INTRODUCTION

Early embryogenesis in Xenopus proceeds from a fertilized eggto a blastula of approximately 4,000 cells in a series of 12 rapidcell divisions. Throughout this period there is no transcriptionalactivity (Prioleau et al., 1995; Hair et al., 1998), and the assemblyof new chromatin from almost continuously replicating DNA islargely dependent on a maternal pool of histones and assemblyfactors. In general, chromatin assembly involves the associationof pre-acetylated core histones with replicating DNA andsubsequent stabilization of nucleosomes by histone deacetylation(Verreault et al., 1996). For instance, newly synthesized histoneH4 is acetylated at defined lysine residues by a cytoplasmichistone acetyltransferase (HAT B; Sobel et al., 1994). Sequencingof the amino terminus of newly synthesized histone H4 from arange of different organisms has shown it to be diacetylated, atlysine residues 5 and 12 (Sobel et al., 1995), although yeast HATB activity on histone H4 in vitro shows some variation from thispattern (Kleff et al., 1995; Parthun et al., 1996). Deacetylationwould normally occur soon after histone deposition in newchromatin but information is only now becoming available as tohow histones are accessed by histone deacetylase (HDAC) andhow the deacetylation reaction is regulated.

The aspect of histone deacetylation that has received most

attention recently is its role in repression of the activity ofspecific sets of genes (reviewed by Grunstein, 1997; Wolffe,1997). For instance, associations of HDAC1 (Taunton et al.,1996) with the retinoblastoma protein Rb (Brehm et al., 1998;Magnaghi-Jaulin et al., 1998), or with the corepressor of themammalian Mad/Max complex, mSin3 (Alland et al., 1997;Hassig et al., 1997; Laherty et al., 1997), or with N-CoR, thecorepressor of the thyroid hormone receptor (Alland et al.,1997; Heinzel et al., 1997), all serve to target the deacetylasecomplex to specific chromatin sites. These particularassociations would appear to tether the HDAC complex to thepromoter regions of the relevant subsets of genes, throughinteraction of the corepressors with transcription factors: Rbwith E2F1 (Brehm et al., 1998; Luo et al., 1998); mSin3 withMad-Max (Alland et al., 1997; Hassig et al., 1997; Laherty etal., 1997), and with the thyroid hormone receptor (Alland etal., 1997; Heinzel et al., 1997). Once tethered to the gene, theHDAC activity can target nearby nucleosomes, deacetylatingcore histones and contributing to transcriptional repression.

Various multiprotein complexes containing histonedeacetylases have been described, and some have been isolatedand partially characterized. Complexes include yeast HDAC-A and HDAC-B (Carmen et al., 1996; Rundlett et al., 1996)and a range of complexes containing histone deacetylases

2441Journal of Cell Science 112, 2441-2452 (1999)

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Reversible acetylation of core histones plays an importantregulatory role in transcription and replication ofchromatin. The acetylation status of chromatin isdetermined by the equilibrium between activities of histoneacetyltransferases (HATs) and histone deacetylases(HDACs). The Xenopus protein HDACm shows sequencehomology to other putative histone deacetylases, but itsmRNA is expressed only during early development. BothHDACm protein and acetylated non-chromosomal histonesare accumulated in developing oocytes, indicating that thekey components for histone deposition into new chromatinduring blastula formation are in place by the end ofoogenesis. Here we show that the 57 kDa HDACm proteinundergoes steady accumulation in the nucleus, where it isorganized in a multiprotein complex of approx. 300 kDa. Asecond, major component of the nuclear complex is the

retinoblastoma-associated protein p48 (RbAp48/46), whichmay be used as an adaptor to contact acetylated histonesin newly assembled chromatin. The nuclear complex hasHDAC activity that is sensitive to trichostatin A, zinc ionsand phosphatase treatment. The 57 kDa protein serves asa marker for total HDAC activity throughout oogenesis andearly embryogenesis. The active HDACm complex and itsacetylated histone substrates appear to be kept apart untilafter chromatin assembly has taken place. However,recombinant HDACm, injected into the cytoplasm ofoocytes, not only is translocated to the nucleus, but also isfree to interact with the endogenous chromatin.

Key words: Chromatin, Histone deacetylase, RetinoblastomaAp48/46, Oocyte nucleus

SUMMARY

Maternal histone deacetylase is accumulated in the nuclei of Xenopus

oocytes as protein complexes with potential enzyme activity

James Ryan1, Alexander J. Llinas1, Darren A. White2, Bryan M. Turner2 and John Sommerville1,*1School of Biomedical Sciences, Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16 9TS, UK2Department of Anatomy, University of Birmingham Medical School, Birmingham, B15 2TT, UK*Author for correspondence (e-mail: js15@st-and.ac.uk)

Accepted 11 May; published on WWW 24 June 1999

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HDAC1-3 in vertebrate cells (Grunstein, 1997). HDAC activityhas been identified in protein complexes containing the methylCpG-binding protein MeCP2 together with mSin3, which maytarget deacetylase to methylation sites in the DNA of mammals(Nan et al., 1998) and Xenopus (Jones et al., 1998).Furthermore HDAC activity has been detected in complexescontaining a Snf2 ATPase together with the retinoblastoma-associated protein RbAp48/46 (Wade et al., 1998). The Snf2ATPase activity of the Mi2 protein has been shown recently toremodel nucleosomes in vitro and is isolated in a proteincomplex also containing HDAC1/2 (Tong et al., 1998; Zhanget al., 1998). There thus exists a common nucleosomeremodelling/histone deacetylating complex.

Most of the earlier reports describe protein complexesobtained from tissue or cell homogenates and involve multistepchromatographic separations. To avoid any spurious proteinassociations, the approach taken in the studies reported herehas been to examine the full complement of complexescontained in the nuclei of Xenopus oocytes by hand-isolatingnuclei (germinal vesicles) sealed under oil (Paine et al., 1992)and releasing the contents under physiological conditions.

Xenopus AB21 (data bank accession number: X78454) wasinitially recognized as a homologue of the yeast gene regulatorRPD3 (Vidal and Gabor, 1991), which was later shown also tobe a homologue of human HDAC1 (Taunton et al., 1996).There has now been cloned and identified an additional seriesof putative histone deacetylases, in yeast (Rundlett et al.,1996), Caenorhabditis elegans (Waterston et al., 1992),Drosophila melanogaster (De Rubertis et al., 1996; Johnson etal., 1998) and mouse (Bartl et al., 1997), all of which appearto have a conserved region stretching from near the aminoterminus to more than halfway through the protein sequence.It is likely that this conserved region represents the enzymecore responsible for deacetylase activity (Ladomery et al.,1997; Leipe and Landsman, 1997).

