Histone acetylation during early stages of sea urchin (arbacia punctulata) development

428 Preliminary

notes

frequency distribution of strand lengths (unpublished results) is very broad and not at all similar to the distribution reported here for connecting strands in mitotic fibers. The CHO interphase nucleus is more active in replication and transcription than CHO mitotic chromosomes. It is therefore important to know if the morphological difference in subunit structure reported here can be detected by nuclease digestion studies comparing active and inactive chromatins. We thank Dr D. E. Olins for advice and support, and Miss P. A. Brimer, Dr J. P. O’Neill, and Mrs M. H. Hsie for useful contributions. G. B. H. is postdoctoral investigator supported by subcontract no. 3322 from the Biology Division of Oak Ridge National Laboratory to the University of Tennessee.

B R, van Bruggen, E F J & Arnberg, A C, Biochem biophys res commun 60 (1974) 1365. Woodcock, C L F, J cell bio159 (1973) 368a. ii: Woodcock, C L. Mauuire, D L & Stanchtield. J E, J cell biol 63 (19745377a. 25. Woodcock, C L & Frado, L-L Y, Biochem biophys res commun 66 (1975) 403. 26. O’Neill, J P, Schroeder, C H, Riddle, J C & Hsie, A W, Exp cell res 97 (1976) 213. Received March 3, 1976 Accepted March 29, 1976

Histone acetylation during early stagesof sea urchin (Arbacia punctuhta) development CAROLYN J. BURDICK and BARBARA A. TAYLOR, Department of Biology, Brooklyn College, Brooklyn, NY 11210, and Marine Biological tory, Woods Hole, MA 02543, USA

Labora-

A correlation has been found between histone acetylation and gene activation at the time of gastrulation in the sea urchin, Arbacia punctulata. Between the blastula and gastrula stages, there is a 2.5-fold increase in the rate of acetylation of a histone fraction consisting of slightly lysine-rich and argininerich components, with a marked decrease in the fully differentiated pluteus stage. This pre-gastrular increase in histone acetylation is not correlated with (2) a decrease in the rate of histone deacetylation; (2) a decrease in acetyl coenzyme A pool size; (3) an increase in acetate uptake; (4) histone synthesis. The results thus suggest that increased histone acetylation may be at least one preparative factor for the activation of new genes at gastrulation.

Summary.

References 1. Clark. R J & Felsenfeld. G., Biochemistrv 13 (1974) 3622. 2. Corden, J, Lohr, D & Van Holde, K E, Fed proc (1975) 581, Abstr. no. 2052. 3. Finch, J T, Noll, M & Kornberg, R D, Proc natl acad sci US 72 (1975) 3322. 4. Ham. R G. Proc natl acad sci US 53 (1965) 288. 5. Honda, B M, Baillie, D L & Candid& E P, FEBS lett 48 (1974) 156. 6. - J bib1 chkm 250 (1975) 4643. 7. Hsie, A W & Puck, T T, Proc natl acad sci US 68 (1971) 358. 8. Koa, F T & Puck, T T, Proc natl acad sci US 60 (1968) 1275. 9. Kornberg, R D, Science 184 (1974) 868. 10. Lohr, D & Van Holde, K E, Science 188 (1975) 165. 11. Miller, 0 L & Bakken, A H, Acta endocrinol, suppl. 168 (1972) 155. 12. Noll, M, Nature 251 (1974) 249. 13. Olins, A L, Carson, R D & Olins, D E, J cell biol 64 (1975) 528. 14. Olins, A L & Olins, D E, J cell bio159 (1973) 252a. 15. - Science 183 (1974) 330. 16. Oosterhof, D K, Hozier, J C & Rill, R L, Proc natl acad sci US 72 (1975) 633. 17. Oudet, P, Gross-Bellard, M & Chambon, P, Cell 4 (1975) 281. 18. Robbins, E & Marcus, P I, Science 144 (1964) 1152. 19. Sahasrabuddhe. C G & Van Holde. K E. J biol them 249 (1974j 152. 20. Senior, MB, Olin% A L & Olins, D E, Science 187 (1975) 173. 21. Tres, L L & Kierszenbaum, A L, J cell biol 65 (1975) 258. 22. Van Holde, K E, Sahasrabuddhe, C G, Shaw, Exp CellRes lOO(1976)

