tate and Bird, 1993; Bird, 1992),the exact role of this Bode, J., and Maas, K. (1988)Biochemistry 27,4706-4711 phenomenon in the control of gene expression remains poorly Boyes, J., and Bird, A. (1992)EMBO J. 11,327433 understood. From various experiments, it seems that modifica- Chelly, J., Concordet, J.2, Kaplan, J.-C., and Kahn, A. (1989)Proc. Natl. Acad. U.S. A. 86,2617-2621 tions in themethylation level are not primarily involved in the Delain, D., Meienhofer, M. C., Prom, D., and Schapira, F. (1973)Differentiation 1, rapid changes of gene expression but rather serve to lock an 349-354 expression pattern (activation or repression) after it has been Elgin, S. C. R. (1988)J. Biol. Chern. 263,19259-19262 Gazdar, A. E,Zweig, M. H., Carney, D. N., VanSteirteghen, A. C., Baylin, S. B., and established (Boyes and Bird, 1992; Opdecampet al., 1992; Szyf Minna, J. D. (1981)Cancer Res. 41,2773-2777 et al., 1990). In the aldolase C gene, the methylation pattern, Godbout, R., and Tilghman, S. M. (1988)Genes & Deu. 2,949-956 i.e. two MspUHpaII sites demethylated in the brain andtotally Gordon, J. W., and Ruddle, F. H. (1983)Methodp Eluyrnol. 101, 411433 Gorman, C., Moffat, L., and Howard. B. (1982)Mol. Cell. Biol. 2, 1044-1051 (-3800 bp) or partially (-450 bp) methylated in other tissues, is Gross, D. S., and Garrard, W.T. (1988)Annu. Reu. Biochem. 67,159-197 conserved in the different lines harboring the 12.5ialdC trans- Kahn, A., Cottreau, D., and Dreyfus, J. C. (1980)Pedintr: Res. 14, 1162-1167 gene whose level of expression seems to be more or less corre- Kahn, A,, Cottreau, D., Daegelen, D., and Dreyfus, J. C. (1981)Eur: J. Biochern. 116, 7-12 lated with the number of integrated copies. In contrast, the Keyser, Y. D., Bertagna, X., Lenne, F., Girard, F., Luton, J. P., and Kahn, A. (1985) 5.5iCAT transgene, albeit including the same 5”flanking re- J. Clin. Invest. 76, 1892-1899 gion as the 12.5ialdC transgene, is expressed without any re- Lebherz, H. G., and Rutter W. J. (1969)Biochemistry 8, 109-121 Meehan, R., Lewis, J., Cross, S., Nan, X., Jeppesen, P., and Bird, A. (1992)J. Cell lationship with the copy number. This absence of tissue-specific Sci. 16,9-14 methylation pattern and of copy-dependent expression might Miquerol, L., Lopez, S., Cartier, N., Tulliez, M., Raymondjean, M., and Kahn, A. be related to the lack of aldolase C intragenic or 3’-flanking (1994)J. Biol. Chern., in press Mukai, T., Yatsuki, H., Masuko, S., Arai, Y., Joh, K, and Hori, K (1991)Biochern. element(s) or to the own influence of the procaryotic CAT gene. Biophys. Res. Cornrnun. 174, 1035-1042 In contrastwith the resultsof transgene expression, we have Noonan, K. E., and Roninson I. B. (1988)Nucleic Acids Res. 16,10366 observed that sequences upstream of the position -115 bp were Numazaki, M., Tsutsumi, K-I., Tsutsumi, R., and Ishikawa, K (1984)Eur. J. Biochern. 142, 165-170 inefficient on the activity of aldolaseiCAT constructs in tran- Opdecamp, K, RiviBre, M., MolnB, M., Szpirer, J., and Szpirer, C. (1992)Nucleic siently transfected PC12 cells(Thomas et al., 1993). Such posi- Acids Res. 20,171-178 tive cis-acting sequences, active in thechromosomal context (in Peek, R., Niessen, R., Schoenmakers, J., and Lubsen, N. (1991)Nucleic Acids Res. 19,77433 transgenic animals or in stably transfected cells) but inactive Penhoet, E., Rajkumar, T., and Rubber, W. J. (1966)Proc. Natl. Ad.Sci. U.S. A. when tested by transient transfection assays, have been re- 56,1275-1282 ported in different genes such as those for chicken lysozyme Phi-Van, L., and Stratling, W. H. (1988)EMBO J. 7,655-664 Phi-Van, L., Von Kries, J. P., Ostertag, W., and Stratling, W. H. (1990)Mol. Cell. (Phi-Van and Stratling, 1988; Stief et al., 1989; Phi-Van et al., Biol. 10,2302-2307 1990) or interferon+ (Bode and Mass, 1988). It has Popovici, T., Berwald-Netter, Y., Vibert, M., Kahn, A,, and Skala, H. (1990)FEBS been suggested that thistype of element is important for insu- Lett. 1, 189-193 Sato, K. M., Moms, H. P., and Weinhouse, S. (1972) 178,87%881 lating agene and protected it from the influence of surrounding Schapira, F., Reuber, M. D., and Hatzfeld, A. (1970)Biochem. Biophys. Res. Com- chromosomal sequences. However, the 5”flanking fragment of mun. 40,321327 the aldolase C gene, encompassing from -5500 to -800 bp, Skala, H., V~bert,M., Lamas, E., Maire, P., Schweighoffer, F., and Kahn, A. (1987) Eur: J. Biochem. 163,513-518 although able to stimulate transgene expression, is clearly in- Stief, A,, Winter, D. M., Stratling, W. H., and Sippel, A. E. (1989)Nature 341, sufficient to insulate the 5.5fCAT transgene, whose expression 343-345 remains dependent on the integration site and independent of Susor, W. A,, and Rutter, W. J. (1971)Anal. Biochern. 43, 147-155 Szyf, M., Tanigawa, G., and McCarthy, p. (1990)Mol. Cell. Biol. 10, 439MOO the copy number. Takashi, M., Haimoto, H., Koshikawa, T., and Kato, K. (1990)Arn.J. Clin. Pathol. In conclusion the aldolase C gene represents aparadigm of a 93,631436 family of genes whose expression is ubiquitous in thefetus and Tate, P., and Bird, A. (1993)Current Opin. Genet. Deu. 3,226-231 Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993)Eur: J. Biochern., restricted to the brain in adults, whichbut are reactivatable in in press proliferating non-brain tissues. We have already shown in this Thompson, R. J., Kynoch, P. A. M., and Willson, V. J. C. (1982)Brain Res. 232, paper that thechromatin structure around a brain-specific al- 489-493 Tilghman, S. M., and Belayew, A. (1982)Proc. Natl. Acad. U.S. A. 79,5254-5257 dolase C proximal promoter fragment remains accessible out- Tsutsumi, K.-I., Mukai, T., Tsutsumi, R., Hidaka, S., Arai, Y., Hori, K., and Ishi- side the brain and that this promoter needs, in order to be kawa, K (1985)J. Mol. Biol. 181, 153-160 strongly active in uiuo, the presence of more upstream 5”flank- Vibert, M., Henry, J., Kahn, A., and Skala, H. (1989)Eur. J. Biochern. 181,33-39 Vdleponteau, B., Lundel, M., and Martinson, H. (1984)Cell 39,469478 ing sequences acting in the chromosomal context but not in GMO,S., Hattori, M., Misumi, Y., Yubisui, T., and Sakaki, Y. (1990)J. Biochem. transient expression experiments. (Tokyo) 107,810-816