1147Journal of Cell Science 110, 1147-1158 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS3523

The transition from a late 1-cell mouse embryo to a 4-cellembryo, the period when zygotic gene expression begins,is accompanied by an increasing ability to repress theactivities of promoters and replication origins. Since thisrepression can be relieved by either butyrate orenhancers, it appears to be mediated through chromatinstructure. Here we identify changes in the synthesis andmodification of chromatin bound histones that are con-sistent with this hypothesis. Oocytes, which can represspromoter activity, synthesized a full complement ofhistones, and histone synthesis up to the early 2-cell stageoriginated from mRNA inherited from the oocyte.However, while histones H3 and H4 continued to be syn-thesized in early 1-cell embryos, synthesis of histonesH2A, H2B and H1 (proteins required for chromatin con-densation) was delayed until the late 1-cell stage, reachingtheir maximum rate in early 2-cell embryos. Moreover,

histone H4 in both 1-cell and 2-cell embryos was pre-dominantly diacetylated (a modification that facilitatestranscription). Deacetylation towards the unacetylatedand monoacetylated H4 population in fibroblasts began atthe late 2-cell to 4-cell stage. Arresting development at thebeginning of S-phase in 1-cell embryos prevented both theappearance of chromatin-mediated repression of tran-scription in paternal pronuclei and synthesis of newhistones. These changes correlated with the establishmentof chromatin-mediated repression during formation of a2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates andcore histones are deacetylated.

Key words: Histone, Acetylation, Mouse, Embryo, Repression,Transcription, Zygotic gene expression

SUMMARY

Changes in histone synthesis and modification at the beginning of mouse

development correlate with the establishment of chromatin mediated

repression of transcription

Maria Wiekowski1,2, Miriam Miranda1,3, Jean-Yves Nothias1,4 and Melvin L. DePamphilis1,5,* 1Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110, USA2Schering-Plough Research Institute, Department of Immunology, Kenilworth, NJ 07033-0539, USA3Wyeth-Ayerst Research, 401 North Middle Town Road, Pearl River, NY 10965, USA4Institut Alfred Fessard - CNRS, Avenue de la Terrasse, 91 198 Gif-sur-Yvette, France5National Institute of Child Health and Human Development, Building 6, Room 416, National Institutes of Health, Bethesda, MD20892-2753, USA

*Author for correspondence (e-mail: depamphm@box-d.nih.gov)

INTRODUCTION

During mouse development, formation of a 2-cell embryomarks the transition from maternal to zygotic gene dependence(Fig. 1; reviewed by Schultz, 1993; Nothias et al., 1995; Chris-tians et al., 1995). Transcription stops when oocytes undergomeiotic maturation to form unfertilized eggs. Moreover, degra-dation of maternal mRNA begins and is almost completed bythe 2-cell stage, although synthesis of proteins from maternallyinherited mRNA can still be detected at the 8-cell stage. Fer-tilization triggers completion of meiosis and formation of a 1-cell embryo with two haploid pronuclei, one from each parent,but the onset of zygotic gene expression is a time dependentevent that is delayed for about 24 hours, thereby beginningafter formation of a 2-cell embryo. Transcription of zygoticgenes can begin in late 1-cell embryos, but nascent transcriptsproduced in these embryos are not translated and therefore notproductive; translation is coupled to transcription only during

the 2-cell stage (Nothias et al., 1996). This delay in zygoticgene expression provides a window of opportunity for remod-eling parental chromosomes without accidentally and prema-turely expressing their genes. In fact, one or more factorscapable of repressing DNA transcription and replication areabsent from 1-cell embryos during this remodeling period, andare then produced again prior to the productive transcription ofzygotic genes.

Injection of plasmid-encoded reporter genes into the nucleiof oocytes and early embryos has revealed the presence oftransacting factors that can repress the activities of promoters(Dooley et al., 1989; Martínez-Salas et al., 1989; Wiekowskiet al., 1991, 1993; Majumder et al., 1993; Henery et al., 1995)and replication origins (Martínez-Salas et al., 1988) from 20to >500-fold. While maternal nuclei in both oocytes and 1-cell embryos exhibit this repression (Martínez-Salas et al.,1989; Wiekowski et al., 1991, 1993), the paternal pronucleusexhibits repression only when 1-cell embryos develop beyond

1148 M. Wiekowski and others

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Fig. 1. Events at the beginning of mousedevelopment (adapted from Nothias et al.,1995; Majumder and DePamphilis, 1995).Injection of human chorionic gonadotropin(hCG) activates meiotic maturation.Fertilization activates a time dependentmechanism (zygotic clock) that delayszygotic gene expression for ~24 hours.Thus, addition of aphidicolin to 1-cellembryos prior to the appearance ofpronuclei (18-21 hours post-hCG) arreststhem at the beginning of S-phase, but doesnot prevent zygotic gene expression. Eventsassociated with maternal ( ), paternal ( ), and zygotic ( ) nuclei are indicatedby shaded bars. Open bars indicated periodsof histone synthesis and acetylation.

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S-phase. Application of nuclear transplantation (Henery etal., 1995) and nuclear alteration (Wiekowski et al., 1993)techniques revealed that this ‘repressor’ is absent from thecytoplasm of early 1-cell embryos (not simply excluded fromtheir paternal pronucleus), and is produced in the cytoplasmsometime between S-phase in a 1-cell embryo and formationof a 2-cell embryo where it can operate within any nucleus,regardless of its parental origin or ploidy. The potency of thisrepression then increases as development proceeds to the 4-cell stage.