Investigations of RNA from various developmental stages of the sea urchin by DNA-RNA hybridization methods have suggested that the kinds of RNA synthesized at the blastula stage are identical to those which are already present in the unfertilized egg. Between the blastula and gastrula stages, however, “different” RNAs appear which are characteristic of the later stages of development. This finding has led to the suggestion that new genes may be activated at this time [I, 21. The fact that isolated embryo chromatin retains this stage-specific transcriptional capacity [3] indicates that the control of template specificity resides within the chromatin itself.

Preliminary

It has been suggested that modifications of amino acid residues within the basic chromosomal proteins-the histones-may play an important role in gene regulation by altering the binding properties of these modified histones to DNA. One such modification which has received a good deal of attention is histone acetylation. Argininerich histone fractions f2a 1 and f3 and the slightly lysine-rich histone fractions f2a2 and I2 b are found to be acetylated on specific &-NH* lysyl residues [4-91. Evidence that such acetylation does indeed alter histone binding to DNA has recently been reported [lo, 111. Physiologically, the degree of histone acetylation in different tissues agrees well with the capacity of their chromatin to synthesize RNA [12-161. Increased histone acetylation has been found to accompany gene activation in phytohemagglutinin-stimulated lymphocytes [ 171 and regenerating rat liver [18]. In the present investigation, an attempt has been made to determine whether or not an increase in the rate of histone acetylation is correlated with the apparent activation of new genes between the blastula and gastrula stages of sea urchin (Arbacia punctulata) development. Materials

and Methods

Preparation of embryos. Eggs were obtained from Arbacia punctulata by injection of 0.5 M KCI into the

coelomic cavity, washed three times with filtered sea water, and fertilized with a 0.1% suspension of sperm. Only those cultures in which fertilization was at least 95% were used. Cultures which were to be used for isolation of nuclei from pre-hatching embryos were demembranated immediately after fertilization according to the procedure of Hynes et al. [19]. Otherwise nuclear preparations were highly contaminated with fertilization membranes and cytoplasm. Fertilized eggs were washed free of excess sperm with filtered sea water, and embryos were grown to the desired stages at 20°C. Radioactive labeling. Embryos from various develonmental stages were oulse-labeled for 1 h at 20°C with-either 20 &/ml [Y?]sodium acetate (100 mCi/ mmole: New Enaland Nuclear) or 5 &i/ml PHILleucine (5 Cilmmole; New England Nuclear): The

notes

429

embryo concentration at the time of labeling as determined by a hemocytometer count was the same at all stages of development. In pulse chase experiments, after the labeling period, the culture was divided in half. One half was used immediately for preparation of nuclei as described below. The other half was centrifugated. The embryos were washed three times with filtered sea water, resuspended in filtered sea water containing 2~ 10e3 M cold sodium acetate and incubated for another hour at 20°C. Preparation

of nuclei

and extraction

of histone.

