Micronuclei of Tetrahymena contain two types of histone H3 (histone extraction/two-dimensional gel electrophoresis/nucleosome)

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1 P'roc. Natl. cad. Sci. US Vol. 76, No. 1, pp , October 1979 iochemistry Micronuclei of Tetrahymena contain two types of histone H3 (histone extraction/two-dimensional gel electrophoresis/nucleosome) C. D. LLIS, C. V. C. GLOVER, ND M.. GOROVSKY Department of iology, University of Rochester, Rochester, New York Communicated by Joseph E. Gall, June 25, 1979 STRCT Evidence is presented that micronuclei of Tetrahymena thermophila contain significant amounts of two types of histone H3. One is indistinguishable from that found in macronuclei and the other is unique to micronuclei. The micronucleus-specific H3 has a slightly faster mobility than the common H3 in three different gel systems (both of these species were artifactually lost during procedures for histone preparation in previous studies). oth micronuclear H3s appear to contain a single cysteine residue and are present in sucrose gradientpurified nucleosomes. cid extracts from micronuclei also contain three prominent high molecular weight proteins that also were lost during previous procedures. These proteins are present in extracts from oligomers but are not observed in extracts from mononucleosomes, suggesting that they may be associated with linker regions between nucleosomes. Each vegetative cell of the ciliated protozoan Tetrahymena thermophila (formerly T. pyriformis, syngen 1) contains two types of nuclei, a macronucleus and micronucleus. During sexual reproduction, genetic continuity is maintained through the transcriptionally inactive micronucleus, and the transcriptionally active macronucleus is destroyed. New macronuclei and micronuclei arise from the products of mitotic division of the zygotic nucleus. Thus, these nuclei provide a unique opportunity to study how identical (or closely related) genetic material in the same cell is maintained in different structural and functional states (1). Macronuclei contain each of the five major histone classes H1, H2 (2), H2, H3, and H4, and all of these species display high degrees of secondary modification. On the other hand, H2, H2, and H4 in micronuclei are largely if not entirely unmodified. Furthermore, our previous studies suggested that micronuclei contain little, if any, H1 and H3 (3-6). H3 is a major constituent of nucleosomes from a wide variety of organisms and has been shown to play an essential role in nucleosome structure (7). Electron microscopic (4) and biochemical (3, 4) evidence demonstrates that micronuclear chromatin is organized in nucleosomes that are similar to those in macronuclei. Therefore, the absence of H3 in micronuclei is problematic. If this absence is not artifactual, it suggests that an unusual (and potentially interesting) architecture exists in these nucleosomes. We have failed to observe any unusual interactions between Tetrahymana histones that might allow assembly of nucleosomes in the absence of H3 (2). This led us to reexamine the possibility that micronuclear H3 might have been lost or not resolved from other histones in past analyses. Utilizing improved methods for extracting histones and recent advances in twodimensional gel electrophoresis, we have found that micronuclei contain two types of H3 molecules. One of these is indistinguishable from H3 of macronuclei. The other species is unique to micronuclei. oth species appear to have been artifactually lost in previous studies. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact MTERILS ND METHODS Cell Culture and Isolation of Nuclei. T. thermophila, strain VII, were cultured axenically in enriched proteose peptone as described (8). Cells were harvested at densities not exceeding 5, cells per ml. Macronuclei and micronuclei were isolated according to published procedures (8). Extraction of Histones. Previous procedures. The results presented here are more easily understood if our previous procedures for preparation of histones (9, 1) are summarized. Macronuclei and micronuclei were extracted several times in 2.4 M urea/.2 M H2SO4. The combined acid extract was typically one-fourth to one-half as concentrated as in the new procedure. fter centrifugation, acid-soluble material was precipitated with 4 vol of 95% ethanol at -2 C for at least 12 hr, washed with 95% ethanol, and dried under reduced pressure. Throughout these procedures, unsiliconized glassware was used. New procedures. Macronuclei (at least 2 X 16/1 l of acid extract) and micronuclei (at least 3 X 17/1,ul) were extracted once with.2 M H2SO4 for hr at 4 C. The sulfuric acid solution was added directly to the pelleted nuclei in plastic Eppendorf tubes which then were shaken gently