Repression of promoter activity at the beginning of mousedevelopment appears to be mediated through chromatinstructure, because it can be relieved either by treating the cellswith butyrate or, in cleavage stage embryos (≤2 cells), bylinking the promoter or replication origin to an embryo-responsive enhancer (Wiekowski et al., 1993; Majumder etal., 1993). Butyrate and other inhibitors of histone deacety-lase increase the fraction of hyperacetylated core histones(Yoshida et al., 1995) and thereby stimulate transcriptionfrom specific genes (Seigneurin et al., 1995; Yoshida et al.,1995; Arts et al., 1995), consistent with the fact that tran-scriptionally active eukaryotic genes are generally associatedwith acetylated core histones (Tazi and Bird, 1990; Hebbeset al., 1988, 1994; Jeppesen and Turner, 1993; Sommervilleet al., 1993). In mouse embryos, the fact that butyrate canstimulate promoter activity in the maternal pronucleus(‘repressed’) but not in the paternal pronucleus (‘unre-pressed’) of a 1-cell embryo shows that this stimulationresults from changes in chromatin structure on the injectedplasmid rather than from increased synthesis of transcriptionfactors, a change that would be expected to affect bothpronuclei. In fact, inhibition of histone deacetylase simplystimulates synthesis of transcription dependent proteins at theonset of zygotic gene expression without changing the overallpattern of protein synthesis (Wiekowski et al., 1993; Worradet al., 1995). Enhancers can substitute for butyrate in stimu-

lation of promoter activity only after formation of a 2-cellembryo, because prior to this event, an enhancer specific co-activator activity is missing (Majumder et al., 1997). In vitro,enhancers do not stimulate transcription unless the DNAsubstrate is organized into chromatin (reviewed by Majumderet al., 1993; Paranjape et al., 1994). In vivo, enhancers havelittle, if any, effect on promoters injected into cleavage stageembryos treated with butyrate (Wiekowski et al., 1993;Majumder et al., 1993). Therefore, the primary role ofenhancers is not simply to provide additional transcriptionfactors to facilitate formation of an active initiation complex,but to relieve the repression of weak promoters by chromatin.

Here we report changes in the synthesis and modification ofchromatin bound histones at the beginning of mouse develop-ment that support the conclusion that formation of a 2-cellmouse embryo is accompanied by changes in chromatinstructure that can repress the activity of promoters and repli-cation origins (summarized in Fig. 1). Histone H4 is diacety-lated in 1-cell embryos and then progressively deacetylatedduring early cleavage stages, while synthesis of histones H2A,H2B and H1 does not begin until the late 1-cell stage and relieson maternally inherited mRNA. Somatic histone H1 does notappear until the 4-cell stage (Clarke et al., 1992). These eventshave parallels in frog development where histone H4 also isstored in eggs as a diacetylated form and then progressivelydeacetylated after zygotic gene expression begins at theblastula stage (Dimitrov et al., 1993). Histone deacetylaseinhibitors can then induce expression of specific genes(Almouzni et al., 1994). The linker histone changes from thematernal histone H1 variant (B4) at the mid-blastula transitionto the somatic histone H1 variant at the end of gastrulation,resulting in specific repression of oocyte 5S RNA geneexpression (Bouvet et al., 1994; Kandolf, 1994), and the initialactivation of H1 synthesis occurs entirely by mobilizingmaternal transcripts that then disappear by the early gastrulastage (Woodland et al., 1979).

MATERIALS AND METHODS

Radiolabeling of mouse histonesOocytes and embryos were cultured as previously described (DePam-philis et al., 1988; Wiekowski et al., 1991, 1993). Mouse oocytes wereisolated from 14- to 16-day-old [C57BL/6J × SJL/J]F1 females.Mouse 1-cell embryos were isolated from 8- to 10-week-old[C57BL/6J × SJL/J]F1 females 18 hours after injection of humanchorionic gonadotropin (post-hCG) and mating with [C57BL/6J ×SJL/J]F1 males. Two-cell embryos were isolated from pregnantfemales at 38 hours post-hCG. Nascent histones in mouse oocytes andpreimplantation embryos were characterized as described inWiekowski and DePamphilis (1993). In general, nascent histoneswere labeled with 3H-arginine and 3H-lysine before lysing the cellsin the presence of butyrate to inhibit histone deacetylase, and PMSFand bisulfite to inhibit proteases. A similar cell lysate was preparedfrom mouse fibroblasts and combined with the oocyte or embryolysate in order to provide an excess of unlabeled (carrier) histones.The nuclei were then isolated, disrupted in the presence of EDTA, lowsalt, butyrate and protease inhibitors to release chromatin, and thechromatin was washed and then sonicated before extracting histonesinto acid. The acid soluble fraction was combined with acetone to pre-cipitate histones which were stored at −80°C for not more than 1 weekbefore analysis. Additional details are provided in figure legends. Themobilities and relative amounts of various proteins exhibited somevariability among the three to twenty examples obtained for eachexperiment. Representative patterns are shown in each figure.

Unless otherwise indicated, 1-cell embryos were labeled from 20to 26 hours post-hCG, and 2-cell embryos from 40 to 46 hours post-hCG. Oocytes were maintained in 100 µg/ml dibutryl-cAMP to arrestthem in prophase of meiosis I and labeled for 24 hours. Proliferating3T3 mouse fibroblasts were cultured in Dulbecco’s modified Eagle’smedium supplemented with 10% calf serum and labeled for 24 hours.

Fig. 2. Identification of mouse histones. (A) SDS-gel electrophoresis: hiand then fractionated by electrophoresis in a 12% polyacrylamide-SDS g1993). Individual calf thymus histones (Boehringer-Mannheim) were runmM butyrate. Proteins were visualized by staining gels with Coomassie fibroblasts into 0.4 N sulfuric acid and then fractionated by electrophore0.375% Triton X-100, 5% acetic acid, and 8 M urea gel using a Bio-Rad

A

1149Histone synthesis and transcription repression

Where indicated, 2.5 mM or 10 mM sodium butyrate was added tothe medium 4 hours before addition of labeled amino acids.

RESULTS

Identification of mouse histonesMouse histones were identified by three criteria: their solu-bility in acid, their migration during electrophoresis in poly-acrylamide gels containing sodium dodecyl sulfate (SDS), andtheir migration in polyacrylamide gels containing Triton X-100/acetic acid/urea (TAU). Proteins such as histones with anet positive charge are soluble in acid. Therefore, to establishthe identity of mouse histones, proteins were extracted frommouse fibroblasts into 0.4 N sulfuric acid and then subjectedto SDS-gel electrophoresis in parallel with purified calfthymus histones (Fig. 2A). SDS-gel electrophoresis separatesproteins primarily according to their molecular masses,although proteins with a net positive charge (e.g. histones)sometimes migrate anomalously (von Holt et al., 1989). Themigration rates of mouse histones (H4>H2A>H2B>H3>H1)were indistinguishable from those of calf histones, and wereconsistent with previous studies (Bonner et al., 1980). Basedon amino acid composition, core histones H2A, H2B, H3 andH4 are between 11 and 14.5 kDa (van Holt et al., 1989) butmigrated as though they were 13 to 17 kDa proteins. HistoneH1 is 22 kDa, but migrated as though it was a 34 to 38 kDaprotein.