After labeling or chase periods, embryos were collected by centrifugation and nuclei were prepared by the procedure of Thaler et al. [20]. Histone was extracted with 10 vol of 0.2 N HCI for 1 h at 4°C and precipitated overnight with 10 vol of acetone. The histone precipitate was collected by centrifugation, washed with acetone and ether, and dried. Electrophoresis. Histone electrophoresis was done on cellulose polyacetate strips (Sepraphore III, Gelman Instrument Co.) in 0.05 M barbital buffer, pH 9.0, containing 8 M urea for 1 h at 300 V. Using this method, the histone was separated into two major bands: a faster moving band (fraction I) consisting of lysinerich histone (fl) and a slower moving band (fraction II) consisting of slightly lysine-rich and arginine-rich histones (f2a 1, f2a2, f2b and f3) [17]. In order to determine the specific activities of the protein in each band, the strips were cut in half longitudinally. One half of each strip was stained, and the histone concentration in each band was determined colorimetritally [21]. The corresponding areas on the other half of the strip were added to Bray’s scintillation mixture. Radioactivity was measured in a Beckman scintillation counter. Acetate uptake. To determine the rate of uptake of the tritiated acetate into the embryos themselves, embryos were washed extensively with filtered sea water after a 60 min pu!se with [3H]sodium acetate. Thev were then homogenized in distilled water, and an aliquot of this whole embryo homogenate was added to Aquasol (New England Nuclear). Radioactivity was -determined in a Beckman scintillation counter. Acetyl coenzyme A pool size. Acetyl coenzyme A was extracted from embryos at various stages of development with 5% perchloric acid. The amount of acetyl CoA in these extracts was measured by the method of Lumbers et al. [22].

Results acetylation. Fig. 1 shows the amount of incorporation of [3H]sodium acetate into histone fractions I and II during a 60 min pulse by sea urchin embryos at different stages of development. It can be seen that there is a 2.5fold increase in the rate of acetylation of histone fraction II, consisting of the slightly lysine-rich (f2a2 and f2b) and arginine-rich (f2a 1 and f3)

Histone

Exp CdRes lOO(1976)

430 Preliminary notes 5600 2400

-

2200

-

2000

-

2600

-

2600

-

2400

-

2200

-

2000

-

1600

-

1600

-

t

t

t

t

1400

EARLY BLASTLILA

HATCHED GLASTULA

GASTRULA

PLUTEUS

1200

age of embryos (hours); ordinate: (/Ig. I) [3H]acetate incorporation dpm/pg protein; gg. 2) [3H]L-leucine incorporation dpm/wg protein. A-A, Histone fraction I; O-O, histone fraction II. For each embryo age, the mean of the number of determinations indicated in parenthesis and the range of values obtained is plotted.

Figs 1, 2. Abscissa:

components. This increase starts at about 14 h of development (hatched blastula stage) and reaches a peak at the time of gastrulation (22 h), with a marked decrease in the fully differentiated pluteus stage (48 h). The increase is significant at the 1% level. The rate of acetylation of histone fraction I (lysine-rich histone fl), on the other hand, is minimal and does not change significantly during the developmental period studied. Histone deacetylation. Pulse chase experiments were carried out to determine the rate of deacetylation of histone fraction II by embryos at the early blastula (8 h), hatched blastula (14 h), and gastrula (22 h) stages of development. As seen in table 1, 8 h embryos showed the lowest rate of acetate release from this histone fraction during the 60 min chase period. Even at the later stages of development, however, the acetylated histones in this fraction appeared to be quite stable since the maxExp Cell Rrs 100 (1976)

1000

-

800

-

600

-

400

-

200

0

2

4

6

8 10 t EARLY ELASTULA

12

14 16 t HATCHED BLASTLILA

16

20

22

t GASTAULA

t PLUTEUS

imum rate of deacetylation observed in 1 h was only about 14%. Acetyl coenzyme A pool size. The pool size of acetyl coenzyme A, the immediate precursor for histone acetylation, was measured in embryos at 8, 14, and 22 h of development. Table 2 shows that the number of pmoles of acetyl CoA/embryo was slightly higher in 8 h embryos than in the other two developmental stages studied.