The mRNA encoded by Xenopus AB21 is expressed only inoocytes, remains stable in embryos up to neurula and hassequence characteristics of a maternal message. Its proteinproduct, HDACm (maternal histone deacetylase; Ladomery etal., 1997), is synthesized during oogenesis and decreases inamount after mid-blastula, when the cell cycle slows down toa normal rate and cells start to differentiate. Here we show thatHDACm is a marker for histone deacetylase activity duringearly development and is likely to represent the active subunitthat deacetylates core histones after assembly of earlyembryonic chromatin. The accumulation of HDACm-containing, multiprotein complexes in oocyte nuclei has beenstudied and various properties of the complexes are described.

MATERIALS AND METHODS

Proteins and antibodiesGlutathione S-transferase (GST) fusion proteins were expressed frompGEM (Pharmacia) vectors and isolated on glutathione-Sepharose 4Baccording to the manufacturer’s instructions. The fragments ofXenopus AB21 cDNA used in the constructs have been described(Ladomery et al., 1997). Antibodies to ∆R/∆H and ∆V fusion proteinswere raised in rabbits, as were antibodies directed against thesynthetic peptide CDEKTDSKRVKEETKSV, representing thecarboxy-terminal 16 amino acid residues of HDACm plus anadditional N-terminal cysteine (anti-Cpep). Antibodies to RbAp48

were similarly raised in rabbits using the synthetic peptideCENIYNDEDPEGSVDPEGQGS, representing the carboxy-terminal20 amino acid residues of the human protein (Qian et al., 1993).Rabbit antibodies to mSin3 were obtained from Santa Cruz Biotechand rabbit antibodies to MeCP2 were kindly provided by Dr AdrianBird (University of Edinburgh). All antibodies used gaveimmunoreactive bands corresponding to the predicted sizes of theirantigens on SDS-PAGE of Xenopus nuclear extracts.

Oocyte extractsOvary fragments from Xenopus laevis females were gently mixed for1-2 hours (until the tissue had disaggregated) in calcium-free OR-2medium (Evans and Kay, 1991) containing 2 mg/ml of collagenase(Sigma, Type I). Defolliculated oocytes were washed through severalchanges of calcium-containing medium and allowed to recover for 16-24 hours before being used. Oocytes were sorted into individual stagesaccording to Dumont (1977). Pools of 50 oocytes (stages I-IV) or 25oocytes (stages V and VI) were collected and homogenized by cycling20 times through a pipette tip in three volumes of homogenizationbuffer: 0.1 M KCl, 2 mM MgCl2, 2 mM dithiothreitol, 20 mM Tris-HCl, pH 7.5. After centrifugation at 10,000 g for 15 minutes in aswing-out rotor (Sorvall SW24), the clarified supernatant (SN10) wascarefully removed. Nuclei and cytoplasms were isolated underparaffin oil as described previously (Paine et al., 1992) and wereresuspended in homogenization buffer for further extraction.

ImmunostainingTotal protein, minus pigment and yolk, equivalent to two oocytes,embryos or cytoplasms or to ten nuclei, was separated by SDS-PAGEand transferred to nitrocellulose membranes (Schleicher & Schuell).Blots were incubated for 2 hours at 20°C with IgG (0.2-0.5 µg/ml)isolated from either anti-∆V, anti-∆R/∆H or anti-Cpep antiserum (orwith the antiserum diluted 1:2,000), then incubated with peroxidase-conjugated anti-rabbit IgG (1:10,000, Chemicon) and developed usingthe ECL (Amersham) procedure.

Nuclei, dissected from fixed oocytes or isolated from unfixed oocytesunder oil, were immunostained as whole-mount preparations(Klymkowsky and Hanken, 1991). Oocytes were fixed overnight inDent’s fixative (methanol:dimethylsulphoxide, 4:1) in siliconized watchglasses and nuclei were then dissected out. Alternatively, isolated nucleicontained in paraffin oil were layered over a cushion of Dent’s fixativein a siliconized plastic reaction tube and the phases were gently mixedby inverting the tube for 1 minute, eventually recovering the nuclei fromthe fixative. Fixed nuclei were washed several times over 5 hours inTris-buffered saline (TBS) and then incubated for 24 hour in primaryantisera diluted 1:25 in 95% fetal calf serum (FCS), 5% DMSO. Afterwashing the nuclei again in TBS, they were incubated for a further 24hours in FITC-conjugated goat anti-rabbit IgG (Chemicon) at a dilutionof 1:50 in FCS/DMSO. After further washing in TBS, the nuclei weredehydrated in methanol for 90 minutes and cleared in benzylbenzoate:benzyl alcohol (2:1). Whole-mount nuclei were thenexamined by confocal laser microscopy (BioRad system).

Lampbrush chromosomes were immunostained as describedpreviously (Sommerville et al., 1993), using primary antisera diluted1:500 with 10% FCS in TBS. The secondary antibody was FITC-conjugated goat anti-rabbit IgG (Chemicon) at a dilution of 1:500 inFCS:TBS.

ImmunoprecipitationPorous glass beads linked to protein A (ProSep, Bioprocessing Ltd)were washed in 0.1 M borate buffer (pH 9.0) and 5 µl samples of beadswere incubated with 5 µl of antiserum for 2 hours at 20°C. The beadswere then washed several times in borate buffer before chemicalcrosslinking of IgG to protein A with 20 mM dimethyl pimelimidatein borate buffer for 30 minutes. The beads were next washed in 0.2 Methanolamine (pH 8.0) to block any further crosslinking, then in elutionbuffer (0.1 M glycine, pH 3.0) and finally in TBS containing 0.1%

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Tween 20 (TBST). Samples of 50 oocyte nuclei were then lysed in 0.3ml of TBST containing 1 mg/ml bovine serum albumin: the lysate wasmixed with the antibody-beads for 90 minutes at 20°C. The beads werewashed thoroughly with TBST to remove unbound material and thenwith 4×50 µl samples of elution buffer to remove bound protein.

Injection into oocytes of protein Ab21The pBlueScript cDNA clone AB21 was linearized by digestion withXhoI and run-off transcripts were synthesized using T3 RNApolymerase (mMessage mMachine, Ambion). About 50 µg of cappedRNA, containing the short 5′UTR, the entire coding region and the3′UTR to the poly(A) tail, was generated from 1 µg of template DNA.Samples of this RNA were translated in reticulocyte lysate (Promega)and labelled with 0.6 mCi/ml of [35S]methionine (1000 Ci/mmole,Amersham) according to the manufacturer’s protocol. Protein wasrecovered in the fraction excluded from a Sephadex-G50 spin columnand Ab21 was immunoprecipitated as described above. Sets of 100 mid-vitellogenic (stage III/IV) or late vitellogenic (stage V) oocytes wereisolated and 10 nl samples containing approx. 10 pg of Ab21 wasinjected into the cytoplasm of each oocyte. At various times up to 32hours after injection, nuclei and cytoplasms were isolated from groupsof five oocytes (Paine et al., 1992) and the ratio of concentration ofradioactive protein [N/C] was calculated for each time point asdescribed previously (Vancurova et al., 1995). Proteins truncated at thecarboxyl end, particularly Ab21∆H, which lacks the entire charged taildomain, were synthesized in a similar fashion and also injected into thecytoplasm of oocytes. Synthesis of RNA in injected and non-injectedoocytes was studied by labelling the oocytes in vivo with 0.2 mCi/mlof [3H]uridine (27 mCi/mmole, Amersham). Again, samples of fiveoocytes were isolated at various time points over a 32 hour period.Radioactivity incorporated into RNA was measured for each time point.Lampbrush chromosomes from injected oocytes were prepared asdescribed previously (Sommerville et al., 1993) except that thespreading solution contained 15 mM KCl, 5 mM NaCl, 1 mM MgCl2,0.05% formaldehyde, 1 mM Tris-HCl, pH 7.5 (Gall et al., 1991).