TAU-gel electrophoresis fractionates histones according totheir charge as well as their molecular mass, thereby allowing

stones were extracted from mouse fibroblasts into 0.4 N sulfuric acidel using a Bio-Rad Protean IIxi System (Wiekowski and DePamphilis, in parallel. Where indicated, cells were cultured in the presence of 10

Blue. (B) TAU-gel electrophoresis: histones were extracted from mousesis in a 15% acrylamide/bisacrylamide (19:1, Bio-Rad) gel containing Mini-Protean II Dual Slab Cell (Wiekowski and DePamphilis, 1993).

B

1150 M. Wiekowski and others

identification of post-translational modifications such as acety-lation (Lennox and Cohen, 1989). Since the relative migrationsof histones during TAU-gel electrophoresis are strongly influ-enced by the composition of the gel (Zweidler, 1978), optimalconditions were developed for fractionation of mouse histones(Fig. 2B). Under these conditions, the migration pattern ofmouse histones (H4>H2B>H3>H1>H2A) was consistent withprevious studies (Bonner et al., 1980), and mouse histones co-migrated with their corresponding calf histones.

Acetylation of lysine residues decreases the mobility ofhistones during TAU-gel electrophoresis, because each acety-lation event neutralizes one positive charge on the protein(Ruis-Carrillo et al., 1975). For example, histones extractedfrom mouse fibroblasts treated with butyrate were separatedduring TAU-gel electrophoresis into five bands representinghistone H4 with 0 to 4 acetyl groups (Fig. 2B). In contrast,histones from butyrate treated cells migrated identically tothose from untreated cells during SDS-gel electrophoresis (Fig.2A). The increased amounts of proteins observed with butyratetreated cells reflected increased protein synthesis (Wiekowskiet al., 1993).

Staggered synthesis of core histones in 1-cellembryosThe relatively small size (2.5×10−4 the volume of a frog egg)and low number (~25 zygotes per pregnant female) of embryosavailable restricted analysis of histone composition to nascentproteins that could be labeled by culturing oocytes or embryosin the presence of 3H-lysine and 3H-arginine. Since these twoamino acids comprise 18% to 21% of the amino acids in eachof the five major histones (von Holt et al., 1989), the amountof radiolabel per mole of each histone was nearly equivalent.Histones were then acid-extracted from purified chromatinpreparations in the presence of an excess of unlabeled mouseproteins prepared from 3T3 fibroblasts. Gels were stained firstwith Coomassie Blue to visualize total proteins and thensubjected to fluorography to visualize 3H-labeled proteins.Nascent 3H-histones were identified by superimposing the

Fig. 3. Analysis of nascent histones by SDS-gel electrophoresis. (A) Nashours post-hCG) and 2-cell embryos (40-47 hours post-hCG) were labellysine (2,000 µCi/ml, 90-100 Ci/mmol) for the times indicated post-hCGthen soaked in ‘En3Hance’ (Dupont), dried under vacuum, and exposed histones by fluorography. Nascent histone variants in cell extracts were istained gel (unlabeled mouse 3T3 cell histones added prior to extractionand 2-cell embryos that were cultured either with (+) or without (−) 4 µg

A B

resulting autoradiogram over the stained gel. In addition, 3H-histones from mouse 3T3 fibroblasts were run in parallel lanes.

Fractionation of nascent core histones by SDS-gel elec-trophoresis revealed that the pattern of chromatin bound nascentcore histones changed from oocytes to 1-cell embryos to 2-cellembryos (Fig. 3A). Core histones synthesized in 2-cell mouseembryos closely resembled those synthesized in mouse fibro-blasts. However, the fraction of nascent H2A and H2B was over-represented in oocytes and under-represented in 1-cell embryos,suggesting that the pattern of core histone synthesis changedduring development of fertilized eggs to 2-cell embryos.

Development of mouse embryos can be synchronized bytriggering ovulation through injection of human chorionicgonadotrophin (hCG). Fertilization in vivo occurs ~12 hourspost-hCG, paternal and maternal pronuclei appear ~18 hourspost-hCG, DNA replication begins ~23 hours post-hCGfollowed by the first mitosis and formation of a 2-cell embryoat ~32 hours post-hCG (Fig. 1). In early 1-cell embryos (19-26 hours post-hCG), the amount of nascent histones H3 andH4 was greater than the amount of nascent histones H2A andH2B (Fig. 3B). In 2-cell embryos (40-47 hours post-hCG), theopposite was true: synthesis of H2A and H2B was greater thansynthesis of H3 and H4. Occasionally the amount of nascentH2B in mouse fibroblasts was over represented, which mayreflect changes in histone synthesis as a function of the fractionof cells in S-phase (Osley, 1991).

TAU-gel electrophoresis of these histone preparationsconfirmed that early 1-cell embryos synthesized histones H3and H4, but not H2B (Fig. 4A). Synthesis of histone H2Boccurred concurrent with formation of a 2-cell embryo (~32hours post-hCG). Histone H2A could not be distinguishedfrom other basic 3H-proteins under these TAU-gel conditions.The 1-cell stage was also characterized by synthesis of twounidentified acid-soluble proteins (X and Y, Fig. 4A).

Synthesis of histone H1 begins in late 1-cellembryosPurified histone H1 from calf thymus migrated as two

cent histones in mouse 3T3 fibroblasts, oocytes, 1-cell embryos (19-26ed in the presence of 3H-arginine (400 µCi/ml, 60-70 Ci/mmol) and 3H-. Histones were analyzed as Fig. 2A, except that the stained gels were

to X-OMAT AR film (Kodak) at −80°C in order to visualize 3H-dentified by superimposing the autoradiogram (3H-histones) over the). (B) Nascent histones were labeled for the times indicated in 1-cell/ml aphidicolin.