Table 1. Rate of histone fraction ZZ deacetylation in early sea urchin embryos % Loss of r3H]acetate from histone fraction II after 60 min chase Age of embryos (hours)

Mean %

(No. of determinations)

8 (Early blastula) 14 (Hatched blastula) 22 (Gastrula)

9 13.5

(3) (3)

14.3

(3)

Preliminary

Table 2. Acetyl

coenzyme A pool size of early sea urchin embryos Acetyl coenzyme A pool (pmoles acetyl CoA/embryo) Age of embryos (hours)

Mean

(No. of determinations)

5.6x lo+ 8 (Early blastula) (2) 14 (Hatched blastula) 3.2x 1O-5 (2) 22 (Gastrula) 3.6x lo+ (2)

Acetate uptake. The rate of acetate uptake into sea urchin embryos from 8 h to 22 h of development was measured. The results are shown in table 3. The rate of uptake was found to be approximately the same for embryos from 8 h to 18 h of development. At 22 h, a small increase in rate was observed. This increase, however, is not statistically significant (0.2

O. 1). Histone synthesis. Fig. 2 shows the amount of incorporation of [3H]leucine into histone fractions I and II during a 60 min pulse. A marked decrease in the rate of synthesis of both of these histone fractions by sea urchin embryos during the developmental period studied can be clearly observed. Discussion

In mammalian systems, correlations have been reported between changing patterns of histone acetylation and gene activation [ 17, 181. In the work presented here, a correlation has also been found between increased histone acetylation and gene activation at the time of gastrulation in the sea urchin, Arbacia punctulata. Namely, between the blastula and gastrula stages, a 2.5fold increase in the rate of acetylation of a histone fraction consisting of histones f2a1, f2a2, I2 b, and f3 has been observed. This result is in agreement with Johnson et al. [23]

notes

43 1

who used the sea urchin, Strongylocen trotus purpuratus and reported that the extent of acetylation of histones f2a2 and f2 b is highest at the mesenchyme blastula and gastrula stages in this sea urchin species as well. Histone fl acetylation is minimal in their system as well as in ours. Yukawa et al. [24], on the other hand, using another sea urchin species, Anthocidaris crassispina, have found that an increase in basic protein acetylation does not occur until the post-gastrular period. Although the findings described above strongly suggest a significant pregastrular increase in the rate of acetate incorporation into histone fraction II, there are several alternative explanations for the results observed: (1) More rapid histone deacetylation rates in the early embryos; (2) larger acetyl coenzyme A pool sizes in the early embryos; (3) a pre-gastrular increase in the rate of acetate uptake into the embryos; (4) correlation with histone synthesis. These possibilities were therefore investigated. (1) The rate of release of labeled acetate groups from histone fraction II in a 1 h cold chase was determined using 8, 14, and 22 h embryos. The slowest rate of histone deacetylation was observed in the youngest embryos. It therefore does not appear that

Table 3. Acetate urchin embryos

uptake

into

early

sea

Rate of [3H]acetate uptake (dpmlembryolhour) Age of embryos (hours)

Mean fS.E.

(No. of determinations)

8 (Early blastula) 7.19k2.36 6.%+ 1.30 10 14 (Hatched blastula) 6.97+ 1.60 7.05+ 1.42 18 10.6 f1.44 22 (Gastrula)

(5) (5) (5) (5) (5)

Exp CellReslOO(1976)

