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Translational regulation of histone synthesis in the sea urchin strongylocentrotus purpuratus.

The pattern and schedule of histone synthesis in unfertilized eggs and early embryos of the sea urchin Strongylocentrotus purpuratus were studied using two-dimensional gel electrophoresis. After fertilization there is an abrupt change in the pattern of histone variant synthesis. Although both cleavage-stage (CS) variants. However, after fertilization, both CS and alpha messages are translated. Since alpha histone mRNA isolated from unfertilized eggs can be translated in vitro, the synthesis of alpha histone subtypes appears to be under translational control. Although the synthesis of alpha subtypes is shown here to occur before the second S phase after fertilization, little or no alpha histone is incorporated into chromatin at this time. Thus, early chromatin is composed predominantly of CS variants probably recruited for the most part from the large pool of CS histones stored in the unfertilized egg.

During sea urchin development, histone variants of the HI, H2A, and H2B classes appear sequentially in the chromatin (1-6). Variants of each class are coded by different members of the histone multigene family and differ from each other in primary amino acid sequence (5,(7)(8)(9). Characteristic sets of histones are made and assembled into chromatin at each of several developmental stages. Cleavage-stage (CS) histones are the first to appear (4). They are presumably made during oogenesis and are stored in the unfertilized egg in large quantities (at least several hundred haploid DNA equivalents [10,11]). The CS variants participate in an extensive remodeling of the sperm chromatin after fertilization, which results in a reduction in the nucleosome repeat length (12). Almost immediately after fertilization, the sperm H1 variant is replaced by CS HI. Concomitant with DNA synthesis, substantial amounts of CS H2A and CS H2B accumulate in the chromatin. These protein transitions can take place in the absence of protein synthesis. Later in development the ct variants appear, and, by the morula stage, chromatin is composed predominantly of nucleosomes that contain a histones (1, 4). Synthesis of late histones (designated /3, -/, and 8) is initiated at the mesenchyme blastula stage (1, 4). When sea urchin maternal mRNA is translated in vitro, both a (6,8,13) and CS histones (8) are made. Because both a and CS histone mRNAs are stored in the sea urchin egg, it is surprising that mature eggs contain only CS proteins. There is no detectable storage of a histones (11). These observations suggest that the synthesis of histone subtypes may be under translational control. On the basis of the observation that newly synthesized a variants, incorporated into chromatin, are first detected at about the third S phase after fertilization, others have suggested a qualitative translational control for histone variant synthesis (4,6).
Fertilization of the sea urchin egg stimulates the rate of protein synthesis -15-fold by 2 h postfertilization (14). This is accompanied by an ~30-fold increase in the fraction of ribosomes found in polysomes but not by large changes in the efficiency of translation (defined as the rate of protein synthesis/polysomal mRNA/time) (15). The increase is almost entirely dependent on maternal message since it occurs in actinomycin D-treated embryos (16) and enucleated egg fragments (17). These results taken together suggest a recruitment of untranslated maternal message into polysomes after fertilization. At 2 h after fertilization 90% of the polysomal mRNA is maternal, whereas by gastrula stage virtually all is newly transcribed from embryonic genes (15). Using an isoelectric focusing gel system that does not resolve basic proteins, Brandhorst could detect few obvious differences in the protein species synthesized before and after fertilization (18). Therefore, it was concluded that there are few classes of abundant messages translated after fertilization that are not already being translated and that the major increase in protein synthesis rate that accompanies fertilization is not accomplished by the recruitment of qualitatively new messages but rather by the translation of more mRNAs of the same types (18). However, there is an increasing amount of evidence that suggests that histone mRNAs behave differently than much of the maternal message. Wells et al. (19) have shown that, unlike most messages, the recruitment of H3 mRNA onto polysomes is delayed. Venezsky et al. (20) by in situ hybridization, showed that RNA transcripts complementary to a genomic histone repeat are found in high concentration in sea urchin egg pronuclei. However, a similar high nuclear concentration is not observed for two other abundant maternal mRNAs.
Although histone mRNA is an abundant maternal messenger class (~3 pg/egg), amounting to possibly as much as 8% of the mRNA pool (21), a detailed analysis of histone variant types synthesized before and after fertilization has not been made. In this paper we use a polyacrylamide gel system that is specifically designed to resolve histone variants (22) and to separate them from contaminating proteins (23) in order to examine the pattern of histone synthesis in acid extracts of unfertilized and fertilized eggs. We fred that (a) before fertilization CS histones are the only type ofhistone made, (b) after fertilization or artificial activation both CS and a variants are synthesized, and (c) a synthesis begins before the second S phase, which is earlier than other reports have indicated.

