Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1237-1241, February 1995 Cell Biology

Conservation of deposition-related sites in newly synthesized H3 and H4 (chromatin/nucleosome assembly/deblocking and microsequencing) RICHARD E. SOBEL*, RICHARD G. COOKt, CAROLYN A. PERRY*, ANTHONY T. ANNUNZIATOt, AND C. DAVID ALLIS*§ *Department of Biology, Syracuse University, Syracuse, NY 13244; tDepartment of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030; and *Department of Biology, Boston College, Chestnut Hill, MA 02167 Communicated by Salih J. Wakil, Baylor College of Medicine, Houston, 7X, October 12, 1994

ABSTRACT Newly synthesized H4 is deposited in deposited into macronuclei (4). By stain and by label, histone a diacetylated isoform in a wide variety of organisms. In extracted from these micronuclei is greatly enriched in di- Tetrahymena a specific pair of residues, 4 and 11, have acetylated H4 and mono- and diacetylated forms of H3. been shown to undergo this modification in vivo. In this report, Because micronuclei are transcriptionally inactive, acetylation we demonstrate that the analogous residues, lysines 5 and 12, of newly synthesized H3 and H4 in micronuclei is clearly are acetylated in Drosophila and HeLa 114. These data strongly distinct from transcription-related acetylation in this system. suggest that deposition-related acetylation sites in H4 have Despite evidence suggesting a conserved pattern of depo- been highly, perhaps absolutely, conserved. In Tetrahymena sition-related H4 diacetylation, very few studies have at- and Drosophila newly synthesized histone H3 is also deposited tempted to ascertain the sites of diacetylation in newly syn- in several modified forms. Using pulse-labeled H3 we have thesized H4 in vivo. We previously demonstrated that a specific determined that, like H4, a specific, but distinct, subset of pair of residues in Tetrahymena H4, lysines 4 and 11 (K4/K11), lysines is acetylated in these organisms. In Tetrahymena, is utilized during deposition-related H4 diacetylation (10). lysines 9 and 14 are highly preferred sites ofacetylation in new Similarly, 5 (which is analogous to lysine 4 in Tetrahy- H3 while in Drosophila, lysines 14 and 23 are strongly pre- mena) showed the greatest turnover of acetyl groups in ferred. No evidence has been obtained for acetylation ofnewly S-phase synchronized Physarum (11). Our recent ability to synthesized H3 in HeLa cells. Thus, although the pattern and chemically deblockDrosophila H4 (12) has provided an inroad sites of deposition-related acetylation appear to be highly to examine deposition-related acetylation sites in other sys- conserved in H4, the same does not appear to be the case for tems. We have extended our initial results in Tetrahymena by histone H3. deblocking newly synthesized Drosophila and HeLa H4 and demonstrating that the same residues, lysines 5 and 12, are Modification of histones by acetylation of the E-amino group acetylated. Thus, the K5/K12 pattern of acetylation is likely a of specific lysine residues in the N-terminal domain of all four hallmark property of new H4 in all organisms. core histones is an active metabolic process whose exact In Tetrahymena and Drosophila, newly synthesized histone function remains controversial. The primary focus of much H3 is also deposited in several modified forms. Using the same current research is on how histone acetylation relates to the approach, we have determined the sites of acetylation in newly regulation of gene expression (1, 2). Less attention is being synthesized H3 from Tetrahymena and Drosophila. As is the placed on understanding the biological function of deposition- case with H4, a specific, but distinct, subset of lysines is related acetylation, a reaction first described to affect specific acetylated. Thus, at least in these