432 Preliminary

notes

the apparent increase in histone acetylation period. It is therefore highly unlikely that in the older embryos simply reflects a more what we are observing simply reflects conrapid turn-over of the histone acetyl groups version of the labeled acetate into amino at the earlier stages of development. It acids by the embryos and subsequent inshould also be noted that in Arbacia the corporation into histone during the pulsemaximum turnover rate of the acetylated labeling period. The marked decrease in histone which was observed at the gastrula histone synthesis between the blastula and stage (14 %/h) was much lower than that re- gastrula stages in Arbacia is in agreement ported by Johnson et al. [23] in Strongylowith results obtained using Strongylocentrotus gastrulae (about 50 %/h). Such centrotus [27-291. It supports the conslow rates of histone deacetylation are not clusion that histones are synthesized unknown, however, in other systems [16, concomitantly with DNA [27-3 11, since the 25, 261. rate of cell division slows markedly after (2) Acetyl coenzyme A is the immediate the blastula stage of development [32]. It therefore appears that there is, indeed, precursor for histone acetylation. Pool sizes of this substance were measured in em- a significant increase in the rate of incorbryos at the early blastula (8 h), hatched poration of acetate into slightly lysine-rich blastula (14 h), and gastrula (22 h) stages. and arginine-rich histone between the blasThe amount of acetyl CoA/embryo was tula and gastrula stages of Arbacia puncfound to be slightly higher in the 8 h em- data development, correlated with the apbryos. This difference in pool size is not parent activation of new genes at this time. large enough, however, to account for the While the results obtained do not establish 2.5fold increase in histone acetylation at a cause and effect relationship between the the later stages of development simply on two events, they certainly strongly suggest the basis of a lower specific activity of the that increased histone acetylation may be at least one preparative factor for the activaacetate donor in the younger embryos. (3) The rate of acetate uptake into em- tion of new genes at the gastrula stage of bryos from 8 h to 22 h of development was sea urchin development. determined. No statistically significant This work was supported in part by the City Unichange in acetate uptake was noted during versity of New York Faculty research award no. 1587. this embryonic period encompassing early blastula, hatched blastula, and gastrula stages. We can therefore rule out the possibility that the pre-gastrular increase in References 1. Whitely, A H, McCarthy, B J & Whitely, H R, histone acetylation observed is simply due Proc natl acad sci US 55 (1966) 519. to an increase in the rate of acetate uptake 2. Glisin, V R, Glisin, M V & Doty, P, Proc natl acad sci US 56 (1966) 285. into the embryos at this stage in the devel3. Chetsanga, C J, Poccia, D L, Hill, R J & Doty, P, opmental process. Cold Spring harbor symp quant bio135 (1970) 629. 4. Vidali, G, Gershey, E L & Allfrey, V G, J biol (4) The observed increase in the rate of them 243 ( 1%8) 6361. histone acetylation is not correlated with 5. Gershev. E L. Vidali. G & Allfrev. V G. J biol them 243 (1%8) 5018.’ histone synthesis, as seen by the fact that 6. DeLanae. R J. Fambrouah. D M. Smith. E L & the rate of incorporation of [3H]leucine Bonne< J, J biol them 24;i (lW9) 3 19. 7. - Ibid 244 (1969) 5669. into histone was found to undergo a marked 8. Sanders, L A, Schechter, N M & McCarty, K S, decrease during the same developmental Biochemistry 12 (1973) 783. aI

Exp Cell Res 100 (1976)

Preliminary 9. Jackson. V. Shires. A, Chalklev, R & Granner, D K, J biol &em ZSO’(1975)4856.10. Allfrey, V G, Faulkner, R & Mirsky, A E, Proc natl acad sci US 5 1 (1964) 786. 11. Adler, A J, Fasman, G D, Wangh, L J & Allfrey, V G, J biol them 249 (1974) 2911. 12. Wangh, L A, Ruiz-Carrillo & Allfrey, V G, Arch biochem biophys 150 (1972) 44. 13. Berlowitz, L & Palotta, D, Exp cell res 71 (1972) 45. 14. Gorovsky, M A, Pleger, G L, Kleevert, J B & Johmann, C A, J cell bio157 (1973) 773. 15. Joachim, H. Cell differ 4 (1975) 123. 16. Sarkander. H I. Fleischer-Lambroooulos. H & Brade, W P, FEBS lett 52 (1975) 40. 1 17. Poeo. B G T. Allfrev. V G & Mirskv. A E. Proc na; acad sci US 55 (1966) 805. ’ 18. Pogo, B G T, Pogo, A 0, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 59 (1%8) 1337. 19. Hynes, R 0 &Gross, P R, Dev biol21 (1970) 383. 20. Thaler, M M, Cox, C L & Villee, C A, J cell bio142 (1969) 846. 21. Tidwell, T, Allfrey, V G & Mirsky, A E, J biol them 243 (1968) 707. 22. Lumbers, J, Threlfall, C J & Stoner, H B, Anal biochem 31 (1969) 21. 23. Johnson, A‘W, Wilhelm, J A & Hnilica, L S, Biochim bionhvs acta 295 (1973) 150. 24. Yukawa, O’&-Koshihara,‘ H, Dev biol 33 (1973) 477. 25. Byvoet, P, Biochim biophys acta 160(1968) 217. 26. Shepherd, G R, Biochim biophvs _ - acta 299 (1973) 485: 27. Orengo, A & Hnilica, L A, Exp cell res 62 (1970) 331. 28. Moav, B & Nemer, M, Biochemistry 10(1971)881. 29. Seale, R L & Aronson, A I, J mol biol 75 (1973) 633. 30. Gallwitz, D & Mueller, G C, J biol them 244 (1969) 5947. 31. Kedes, L H, Gross, P R, Cognetti, G & Hunter, A L, J mol bio145 (1969) 337. 32. Harvey, E B, The American Arbacia and other sea urchins. Princeton University Press, Princeton, NY (1956). Received March 10, 1976 Accepted April 7, 1976