Enzyme assayDeacetylase substrates were prepared essentially as described bySendra et al. (1988). Purified rat liver histones (2 mg) or a peptidecorresponding to the 18 amino-terminal residues of histone H4 (1 mg)were dissolved in 0.5 ml of 50 mM sodium borate, pH 9.0, and mixedwith 6 mCi of [3H]acetic anhydride (Amersham, 8.9 Ci/mmole, 12mCi/ml in dioxane). After 3 hours at 0°C the mixture was adjusted to0.25 M HCl and the peptides precipitated with 12 volumes of acetone,washed three times in cold acetone and dried under vacuum. Histonedeacetylase activity was assayed according to Li et al. (1996). Up to100 µl of nuclear extract was mixed with 150 µl of assay buffer (25mM sodium phosphate/citric acid pH 7.0), 20 µl of 3H-acetylatedpeptide or histone mix (about 1.2×106 dpm, dissolved in assay buffer)and dH2O up to a total volume of 300 µl. After 1 hour at 37°C thereaction was stopped by adding acetic acid and HCl to 0.12 M and0.72 M, respectively, followed by 2 ml of ethyl acetate (Sigma).Samples were then vortexed and centrifuged at 9000 g for 1 minute.Half the volume of ethyl acetate was removed and the dissolved[3H]acetate was measured by scintillation counting. Samples weredephosphorylated by adding 2 i.u. alkaline phosphatase (Sigma, typeIII) to 2.5 µl of embryo extract, or 20 µl of oocyte extract, adjustingto pH 8.3, and incubating for 1 hour at 22°C prior to assay.

Gradient analysisClarified supernatants (SN10 fractions) were layered on 10%-25%glycerol gradients made up in 0.1 M KCl, 2 mM MgCl2, 2 mMdithiothreitol, 0.2% Nonidet P-40, 20 mM Tris-HCl, pH 7.5. Aftercentrifugation at 30,000 or 36,000 rpm in a 6×5 ml swing-out rotor(Beckman SW55Ti) at 0°C for 16 hours, the tube contentswere fractionated. Fractions were analysed for HDACm (byimmunoblotting) and HDAC activity (by the in vitro assay). Size was

calculated using protein standards in parallel gradients: haemoglobin(67 kDa); IgG (160 kDa) or alcohol dehydrogenase (180 kDa);apoferritin (443 kDa); microglobulin (670 kDa); IgM (960 kDa). Theprotein standards were detected in gradient fractions by staining gelswith Coomassie Brilliant Blue after SDS-PAGE.

RESULTS

HDACm and potential histone deacetylase activityare accumulated through oogenesisThe maternal histone deacetylase, HDACm, consists of aregion highly conserved between various putative deacetylases(Ladomery et al., 1997) plus a more variable region terminatingwith a sequence of 100 residues rich in charged side chains(Fig. 1A). It is hypothesized that the conserved regionrepresents the enzyme core and the variable region contains

Fig. 1. (A) Representation of the HDACm protein showing thelocation of the conserved region and the variable region, with thehighly charged tail domain boxed black. Parts of the proteinsequence used to raise the antibodies anti-∆R/∆H, anti-∆V and anti-Cpep are shown below. (B) Immunoblots of soluble extract from theequivalent of two stage VI oocytes, using anti-∆R/∆H, anti-∆V andanti-Cpep. The same extract is blotted with antibodies raised againstthe α-subunit of casein kinase II (CK2α) as a control. (C) Specificimmunoprecipitation of HDAC activity from a stage VI oocyteextract. Samples eluted from anti-Cpep resin (black columns) arecompared with samples eluted from control resin (cross-hatchedcolumns). Unbound (UB), first wash (W1), second wash (W2), thirdwash (W3) and resin-bound (bound) fractions were assayed forHDAC activity and expressed as a percentage of the total activityapplied to the resin.

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regulatory sequences, which may be specific to the particulardeacetylase. Antibodies were raised against GST-HDACmfusion proteins containing the conserved region (anti-∆R/∆H)and the charged tail domain (anti-∆V), and against a syntheticpeptide representing the carboxy-terminal 16 amino acidresidues of HDACm (anti-Cpep). All three antibodiesrecognize a protein of 57 kDa present in extracts from Xenopusoocytes separated by SDS-PAGE (Fig. 1B).

The immunoblotting profiles were complemented by an invitro assay (Li et al., 1996) to monitor levels of histonedeacetylase (HDAC) activity in various oocyte and embryoextracts. Although free histones were used as the substrate,recent assays using nucleosomal histones have shown nosubstantial difference in HDAC specificity for singlenucleosomal, as opposed to free, histones (Zhang et al., 1998).

To confirm that the HDACm protein is itself associatedwith the HDAC activity detected in oocyte extracts,immunoprecipitation experiments were carried out using anti-Cpep. Of the total amount of enzyme activity incubated withaffinity-selected IgG, over 50% was recovered bound to proteinA-Sepharose after thorough washing of the resin (Fig. 1C). Thiscompares with 0.6% of the activity bound to the resin in theabsence of specific IgG. The affinity-bound fraction retained itsactivity while still coupled to the resin and, in all, about 95%of the input activity was recovered from the accumulatedfractions, indicating the robust nature of the enzyme.

Coincidence in the expression of the HDACm antigen with

recovered HDAC activity was examined further using extractstaken from oocytes at different stages of development. As canbe seen (Fig. 2A), the amount of HDACm per oocyte increasessteadily through the course of oogenesis, to reach a peak infull-grown (stage VI) oocytes. It was shown previously that thisamount of immunoreactive protein remains fairly constantthrough oocyte maturation (M), fertilization and the rapid cellcleavage stages of early embryogenesis, but decreases afterblastula (Ladomery et al., 1997). Measurement of HDACactivity in the same samples (Fig. 2B) shows that the profile ofactivity through oogenesis and early embryogenesis is verysimilar to that of the HDACm antigen itself. Although thelevels of enzyme activity are similar in oocytes and earlyembryos, these values represent approx. 4,000-fold higherlevels in oocytes compared to mid-blastula embryos on a percell basis. The presence of such vast excess of HDACm inoocytes emphasizes the degree of control over HDAC activitythat must be exerted during oogenesis when thetranscriptionally active lampbrush chromosomes maintainacetylated forms of histone H4 (Sommerville et al., 1993) andstored histone H4 is being accumulated in a diacetylated state(Almouzni et al., 1994).