organisms, a nonrandom histones during synthesis and deposition onto replicating chro- acetylation pattern is observed for both of the arginine-rich matin (3, 4). core histones that are the first to be deposited during stepwise Biochemical analyses of deposition-related acetylation is chromatin assembly (13). In contrast, we find no evidence for hampered by the fact that only a fraction of the total histone deposition-related acetylation of H3 in HeLa cells. is affected and once newly synthesized histone is deposited into nuclei, the pattern of acetylation is remodeled to fulfill tran- AND METHODS scription-related functions. In most systems, deposition- MATERIALS related acetylation is witnessed only by administering a short Cell Culture, Labeling Conditions, Nucleus Isolation, and pulse oflabel to preferentially label newly synthesized histones. Histone Extraction. Tetrahymena. Genetically marked strains Using this approach, numerous studies have reported that of Tetrahymena thermophila were used in all experiments newly synthesized H4 is deposited as a modified isoform (3-9). reported here. Cells were grown, starved, and mated as This modification, although poorly understood, occurs in described (14). More than 85-90% pairing was observed at 3-4 organisms ranging from protozoa to humans and thus appears hr in all experiments. Where appropriate, cells were pulse- to be highly conserved. labeled for 2-30 min with [3H]lysine (2 ,gCi/ml; 100 mCi/ In the ciliated protozoan Tetrahymena, deposition-related mmol; 1 Ci = 37 GBq) at 5 hr of conjugation. Highly purified acetylation is particularly clear because of the separation populations of micronuclei were isolated (15) with modifica- between germ-line and somatic nuclei. Each vegetative cell tions (16) and reversal of formaldehyde cross-links, and re- contains a transcriptionally active, somatic macronucleus that covery of acid-soluble protein was as described (17). governs the phenotype of the cell and a transcriptionally inert, Drosophila. Kc cells were grown in D-22 medium at 24°C. germinal micronucleus that is responsible for genetic conti- During logarithmic phase, cells were placed in Schneider nuity. During conjugation, the sexual stage of the life cycle, medium lacking lysine and yeastolate (GIBCO) for 6-8 hr at essentially all of the newly synthesized H3 and H4 is selectively room temperature to deplete endogenous lysine pools. Cells deposited into micronuclei; little if any new H3 or H4 is were concentrated in the same medium and labeled for 5-60 min with [3H]lysine (100 ,uCi/ml) in the presence of trapoxin The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: RP, reverse phase; PTH, phenylthiohydantbin. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1237 Downloaded by guest on September 30, 2021 1238 Cell Biology: Sobel et aL Proc. NatL Acad Sci USA 92 (1995) (100 ,ug/ml) to inhibit histone deacetylation (18). At the end lysine cpm was recovered at the appropriate cycles of micro- of the labeling period, an excess of ice-cold D-22 medium was sequencing and subjected to the analyses outlined in Fig. 1. added, cells were collected, and nuclei were prepared accord- Newly Synthesized Drosophila and HeLa H4 Is Diacetylated ing to Sobel et al. (12). Acid-soluble protein was recovered as Using Lysines 5 and 12. Using a [3H]lysine pulse label to described above. selectively follow newly synthesized H4, we sought to deter- HeLa. HeLa cells were pulsed for 2-10 min in the presence mine the in vivo sites of deposition-related H4 diacetylation in of 50 mM sodium butyrate with [3H]lysine as described (19). organisms as diverse as flies and humans to see if the sites used Nuclei were isolated according to Annunziato and Seale (20) under these conditions match the K4/K11 pattern previously except that 5 mM sodium butyrate was included in HeLa