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Regulation of Zygotic Gene Activation in the Preimplantation Mouse Embryo: Global Activation and Repression of Gene Expression¹

^a Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018 ^b Kunming Institute of Zoology, Kunming, Yunnan Province, China

ABSTRACT

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A (an inhibitor of histone deacetylases) or aphidicolin (an inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

developmental biology, early development, embryo, gene regulation

INTRODUCTION

Following fertilization of mouse eggs, the maternal-to-zygotic transition, which initiates during the one-cell stage, is clearly evident by the two-cell stage [1, 2]. One outcome of this transition is that transcripts that are common to the oocyte and embryo and that are degraded during oocyte maturation following fertilization are replaced with zygotic transcripts (e.g., actin) [3]. In addition, a dramatic reprogramming of gene expression occurs during this transition, and this reprogramming is likely the molecular foundation for transforming the highly differentiated oocyte into the totipotent blastomeres of the early cleavage stage preimplantation embryo. Although the molecular basis for how this reprogramming occurs is still poorly understood, the transition is accompanied by a more efficient use of TATA-less promoters [4, 5]. This shift in promoter utilization could constitute a major mechanism for the reprogramming. In addition, the reprogramming of some genes requires the first round of DNA replication [6, 7].

Several lines of evidence indicate that superimposed on genome activation and the attendant reprogramming of gene expression is the development of a chromatin-mediated transcriptionally repressive state. A requirement develops by the two-cell stage for an enhancer for the efficient expression of a plasmid-borne luciferase reporter gene driven by the tk promoter [8–11]. This requirement, which is also observed in rabbit preimplantation embryos [12], is ascribed to relieve the repression that develops during the two-cell stage [13–15]. Moreover, this requirement becomes more pronounced with further development [13]. The development of the transcriptionally repressive state is likely at the level of chromatin structure and not in the activity of the transcription machinery per se [13], because inducing histone hyperacetylation, which is linked with the conversion of transcriptionally repressive chromatin into transcriptionally permissive chromatin [16, 17], relieves this requirement for an enhancer [11, 18]. The repression observed for the plasmid-borne reporter gene is also observed for the expression of endogenous genes. For example, the expression of eIF-1A and the transcription-requiring complex (TRC) increases between the one-cell and mid two-cell stages, and then decreases by the late two-cell/four-cell stage and inducing histone hyperacetylation by treating the embryos with an inhibitor of histone deacetylases prevents this decrease [6].

The repression is likely to be global and not restricted to a very small portion of the genes that are expressed in the two-cell embryo. The total amount of BrUTP incorporated by permeabilized two-cell blastomeres in G2 obtained from embryos treated with a histone deacetylase inhibitor is 50% higher than their untreated counterparts [19]; under these conditions, BrUTP incorporation reflects the activity of RNA polymerase. The increase in transcription in response to histone hyperacetylation is interpreted to reflect a global relief of repression.

The second round of DNA replication also appears coupled with the formation of the transcriptionally repressive state. Inhibiting the second round of DNA replication by treating the embryos with aphidicolin prevents the decrease in eIF-1A transcript abundance and the decrease in the relative rate of TRC synthesis that normally occurs by the late two-cell stage/early four-cell stage [6]. Likewise, permeabilized two-cell blastomeres treated with aphidicolin to inhibit the second round of DNA replication but which are chronologically in G2, incorporate about 50% more BrUTP than their untreated counterparts when expressed on a chromosome basis [19]; the treated embryos are two-cell, whereas the untreated embryos are four-cell. Again, the increase in transcription in response to inhibiting DNA synthesis is interpreted to reflect a global relief of repression.

Essentially no information exists regarding the spectrum of genes that are subject to this repression that develops during the two-cell stage. We report here results of an mRNA differential display analysis in which we examined genes whose expression transiently increases during the two-cell stage or genes that become activated during zygotic gene activation and the effect of inducing histone hyperacetylation and inhibiting the second round of DNA replication on their expression. Results of the studies indicate that 45% of the genes analyzed are subject to repression. Moreover, inhibiting the development of the transcriptionally repressive state by inducing histone hyperacetylation inhibits development beyond the two-cell stage, and suggests that the repressive state may dictate the pattern of gene expression that is required for successful development.