HDACm is accumulated in the nuclei of growingoocytes The ability to collect hand-isolated nuclei and cytoplasmssealed under oil and to examine them microscopically (Paine

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Fig. 2. Levels of soluble HDACm protein and HDAC activity in oocytes at different stages of development. (A) Immunoblot, using anti-Cpep,of extracts from different stage oocytes. Each track contains the protein equivalent of two oocytes. Stages are from I to VI plus stage VI oocytesmatured (VIM) by treatment with progesterone for 16 hours. The fusion protein GST-∆V (∆V; 0.1 µg) is used as a positive control at 48 kDa.(B) Histone deacetylase activity assayed in extracts from oocytes (stages as in A) or embryos (C, 8-cell; B, mid-blastula; G, gastrula). Activityis expressed as dpm of [3H]acetate released from histone by extracts equivalent to a single oocyte or embryo (total column height). Eachcolumn height represents the average of two assays. Also indicated is the relative amount/cell (%, black column height), which becomesincreasingly smaller to disappearing (G) as cell divisions increase in number. (C) Immunoblot, using anti-Cpep, of nuclear (N) and cytoplasmic(C) proteins from stage III to stage VI oocytes. Each track contains the protein equivalent of ten nuclei or two cytoplasms. (D) Histonedeacetylase activity assayed in extracts from nuclei and cytoplasms. Activity, expressed as dpm, is normalized to values per nucleus and percytoplasm.

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et al., 1992), ensures accurate estimates of the relative amountsof materials in the two compartments. Immunoblots,comparing the relative contents of soluble HDACm in thenucleus and cytoplasm of oocytes at different stages ofdevelopment (stage III to stage VI), show that HDACm ispresent at much higher concentrations in the nucleus. Thisindicates that the 57 kDa HDACm protein undergoestranslocation into the nucleus after synthesis (Fig. 2C). Due tothe large disparity between the total amounts of proteinrecovered, loading on the gels is 10 nuclei/track and 2cytoplasms/track. Nevertheless, it is evident that the totalamount of HDACm in the nucleus is still much higher than inthe cytoplasm. On assaying HDAC activity from the sameextracts, and normalizing to values per nucleus and percytoplasm, it is confirmed that there is much more activity ina single nucleus than in a single cytoplasm, with the expectedincreases in amounts between oogenic stages (Fig. 2D). Interms of concentration of both HDACm protein and HDACactivity, the relative values for nuclei would be even higher,due to the 50:1 ratio of available cytoplasmic: nuclear volumes.In full-grown (stage VI) oocytes, it is estimated that theconcentration of HDAC activity is at least 400-fold higher inthe nucleus than in the cytoplasm.

Characterization of HDAC activity present in oocyteand embryo extractsTwo types of HDAC activity found in yeast cells (Carmen etal., 1996) can be differentiated on the basis of sensitivity to thespecific inhibitor trichostatin A (TSA; Ikegami et al., 1993).Whereas HDAC-A is inhibited by 80% in the presence of 10nM TSA, HDAC-B is inhibited by less than 20% under thesame assay conditions (Carmen et al., 1996). On titratingHDAC activity with increasing concentration of TSA, extractsfrom either oocytes or embryos show a single transition ofsensitivity, developing approx. 80% inhibition at 0.5 ng/ml(approx. 2 nM) TSA (Fig. 3A). Therefore the HDAC activitypresent in Xenopus oocytes and early embryos appears to bemainly, if not exclusively, of the HDAC-A type, that is of thevertebrate HDAC1 class.

It has been noted previously that yeast deacetylase issensitive to the divalent cation Zn2+ (Carmen et al., 1996).HDAC activity in extracts from both oocytes and embryos isabout 80% inhibited by 1 mM ZnCl2 (Fig. 3B). No similareffect is obtained with Mg2+, indicating that excess zinc maybe specifically disrupting the structure of HDACm. A possiblerole of Zn2+ in the structure of deacetylases has been suggestedpreviously (Ladomery et al., 1997; Johnson et al., 1998).Furthermore, it has been reported (Brosch et al., 1992) that thesubstrate specificity of histone deacetylase in Zea mays isinfluenced by treatment with alkaline phosphatase (AP).Treatment of oocyte and embryo extracts with 50 units/ml APat 22°C for 60 minutes results in a loss of 60-90% of the HDACactivity in the in vitro assay (Fig. 3C). Endogenous HDACactivity in nuclear extracts was not increased by preincubationwith ATP to a final concentration of 5 mM (not shown).

HDACm is a component of a multimolecularcomplex with histone deacetylase activityYeast HDAC-A and HDAC-B activities are associated withprotein complexes of approx. 350 and 600 kDa, respectively(Carmen et al., 1996). Various sizes of protein complex

containing HDAC activity have been described for extractsfrom vertebrate cells, ranging from 220 kDa (Li et al., 1996)to 700 kDa (Jones et al., 1998) and even 1.0-1.5 MDa (Wadeet al., 1998). However, the larger complexes described havebeen isolated from whole tissue or cell homogenates that havegone through a series of chromatography steps, includingexposure to ion-exchange resins. Limitations of theseprocedures are that the chance of spurious interactions betweenproteins is increased during shifts in ionic conditions, and that,due to extensive selection, the final product is representativeonly of a particular subclass of complex. To obtain acomprehensive measure of the relationship between molecularsize and deacetylase activity in Xenopus oocytes, wholeoocytes, nuclei and cytoplasms were separated under near-physiological conditions by rate-zonal centrifugation. Clarifiedsupernatants were layered on linear 10%-25% glycerolgradients, which were centrifuged until a 19S marker (IgM)approached, or reached, the bottom of the tubes. The gradientswere then fractionated and analysed for HDACm content (byimmunoblotting) and histone deacetylase activity (using the invitro assay).