buffer determined for Tetrahymena (10). Using a modification of a A. Acid-soluble proteins were obtained as described above. procedure reported by Wellner et al. (22), we have recently Reverse-Phase (RP) Purification of Histones. Separation of shown that Drosophila H4 can be deblocked to permit direct Drosophila and HeLa core histones was as described (12). As microsequence analysis of acetylation sites (12). We reasoned with Drosophila histones, HeLa H4 coelutes with H2A under that it should be possible to deblock HeLa H4 by this same these conditions. HeLa H3, on the other hand, elutes as two procedure. highly purified H3 subtypes. Based on mobility in Triton/acid/ Presented in Fig. 2 and Table 1 are the data from an analysis urea gels and the published order of elution of butyrate- with pulse-labeled Drosophila and HeLa H4. Several results treated histones from a similar RP-HPLC (21), we are equat- are noteworthy. First, the expected N-terminal sequence of ing the first H3 peak with H3.2/H3.3 and the second peak with both H4s was obtained during sequencing, demonstrating that H3.1. All fractions recovered from RP-HPLC were dried the deblocking treatment with trifluoroacetic acid was suc- under vacuum and stored dry until needed. cessful. Second, with both H4s, the majority of the [3H]lysine Gel Electrophoresis and Electrophoretic Transfer. SDS and label recovered at positions 5 and 12 elutes at the position of acid/urea (AU) gels used in this study were as described (4, 12). Acetylated isoforms from AU gels or in some cases SDS 5 hr Mating Tet Drosophila, HeLa gels were directly electroblotted from these gels onto Immo- I bilon-PSQ membranes (Millipore). No evidence was found for any selective transfer/retention of specific acetylated histone subspecies. K7s Deblocking of Drosophila and HeLa H4 and Automated Microsequencing Procedures. Pulse-labeled H3 and H4 from pulse-label with[3H]lysine Tetrahymena, Drosophila, or HeLa were recovered by RP- RP-HPLC /gel - blot HPLC and, where appropriate, were microsequenced directly. purify (deblock) Where indicated, histones were first electrophoresed in a SDS sequence H3 or H4 or AU gel before being transferred to Immobilon and se- quenced directly off of the membrane. Deblocking of Dro- II "ico | sophila and HeLa H4 was similar to that described earlier for Drosophila H4 (12) except that, after SDS gel electrophoresis -*- repetitive and electroblotting to remove contaminating H2A, H4 was yield deblocked by incubating the Immobilon membrane directly in 100% trifluoroacetic acid. cycle no.0 f4/5 7/8 11/12 15/16 Microsequencing was as described (12). Since H3 is not blocked in Tetrahymena, Drosophila, and HeLa, deblocking (Recver cp7m steps were not needed. In each sequence analysis, 75% of each cycle was routed to the in-line phenylthiotrydantoin (PTH) analyzer (Applied Biosystems, model 120A) for residue iden- RP-HPLC tification and was collected for further analyses as described ^, |cycle Eiycle no. below. In all cases, the appropriate N-terminal sequence of H4 or H3 was obtained. Fractions from the PTH analyzer were collected into scintillation vials at 1-min inter- vals using an external fraction collector. Under these condi- tions, the PTH derivative of acetyllysine (eluting from the column at fraction 15) is well separated from that of unmod- ified lysine (which elutes at fraction 29). FIG. 1. Strategy for identification of acetylation sites in newly synthesized H3 and H4. (1) Five-hour mating Tetrahymena or expo- nentially growing Drosophila or HeLa cells were pulse-labeled with RESULTS [3H]lysine in vivo. After nucleus isolation, total histone was extracted and H3 and H4 were purified by RP-HPLC and/or gel electrophoresis Strategy for Identification of Acetylation Sites in Newly and subsequently electroblotted to Immobilon membrane. In