MATERIALS AND METHODS

Oocyte and Embryo Collection and Embryo Culture

Germinal vesicle-intact oocytes were collected from eCG-primed CF-1 mice as previously described [20]; 0.2 mM 3-isobutylmethylxanthine was present in the collection medium to maintain meiotic arrest. The collecting medium was bicarbonate-free minimal essential medium (MEM; Earles salts) supplemented with pyruvate (100 µg/ml), gentamicin (10 µg/ml), polyvinylpyrrolidone (3 mg/ml), and 25 mM Hepes pH 7.2 (MEM/PVP). One-cell embryos were collected from superovulated CF-1 mice mated to B6D2F1/J males as previously described [5]; the collecting medium was bicarbonate-free MEM/PVP. Embryo culture was conducted in KSOM medium plus amino acids [21] at 37°C in an atmosphere containing 5% CO₂, 5% O₂, and 90% N₂.

For mRNA differential display, one-cell embryos with one or two visible pronuclei were randomly assigned to five different treatment groups as follows: 1) 43-h controls: one-cell embryos were allowed to develop in vitro until 43 h post-hCG, which is about mid two-cell stage; 2) -amanitin treatment: one-cell embryos were incubated with 24 µg/ml -amanitin, which inhibits RNA polymerase II-dependent transcription, until 43 h post-hCG; these treated embryos cleaved to the two-cell stage; 3) 63-h controls: one-cell embryos were cultured until 63 h post-hCG, at which time most of the embryos had reached the four-cell stage; 4) aphidicolin treatment: 32–33 h post-hCG, one-cell embryos, which had completed DNA replication and were in G2/M, were transferred to KSOM + amino acids containing 3 µg/ml aphidicolin, which is a specific inhibitor of replicative DNA polymerases, and then cultured until 63 h post-hCG; these treated embryos cleaved to the two-cell stage, but not to the four-cell stage; and 5) trichostatin A (TSA) treatment: one-cell embryos were incubated with 33 nM TSA, which is an inhibitor of histone deacetylases [22], until 63 h post-hCG; these embryos cleaved to the two-cell stage but not to the four-cell stage (see Results). Oocytes were used to establish basal transcript levels. Twenty oocytes/embryos were used for each treatment group.

Following culture, the embryos from the different treatment groups were washed three times in MEM/PVP and transferred in <5 µl to 100 µl of lysis buffer (4 M guanidine thiocyanate, 1 M 2-mercaptoethanol, and 0.1 M Tris-HCl pH 7.4). The embryos from each treatment group from five separate experiments (i.e., 100 embryos) were pooled into a single tube containing lysis buffer. The samples were stored at -20°C prior to RNA isolation.

To study the effect of TSA on embryo development, increasing concentrations of TSA (see Figs. 2 and 4) were added to one-cell embryos in G2/M. The embryos were cultured in KSOM + amino acids at 37°C in an atmosphere containing 5% CO₂, 5% O₂, and 90% N₂. The embryos were scored for development over the next 2 days. At least 20–30 embryos were used for each treatment group.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Effect of increasing concentrations of TSA on development of one-cell embryos. TSA was added at the indicated concentrations to mid-late one-cell embryos and cultured for 1 day (A) or 2 (B) days. Solid bars, one-cell embryos; open bars, two-cell embryos; shaded bars, four-cell embryos; stippled bars, eight-cell embryos. The experiment was conducted three times and the data are expressed as the mean ± SEM