Supernatants from full-grown (stage VI) oocytes showHDAC activity sedimenting over a fairly broad range, with apeak at approx. 360 kDa (Fig. 4A). A slightly slowersedimenting (approx. 300 kDa) peak of activity is obtainedfrom nuclei hand-isolated from oocytes under oil (Fig. 4A). Ingeneral, the active complexes obtained from nuclei were moreconsistent in size and composition than complexes obtainedfrom homogenized oocytes. Immunoblot analysis of fractionsfrom both isolated nuclei (Fig. 4C) and cytoplasms (Fig. 4D)confirms that the 57 kDa protein in the approx. 300 kDacomplex is mostly nuclear in origin. Pretreatment of stage VIoocytes with progesterone for 16 hours, which leads to thecompletion of oocyte maturation (including nuclearbreakdown), has no further effect on the sedimentationproperties of the HDAC complex (Fig. 4B). Although theHDACm-containing complex detected in oocytes or nucleiappears as a single, major form, supernatants fromhomogenates of early embryos (at mid-blastula) show a moreheterodisperse sedimentation profile (Fig. 4E), indicating thatsome larger complexes may have formed by this stage.

Fig. 3. Characteristics of oocyte (stage VI) and embryo (blastula)HDAC activity. (A) Sensitivity to trichostatin A (TSA).(B) Sensitivity to zinc ions. (C) Sensitivity to treatment with alkalinephosphatase. Soluble extracts from stage VI oocytes (black columns)and blastulae (hatched columns) were incubated in the absence (−) orpresence (+) of alkaline phosphatase (2 i.u./µl of extract).

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Furthermore, slower-migrating, immunoreactive bandsindicate that HDACm is subject to protein modification atblastula. One modification which has been studied isphosphorylation: the GST-∆V fusion protein is phosphorylatedat specific sites by nuclear casein kinase II (CK2) and slowermigrating forms of HDACm are recovered from the nuclei ofmaturing oocytes and early embryos (A.J.L., J.R. and J.S., inpreparation).

Identification of RbAp48/46 as a second componentof the nuclear HDAC complexIt is unlikely that the HDAC activity sedimenting at a rateindicating a particle mass of approx. 300 kDa consists solelyof a multimer of the 57 kDa HDACm protein. Associations ofHDAC1 with other proteins have been described: for instancewith the retinoblastoma-associated protein p48 (RbAp48/46,Taunton et al., 1996), with the transcription corepressor mSin3(Alland et al., 1997) and with the methylated DNA-bindingprotein MeCP2 (Nan et al., 1998; Jones et al., 1998). In orderto check if these and other candidate partners are present in theoocyte nuclear particles, glycerol gradient fractions wereimmunoblotted with a range of different antibodies. In additionto anti-RbAp48, anti-mSin3 and anti-MeCP2, antibodies thatspecifically recognize histone H4 acetylated at lysine 12(Turner et al., 1989) were used to check the sedimentation rateof the potential HDACm substrate. Positions in the gradientsof the histone chaperones N1/N2 and nucleoplasmin, whichrespectively form complexes with H3/H4 of 120 kDa and withH2A/H2B of 130-170 kDa (Kleinschmidt et al., 1985), weredetected by phospholabelling of the nuclear sample (notshown), and the sedimentation rate of complexes containingnucleoplasmin was also detected by immunoblotting. Oncomparing the various immunoblots (Fig. 5), it can be seen thatthe only substantial overlap in distribution is between HDACmand RbAp48/46, making it possible that most HDACmparticles also contain RbAp48/46. Protein complexescontaining HDAC and mSin3 (Alland et al., 1997) and HDAC,Sin3 and MeCP2 (Nan et al., 1998; Jones et al., 1998) havebeen described previously. However, in the preparationsderived from isolated nuclei, it is seen that the overlaps indistribution are marginal and that only minor amounts ofHDACm can be present in particles containing either Sin3 orMeCP2 (Fig. 5). As described previously (Kleinschmidt et al.,1985), N1/N2 and nucleoplasmin both sediment in proteincomplexes smaller (<200 kDa) than those reported here thatcontain HDACm. It is not surprising, then, that acetylatedhistone H4 sediments in the same size range as its chaperone,N1/N2, and in a particle not associated with its deacetylase.

The possible association of RbAp48/46 with HDACm wasfurther investigated by immunoprecipitation. Anti-Cpep wasbound and chemically crosslinked to protein A immobilizedon glass beads. After incubation of the antibody beads withan extract from hand-isolated nuclei, and after thoroughwashing to remove fortuitously bound protein, RbAp48/46was detected by immunoblotting a fraction eluted with 0.1 Mglycine, pH 3.0 (Fig. 6A). Although HDACm was also eluted,most remained bound to the anti-Cpep beads and could bedetected in later washes using SDS-PAGE sample buffer (notshown). The reciprocal immunoprecipitation was also carriedout: HDACm being bound, along with RbAp48/46, to anti-RbAp48 beads (Fig. 6B). These results indicate that HDACm

and RbAp48/46 are components of a common particle presentin oocyte nuclei. Since less than 20% of the total RbAp48/46was bound by anti-Cpep beads and more than 80% of the totalHDACm was bound by anti-RbAp48 beads, it can beconcluded that that most of the HDACm present in nuclearextracts has associated RbAp48/46, whereas most of theRbAp48/46 may exist in particles lacking HDACm.Immunoblotting of nuclear and cytoplasmic extracts withanti-RbAp48 shows that RbAp48/46 accumulates in oocytenuclei through oogenesis in much the same way as HDACm,with a major increase in amount between stages III and IV(Fig. 6C). The progressive increase in HDAC activity duringoogenesis may well result from a steady assembly in the

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Fig. 4. Sedimentation analysis of protein complexes that containHDACm protein and HDAc activity in full-grown oocytes, maturedoocytes and early embryos. (A) Activity profiles of soluble extractsfrom 50 stage VI oocytes (white squares) and from 100 nucleiisolated from stage VI oocytes (black circles). Peak sedimentationpositions of molecular mass markers (kDa) are shown (crosses).(B) As A, with extracts from stage VI oocytes treated withprogesterone for 0 hours (white squares) and 16 hours (blacksquares). Only matured oocytes were selected for the 16 hoursample. (C) Immunoblot, using anti-Cpep, of stage VI nuclearmaterial recovered from alternative fractions in A. The materialpelleted in the gradient (P) and the fusion protein GST-∆V (0.1 µg)at 48 kDa are included in the blot. (D) As C, but from a gradient usedto separate 50 stage VI cytoplasms. This blot is overexposed to showthe small amount of the 57 kDa protein isolated from the cytoplasm.(E) As C, but from a gradient used to separate the extract from 50blastula-stage embryos.

kDa (+

–––––+)

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nucleus of complexes containing both HDACm andRbAp48/46.

Immunoprecipitates of nuclear extracts with anti-Cpep gaveonly weak signals on blotting with anti-Sin3 and anti-MeCP2(not shown). Since complexes containing Sin3, MeCP2 andHDAC enzyme activity have been isolated from large-scaleovarian preparations (Jones et al., 1998), such complexes mustbe relatively minor to the HDACm-RbAp48/46 complexesdetected here in nuclear isolates.