1151Histone synthesis and transcription repression

prominent bands during SDS-gel electrophoresis (Fig. 2A).The same bands were also identified in the acid solubleproteins extracted from mouse 3T3 fibroblasts (Figs 2A,3A,B), and they were the only acid-soluble proteins that cross-reacted with a monoclonal antibody specific for histone H1 ina western blot (data not shown). These two histone H1 variantswere synthesized in mouse oocytes (Fig. 3A). However,following fertilization, synthesis of histone H1 was not evidentuntil formation of a 2-cell embryo, and then only the fastermigrating H1 variant was observed (Fig. 3A). When 1-cellembryos were arrested at the beginning of their S-phase withaphidicolin (a specific inhibitor of replicative DNA poly-merases), histone H1 synthesis began between 26 and 40 hourspost-hCG, the time when 1-cell embryos normally underwentcleavage into 2-cell embryos (Fig. 3B). This conclusion wasconfirmed by fractionating nascent histones by TAU-gel elec-trophoresis. A prominent band of nascent histone H1 appearedbetween 26 and 30 hours post-hCG (Fig. 4A).

Acid-soluble proteins from early 1-cell embryos (19-26hours post-hCG) generally contained one or two faint bandsthat migrated in the vicinity of histone H1 during SDS-gel elec-trophoresis (Fig. 3) and 8 distinct bands that migrated in thevicinity of histone H1 during TAU-gel electrophoresis (Fig. 4).Thus, early 1-cell embryos either failed to synthesize histoneH1, or synthesized their own histone H1 variants. To distin-

Fig. 4. Analysis of nascent histones by TAU-gelelectrophoresis. One-cell and 2-cell embryos were labeledfor the times indicated before nascent histones wereanalyzed by TAU-gel electrophoresis. (A) Histonessynthesized in embryos developing in vitro. (B) Histonessynthesized in embryos cultured with 4 µg/ml aphidicolin.(C) Histones synthesized in embryos cultured either with(+) or without (−) 11 µg/mL of α-amanitin.

A

guish between these two possibilities, nascent 3H-histones inembryos from the 1-cell to 8-cell stage were extracted with per-chloric acid and then fractionated by TAU-gel electrophoresisin parallel with 3H-histones that had been extracted withsulfuric acid from 2-cell embryos. While all histone subtypesare soluble in sulfuric acid, histone H1 is selectively solubi-lized in perchloric acid (Johns, 1964; Ohsumi and Katagiri,1991). The efficiency of this extraction procedure wasmonitored by staining the gels with Coomassie Blue tovisualize total protein (Fig. 5A). Comparison of these data withthe results of fluorography of the same gel revealed thatsynthesis of 3H-histone H1 began in late 1-cell embryos (24-29 hours post-hCG), reaching its maximum rate in early 2-cellembryos (36-41 hours post-hCG). The rate of histone H1synthesis was reduced in late 2-cell embryos (43-48 hours post-hCG), and then restored again in 4-cell and 8-cell embryos(Figs 5B and 7B). This change in the rate of histone H1synthesis may reflect its transition from maternal to zygoticgene dependence.

Histone synthesis in fertilized eggs is independentof cell cleavage, DNA replication and DNAtranscriptionIn most somatic cells, histone synthesis is restricted to S-phase,that period during cell proliferation when DNA replication

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1152 M. Wiekowski and others

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Fig. 5. Identification of histone H1 by selective extraction inperchloric acid. Histones were labeled in 1-cell, 2-cell and 4-cellembryos for the times indicated and then extracted using perchloricacid. Early 1-cell embryos were labeled from 19 to 24 hours post-hCG, late 1-cells from 24 to 29 hours, early 2-cells from 36 to 41hours, late 2-cells from 43 to 48 hours, and 4-cells from 57 to 62hours. For comparison, total histones were extracted from 2-cellembryos using sulfuric acid. Histones were fractionated by TAU-gelelectrophoresis. Except for the first lane (No Embryos), each lanecontained 80 embryos. (A) Total proteins stained by CoomassieBlue. (B) Nascent proteins visualized in the same gel byfluorography.

occurs. When DNA replication is interrupted, histone mRNArapidly disappears from the cytoplasm (Osley, 1991). Todetermine whether histone synthesis in fertilized mouse eggswas also dependent on DNA replication, 1-cell embryos werecultured in the presence of aphidicolin, a specific inhibitor ofthe replicative DNA polymerases.

The pattern of histone synthesis was unaffected by aphidi-colin. The relative synthesis of core histones in early 1-cellembryos (19-26 hours post-hCG) was unchanged (Fig. 3).Moreover, the pattern of core histone synthesis between 26 and40 hours post-hCG changed to the pattern observed in 2-cellembryos, even though aphidicolin prevented these cells fromundergoing DNA replication and cleavage into 2-cell embryos.Histone H1 synthesis began between 26 and 30 hours post-hCG in both developing (Fig. 4A) and S-phase arrested 1-cellembryos (Fig. 4B). Similarly, synthesis of histone H2B was notevident until the time when 1-cell embryos normally became2-cell embryos, regardless of the presence or absence of aphidi-colin; by ~30 hours post-hCG, the pattern of nascent core

histones in arrested 1-cell embryos was indistinguishable fromthat in 2-cell embryos (Fig. 4). Histone synthesis was easier toobserve in S-phase arrested 1-cell embryos, because theyunderwent a reduction in overall protein synthesis that beginsat 40 hours post-hCG (Wiekowski et al., 1991). These datademonstrate that regulation of histone synthesis in fertilizedmouse eggs is independent of DNA replication and cellcleavage. The same was true for protein bands X and Y thatwere unique to 1-cell embryos.

Addition of α-amanitin, a specific inhibitor of RNA poly-merases II and III, to developing 1-cell embryos did not affectthe pattern of core histone synthesis (Fig. 4C), although thesame concentration of α-amanitin prevented synthesis of thetranscription requiring complex of proteins that marks theonset of zygotic gene expression (Nothias et al., 1996).Therefore, synthesis of histones in 1-cell and early 2-cellembryos must be directed by maternal mRNA rather thanzygotic mRNA. This would account for the apparent inde-pendence of histone synthesis from cell cycle events in fer-tilized eggs. The same appeared true for all acid-solubleproteins, included bands X and Y that were unique to 1-cellembryos.