RNA Isolation for Differential Display

For the different treatments, total RNA was isolated from 100 oocytes/embryos and subjected to mRNA differential display as described previously with a minor modification [23]. Briefly, before RNA extraction, 20 µg of glycogen (Roche Diagnostics Corporation, Indianapolis, IN) was added to the samples as a carrier. Total RNA was precipitated from the lysis buffer by adding 8 µl of 1 M acetic acid, 5 µl of 2 M potassium acetate, and 300 µl of 100% ethanol and incubating at -20°C overnight. The nucleic acids were pelleted by centrifugation at 14 000 x g for 15 min and the pellets washed with 300 µl of ice-cold 75% ethanol. The pellets were then resuspended in 20 µl of resuspension buffer (40 mM Tris-HCl pH 7.9, 10 mM NaCl, and 6 mM MgCl₂) containing 20 units of recombinant RNasin. Contaminating DNA was digested by adding 1 unit of RQ1 RNase-free DNase (Promega, Madison, WI) and incubating the sample at 37°C for 30 min. After adding 50 µl of diethyl pyrocarbonate (DEPC)-treated water, the samples were extracted with 80 µl of resuspension solution-saturated phenol (Fisher Scientific, Pittsburgh, PA). The samples were vortexed and centrifuged for 8 min at 14 000 x g, and the aqueous phase was transferred to a new 0.5-ml tube. RNA was reprecipitated by adding 8 µl of 3 M potassium acetate (pH 5.2) and 300 µl of 100% ethanol, and the sample was then incubated at -20°C overnight. Samples were then pelleted by centrifugation at 14 000 x g for 15 min at 4°C and washed with 300 µl of ice-cold 75% ethanol. Pellets were dissolved in 10 µl of DEPC-treated water containing 10 units of recombinant RNasin and subjected to differential display.

Messenger RNA Differential Display

Differential Display was performed using the RNAimage kit (GenHunter Corporation, Nashville, TN) and reverse transcription (RT) and polymerase chain reaction (PCR) were performed according to the manufacturer's instructions; [³⁵S]dATP was used as the radiolabeled deoxyribonucleotide. RT was conducted on the total amount of RNA recovered, and duplicate PCR reactions were conducted on 1/10 of the total reverse transcribed sample. Anchor primers were H-T₁₁G (5'-AAGCTTTTTTTTTTTG-3'), H-T₁₁C (5'-AAGCTTTTTTTTTTTC-3'), or H-T₁₁A (5'-AAGCTTTTTTTTTTTA-3'). Arbitrary primers were H-AP1 (5'-AAGCTTGATTGCC-3'), H-AP2 (5'-AAGCTTCGACTGT-3'), H-AP3 (5'-AAGCTTTGGTCAG-3'), H-AP4 (5'-AAGCTTCTCAACG-3'), H-AP5 (5'-AAGCTTAGTAGGC-3'), H-AP6 (5'-AAGCTTGCACCAT-3'), H-AP7 (5'-AAGCTTAACGAGG-3'), or H-AP8 (5'-AAGCTTTTACCGC-3'). A total of 24 different combinations of primer pairs were used. The ³⁵S-radiolabeled amplicons were resolved on a 6% DNA sequencing gel in which 5 µl of the 20-µl reaction mixture was applied. The gels were dried without fixation onto filter paper and then subjected to autoradiography using Kodak XAR-5 x-ray film at room temperature. The exposure time was usually 4–5 days.

Reamplification, Cloning, and Sequencing of cDNA

The bands of interest were cut from dried differential display gels with a sterile blade and then rehydrated in 100 µl of DEPC-treated water for 10 min at room temperature. The samples were then incubated in boiling water for 15 min. After pelleting the gel and paper debris by centrifugation at 14 000 x g for 2 min, the DNA was precipitated by adding 8 µl of 3 M potassium acetate and 2.5 µl glycogen (20 mg/ml); the sample was held at -20°C overnight. DNA was collected by centrifugation at 14 000 x g for 15 min at 4°C, washed with 200 µl of ice-cold 85% ethanol, and then resuspended in 10 µl of DEPC-treated water. The DNA was then reamplified using the same primer combination and PCR conditions used for mRNA differential display, but with the following modifications: the reaction volume was 40 µl and contained 20 µM dNTPs (radiolabeled [³⁵S]dATP was not included) and the reactions were performed for 40 cycles using the following parameters: 94°C for 30 sec, 40°C for 3 min, and 72°C for 30 sec in which the last cycle was followed by a 5-min extension at 72°C. The PCR product was visualized by ethidium bromide staining following electrophoresis in a 1.5% agarose gel.

The amplified PCR products were cloned into a pT-Adv plasmid vector using the AdvanTAge PCR cloning Kit (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer's instructions. Plasmids containing the amplified PCR products were extracted using the QIAprep Spin Miniprep Kit (QIAGEN Inc., Chatsworth, CA) according to the manufacturer's instructions and subjected to automatic DNA sequencing.

