Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1237-1241, February 1995 Cell Biology Conservation of deposition-related acetylation sites in newly synthesized histones 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 histone 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, lysines 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, lysine 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.