Stimulation of cellular RNA synthesis in mouse-kidney cell cultures infected with SV40 virus P. MAY, E. MAY and J. BORDE, Znstifut de Recherches Scientifiques France

sur le Cancer, 94800 Villejuif*

In confluent primary mouse-kidney cell cultures, abortive infection with SV40 has been demonstrated to cause an increase in the bulk of cel-

Summary.

notes

433

lular RNA (mainly rRNA). However, the increase in the rate of rRNA synthesis is not involved in the initiation of the virus-induced cellular DNA replication since after actinomycin D treatment (0.05 Fg/ml, from 6 to 9 h p.i.) the onset of cellular DNA replication takes place at a time when the rate of rRNA synthesis is still depressed.

Simian virus 40 (SV40) induces in confluent, “contact-inhibited” primary mousekidney cell cultures an abortive infection that leads to the replication of the cell chromatin and mitosis while no detectable amounts of viral progeny DNA or capsid protein are produced [l-4]. In this paper, we show that infection of confluent primary mouse-kidney cell cultures with SV40 stimulates the synthesis of the. bulk of cellular RNA (mainly rRNA). To decide whether the activation of rRNA is involved in the virus-induced initiation of cellular DNA replication, an analysis of recovery times of the macromolecular syntheses after actinomycin D treatment has been performed. Materials

and Methods

Primary mouse-kidney cell cultures, prepared from lo-day-old CR-l mice, were grown in large lOO-mm plastic Petri dishes or, when indicated,-in 30-mm plastic Petri dishes containing 22 X 22 mm glass coverslips (coverslip cultures). The cultures were infected with 0.4 ml (or 0.15 ml in 30-mm Petri dishes) of plaque purified non-defective wild-type SV40 [ 1, 21. All viral preparations contained about 2X 108PFU/ml. The results were the same whether crude viral lysates or highly purified viral preparations were used for infection. The rate of [5-3H]TdR incorporation was determined bv labellina coverslio cultures for 1 h at the times indicated inResults as described in ref. [ 11. SV40-infected and mock-infected mouse-kidney cells were incubated for 1 h at the appropriate times with 10 &i [5-3H]uridine ([3H]U, CEA, France, 20 Ci/ mmol) and 5 pg/ml unlabelled uridine (Calbiochem, A Grade) in 5 ml of medium. The cultures were then washed consecutively with ice-cold phosphate buffer saline (PBS) and cold 5 % trichloroacetic acid (TCA). After centrifugation at 16000 g for 5 min, DNA and RNA were extracted from the pellet according to Schmidt & Thannhauser r51 and the RNA content was determined by the orcinoi method with yeast RNA (grade, XI, Sigma) used as standard. A 100~~1aliquot of the alkaline hydrolysate containing the nucleotides derived from the RNA was mixed in 2 ml of ethanol and its radioactivity was determined. Exp Cell Res 100 (1976)