For computational analysis, vector contaminating sequences were removed and then all of the remaining sequences were analyzed with the Contig Assembly Program (CAP) at BCM Search Launcher to identify redundant clones. All sequences were then subsequently analyzed with RepeatMasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html) to identify retroviral, transposons, and other repeat sequences. Finally, all of the sequences were submitted for sequence similarity search using the BLAST2 algorithm at National Center for Biotechnology Information.

Verification of Amplicon Expression in Two-Cell Mouse Embryos

To verify that the amplicons detected by mRNA differential display were in fact expressed in the two-cell embryo, RT-PCR using gene-specific primers was conducted. RNA was isolated as described above and 100 embryo equivalents were subjected to RT. The RT reaction consisted of 25 µg/ml oligonucleotide (dT)_12–18, buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂, and 10 mM dithiothreitol), 0.5 mM dNTPs, 2 U/µl recombinant RNasin, and 10 U/µl of Superscript II Reverse Transcriptase in a final volume of 20 µl. The reaction mixture was incubated at 42°C for 50 min followed by 15 min at 70°C. PCR was conducted on five embryo equivalents with gene-specific primers based on the DNA sequence obtained for the amplicon in question. The conditions for PCR were established for each amplicon.

Semi-Quantitative RT-PCR Assay

The relative abundance for one of the transcripts (clone B003) identified from the differential display analysis was determined by a semiquantitative RT-PCR assay [24]. Two-cell and four-cell embryos, as well as TSA- and aphidicolin-treated embryos were collected as described in Oocyte and Embryo Collection and Embryo Culture, and RNA was isolated as previously described [24]; 200 oocytes/embryos were in each group. Prior to the isolation of RNA, 0.125 pg of rabbit globin mRNA (BRL) was added per oocyte/embryo. The globin mRNA serves as a control to normalize for RNA recovery and the efficiency of RT-PCR reactions, which are performed in the linear range when the amount of PCR product is plotted as a function of cycle number [24].

Two hundred oocyte/embryo equivalents were subjected to RT using oligo(dT) as described earlier (Verification of Amplicon Expression in Two-Cell Mouse Embryos). PCR for clone B003 was performed on two oocyte/embryo equivalents using the following program: 94°C for 3 min, followed by 32 cycles at 94°C for 30 sec, 54°C for 1 min, and 72°C for 30 sec; the last cycle was followed by a 5-min extension at 72°C. The PCR reactions were conducted in the presence of [-³²P]dCTP (3000 Ci/mmol, Amersham). The 5' sense primer for clone B003 was 5'-TTGTGAACAAACAAACAAAGGAACA-3' and the 3' antisense primer was 5'-CAGAAGAAGAAGAAGAAGTGAGA-3'. Following PCR, the products were subjected to electrophoresis in a 6% polyacrylamide gel. The gel was dried, exposed in a phosphorImager cassette, and the signal quantified using the Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The relative abundance of transcripts for B003 were calculated as previously described [24].

Immunocytochemical Detection of Hyperacetylated Histone H4 and Incorporated BrdU

Immunocytochemical detection of hyperacetylated histone H4 with an antibody that is specific for acetylated lysine 5 was performed as previously described [25]. For BrdU labeling, two-cell embryos 40 h post-hCG were incubated in KSOM containing 10 µM BrdU for 1 h. The embryos were then fixed and processed for BrdU incorporation by immunocytochemical detection as previously described [26].

RESULTS

Messenger RNA Differential Display Analysis: Criteria for Amplicon Selection

Differential display is known to have several drawbacks (e.g., it entails somewhat arbitrary criteria for selecting amplicons that will be analyzed further, the amplicons are typically short in length and hence provide a minimum of information for sequence analysis and comparison, is biased toward detecting more abundant transcripts, and generates a substantial fraction of false positives). Nevertheless, we used this approach to identify genes that are repressed following genome activation, because we have successfully used it to identify genes whose expression transiently increases during the two-cell stage [6]. In our studies, mRNA differential display was performed on oocytes, two-cell embryos, one-cell embryos cultured to the two-cell stage in the presence of -amanitin, two-cell embryos treated from the late one-cell stage with TSA, an inhibitor of histone deacetylases [22], two-cell embryos treated with aphidicolin to inhibit the second round of DNA replication, and late two-cell/early four-cell embryos. Twenty-four pairs of 5' primers and anchored 3' primers were used and each experiment was conducted in duplicate.