Localization of HDACm in oocyte nucleiImmunostaining of whole-mount oocytes (not shown) andhand-isolated nuclei (Fig. 7), shows that HDACm (Fig. 7A)and RbAp48/46 (Fig. 7B) are located mainly around theinternal margins of the nucleus, with similar, but not identicaldistributions. Whereas HDACm generally has an asymmetricdistribution (Fig. 7A), RbAp48/46 appears to form acontinuous border (Fig. 7B). Neither protein could be detectedon the active chromatin that constitutes the lampbrushchromosomes in stage IV oocytes and lies internally in thenucleoplasm (not shown). This contrasts with the distributionof acetylated histone H4, which occurs throughout the

nucleoplasm in a largely punctate fashion and on the chromatin(Fig. 7C), which is located towards the centre of the nucleusas detected by DNA staining (Fig. 7D). These results indicatethat most of the HDACm-containing particles are located awayfrom the chromatin and show no particular association withparticles containing stored, acetylated histone H4. However,the considerable overlap seen in the location of HDACm withrespect to RbAp48/46 is compatible with the proposition thatthe HDACm particles contain a subset of the nuclearRbAp48/46 molecules.

Distributions similar to those shown here were also obtainedby immunostaining sectioned ovary with anti-∆V and anti-Cpep (for HDACm) and with anti-RbAp48 (not shown).However, unlike the restricted location of HDACm andRbAp48/46 seen in whole-mount nuclei, anti-Sin3 gives amore uniform nucleoplasmic staining, similar to that shown foracetylated histone H4. This would indicate that only a smallproportion of the Sin3 protein present in oocyte nuclei isassociated with HDACm. Since most of the immunostainingwith anti-MeCP2 activity is located in the cytoplasm of stageVI oocytes (not shown), we would conclude that inclusion ofMeCP2 in HDACm complexes (Jones et al., 1998) might occurto a greater extent at a later stage of development.

Injection of recombinant HDACm into the cytoplasmof oocytes results in its nuclear import, binding tochromatin and chromosome condensationFrom our earlier work (Sommerville et al., 1993) and theexperiments described here, it appears that stored HDACactivity cannot easily access the endogenous hyperacetylatedchromatin present in transcriptionally active lampbrush

Fig. 5. Detection of various proteins in an oocyte nuclear extractseparated by rate sedimentation. The same gradient fractions wereimmunoblotted with anti-Cpep (HDACm); antibodies raised againsta C-terminal peptide corresponding to RbAp48; antibodies tomammalian Sin3, which detects two forms in Xenopus oocytes, asdescribed previously (Jones et al., 1998); antibodies raised againstmouse MeCP2, which recognizes a single band of the appropriatesize (81 kDa); antibodies detecting Xenopus nucleoplasmin (Npl);and antibodies specific for acetylated lysine 12 of histone H4(H4Ac12). The positions of sedimentation markers are indicated(kDa).

Fig. 6. Coprecipitation of HDACm and RbAp48/46 with antibodiesto HDACm and RbAp48. Nuclear extracts from stage VI oocyteswere incubated with anti-Cpep (A) and anti-RbAp48 (B), both ofwhich had been immobilized on protein A beads. Unbound (UB) andeluted (E) fractions were immunoblotted with anti-Cpep and anti-RbAp48. Xenopus RbAp48/46 has apparent molecular masses of 55and 53 kDa on SDS-PAGE. (C) Immunoblot, using anti-RbAp48, ofcytoplasmic (C) and nuclear (N) proteins from stage III-V oocytes.Each track contains the protein equivalent of ten nuclei or twocytoplasms.

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chromosomes. However, it has been shown recently thathistone deacetylase expressed from mRNA injected into thecytoplasm of Xenopus oocytes leads to strong repression oftranscription from a DNA template injected into the nucleus(Wong et al., 1998). In these experiments transcription wasdriven by the TRβA (thyroid hormone receptor) promoterfollowing assembly of its DNA into chromatin. Therefore, wewished to examine the effect on lampbrush chromosomes ofoverexpressing HDACm.

Recombinant Ab21 protein and a truncated version lackingthe carboxy-terminal domain (Ab21∆H) were synthesized in areticulocyte lysate system (Fig. 8A). The identity of the Ab21protein was checked by immunoprecipitation (Fig. 8A).Injection of 35S-labelled Ab21 into the cytoplasm of stage Voocytes resulted in a tenfold concentration of the protein in the

nucleus after 24 hours (Fig. 8B), the kinetics of nuclear uptakebeing similar to those described for nucleoplasmin (Vancurovaet al., 1995). In contrast, Ab21∆H failed to concentratesubstantially in the nucleus (Fig. 8B), a result explained by theabsence of the region containing the putative nuclearlocalization signal of HDACm from the truncated protein.

The effect of nuclear uptake of Ab21 on endogenoustranscription was checked by examining metabolic labelling ofinjected oocytes with [3H]uridine (Fig. 8C). Whereas non-injected oocytes, and injected oocytes treated with TSA,continue to incorporate radiolabel into RNA up to 32 hoursafter injection of Ab21, non-treated, injected oocytes fail to

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Fig. 7. Immunostaining of intact, hand-isolated nucleiviewed by confocal microscopy. Nuclei, isolated underoil from stage VI oocytes, were fixed andimmunostained using: (A) anti-Cpep; (B) anti-RbAp48; (C) anti-H4Ac12; (D) propidium iodide. Thefluorescent image is provided by fluorescein-conjugated anti-rabbit IgG used as the secondaryantibody. The optical sections shown arerepresentative of several images seen through theapproximate centre of the nuclei. The fluorescentpatch at the top left of B is the cap of an adjacentnucleus.

Fig. 8. Nuclear import of Ab21 protein and its effect on RNAsynthesis in oocytes. (A) Autoradiograph showing that Ab21 and its∆H truncation are substantially the only proteins synthesized fromtheir respective RNA transcripts in the reticulocyte lysate system.The 57 kDa Ab21 protein is efficiently immunoprecipitated withanti-Cpep: unbound (UB) and bound and eluted (E) fractions areshown. The composition of the two proteins is indicated to the rightof the gel autoradiographs: the carboxy-tail domain that contains theputative nuclear localization signal is boxed black. (B) Injection ofradiolabelled recombinant proteins into the cytoplasm of stage Voocytes results in much higher levels of nuclear accumulation ofAb21 (filled circles) than of Ab21∆H (open squares). Each pointrepresents the nuclear:cytoplasmic ratio of concentration ofradioactivity, [N]/[C], in a sample of five oocytes. Similar amounts oftotal radioactive protein were recovered from all samples injectedwith Ab21 (crosses). (C) Incorporation of [3H]uridine into RNA innon-injected oocytes (open squares) and in oocytes injected withAb21, untreated (filled circles) and treated with 5 ng/ml of TSA(open circles). Each time point represents radioactivity recoveredfrom samples of five oocytes.