the case Synthesized H3 and H4. In these experiments, Tetrahymena, of Drosophila and HeLa H4, the protein was deblocked while immo- Drosophila, or HeLa cells were pulse-labeled with [3H]lysine as bilized on the membrane prior to microsequencing. (II) H3 and H4 a means to study deposition-related acetylation without com- were microsequenced and 75% of the material collected at each cycle plications from transcription-related acetylation, and in all was used for amino acid identification. At the position of each experiments histones were extracted from isolated nuclei. acetylatable lysine (shown here for H4 only), the [3H]lysine cpm was Thus, these experiments utilize H3 and H4 that is newly collected and analyzed further as in stepIIIbelow. The remaining 25% synthesized and deposited into replicating chromatin. Because of the material collected at each cycle was saved for direct scintillation cells are labeled in vivo with the 3H label is not counting of the cpm recovered. In all cases, the correct amino acid [3H]lysine, sequence was obtained. Dashed line, repetitive yield. (III) At the directly in the acetyl modification itself but is instead present position of each acetylatable lysine, [3H]lysine was subjected to at all positions where lysines exist in the H3 or H4 sequence. RP-HPLC using gradient conditions that separate the PTH derivative Thus, determination of the cpm released at each cycle of of acetyllysine (eluting at position 15) from unmodified lysine (eluting automated sequencing does not define which residues are at position 29). Typically, two or three fractions on each side of these acetylated. To determine which lysines are acetylated, [3H]- peaks were collected and assayed for background cpm. Downloaded by guest on September 30, 2021 Cell Biology: Sobel et aL Proc. Natl. Acad ScL USA 92 (1995) 1239

Tetrahymena di H4 Drosophila di H4 Human di H4 results suggest that this H4 acetylation pattern plays an im- portant functional role during chromatin assembly that has yet to be determined. Nonrandom Acetylation of Newly Synthesized H3 in Tetra- hymena and Drosophila. Giancotti et al. (8) reported that H3 synthesized in early Drosophila embryos is modified, although -LiaI. oI I O~~~~~~- l-a--A the nature of this modification was not determined. Using 20- 2 6 pulse-labeled Drosophila Kc cells, we have confirmed their 7 3 8 findings and shown that the majority of the pulse label in H3 migrates in AU gels as a diacetylated form with a minor Ex1 amount of the label in the monoacetylated isoform (data not shown). Similarly, the majority of newly synthesized H3 enters Tetrahymena micronuclei in either a di- or a monoacetylated L isoform during periods of active DNA replication (4). As far XoL ot as we are aware, no information exists as to whether a specific subset of lysines is being acetylated in newly synthesized H3 under these conditions. To address this question, pulse-labeled diacetylated H3 from Tetrahymena or Drosophila was microsequenced and analyzed by the strategy presented in Fig. 1. As with newly synthesized H4, a nonrandom pattern of deposition-related H3 acetylation emerges in both organisms (Fig. 3). In Tetrahy- Fractionno. Fractionno. Fractionno. mena, the majority of the [3H]lysine label recovered at posi- tions 9 and 14 elutes at the position of acetyllysine and displays FIG. 2.K4/KS and K11/K12 are the preferred sites of diacetyla- an average acetyllysine/lysine ratio of 2.9 (Table 2). In Dro- tion in newly synthesized Tetrahymena, Drosophila, and HeLa H4. sophila, however, lysines 14 and 23 are the preferred sites of Pulse-labeled Drosophila (Center) and HeLa (Right) H4 was recovered and analyzed as outlined in Fig. 1. In both cases, AU gel analyses showed that essentially all of the [3H]lysine label migrates at the position of diacetylated H4, and therefore isolation