The criteria for amplicon selection were as follows: Only amplicons whose pattern of expression was similar in both runs were considered for further analysis; this number was 1649. Of these, an increase in amplicon intensity had to occur between the oocyte and two-cell stage and -amanitin had to inhibit this increase (i.e., the amplicon was likely a product of zygotic gene activation). Of these we pursued only amplicons that revealed a decrease in intensity between the two-cell and four-cell stages (transiently expressed) or those in which the intensity increased between the two-cell and four-cell stages (constitutively activated). The ability of either inducing histone hyperacetylation with TSA or inhibiting the second round of DNA replication with aphidicolin to inhibit the decrease of the transiently expressed amplicons or to increase the band intensity of constitutively expressed amplicons was then examined. For the constitutively expressed amplicons, changes in band intensity of at least 30% were analyzed. The basis for selecting 30% was that this difference could readily be detected by visual examination of the autoradiograms and was corroborated by scanning the autoradiograms (data not shown). As presented in the Introduction, we infer that the decrease in expression of a transiently expressed gene or the ability of either TSA or aphidicolin either to prevent this decrease or to increase the expression of constitutively activated genes reflects genes that are subject to repression during the development of the transcriptionally repressive state.

Of the 1649 amplicons examined, 265 were classified according to the aforementioned criteria and representative patterns of some of the expression profiles are shown in Figure 1A. Several findings supported our view that the differential display data were representative, at least to a first approximation, of changes in gene expression that occur during these developmental stages. First, DNA sequence analysis of clone B023 (see below) revealed it to be eIF-1A, which we previously identified as a transiently expressed gene using mRNA differential display with different primers and confirmed by an independent RT-PCR assay [6]. Second, the observation that both TSA and aphidicolin prevented the decrease in amplicon intensity between the two-cell and four-cell stages was previously demonstrated by an RT-PCR assay using gene-specific primers [6]. Lastly, using our RT-PCR assay and gene-specific primers for clone B003, a decrease in transcript abundance between the two-cell and four-cell stages was observed and TSA, but not aphidicolin, prevented this decrease (Fig. 1B).

View larger version (46K): [in this window] [in a new window]   FIG. 1. A) Expression profile of representative amplicons observed following differential display. Regions of the autoradiogram are shown for selected amplicons (see Table 3 for further information about each amplicon). Yes denotes that TSA, aphidicolin, or both either inhibited the decrease for the transiently expressed amplicons B003, B023, B016, or increased the expression of amplicon B028, whereas No denotes that aphidicolin did not inhibit the decrease for the transiently expressed amplicon B003. Oo, Oocyte; 2C, two-cell embryo; Am, one-cell embryo cultured in the presence of -amanitin to the two-cell stage; 4C, four-cell embryo; TSA, one-cell embryo cultured in the presence of TSA to a time that corresponded to the four-cell stage; Aph, embryos in which the second round of DNA replication was inhibited and cultured to a time that corresponded to the four-cell stage. B) Expression profile of B003 using gene-specific primers and RT-PCR. The data, which are normalized to the amount of globin, are expressed relative to the amount of B003 transcript in the two-cell embryo

DNA Sequence Analysis of Selected Amplicons

We analyzed all 40 of the transiently expressed amplicons, as well as 18 constitutively expressed amplicons whose expression was increased by TSA/aphidicolin (Table 1). Of the 58 amplicons that were pursued, we were unable to amplify 5. Of those that could be amplified, DNA sequence analysis revealed that three corresponded to mitochondrial genes, and two were due to bacterial contamination. Another amplicon (192 base pairs [bp]) that was classified as being transiently expressed and was similar to the Mus musculus CBP/p300-interacting transactivator with a glu/asp-rich carboxy terminal domain (accession number NM010825, P < 7E-79) was also detected as a constitutively expressed amplicon (156 bp, P < 2E-63, accession number U68384). These differences in classification could reflect an artifact of the approach or possibly the presence of two promoters that respond differentially to the treatments. Nevertheless, we omitted these two amplicons from further analysis. Thus, of the 58 amplicons analyzed, we classified 20% (12/58) as false positives.