MATERIALS AND METHODS
Strongylocentrotus t~puratus were obtained from Pacific Bio-marine Laboratories Inc., Venice, CA. Urchins were maintained in a refrigerated aquarium and fed ]aminaria coficcted at the Marine Biological Laboratory, Woods Hole, MA.

aH(N)]lysine [New England
Nuclear, Boston, MA]; 64.5 Ci/mmol) in a conical 1.5-ml Eppendorf tube (Brinkman Instruments, Inc., Westhury, NY) that was rotated at 30 rpm at 150C for the times specified in the figure legends. To each 1 ml of culture, 25/d of I M HEPES pH 7.3, dissolved in MPFSW, was added to buffer the 0.01 N HC1 in which the isotype is packaged. More than 90% of the eggs could be fertilized after the incubations.
Monospermic embryos were cultured as previously described (24) with 50 ~Ci/mi of [~l-l]lysine for the periods specified in the figure legends.
Eggs were artificially activated with N-9 seawater, which was prepared by adjusting ordinary seawater to pH 9.0 with NI-hOH (25). In these experiments, eggs were fn'st cultured with 100 #Ci/mi [~H]lysine in N-9 seawater for i h. At the end of this time, eggs were washed twice with MPFSW and then cultured in ordinary MPFSW containing this same amount of isotope, for the periods given in the figure legends.
To produce actinomycin D-treated blastulae, eggs were incubated in 25 pg/ ml actinomycin D (grade 1; Sigma Chemical Co.) dissolved in MPFSW for 90 rain before fertilization. Each lot of actinomycin D was previously tested. Eggs were then pelleted by hand centrifugalion and fertilized in MPFSW. Fertilized eggs were washed to remove excess sperm, washed twice in 25 t~g/ml actinomycin D in MPFSW, and cultured in this solution in the dark to the blastula stage (5,26,27), Nuclei from blastulae grown in actinomycin were isolated by Dounce homogenization in 0.075 M NaC1, 25 mM Tris, I mM EDTA pH 7.6 solution containing 0.01% Triton X-100 as described in Poccia et al. (24).
Polyspormic fertilization was done as previously described (11). The degree of polyspermy was determined in eggs fixed in Caruoy's solution (ethanol:acetic acid 3:1). An aliquot of fixed eggs was dried on microscope slides, and nuclei were stained with 1% orcein in 75% acetic acid. The number of male pronuelei was counted under bright-field optics equipped with a green filter.

RAPID COMMUNICATIONS
For acid extraction, both eggs and embryos were washed free of exogenous label, using gentle hand centrifugation (~ 1,000 rpm for 10 s) with cold MPFSW. Eggs were disrupted by the addition of 0.ff75 M NaCI, 0.025 M EDTA, 0.01 M Tris pH 8.0, 0.1% Triton, 0.1 nLM PMSF, 10 mbl DTT, followed by Dounce homogenization and then extracted with an equal volume of 0.8 N HeSO4 at 0°C for 8 h, with occasional vortexing. All subsequent steps were done in 1.5-ml Eppendorf test tubes. The extract was centrifuged at 10,000 g for 30 rain to remove acid-insoluble material and the superuatant was made 20% in trichloroacetic acid (TCA) by adding 100% (wt/vol) TCA solution. The pellet was reextracted with 0.4 N sulfuric acid and centrifuged. This supernatant was made 20% in TCA and pooled with the first extract. After 30 rain on ice, the precipitate was pelleted, washed twice with acetone, 0.1% H2SO4 at -20°C, and washed several times with cold acetone. The protein pellet was dried at room temperature in vacuo before resuspeusion in sample buffer. Nuclei were twice acid-extracted in 0.4 N H~SO4, and the combined superuatants were processed in the same way described above for the acid extracts.

Electrophoretic Analysis of CS and ~ Variants
To analyze histone synthetic patterns in whole eggs and embryos, a high-resolution electrophoretic system is required. A system has been described (23) that separates HI and core histone variants on the same slab, even when large amounts of contaminating basic proteins are present in the sample. Electrophoresis on Triton/6 M urea gels in the first dimension results in preferential retardation of the histones (22), which then migrate off the diagonal (away from contaminating ribosomal proteins) upon SDS gel electrophoresis in the second dimension.