of the diacetylated isoform was not necessary. [3H]Lysine cpm recovered from cycles 5, 8, 12, and 16 were further analyzed by RP-HPLC under conditions that separate acetyllysine (fraction 15; see left asterisk under each plot) from unmodifiedaysine (fraction 29; see right asterisk under each plot). The expected N-terminal sequence of both H4s was obtained. For comparison, the data obtained for pulse-labeled Tetrahymena H4 was replotted from that originally presented in figure 4A of Chicoine et al. (10). Because of a deletion at position 3 in the Tetrahymena H4 (as compared to other H4s) the analogous lysines are at 4, 7, 11, and 15. acetyllysine (see asterisk on left of each plot) and exhibits an average acetyllysine/lysine ratio of 2.3 (Table 1). In contrast, x E the majority of the [3H]lysine cpm recovered at positions 8 and 0l. 16 elutes at the position of unmodified lysine (asterisk on right a of each plot) with an average acetyllysine/lysine ratio of 0.34. 6 For comparison, the data originally described for newly _ synthesized Tetrahymena H4 (taken from figure 4 of ref. 10) is & also presented in Fig. 2. It is clear that lysines 4 and 11, the residues analogous to 5 and 12 in other H4s, are the exclusive sites of diacetylation in Tetrahymena H4. We conclude that the in vivo pattern and sites of diacetylation in newly synthesized H4 are nonrandom and that K4/KS and K11/K12 are the exclusive sites of acetylation in cell types ranging from proto- zoa to flies to humans. Thus, this KS/K12 pattern of acetyla- tion site usage is highly, perhaps absolutely, conserved. These Table 1. Acetyllysine/lysine ratio in newly synthesized H4 Site of acetylation H4 5 8 12 16 Fraction no. Fraction no. Drosophila 2.54 0.38 2.52 0.68 FIG. 3. Newly synthesized H3 in Tetrahymena and Drosophila displays a nonrandom pattern acetylation site utilization. Pulse-labeled HeLa 2.28 0.14 1.65 0.31 Tetrahymena diacetylated H3 (extracted from micronuclei isolated Data shown are the same as those used to plot Fig. 2. Site refers to from 5-hr mating cells, electrophoresed in an AU gel, and electro- position of potentially acetylated lysines in each histone being se- blotted to Immobilon; Left) and total Drosophila H3 (recovered by quenced and to the cycle collected for RP-HPLC to separate acetyl- RP-HPLC; Right) were microsequenced and analyzed as in Fig. 1. lysine from lysine. Typically, two fractions from the RP-HPLC con- [3H]Lysine cpm recovered from cycles 4, 9, 14, 18, 23, and 27 were tained the majority of the counts at the position oflysine or acetyllysine collected and further analyzed by RP-HPLC under conditions that and were summed. cpm from the other six to eight fractions were separate acetyllysine from unmodified lysine exactly as described in averaged and subtracted as background. Fig. 2. Downloaded by guest on September 30, 2021 1240 Cell Biology: Sobel et aL Proc. Natl. Acad Sci. USA 92 (1995)

Table 2. Acetyllysine/lysine ratio in newly synthesized H3 Human H3.2V3.3 Human H3.1 Site of acetylation H3 4 9 14 18 23 27 Tet-di* 0.02 4.06 1.73 0.03 0.09 0.09 Tet-mono* 0.04 2.06 0.50 0.16 0.13 0.06 Dros-di 0.01 0.04 2.10 0.32 2.00 0.22 Dros-mono* 0.04 0.08 0.55 0.15 0.97 0.30 HeLa H3.1 ND 0 0.05 0 0 0 HeLa H3.2/3 ND 0 0.11 0.06 0 0 62 1 2.5. Data shown are the same as those used to plot Figs. 3 and 4. Site refers to position ofpotentially acetylated lysines in each histone being sequenced and to the cycle collected for RP-HPLC to separate acetyllysine from lysine. Typically, two fractions from the RP-HPLC .ff18 contained the majority of the counts at the position of lysine or E | acetyllysine and were summed. cpm from the other six to eight fractions were averaged and subtracted as background. ND, this cycle was not collected for analysis. Tet, Tetrahymena; Dros, Drosophila; di, 2 2 diacetylated; mono, monoacetylated. *These