The results of DNA sequence analysis of the remaining amplicons are tabulated in Tables 2–4. It should be noted that the sequence of clone B028 was also detected two other times using different primers, and that the sequence in clone B054 was also detected with another set of primers. The data presented in Table 3 counted each of these sequences only one time. Nine amplicons revealed no apparent sequence homology with any of the extant databases. To verify that the amplicons corresponded to expressed sequences, gene-specific primers were designed and RT-PCR was conducted using two-cell embryo RNA to determine if an amplicon of the correct size was observed. Results of these experiments indicated that of the nine amplicons, seven were detected in two-cell embryos using gene-specific primers (i.e., the degree of false positives of this class of genes was likely to be moderately low). Thus, 15% (7/43) of the amplicons sequenced corresponded to novel genes, assuming that each of the sequences corresponded to a separate transcript. Activation of the genome appeared relatively indiscriminate, because the repetitive sequences comprised 25% (9/34) of the sequences that were representative of a transient expression profile (Table 3).

Thirty-four of the amplicons subjected to DNA sequencing were classified as transiently expressed and nine as constitutively expressed that were stimulated by TSA/aphidicolin (Table 4). For the transiently expressed amplicons, the repression observed, as inferred from the decrease in amplicon intensity between the two-cell and four-cell stages, was relieved by inducing histone hyperacetylation with TSA or inhibiting the second round of DNA replication with aphidicolin for 70% (24/34) of the amplicons. Of interest is that eight of the nine repetitive sequences displayed this response.

We also scored other constitutively expressed amplicons (Table 4), but these were not amplified and sequenced. Results shown in Table 1 indicated that about 20% of the amplicons analyzed represented false positives. Assuming that this incidence of false positives was representative, one can calculate the number of bona fide amplicons for the other constitutively expressed categories (Table 4, numbers in parentheses). The data shown in Table 4 suggest that the repression that develops during the two-cell stage was quite extensive. Based on our aforementioned criteria for what reflects the development of the transcriptionally repressive state (i.e., all transiently expressed amplicons, as well as constitutively expressed amplicons responsive to TSA/aphidicolin), 45% [(34 + 22 + 35 + 9)/(34 + 174 + 9) = 100/217); see Table 4 for origin of numbers] of the amplicons experienced repression during the time of genome activation.

Effect of Trichostatin A on Development

Results of the mRNA differential display analysis indicated that between the two-cell and four-cell stages a global repression of transcription occurred. This repression could dictate the final pattern of gene expression that is required for further development. Thus, preventing the development of the transcriptionally repressive state by inducing histone hyperacetylation with TSA would be expected to prevent cleavage of two-cell embryos to the four-cell stage. In fact, such was the case. Culture of one-cell embryos in the presence of increasing concentrations of TSA did not prevent cleavage to the two-cell stage, but did inhibit cleavage of two-cell embryos to the four-cell stage (Fig. 2). It should be noted that treatment of one-cell embryos with either 10 nM or 25 nM TSA resulted in a concentration-dependent increase in acetylation of histone H4 following culture to the two-cell stage (Fig. 3); acetylation was assayed by using an antibody specific for histone H4 acetylated on lysine 5 [25]. The addition of TSA to early two-cell embryos inhibited cleavage to the four-cell stage, whereas its addition to mid- to late-two-cell embryos did not inhibit cleavage to the four-cell stage but did inhibit further development to the eight-cell stage (Fig. 4). The ability of these latter embryos to cleave to the four-cell stage likely reflected the inability of the TSA treatment to induce a sufficient increase in histone acetylation in this shortened period to relieve the developing transcriptional inhibition.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Effect of increasing concentrations of TSA on histone acetylation in two-cell embryos. One-cell embryos were cultured in the absence (A) or presence of 10 nM TSA (B) or 25 nM TSA (C) until the two-cell stage, at which time they were fixed and processed for immunocytochemical detection of hyperacetylated histone H4 with an antibody that recognizes histone H4 acetylated on lysine 5. Shown are representative confocal images. The enhanced staining at the periphery of the nucleus has been observed previously [25]