Core histones are acetylated in 1-cell and 2-cellembryosAcetylated core histones are frequently associated with tran-scriptionally active genes, and the extent of core histone acety-lation is reflected by the acetylated state of chromatin boundhistone H4, the core histone most easily characterized. TAU-gel electrophoresis fractionated histone H4 into five distinctspecies containing from zero to four acetyl groups (H4, H4Ac1,H4Ac2, etc.). In order to provide internal standards of acety-lated histone H4, a separate group of oocytes and embryoswere cultured in the presence of 2.5 mM butyrate (an inhibitorof histone deacetylase) before and during the radiolabelingperiod, conditions that were previously shown to produce themaximum stimulation of plasmid-encoded promoter activity(Wiekowski et al., 1993). Thus, individual acetylated forms ofnascent histone H4 were identified by comparing the 3H-histone H4 pattern in untreated cells with that in butyratetreated cells and by superimposition of fluorograms ontoCoomassie Blue stained gels in order to determine the positionof 3H-histones with those of the unlabeled carrier histonesprovided by mouse fibroblasts.

Mouse fibroblasts provided convenient standards for com-parison with oocytes and embryos. The bulk of both nascent(3H-labeled protein) and total (Coomassie Blue stainedprotein) histone H4 in mouse fibroblasts was either H4 orH4Ac1 (Fig. 6), and treatment of fibroblasts with butyrateproduced an equal distribution of all five forms of acetylatedhistone H4 (0-4, Fig. 6). Mouse oocytes were similar to mousefibroblasts in that oocyte chromatin contained histone H4,H4Ac1 and H4Ac2, but differed from fibroblasts in thatbutyrate converted all oocyte chromatin bound histone H4 toH4Ac2-4. However, after fertilization, all of the chromatinbound histone H4 synthesized in either 1-cell or 2-cell embryoscontained at least 2 acetyl groups, and butyrate treatmentproduced significant amounts of H4Ac3 and H4Ac4. Therefore,histone H4 synthesized in 1-cell and 2-cell embryos was hyper-acetylated relative to histone H4 synthesized in oocytes andfibroblasts. A reduction in histone H4 acetylation began in 4-

1153Histone synthesis and transcription repression

Fig. 6. Acetylation of nascent histone H4. Embryos were culturedwith 3H-arginine and 3H-lysine in the presence or absence of 2.5 mMbutyrate: 1-cell embryos (19-26 hours post-hCG), 2-cell embryos(40-47 hours), 4-cell embryos (57-62 hours), and 8-cell embryos (69-74 hours). Oocytes from 14-day-old females were labeled for 24hours. Mouse 3T3 fibroblasts were labeled for 20 hours. Nascenthistones were fractionated by TAU-gel electrophoresis. Total proteinswere stained with Coomassie Blue (CB lanes), and 3H-proteinsvisualized by fluorography (remaining lanes). Positions of histonesH4Ac0 to H4Ac4 are indicated. The CB lanes contain two bands(histone H4 and H4Ac1), the concentration of which sometimesexceeds the limits of gel resolution.

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cell and 8-cell embryos. These changes were quantified bydetermining the fraction of 3H-histone H4 at various stages indevelopment that contained two or more acetyl groups (Fig.7B).

To determine whether or not the acetylated state of nascenthistone H4 reflected changes in the overall acetylation patternof histone H4, cells were stained first with a rabbit antiserumdirected against acetylated histone H4 and then with FITC-con-jugated anti-rabbit IgG to visualize its intracellular localiza-tion. Quantifying the relative amounts of fluorescence/µm2

observed in each type of nucleus (Fig. 8) confirmed conclu-sions derived from analysis of nascent histone H4 (Fig. 7). Thenuclear concentration of acetylated histone H4 was essentiallythe same in 1-cell and 2-cell embryos, and its amount and dis-tribution in paternal and maternal pronuclei in the same 1-cellembryo as well as the remaining polar body were indistin-guishable. The concentration of acetylated histone H4 in 1-celland 2-cell embryos was 2.5-fold greater than in oocytes orfibroblasts. Treatment with butyrate increased the nuclear con-centration of acetylated histone H4 from 1.7-fold to 7.4-fold,and resulted in a uniform distribution throughout the nucleusin each cell type. Moreover, butyrate increased the nuclear con-centration of acetylated histone H4 in oocytes and 2-cellembryos about 2.5 times more effectively than in 1-cell

Fig. 7. Changes in promoter activity, nascent histone H4 acetylationand histone H1 synthesis that accompany the onset of zygotic geneactivation. (A) A plasmid (ptkluc) containing the HSV thymidinekinase (tk) promoter driving the firefly luciferase gene was injectedinto the maternal nucleus of oocytes, either the maternal or paternalnucleus of 1-cell embryos, or the zygotic nucleus of 2-cell embryos.Oocytes were arrested in prophase of their first meiosis. One-cellembryos were arrested as they entered S-phase. Some injected 2-cellembryos were arrested in S-phase (36-41 hours post-hCG), some asthey entered S-phase at the 4-cell stage, and some were allowed tocontinue development to the 8-cell stage (data from Wiekowski et al.,1991, 1993). ‘nd’ means experiment was not done. (B) The fractionof radiolabel in each of the five forms of nascent 3H-histone H4 wasdetermined using either densitometry of fluorograms, or directscanning of TAU-gels in a phosphorimager (e.g. Fig. 6 and others).The fraction of nascent histone H4 that contained 2, 3 or 4 acetylgroups was calculated for each cell type. Each lane contained 80oocytes or embryos. (C) The relative amounts of nascent histone H1extracted in perchloric acid (e.g. Fig. 5 and others) were calculatedrelative to early 2-cell embryos where the greatest amounts of 3H-histone H1 were routinely observed. The fraction of cells stainedwith antiserum against mouse somatic cell histone H1 are data fromClarke et al. (1992).

embryos. The ability of butyrate to stimulate the activity of aplasmid-encoded promoter (HSV thymidine kinase promoter)injected into the nuclei of oocytes or embryos, or electropo-rated into fibroblasts paralleled its ability to increase thenuclear concentration of acetylated histone H4 (Fig. 8B), con-sistent with the hypothesis that butyrate stimulates promoteractivity in mouse oocytes and preimplantation embryos byreducing chromatin mediated repression through increasedacetylation of core histones.