2449Histone deacetylase in oocytes

incorporate much after 10 hours. It appears that, at about 10hours after injection of Ab21, sufficient de novo HDAC activityhas accumulated in the nucleus to cause a run-down of RNAsynthesis. However, a more direct approach to examining theeffects of injected Ab21 on transcription in oocytes is throughobservation of their lampbrush chromosomes, structures whosetranscriptional activity is manifested as multiple loopsextending from axes of condensed chromatin.

Lampbrush chromosomes from mid-vitellogenic (stageIII/IV) oocytes are not immunostained with any of theantibodies directed against HDACm nor with anti-RbAp48,even after injection with Ab21∆H (Fig. 9, top row). However,chromosomes isolated from oocytes at 24 hours after injectionof Ab21 are clearly immunostained with anti-∆V (Fig. 9,middle row left). This immunostaining is not uniform

throughout the chromatin; rather it shows reactive foci alongthe axes of the chromosomes. In addition, the chromosomesshow extensive loop retraction and considerable foreshorteningcompared with chromosomes isolated from non-injectedoocytes or oocytes injected with Ab21∆H (Fig. 9, top row).These changes are indicative of widespread inactivation oftranscription. Incubation of oocytes injected with Ab21 in thepresence of 5 ng/ml of TSA appears to inhibit loop retractionand chromosome condensation, while immunoreactivityremains. However, the signal does appear fainter due to itsdistribution over the much longer chromosomal axes. Since, aswe have confirmed (Fig. 3A), TSA is a highly specific inhibitorof HDAC activity in oocytes, it would seem that the observedchromosome foreshortening is due to promiscuous activity ofthe recombinant HDACm. It is not clear how ectopicallysupplied HDACm might escape the normal restraints andlocate on endogenous chromatin. The most likely possibility isthat it first interacts with excess RbAp48/46, which itself canbind chromatin. To examine this possibility, chromosomesfrom oocytes injected with Ab21 were also tested for de novoimmunostaining using anti-RbAp48. As seen in Fig. 9 (right-hand columns), anti-RbAp48 gives immunostaining patternswhich are essentially the same as the patterns obtained withanti-∆V (left-hand columns). Again, injected oocytes treatedwith TSA retain immunostained protein on their chromosomalaxes, but the chromosomes, themselves, are not noticeablyaltered. These results are consistent with the interpretation thatexcess HDACm, injected into oocytes, is rapidly imported intothe nucleus and, at some stage, associates with endogenousRbAp48/46. That injected Ab21 interacts with endogenousRbAp48/46 is confirmed by immunoprecipitation of 35S-labelled Ab21 from nuclear extracts with anti-RbAp48 (notshown). Protein complexes containing these two componentsare then free to interact with the chromatin of lampbrushchromosomes, bringing about premature loop retraction andchromosome condensation. That these changes are dependenton deacetylase activity is suggested by their sensitivity to TSA.

DISCUSSION

Relationship between the HDACm protein and HDACactivityIn all of the samples tested from Xenopus oocytes and earlyembryos, there is a direct correspondence between the amountof the 57 kDa HDACm protein detected and the level of HDACactivity assayed. This relationship is apparent in a range ofdifferent situations, particularly in the developmentalexpression pattern and in sedimentation characteristics. Theobservation that over 50% of recovered HDAC activity isimmunoprecipitated by antibodies directed against thecarboxy-terminal peptide of HDACm indicates that HDACm isthe major HDAC activity present in oocytes. No othercrossreacting bands are detected on immunoblotting solubleextracts from oocytes with antibodies directed against regionsof HDACm conserved in all other histone deacetylasesdescribed (Ladomery et al., 1997).

HDAC complexes in oocytesIn several respects, the HDAC complexes described here aresimilar to the HDAC-A complexes described for S. cerevisiae(Carmen et al., 1996; Rundlett et al., 1996). In yeast, two

Fig. 9. Morphology and immunostaining of lampbrush chromosomesisolated from stage IV oocytes injected with recombinant proteinsAb21 and Ab21∆H. Oocytes were injected with similar amounts(approx. 10 pg) of Ab21∆H (top row) and Ab21 (middle and bottomrows). Chromosomes shown on the bottom row are representative ofpreparations from injected oocytes treated with TSA. The variouschromosome preparations were immunostained with either anti-∆V(left-hand columns) or anti-RbAp48 (right-hand columns). Phasecontrast (Ph) partners of the immunofluorescent images are shown.Each panel shows a representative chromosomal bivalent. In thephase-contrast images, the chromosomes appear either extended,extending well out of the field shown, with fuzzy fringes of lateralloops (top and bottom rows), or contracted, contained within the fieldshown, with largely loop-free chromomeric axes (middle row). Thelarge, refractile structures are nucleoli and the smaller, darkstructures are snurposomes, neither of which show significantimmunostaining.

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distinct types of complex are found: HDAC-A, which has anestimated mass of approx. 350 kDa, deacetylates all four corehistones, and is strongly inhibited by TSA; and HDAC-B,which has a mass of approx. 600 kDa and is relativelyinsensitive to TSA. Most of the 57 kDa HDACm antigen andHDAC activity recovered from late-vitellogenic (stage V/VI)oocytes are found in a peak of material sedimenting withmasses of 300-360 kDa (Figs 4 and 5) and all of the samplesextracted from oocytes and embryos show sensitivity to TSAcomparable to that of yeast HDAC-A, with 80% inhibition ofHDAC activity at a concentration of 0.5 ng/ml (approx. 2 nM)TSA.

Several different types of large multiprotein complexcontaining HDAC activity have been isolated from vertebratecells. The complexity of much of the HDAC activity targetedto specific genes derives from the ability of mSin3 to interactwith both HDAC1/2 and a variety of transcriptionalcorepressors (Alland et al., 1997; Heinzel et al., 1997; Nagy etal., 1997). Other targeted complexes contain the retinoblastomaprotein, Rb, and HDAC1/2, possibly in association withRbAp48/46 (Brehm et al., 1998; Magnaghi-Jaulin et al., 1998;Luo et al., 1998). The coupling of HDAC activity with ATP-dependent nucleosomal remodelling activity generates evenlarger complexes, which contain Snf2-related factors inaddition to HDAC1/2 and associated proteins (Wade et al.,1998; Tong et al., 1998; Zhang et al., 1998).