View larger version (28K): [in this window] [in a new window]   FIG. 4. Effect of increasing concentrations of TSA on development of two-cell embryos. TSA was added at the indicated concentrations to early two-cell embryos (A) or late two-cell embryos (B). The embryos were then cultured for an additional day and then scored for development. Open bars, two-cell embryos; shaded bars, four-cell embryos; stippled bars, eight-cell embryos. The experiment was conducted three times and the data are expressed as the mean ± SEM

A simple explanation for the ability of TSA to inhibit cleavage of two-cell embryos to the four-cell stage when added to either one-cell or early two-cell embryos was that it inhibited DNA replication. BrdU labeling studies demonstrated that this was not the case, because adding TSA to early two-cell embryos had no effect on the percentage of embryos that incorporated BrdU (data not shown). In addition, it should be noted that TSA does not inhibit DNA replication in one-cell embryos and in fact stimulates the completion of S phase in these embryos [26].

The inhibitory effect of TSA on cleavage to the four-cell stage was not reversible. Transfer of two-cell arrested, TSA-treated embryos that were chronologically at the four-cell stage to TSA-free medium did not result in cleavage to the four-cell stage (data not shown). The lack of reversibility was likely due to the uncoupling of appropriate gene expression patterns with cell cycle progression.

DISCUSSION

The results presented here buttress the growing sentiment that activation of the mammalian genome is accompanied by a global change in gene expression on which is superimposed the formation of a transcriptionally repressive state. The sum of these two opposing processes—activation and repression—would direct the final pattern of gene expression that manifests itself as a dramatic reprogramming of gene expression during the maternal-to-zygotic transition. This final pattern of gene expression is likely requisite for further development. Although the expression of a somatic histone H1 is an attractive candidate to initiate the formation of the transcriptionally repressive state [27], the effect of microinjected somatic histone H1 on gene expression in two-cell embryos is not consistent with this proposal [28].

We did not examine the function of any of the genes identified in this study because the overarching objective was to assess global changes in gene expression patterns in order to deduce general principles underlying the reprogramming of gene expression that accompanies activation of the embryonic genome. Despite the limitations of mRNA differential display (see Results), we believe that our results are representative, even though a small number of amplicons was analyzed. We observe that 16% (7/43) of the amplicons are not observed in any of the extant databases. This frequency of novel genes is very similar to that found in a recent study that employed a large-scale cDNA analysis of gene expression in the preimplantation mouse embryo [29]. In particular, of some 3687 cDNAs sequenced from two-cell embryo cDNA libraries, about 22% (812/3687) appear to be novel (i.e., the sequence does not match to that in any other library). Moreover, we note that 21% (9/43) of the amplicons we analyzed correspond to repetitive sequences, and this is similar to the 17% (619/3687) observed in the aforementioned study. Last, we analyzed 1649 amplicons that generated reproducible banding patterns. Of these, 2% (34/1649) displayed the transient expression profile. Assuming that 20% of these amplicons are false positives, then 2.5% (34/1319) displayed the transient expression profile. This percentage is also in good agreement with the large-scale cDNA sequence analysis study that detected 3% of cDNAs whose expression is restricted to the two-cell stage [29], as well as the results of a two-dimensional gel electrophoresis study that analyzed 1500 polypeptides and noted that 2.5% (38/1500) display a transient increase in expression during the two-cell stage [30]. The concordance of our results with those of these other studies that encompass a greater scope than our study suggests that the conclusions drawn from our study are based on reasonable extrapolations.

Activation of the embryonic genome is accompanied by the expression of a large number of novel genes (16%) as well as repetitive sequences (21%, 9/43). The activation of these two classes of genes is consistent with a global gene activation that is relatively promiscuous. In fact, the frequency of detecting repetitive sequences in the large-scale cDNA analysis is highest at the two-cell stage [29]. Thus, genes that are expressed at the outset may simply be those whose promoters are (or become) accessible to the transcription machinery and for which the existing complement of maternally derived transcription factors is sufficient to promote at least basal levels of transcription. This opportunistic transcription may be due to the extensive remodeling of chromatin structure that occurs during this transition (e.g., protamine-histone exchange, changes in histone acetylation [25, 31], expression of somatic histone variants, and other DNA-binding proteins [27, 32, 33]).