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Fig. 8. Changes in the amount of nuclear acetylated histone H4reflected changes in promoter activity. (A) Oocytes, 1-cell embryos,2-cell embryos and fibroblasts that were either untreated or treatedwith butyrate as described above were stained with rabbit antiserumR6/5 specific for histone H4 acetylated at lysine 5 (Turner andFellows, 1989), and the stained regions were visualized by labelingthe cells with FITC-conjugated anti-rabbit IgG as described byWorrad et al. (1995). No preferential labeling of nuclei was observedwith preimmune serum from the same rabbits. Late 1-cell mouseembryos were stained in parallel with either mouse oocytes, late 2-cell embryos or 3T3 fibroblasts and then examined immediately byconfocal microscopy. The amount of fluorescence/µm2 was measuredin 20 to 40 nuclei. The mean fluorescence intensity for each pair wasthen normalized to the average amount of fluorescence observed inthe paternal pronucleus (pPN) of 1-cell embryos observed in eachexperiment in order to compare the relative concentrations ofacetylated histone H4. Error bars indicate s.e.m. mPN indicatesmaternal pronucleus. (B) The ratio of histone H4Ac2-4 present innuclei of untreated cells to nuclei of cells cultured in butyrate wascalculated from the data in A. The ratio of luciferase activityproduced from ptkluc injected into untreated oocytes or embryoscompared to embryos cultured in 2.5 mM butyrate was calculatedfrom the data of Wiekowski et al. (1993) or from the results of ptklucelectroporated into mouse 3T3 fibroblasts.

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Fig. 9. Aphidicolin delays the ability of embryos to represstranscription. One-cell mouse embryos were cultured with or without4 µg/ml aphidicolin. At 25 hours post-hCG, either ptkluc (h, s) orpF101tkluc (j, d) was injected into the paternal pronucleus. At thetimes indicated, embryos were segregated into 1-cell embryos withtwo pronuclei (h, j) and 2-cell embryos (s, d). Each group wasassayed for the amount of luciferase mRNA produced as describedby Nothias et al. (1996). The results were calculated relative to themaximum level of luciferase mRNA per embryo observed.Maximum luciferase activity was observed routinely in S-phasearrested 1-cell embryos.

Aphidicolin suppresses expression of repressoractivityPrevious studies (see Introduction) have shown that repressoris absent from the paternal pronucleus and cytoplasm of S-phase arrested 1-cell embryos. The maternal pronucleusexhibits some repressor activity that presumably is inheritedfrom the oocyte nucleus. Since repressor is present in bothnuclei and cytoplasm of 2-cell embryos, arresting DNAsynthesis in 1-cell embryos appears to prevent expression ofrepressor activity. To test this hypothesis, plasmids encoding

the firefly luciferase gene were injected into the paternal pronu-cleus of 1-cell mouse embryos cultured either in the absenceor presence of aphidicolin, and the amount of luciferase mRNAproduced was measured as a function of time elapsed. Theluciferase gene was linked either to the herpes simplex virusthymidine kinase (tk) promoter alone (ptkluc) or to the tkpromoter with the F101 polyomavirus enhancer 600 bpupstream (pF101tkluc). Luciferase gene expression isdependent on a linked promoter, and the F101 enhancerproduces the highest levels of luciferase enzyme activity inmouse cleavage stage embryos (Nothias et al., 1995; Majumderand DePamphilis, 1995). A quantitative assay for luciferasemRNA (Nothias et al., 1996) revealed directly the effect ofembryo development on luciferase gene transcription. Controlswere carried out to confirm that deletion of the tk promotereliminated luciferase gene transcription, and that the F101enhancer stimulated tk promoter activity when plasmids wereinjected into 2-cell embryos (Nothias et al., 1996, and data notshown), consistent with previous studies in which luciferaseenzyme activity was measured (Henery et al., 1995).

In the absence of aphidicolin (Fig. 9A), most (~90%) of theinjected 1-cell embryos developed into 2-cell embryos andluciferase gene transcription was strongly repressed, regardlessof the presence or absence of the F101 enhancer whichfunctions efficiently in 2-cell embryos. In contrast, both ptklucand pF101tkluc were actively transcribed in 1-cell embryos

1155Histone synthesis and transcription repression

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Fig. 10. Arresting 1-cell embryos in S-phase reduces proteinsynthesis, including histone H1. (A) Groups of 50 S-phase arrested1-cell embryos were cultured in the presence of [35S]methionine for5 hour periods ending at the times indicated. Total protein synthesiswas measured as acid insoluble 35S-labeled material. Other S-phasearrested 1-cell embryos were injected with ptkluc at the timeindicated and luciferase activity was measured at various timesthereafter. Each point is the average of 35 to 50 embryos. (B and C)Histone H1 synthesis was measured in S-phase arrested 1-cellembryos (4 µg/ml aphidicolin added 19 hours post-hCG), in early 2-cell embryos that had developed from 1-cell embryos in vitro, and inearly 2-cell embryos that had developed from 1-cell embryos in vitroin the presence of 25 µg/ml α-amanitin added at 27 hours post-hCG.(B) Gel stained with Coomassie Blue. (C) Fluorogram of same gel.Each lane contains 80 embryos. The H2SO4 extract contained 20 2-cell embryos. Conditions were as in Fig. 5.

that did not develop into 2-cell embryos. These results wereconsistent with previous studies in which luciferase geneexpression was monitored by luciferase enzyme activity(Henery et al., 1995), and were interpreted as ‘irreversible’repression that occurred before enhancer activation factorswere present in sufficient quantity to prevent repression. Sinceplasmid DNA does not replicate in these embryos, therepressed state cannot be reprogrammed at a later time.

In the presence of aphidicolin (Fig. 9B), most of the injected1-cell embryos remained as 1-cell embryos. These S-phasearrested 1-cell embryos also actively transcribed the injectedluciferase gene, but they did so about 20 times more efficientlythan 1-cell embryos in the absence of aphidicolin. Moreover,the small fraction of 2-cell embryos that formed in the presenceof aphidicolin (<10%) also transcribed the injected gene andto the same extent as did the S-phase arrested 1-cell embryos,regardless of the presence or absence of the F101 enhancer.These data are consistent with the hypothesis that aphidicolinprevented expression of repressor activity and thus allowedtranscription, regardless of whether or not cell cleavageoccurred.