The Pattern of Protein Synthesis Changes after Fertilization
The standards shown in Fig. l were used to identify histone variants synthesized in the unfertilized sea urchin egg. Acidsoluble proteins labeled with [3H]lysine were extracted from unfertilized eggs and electrophoresed. A fluorogram of the gel is shown in Fig. 2A. The CS HI, CS H2A, and CS H2B are labeled. Even in overexposed gels, no a variants are present. In addition to bands that comigrate with the CS proteins, we see proteins whose mobilities correspond to O, P, and M described above. All of these newly synthesized proteins are soluble in 0.4 N H2SO4, all contain lysine, and all comigrate with known standards, but additional protein chemistry will be necessary  to confirm their identity. Fig. 3 shows that the unfertilized eggs make the same proteins when the labeling time is of different lengths. Fig. 3 A is a 2-h labeling and Fig. 3 B is a 4-h labeling pattern.
Using one-dimensional SDS gels, Ruderman and Schmidt (32) showed the unfertilized egg to be active in histone synthesis. However, since both CS-and a-histone mRNAs are present in the egg, it was uncertain just which of the histone types was synthesized. Our data show that only CS mRNAs are translated by the unfertilized egg; a proteins either are not synthesized or are made in insignificant amounts. These data are consistent with the observation that several hundred haploid equivalents of CS proteins are stored in the urchin egg (11), whereas no a storage is detected. If a histories were synthesized at levels comparable to CS variants in the unfertilized egg, a histories would have to be preferentially degraded.
If a histories are not made before fertilization, when is a mRNA first translated? Earlier reports show that a proteins are the predominant histone variants made and incorporated into chromatin at the third S phase after fertilization (4). However,  using an isotope of higher specific activity, the two-dimensional gel system, and whole cell extracts, we reexamined the time after fertilization when a synthesis begins.
In S. purpuratus grown at 15°C, the first S period is at 30--45 rain postfertilization, the second S period at 105-125 min, the first cell division at 100-120 min, and the third S period at 170--185 min (33). Fig. 2B shows histones labeled with [SHllysine continuously from 5-70 rain postfertilization (30 min before second S phase). A striking change is apparent. In addition to the continued synthesis of CS proteins, a mRNA translation has commenced. In the H2A class, the a H2A becomes the predominant variant synthesized. In the H2B class, a H2B is the major protein. The rapidity of the change suggests that the appearance of a is not due to the translation of a small amount of newly synthesized RNA (34). Fig. 4 shows that the CS to a transition can also be triggered by artificial activation with pH 9.0 ammonia-seawater (26). The pattern of proteins made is very similar to that seen after fertilization. Thus there is no apparent role of the sperm in control of the histone synthetic switch that cannot be mimicked chemically. Preliminary results show that this early appearance of a histones occurs even in the presence of actinomycin D (data not shown).

a Histones Are N o t a Major Component of Male Pronuclear Chromatin
The synthesis of histones well before their incorporation into replicating chromatin is unlike the situation reported for many other cell types, in which historic and DNA synthesis are more or less tightly coupled within a given cell cycle and nonchromosomal histone pools are believed to be small or nonexistent (35). The oocytes and early embryos of sea urchins and frogs differ in that historic and DNA synthesis are uncoupled and substantial pools are maintained (10, 11, 36-39).

R^P~O CO~MUN~C^T~ONS
Although synthesis, of the c~ variants occurs within the first cell cycle, there is no detectable incorporation of a histones into the male pronuclear chromatin during this time, as judged by Coomassie Blue staining ( Fig. 1B and Poccia et al. [10]). However, later in the morula stage, a becomes the predominant historic variant type in the chromatin (1). Since there are enough CS histones stored in the unfertilized egg to package all the DNA made up to at least the blastula stage, but blastula chromatin contains largely a variants, at some point in development a incorporation into chromatin must predominate over CS incorporation.
It is possible that the flow of a histones, newly synthesized affter fertilization, through a large pool of mostly preformed CS histones, results in a substantial dilution effect. This would delay the appearance of significant levels of a in the chromatin until after the first few cell cycles, when the composition of the pool may change, reflecting greater input of a forms and perhaps withdrawal of CS forms. Although sea urchin histones, once they are incorporated into chromatin, are long-lived (2,40), nothing is known about turnover of the stored histone pool.
A second possibility for the delay between a histone synthesis and appearance in the chromatin is that a variants, once made, are sequestered until the third S phase when they are released and assembled into chromatin. Thus chromatin in the third cycle would undergo a qualitative, rather than a simple quantitative transition in its composition (cf. reference 4).
To clarify these issues, it is essential to compare, at several developmental stages, the histone composition of the chromatin with its cognate histone pool. Clearly, further study on the fate of the stored CS proteins is necessary to determine whether they are degraded, rendered assembly incompetent, or assembled into chromatin at a later developmental stage. In some earlier papers (4, 11) labeled a histories can be seen in extracts of nuclei isolated at the two-cell stage. However, no careful quantitative measurements of the ratio of a to CS variants in embryonic nuclei of the first three or four cycles after fertilization are yet available. Such ratio measurements are hampered by contamination with nonchromosomal proteins (including histones from the pool) and low nuclear yields.
There are many possible mechanisms for the translational regulation of a histone mRNA. An old idea is that specific proteins might "mask" the mRNAs in the unfertilized egg. At fertilization or later in development there would be a selective unmasking dictated by the maternal program. Recent work, however, suggests another possible mechanism. A significant amount ofhistone gene transcripts, at least 12%, is sequestered in the female pronucleus and is later released into the cytoplasm (20). The observed delayed recruitment of H3 histone message (19) is consistent with this observation. It is likely, in light of our results, that much of histone mRNA recruitment is of mRNA. We are now testing these ideas. If a message is sequestered in the female pronucleus, we would expect that the message isolated from nucleated egg halves would code for both a and CS histones, while mRNA from annucleate merogons would only code for CS variants.
L. Herlands would like to thank Jo¢l Rosenbaum for his guidance.
This work is supported in part by National Institutes of Health grants GM17383 and HD12982 and the Harry Winston Foundation.