acetylated isoforms were blotted to nitrocellulose from acid/ 23 2 urea gels. acetylation with pulse-labeled H3 (displaying an average acetyllysine/lysine ratio of 2.1; Table 2). Similar data collected from the monoacetylated isoform of pulse-labeled Tetrahy- I~~~~~~~ mena and Drosophila H3 indicate that lysines 9 and 23 are the 27 2 preferred sites of monoacetylation, respectively (Table 2). We conclude that a nonrandom but distinct subset of lysines, 0.5 residues 9 and 14 in Tetrahymena and 14 and 23 in Drosophila, is preferentially acetylated in newly synthesized H3. Thus, the * * * * only deposition acetylation site in common between protozoa Fraction no. Fraction no. and flies is K14. FIG. 4. Newly synthesized HeLa H3 subtypes are not appreciably Similar Analyses Fail to Detect Deposition-Related Acety- acetylated under conditions where new H4 is diacetylated. RP-HPLC lation of HeLa H3. We wondered if any evidence for acetyla- purified H3.2/H3.3 (Left) and H3.1 (Right) were recovered and tion of newly synthesized HeLa H3 could be obtained by the microsequenced and [3H]lysine cpm recovered from cycles 9, 14, 18, strategy outlined in Fig. 1. As a test, pulse-labeled HeLa H3 23, and 27 was collected and further analyzed as described in Figs. 2 subtypes (labeled for 9 min in the presence of butyrate) were and 3. In both cases, the expected N-terminal sequence of human H3 purified by RP-HPLC, directly microsequenced, and analyzed was obtained. as with Tetrahymena and Drosophila H3. With H3.2/H3.3 and (HATs) responsible for adding deposition-related acetyl H3.1, the proper N-terminal sequence of HeLa H3 was groups onto new H4 will be highly conserved. It is not yet clear obtained, confirming our assignment that these polypeptides whether all cytosolic, type B HAT activities catalyze acetyla- are H3 subtypes (Fig. 4). At positions 9, 14, 18, 23, and 27, the tion at both lysines 5 and 12 in H4 in vivo. Experiments with known positions of H3 acetylation in mammalian cells, the vast HAT B activities from various sources have demonstrated a majority of the [3H]lysine recovered elutes at the position of strong preference for lysines 5 and/or 12 in vitro (12, 25). unacetylated lysine (see also Table 2). Lysine 4 is believed to Based on our in vivo data, it seems likely that acetylation at be methylated (23, 24) and was not included in this analysis. 5 12 will be a hallmark of Thus, in keeping with our AU gel analyses, no evidence was lysines and/or property B-type obtained for acetylation of newly synthesized H3.2/H3.3 or acetylases. However, because our experiments have specifi- H3.1 under these conditions. Thus, although the pattern and cally examined the population of H4 that is both newly sites of deposition-related acetylation in H4 appear to be synthesized and deposited into nuclei, we cannot rule out the highly conserved between widely divergent species, the same formal possibility that nuclear type A HATs also participate in does not appear to be the case for H3. establishing the K5/K12 pattern of deposition-related acety- lation. Second, we have determined that, at least in Tetrahymena DISCUSSION and Drosophila, newly synthesized H3 is deposited in isoforms In this study, the pattern of acetylation sites used in newly that are acetylated at a specific, but distinct, set of lysines. synthesized H3 and H4 has been analyzed in three widely Interestingly, lysines 9 and 14 in Tetrahymena are also pre- divergent cell types. Two new findings have been made. First, ferred sites of acetylation in the steady state population of by deblocking pulse-labeled Drosophila and HeLa H4, we have macronuclear H3 presumably acetylated for a transcription- established that a specific pair of lysines, residues 5 and 12, are related function (10). Similarly, lysines 14 and 23 are highly the