Our results also support the previous conclusion that a transcriptionally repressive state develops during the two-cell stage [6, 11, 13, 19]. Although these previous studies revealed critical insights regarding underlying mechanisms involved in the formation of the transcriptionally repressive state (i.e., it is mediated at the level of chromatin structure), they provided little information regarding the identity of the genes that are repressed. We note that all classes of genes (known genes, genes matched to the databases of unknown function, or potentially novel genes, and repetitive sequences) are subject to repression during the two-cell to four-cell transition. Using our criteria for what constitutes the manifestation of gene repression (see Results), the repression is quite extensive, because 46% (100/217) of the amplicons analyzed are repressed. Of the transiently expressed amplicons, which by our criteria display an inherent repression, inducing histone hyperacetylation (which relieves the repression), or inhibiting the second round of DNA replication (which prevents the formation of the repressive state), or both, relieves the repression of 70% of these genes. Nevertheless, the response is differential in that some genes only respond to inducing histone acetylation, other genes only respond to inhibiting the second round of DNA replication, whereas others respond to both events. This suggests that the mechanism of repression is likely to be complex. For genes that are sensitive to TSA, inducing histone hyperacetylation may result in the formation of transcriptionally permissive chromatin in the region of the promoter and hence facilitate the assembly of a productive transcription complex on that promoter. For genes that are sensitive to aphidicolin, inhibiting the second round of DNA replication may permit retention of productive transcription complexes assembled on the promoter that would otherwise be displaced by the ensuing round of DNA replication. Once the transcription machinery is displaced from its promoter, a change in chromatin structure over the promoter would occur and restrict subsequent access of the transcription machinery. Not enough genes have been analyzed and classified with respect to the effect of TSA and aphidicolin on their expression, however, to ascertain if a pattern exists regarding what types of genes are susceptible to being repressed, and if so, if their repression can be relieved.

The development of the transcriptionally repressive state may be essential for further development, because relieving this state by inducing histone hyperacetylation, which would maintain the expression of many genes that normally undergo repression, inhibits cleavage of two-cell embryos to the four-cell stage. It is possible, however, that this inhibition merely reflects a perturbation of cell cycle regulation as a consequence of maintaining histone hyperacetylation and is not linked to the developmental program (e.g., inducing histone hyperacetylation in somatic cells can inhibit cell proliferation) [34]. Nevertheless, it should be noted that treatment of Xenopus laevis embryos with 30 nM TSA, which results in histone hyperacetylation, does not prevent cell division or differentiation prior to gastrulation, and also permits substantial anterior development [35]. Furthermore, TSA treatment does not inhibit cleavage of one-cell embryos to the two-cell stage. Further work is clearly required to determine the molecular basis of how TSA inhibits development of embryos beyond the two-cell stage.

A function of the repressive state may be to sculpt the newly generated gene expression profile such that it is now compatible with further development. As described above, activation of the genome may initially be a relatively opportunistic process due to the extensive remodeling of chromatin structure, and hence the genes that are expressed are simply those for which the necessary transcription factors are present and for which the promoter is accessible. A consequence of such a global activation is that while genes with strong promoters and/or enhancers would be preferentially expressed, many other genes may be inappropriately (i.e., opportunistically) expressed (especially at basal levels of transcription) during this transition. The development of a transcriptionally repressive state could preferentially reduce the expression of these genes, but permit the continued expression of genes that are regulated by strong promoters/enhancers. The expression of these genes would, therefore, be critical for continued development.

ACKNOWLEDGMENTS

We thank Mariam Barkran for helping with the experiments presented in Figures 2 and 4.

FOOTNOTES

First decision: 5 January 2001.

¹ This research was supported by grant HD 22681 from the National Institutes of Health to R.M.S. J.M. was supported by the Major State Basic Research Development Program (G2000016108) of the Chinese Academy of Sciences to the Kunming Institute of Zoology, Kunming, Yunnan Province, China.

² Correspondence: Richard Schultz, Department of Biology, University of Pennsylvania, 415 University Avenue, Philadelphia, PA 19104-6018. FAX: 215 898 8780; rschultz{at}mail.sas.upenn.edu

Accepted: January 26, 2001.

Received: December 5, 2000.

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