Aphidicolin suppresses histone synthesisExperiments described above reveal that synthesis of histonesH1, H2A and H2B begins in late 1-cell embryos, and thatdeacetylation of core histones begins in late 2-cell or 4-cellembryos (Fig. 6), suggesting that one or both events contributesto appearance of repressor activity upon formation of 2-celland 4-cell embryos (Fig. 7). Moreover, repressor was notproduced when 1-cell embryos were cultured in the presenceof aphidicolin, regardless of whether or not they underwentcleavage, suggesting that aphidicolin may affect histonesynthesis or modification. However, no changes were detectedin either the types or relative amounts of histones (or otherbasic proteins) synthesized in 1-cell embryos cultured in thepresence of aphidicolin (Figs 3, 4). What did change in thepresence of aphidicolin was the total amount of proteinsynthesis. The translational capacity of 1-cell embryos culturedin aphidicolin rapidly decreased between 35 and 40 hours post-hCG (Fig. 10A), that time when early 2-cell embryos hadformed and zygotic gene expression had begun (Fig. 1). By thetime development would have reached the 4-cell stage, proteinsynthesis was effectively stopped.

The effect of aphidicolin on total protein synthesis was alsoevident with histone synthesis. For example, the amount ofhistone H1 synthesized in 1-cell embryos that developed intoearly 2-cell embryos in the absence of aphidicolin was ~4-foldmore than the amount synthesized in S-phase arrested 1-cellembryos cultured for the same length of time in the presenceof aphidicolin (Fig. 10B and C). This H1 synthesis resultedfrom maternally inherited mRNA, since it was not inhibited byα-amanitin (Fig. 10C). Increased H1 synthesis in the presenceof α-amanitin presumably resulted from increased stability ofhistone mRNA.

DISCUSSION

Chromatin structure can repress transcription, and the extent ofthis repression depends on at least three parameters: thepresence of a complete histone octamer, low acetylation of core

histones, and the presence of a linker histone such as H1(Paranjape et al., 1994). Although H3 and H4 alone canorganize DNA into nucleosome-like structures, the DNAremains accessible to transcription factors. Addition of H2Aand H2B begins to mask the DNA from transcription compo-nents such as RNA polymerase II (Baer and Rhodes, 1983) andTFIIIA (Hayes and Wolffe, 1992), but addition of histone H1,which requires the prior addition of H2A and H2B (Hayes etal., 1994), condenses chromatin into a 30 nm fiber that

1156 M. Wiekowski and others

represses transcription (Bouvet et al., 1994; Juan et al., 1994;Kandolf, 1994; Paranjape et al., 1994; O’Neill et al., 1995).Both acetylation of core histones (Lee et al., 1993; Vettese-Dadey et al., 1996) and removal of histone H1 (Juan et al.,1994; Ura et al., 1995) can facilitate the ability of some tran-scription factors to bind to chromatin, and together these twoparameters strongly facilitate transcription (Ura et al., 1997).

The transition from a late 1-cell mouse embryo to a 4-cellembryo, the period wherein zygotic gene expression begins, isaccompanied by an increasing ability to repress the activitiesof promoters and replication origins (see Introduction). Thisrepression can be relieved by either butyrate or enhancers,suggesting that it is mediated through chromatin structure. Infact, changes in the synthesis and modification of chromatinbound histones at the beginning of mouse development (sum-marized in Fig. 1) are consistent with this hypothesis: repres-sion was greatest when chromatin contained all five newly syn-thesized histones and core histones were minimally acetylated(growing oocytes and ≥late 2-cell embryos).

Histone synthesis in mouse preimplantationembryosAll five major histones were synthesized in mouse oocytes, butonly histones H3 and H4 were synthesized in early 1-cellembryos. Synthesis of histones H2A, H2B and H1 did notresume until the late 1-cell/early 2-cell stage. Histone synthesisin 1-cell and early 2-cell mouse embryos was independent ofboth DNA replication, DNA transcription and cell cleavage(Figs 3, 4). Therefore, these histones were translated frommRNA inherited from the oocyte. Histone H1 synthesized in1-cell and 2-cell embryos was identified by its mobility duringSDS and TAU gel electrophoresis that was indistinguishablefrom histone H1 isolated from mouse fibroblasts (Figs 3, 4),and by the fact that histone H1 could be selectively extractedwith perchloric acid (Fig. 5). Intriguingly, this early form ofhistone H1 was not detected using anti-H1 antibodies untilformation of a 4-cell embryo during either mouse (Clarke etal., 1992) or bovine (Smith et al., 1995) development. Sincethese antibodies were made against somatic cell histone H1,these results suggest the existence of both maternal and zygotichistone H1 variants. In other studies, addition of α-amanitin tolate 2-cell embryos (G2-phase) prevented expression ofsomatic histone H1 at the 4-cell stage (Clarke et al., 1992),suggesting that synthesis of somatic histone H1 begins duringthe second phase of zygotic gene activation (Nothias et al.,1995). Moreover, synthesis of somatic histone H1 was sensitiveto inhibition of DNA replication at the 4-cell stage, consistentwith coupling of somatic histone gene expression to DNAreplication during the third cell proliferation cycle (Clarke etal., 1992). These results reveal that histone H1 is first expressedfrom maternally inherited mRNA in the late 1-cell and early 2-cell stages of mouse development, followed by a transition toexpression from zygotic genes in the late 2-cell to 4-cell stages.

Consistent with a transition from maternal to zygotic histonegene expression, mRNAs for histones H2A, H2B and H3 havebeen identified in fertilized mouse eggs, and the levels of thesemRNAs have been observed to decrease ~10-fold by the mid2-cell stage (Giebelhaus et al., 1983; Graves et al., 1985). Insomatic mammalian cells (Osley, 1991), histone synthesis isregulated by activating translation of histone mRNA present inG1-phase by the onset of S-phase, by coupling synthesis of

new histone mRNA to DNA replication, and by coupling thestability of histone mRNA to DNA replication (histone mRNAis rapidly degraded after completion of S-phase). Therefore,translation of maternally inherited histone mRNA may beactivated by S-phase in 1-cell embryos, while transcription ofhistone genes in the zygote may be coupled to S-phase in the2-cell embryo. During the long G2-period in 2-cell embryos,maternally inherited histone mRNA will be degraded, and the4-cell stage will be dependent primarily on transcription ofzygotic histone genes. This would account for the observationthat histone synthesis occurs independently of DNA replica-tion in 2-cell embryos (Figs 3, 4; Kaye and Church, 1983), butthat these two events are coupled by the blastocyst stage (Kayeand Church, 1983).