The 300-360 kDa protein complex described here is smallerthan others described, even those with apparently specificfunctions in Xenopus early development (Jones et al., 1998;Wade et al., 1998). The complex that may recruit histonedeacetylase to methylated DNA consists primarily of MeCP2,Sin3 and HDAC activity, giving an estimated mass of 700 kDa(Jones et al., 1998); the complex expressing Snf2 ATPaseactivity has six major subunits, including Mi-2, RbAp48/46and HDAC, but substoichiometric amounts of Sin3, giving anestimated mass of 1.0-1.5 MDa (Wade et al., 1998). Of thevarious antibodies we used in this study, including anti-Sin3,anti-MeCP2 and anti-RbAp48, only anti-RbAp48 gave majoroverlap in sedimentation, coincidence in immunolocalizationand coprecipitation with HDACm. The apparent discrepancyin Xenopus HDAC particle size can be explained by twoconsiderations: first, large particles are selected by multistepchromatography, whereas our approach has been to show thetotal content of material isolated from nuclei; second, varioustypes of large complex are most likely assembled, whenrequired, from simpler units. The 300-360 kDa complex maywell represent a minimal active complex – a storage form,which can be added to, or adapted, for the purposes of specific,targeted, functions. We have not identified any constituents ofthe 300-360 kDa particle other than HDACm and RbAp48/46.Protein crosslinking experiments (not shown) indicate thateach 300-360 kDa complex contains more than one moleculeof HDACm, yet multimers of HDACm, alone, appear not to besufficient for HDAC activity. As has been shown previously(Verreault et al., 1996), RbAp48/46 interacts directly withhistone N termini and may be necessary to bring thedeacetylase into contact with its acetylated substrates. Wesuggest that a simple complex of HDACm and RbAp48/46 maybe sufficient for the activities that are described here. It ispossible that this complex represents a storage form ofdeacetylase activity unique to oocytes. Further studies will be

required to discover the mechanisms of incorporatingHDACm-RbAp48/46 into the Mi-2-containing nucleosomalremodelling complex (Wade et al., 1998) that may operate inearly embryogenesis.

Sequestering of HDAC activity during oogenesisAccumulation of HDACm-containing complexes, along withtheir associated HDAC activity, presents a problem for oocytesin handling such enormous amounts of potential activity. Asingle full-grown oocyte contains as much HDAC activity as4,000 embryonic cells at blastula. Yet oocytes containchromatin, in the form of lampbrush chromosomes, that ishighly active in transcription, a condition reflected by thewidespread occurrence of hyperacetylated forms of histone H4.On immunostaining lampbrush chromosomes using antibodieswhich recognize specifically the four different acetylation sitesin the amino-terminal region of histone H4 (Turner et al.,1992), three of the sites, Lys8, Lys12 and Lys16, were foundto be acetylated at multiple foci within the chromatin(Sommerville et al., 1993). Furthermore, such extensiveacetylation appeared to be stable, because incubation of theoocytes with the potent inhibitor of histone deacetylases,sodium butyrate, did not increase the signal given by theantibodies to acetylated lysines. Thus it appears that the HDACactivity accumulated in oocytes has little natural access to theendogenous chromatin. Indeed, lampbrush chromosomesisolated from mid-vitellogenic oocytes are not immunostainedusing the anti-HDACm antibodies (Fig. 9).

In addition to the acetylated histones contained in thechromatin, oocytes are accumulating large pools of acetylatedhistones to be used in the formation of new chromatin duringthe 12 cell divisions leading to blastula. It is estimated that eachfull-grown oocyte contains 21 ng of maternal histone H4(Adamson and Woodland, 1974), which remains in adiacetylated form until after incorporation into the newlyreplicated chromatin (Dimitrov et al., 1993; Almouzni et al.,1994). The storage of acetylated maternal histones and theirrapid incorporation into new embryonic chromatin is aphenomenon that has been described in differentdevelopmental systems. For instance, in both sea urchins(Chambers and Shaw, 1984) and starfish (Ikegami et al., 1993)maternal histone H4 is stored in a diacetylated form whichis followed, during blastula formation, by a wave ofdeacetylation. All of these observations serve to emphasize theneed to keep apart stored HDAC activity and stored acetylatedhistones until after deposition in embryonic chromatin.

The observation that extracts taken from all stages of earlyXenopus development have an immediately available histonedeacetylase activity would argue that sequestration of HDACactivity from stored histones involves compartmentalizationwithin the intact cells rather than the regulated expressionof inhibitory factors. Studies using chicken immatureerythrocytes claim an association of nuclear HDAC activitywith nuclear matrix material (Li et al., 1996). While we haveno evidence for such an association, it is interesting to note thatimmunoreactivity in oocytes occurs around the internalmargins of the nucleus and not throughout the nucleoplasm. Asmentioned earlier, oocyte chromatin, organized as lampbrushchromosomes, is located internally in the nucleus and remainsunstained with anti-HDACm. Therefore, even in the nucleus,restraints may be placed on the ability of HDACm to make

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2451Histone deacetylase in oocytes

contact with the chromatin and possibly also with the storedmaternal histones. To some degree, the physical separation ofchromatin and components of the active HDAC complex(HDACm and RbAp48/46) is confirmed by the in situimmunostaining shown here (Fig. 7). However, thedistributions of HDAC and stored histones, although different,do overlap and an additional explanation is required. Onepossibility is that the stored histones are protected fromdeacetylation by their close association with chaperones suchas N1/N2 and nucleoplasmin, perhaps resulting in occlusion ofthe acetylated histone tails.

The reason for accumulation of HDACm in the oocytenucleus is not altogether clear, but the nuclear envelope mayserve as a final point of assembly of the active HDAC complex.This complex could then be distributed among the earlyembryonic cells, through cell divisions, along with componentsof the envelope, even to target individual chromosomes(Lemaitre et al., 1998). However, it is also possible thatHDACm has an additional role in deacetylating proteins otherthan histones in the oocyte nucleus: as yet no information isavailable on other types of acetylated protein in oocytes.

Effects of overexpressing HDACmWith the in vitro deacetylation assays used in this work, wehave been unable to obtain significant HDAC activity inpreparations of recombinant HDACm expressed in bacteria asGST fusion proteins. Also, HDACm (Ab21) synthesized inreticulocyte lysates fails to generate any enzyme activity(results not shown). These observations suggest that, as withother systems studied, HDACm alone is insufficient, andcatalytic activity can only be gained through association withother proteins and possibly also, in view of our resultswith phosphatase treatment (Fig. 3C), by phosphorylation.Nevertheless, injection of additional recombinant HDACm(Ab21) into oocytes does appear to be sufficient for recruitmentof deacetylase activity in vivo. The corresponding location ofAb21 and RbAp48 on endogenous chromatin (Fig. 9) suggeststhat both are required for the de novo activity observed. Theseresults are not at odds with the deacetylase activity recordedon minichromosomes, after injection into oocytes of RNAencoding HDAC protein (Wong et al., 1998). It is concludedthat ectopically expressed HDACm escapes the restraintsimposed on endogenously expressed HDACm and that itsuncontrolled activation can lead to premature condensation oflampbrush chromosomes, a process that naturally takes placeonly towards the end of oogenesis.

We thank Minori Yoshida for the gift of TSA and Adrian Bird forantibodies to MeCP2. This work was supported by grants to J.S. andB.M.T. from The Wellcome Trust.

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