exclusive sites of deposition-related acetylation in newly preferred acetylation sites in the bulk population of mamma- synthesized, diacetylated H4. These results extend our initial lian H3 (21, 26) and our analyses have confirmed these data results in Tetrahymena and demonstrate that the pattern and with both Drosophila and HeLa H3. Thus, in both of these acetylation sites in newly synthesized H4 are identical between organisms the deposition-related H3 acetylation profile closely protists, flies, and humans. Conservation of this K5/K12 mimics the acetylation profile exhibited by the steady state, pattern of deposition-related H4 acetylation argues for a nuclear H3 population. This situation differs from what is functional, yet poorly understood, role of acetylation at these observed with H4 where the K5/K12 pattern of deposition- two specific lysines. related acetylation is clearly distinct from the pattern of bulk This high degree of conservation of the K5/K12 H4 acety- nuclear acetylation (which is dominated by monoacetylation at lation pattern also suggests that the histone acetyltransferases K16 in most organisms; see ref. 26 for a review). Although the Downloaded by guest on September 30, 2021 Cell Biology: Sobel et aL Proc. NatL Acad Sci USA 92 (1995) 1241 significance behind this difference is not clear, pulse-chase 1. Turner, B. M. (1993) Cell 75, 5-8. experiments carried out in both Tetrahymena (26) and Dro- 2. Lee, D. Y., Hayes, J. J., Pruss, D. & Wolffe, A. P. (1993) Cell 72, sophila (R.E.S. and C.D.A., unpublished observations) show 73-84. clearly that deposition-related acetyl groups in both H3 and H4 3. Ruiz-Carrillo, A., Wangh, L. J. & Allfrey, V. G. (1975) Science turn over completely before these histones become remodeled 190, 117-125. 4. Allis, C. D., Chicoine, L. G., Richman, R. & Schulman, I. G. into transcription-related acetylation patterns. (1985) Proc. Natl. Acad. Sci. USA 82, 8048-8052. In our experiments with HeLa cells, no evidence has been 5. Jackson, V., Shires, A., Tanphaichitr, N. & Chalkley, R. (1976) obtained for deposition-related acetylation of newly synthe- J. Mo. Biol. 104, 471-483. sized H3. Several trivial explanations could account for this 6. Woodland, H. R. (1979) Dev. Bio. 68, 360-370. negative result. First, it is possible that new H3 is acetylated in 7. Chambers, S. A. M. & Shaw, B. R. (1984) J. Biol. Chem. 259, HeLa cells but is rapidly deacetylated. This is unlikely, how- 13458-13463. ever, since sodium butyrate, an effective inhibitor of histone 8. Giancotti, V., Russo, E., Cristini, F. d., Graziosie, G., Micali, F. deacetylases in mammalian cells (27, 28), was included & Crane-Robinson, C. (1984) Biochem. J. 218, 321-329. throughout the entire labeling period. Moreover, essentially all 9. Harisanova, N. T. & Ralchev., K. H. (1986) Cell Differ. 19, of the new H4 migrates as a diacetylated isoform under these 115-124. conditions. If deacetylation is a factor, H4 acts differently or 10. Chicoine, L. G., Schulman, I. G., Richman, R., Cook, R. G. & is immune to this phenomenon. Allis, C. D. (1986) J. Biol. Chem. 261, 1071-1076. in our 11. Pesis, K H. & Matthews, H. (1986)Arch. Biochem. Biophys. 251, Second, all of analyses pulse labeling with [3H]lysine 665-673. has been used to distinguish new H3 from the larger pool of 12. Sobel, R. E., Cook, R. G. & Allis, C. D. (1994)J. Biol. Chem. 269, preexisting histone and in all experiments nuclear histones 18576-18582. have been analyzed. We cannot rule out the possibility that this 13. Wolffe, A. P. (1991) J. Cell Sci. 99, 201-206. strategy does not permit us to detect a low level of acetylation 14. Martindale, D. W., Allis, C. D. & Bruns, P. J. (1982) Exp. Cell on a small population of newly synthesized H3. Using ex- Res. 140, 227-236. tremely short labeling times, Jackson et al. (5) suggested new 