Establishment of chromatin mediated repression atthe beginning of mouse developmentVirtually all of the chromatin bound nascent histone H4 in fer-tilized mouse eggs and 2-cell embryos was diacetylated (Figs6, 7), and immunofluorescence staining with antibodiesdirected against acetylated histone H4 revealed that the con-centrations of total nuclear H4Ac2-4 in 1-cell and 2-cellembryos was at least twice those in oocytes or fibroblasts (Fig.8). Moreover, synthesis of histones H2A, H2B and H1 did notbegin until the late 1-cell/early 2-cell stage and somatic histoneH1 was not detected by immunofluorescence staining until the4-cell stage in either mouse or bovine embryos (Clarke et al.,1992; Smith et al., 1995). Since hyperacetylated core histonescan faciliate assembly of nucleosomes onto nonreplicatingDNA (Cotten and Chalkley, 1985), and H4Ac2 is used specif-ically in the assembly of nucleosomes at replication forks(Sobel et al., 1995; Kaufman et al., 1995), fertilized eggs arepoised to both remodel and replicate parental chromosomes.However, DNA injection experiments (see Introduction) haveshown that chromatin assembled under these conditions (early(G1-phase) 1-cell embryos) exhibit reduced levels of chromatinmediated repression, consistent with the combined effects ofthe presence of H4Ac2 and the absence of histone H1 on tran-scription of chromatin assembled in vitro (Ura et al., 1997).Therefore, prior to zygotic gene expression, newly assembledchromatin must be modified in order to repress transcription sothat genes can be activated selectively during the subsequentphases of development.

DNA injection experiments reveal that repression isreestablished following S-phase in 1-cell embryos andincreases as development proceeds to the 4-cell stage (seeIntroduction). This appears to be accomplished in two stages.First, the appearance of chromatin bound nascent histonesH2A, H2B and H1 began just prior to formation of a 2-cellembryo. Thus, although nuclei in both 1-cell and 2-cellembryos contained similar concentrations of H4Ac2-4 (Fig.8), repression of transcription will be brought on by theincreasing presence of histone H1 (Ura et al., 1997). Second,the level of acetylation in chromatin bound histone H4 beganto decrease in 4-cell embryos and was nearly complete in 8-cell embryos (Fig. 6). This reduction in core histone acetyla-tion should facilitate establishment of a repressive chromatinstructure. In fact, increasing the concentration of H4Ac2-4 byinhibition of histone deacetylase with butyrate, trichostatin Aor trapoxin also increases the amount of hyperacetylatedhistone H4 in 2-cell to 8-cell embryos (Fig. 8; Worrad et al.,

1157Histone synthesis and transcription repression

1995; Thompson et al., 1995), consistent with the effect ofthese inhibitors on stimulating promoter activity in eitherinjected plasmids (Wiekowski et al., 1993; Majumder et al.,1993) or transgenes (Thompson et al., 1995). Stimulation wasgreatest in 4-cell embryos. When the rate of protein synthesis,including histone synthesis, was suppressed by arresting 1-cell embryos at the beginning of S-phase (Fig. 10), theirability to repress transcription from an injected gene did notmaterialize, even when some embryos slipped through theircheck point controls and formed a 2-cell embryo (Fig. 9).Therefore, protein synthesis is required in late 1-cell embryosin order to establish chromatin mediated (i.e. butyratesensitive) transcription repression in cleavage stage embryos.The fact that synthesis of histones H2A, H2B and H1 did notbegin until the late 1-cell/early 2-cell stage was consistentwith their role in chromatin-mediated repression, althoughother proteins also may be involved. Thus, the extent of tran-scription repression (Fig. 7A) is increased as the amount oflinker histone is increased (Fig. 7C) and the amount ofdiacetylated core histone is decreased (Fig. 7B; Fig. 8, top).Together, these changes in histone synthesis and modificationare consistent with a developmental acquisition of chromatinmediated repression which, in the mouse and perhaps othermammals as well, begins in the late 1-cell embryo and iscompleted by the 4-cell stage.

One puzzle is why the maternal pronucleus in an S-phasearrested 1-cell embryo can repress transcription from aninjected promoter, but not the paternal pronucleus(Wiekowski et al., 1993; Nothias et al., 1996). This differenceappears to be due to chromatin-mediated repression becausebutyrate can stimulate promoter activity in the maternalpronucleus to the level observed in the paternal pronucleus,but butyrate does not stimulate promoter activity in thepaternal pronucleus (Wiekowski et al., 1993). Surprisingly,both pronuclei contain similar concentrations of H4Ac2-4(Fig. 8). Therefore, the maternal pronucleus must inherit oneor more additional proteins from the oocyte, such as histonesH2A, H2B and H1, that allow repression to occur. Since thepaternal pronucleus is derived from the sperm, it must beprovided with histones that are either inherited from theoocyte or synthesized after fertilization from maternalmRNA. Based on synthesis of histone H4, sufficient corehistones are synthesized in oocytes to support two or threerounds of DNA replication (Wassarman and Mrozak, 1981).However, this free histone pool may be sequestered withinthe maternal nucleus by chaperone proteins used in chromatinassembly, as appears to be the case with the much largerhistone pool in amphibian oocytes (Patterton and Wolffe,1996). Thus, following meiotic maturation and fertilization,the fraction and composition of the maternal histone poolavailable to the paternal pronucleus remains to be determined.Chromatin assembled in the two pronuclei could differ incomposition and thereby provide the fertilized egg with anopportunity for genomic imprinting by masking sequences inone genome but not in the other.

Monoclonal antibodies directed against mammalian histone H1were provided by John Brenneman (University of California, Davis)and Missag Parseghian (University of California, Irvine). Anti-serumagainst acetylated histone H4 was provided by Bryan Turner (Uni-versity of Birmingham Medical School, Birmingham, UK).

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(Received 16 December 1996 - Accepted 6 March 1997)