15. Allis, C. D. & Dennison, D. K (1982) Dev. Bio. 93, 519-533. H3 was modified in a rat hepatoma cell line. In most mam- 16. Wang, T. & Allis, C. D. (1993) Mol. Cell. Biol. 13, 163-173. malian cells, however, new H3 seems to be deposited mainly in 17. Madireddi, M. T., Davis, M. C. & Allis, C. D. (1994) Dev. Biol. an unmodified form (3, 6, 29). Given the clear evidence for 165, 418-431. deposition-related acetylation of new H3 in Tetrahymena and 18. Kijima, M., Yoshida, M., Sugita, K., Horinouchi, S. & Beppu, T. Drosophila, it will be important to examine this issue in other (1993) J. Biol. Chem. 268, 22429-22435. systems. 19. Perry, C. A. & Annunziato, A. T. (1989) Nucleic Acids Res. 17, 4275-4291. The strong evidence for conservation ofthe K5/K12 pattern 20. Annunziato, A. T. & Seale, R. L. (1983) J. Biol. Chem. 258, of deposition-related acetylation sites in new H4 suggests that 12675-12684. acetylation of these specific lysines plays an important, yet 21. Marvin, K. W., Yau, P. & Bradbury, E. M. (1990) J. Biol. Chem. undetermined, role in histone deposition and/or chromatin 265, 19839-19847. assembly. Given that the K5/K12 pattern exists in protozoa, 22. Wellner, D., Paneerselram, C. & Horecker, B. L. (1990) Proc. flies, and humans, it seems likely that lysines 5 and 12 are also Natl. Acad. Sci. USA 87, 1947-1949. acetylated in newly synthesized H4 in yeast. In yeast, however, 23. Wu, R. S., Panusz, H. T., Hatch, C. L. & Bonner, W. M. (1984) genetic experiments have converted lysines 5 and 12 in H4 into CRC Crit. Rev. Biochem. 20, 201-263. arginines, which do not undergo acetylation (30). Surprisingly, 24. Morgan, B. A., Mittman, B. A. & Smith, M. M. (1991) Mol. Cell. however, no strong phenotypic effects were observed in the Biol. 11, 4111-4120. K5R/K12R H4 mutant. 25. Mingarro, I., Sendra, R., Salvador, M. L. & Franco, L. (1993) J. Biol. Chem. 268, 13248-13252. Along this line, the acetylation status of newly synthesized 26. Thorne, A. W., Kmiciek, D., Mitchelson, K., Sautierre, P. & H3 in yeast becomes a potentially important consideration. It Crane-Robinson, C. (1990) Eur. J. Biochem. 193, 701-713. is well established that the (H3)2(H4)2 tetramer is the funda- 27. Riggs, M. G., Whittaker, R. G., Neumann, J. R. & Ingram, V. M. mental unit of the nucleosome core particle (31, 32) and that (1977) Nature (London) 268, 462-464. a delicate balance of these two histones is required for viability 28. Cousens, L. S., Gallwitz, D. & Alberts, B. M. (1979) J. Biol. in yeast (33, 34). Moreover, the simultaneous presence of Chem. 254, 1716-1723. N-terminal domains of both H3 and H4 are essential for 29. Cousens, L. S. & Alberts, B. M. (1982) J. Biol. Chem. 257, viability in yeast (24, 35) even though certain acetylation sites 3945-3949. in H3 and H4 are not equivalent (35). If, in yeast, newly 30. Megee, P. C., Morgan, B. A., Mittman, B. A. & Mitchell-Smith, M. (1990) Science 247, 841-845. synthesized H3 is acetylated at specific sites, as is the case for 31. van Holde, K. E. (1989) in Chromatin, Springer Series in Molec- H3 in Tetrahymena and Drosophila, it seems conceivable that ular Biology, ed. Rich, A. (Springer, New York), pp. 416-420. deposition sites in both H3 and H4 must be mutated before 32. Wolffe, A. (1992) Chromatin: Structure and Function (Academic, strong phenotypic effects are observed. San Diego), pp. 85-99. 33. Kim, U.-J., Han, M., Kayne, P. & Grunstein, M. (1988) EMBO We wish to thank M. Yoshida from the University of Tokyo for the J. 7, 221-2219. generous gift of trapoxin. This research was supported by grants from 34. Grunstein, M. (1990) Annu. Rev. Cell Biol. 6, 643-678. the National Institutes of Health to A.T.A. (GM 35837) and to C.D.A. 35. Johnson, L. M., Fisher-Adams, G. & Grunstein, M. (1992) (HD 16259). EMBO J. 11, 2201-2209. Downloaded by guest on September 30, 2021