overnight. fter centrifugation (16, X g for 15 min) the acid-soluble material was precipitated by addition of 1% trichloroacetic acid to a final concentration of 2% and chilling for 15 min at WC. The precipitate was collected, washed once with.24 M HCl in acetone and once with acetone, and dried under reduced pressure. Throughout these procedures, only plastic or siliconized glassware (Siliclad; Clay dams) was used. Gel Electrophoresis. Two-dimensional polyacrylamide gel electrophoresis. Two types of first-dimension gel (11. cm long) were used in this study: one, designated "acid/urea," was the 12% acrylamide/6 M urea gel made according to Panyim and Chalkley (11); the other, designated "Triton/acid/urea," was a 12% acrylamide/6 M urea gel prepared as above except that Triton DF-16 (.38%, Sigma) was included in the gel mixture (12). Samples were prepared by boiling for 1 min in 5% acetic acid/4 M urea/5% (wt/vol) 2-mercaptoethanol/.2% pyronin Y (12). oth types of gel were preelectrophoresed at 2 V for 1-2 hr. Electrophoresis was at 14 V for 6 hr at 25 C (running buffer, 5% acetic acid). First-dimension gels were either frozen or immediately equilibrated for 2 hr in 5 ml of equilibration buffer [62.5 mm Tris-HCl, ph 6.8/1% (wt/vol) glycerol]. One-half hour before the gels were mounted for the second-dimension separation, 2-mercaptoethanol was added to the equilibration buffer at a concentration of.5%. When proteins in the first-dimension gels were to be oxidized, gels were equilibrated for 4 hr in buffer that lacked 2-mercaptoethanol and had air bubbling through it. Sodium dodecyl sulfate (NaDodSO4)/polyacrylamide second-dimension gels were prepared according to Laemmli (13) with the following modifications. The stacking gel (at least 2. bbreviation: NaDodSO4, sodium dodecyl sulfate.

2 4858 iochemistry: llis et al. cm high) was 4% acrylamide (acrylamide-to-n,n' methylenebisacrylamide ratio, 3:.8), and the resolving gel (9. cm) was 22% acrylamide (acrylamide-to-bisacrylamide ratio, 3:.2). First-dimension gels were sealed to the top of the second-dimension gels with 1% agarose in 62.5 mm Tris-HCl (ph 6.8). Electrophoresis was at 25C for 16 V-hr. lthough modified species of the individual histones are partially separated in the first-dimension gels, diffusion during equilibration causes these species to run together so that individual modified species (spots) are difficult to detect in these two-dimensional gels. Second-dimension gels were fixed in 2% (wt/vol) trichloroacetic acid for 1 hr, stained in.7% Coomassie brilliant blue R (Sigma) in 5% methanol/5% acetic acid for at least 1 hr, and destained in 5% methanol/7.5% acetic acid. Long acid/urea gels. Long (25. cm) acid/urea gels were prepared as described above for first-dimension acid/urea gels and were electrophoresed at least 18 hr (2 V, 4C). Fresh running buffer (5% acetic acid) was added to the upper reservoir and electrophoresis was at 25 V (4C) for 28 hr. Gels were stained with fast green and scanned at 63 nm in a Gilford spectrophotometer (14). Isolation and Protein nalysis of Micronuclear Nucleosomes. Micronuclei (typically 2-4 X 18) and [3H]thymidinelabeled micronuclei (1 X 16) were combined and washed twice with digestion buffer (1 mm Tris-HCl, ph 7.5/3 mm MgCl2/1 mm NaCI/.2 mm phenylmethylsulfonyl fluoride) containing.5% Nonidet P-4 and once with digestion buffer alone. Nuclei were resuspended in digestion buffer (1 X 19 nuclei/ml), and CaCl2 was added to a final concentration of.1 mm. Micrococcal nuclease (Sigma) was added to a final concentration of.4 unit/ml, and digestion was allowed to proceed for 3 min at room temperature. Digestion was terminated by adding EDT to a concentration of 1 mm. fter chilling for 2 min at C, nuclei were collected (16, X g, 1 min) and the supernatant (S1) was saved. Pelleted nuclei were lysed by vigorous resuspension in 1 mm EDT at ph 8. for 3 min at C and centrifuged at 16, X g for 1 min. This supernatant (S2) was combined with S1 and loaded (2 X 18 nuclei/gradient) onto 5-ml 5-2% sucrose gradients in 1 mm Tris-HCl, ph 7.5/1 mm EDT/.2 mm phenylmethylsulfonyl fluoride. Gradients were spun at 15, X g (rmax) for 12 hr at 2 C. fter fractionation, an aliquot of each fraction was assayed for radioactivity to determine the profile of micronuclear DN across the gradient. Regions of the gradient corresponding to mononucleosomes (possibly including some dimers) and oligomers were pooled separately, made 1 mm in MgCI2, and collected at 25, X g (rmax) for 24 hr. pproximately 8-9% of the 3H-labeled DN from the mononucleosome region of the gradient and essentially all of the DN from the oligomer region was pelleted by these procedures. cid-soluble material was extracted from these pellets exactly as described above (new procedure). RESULTS Fig. 1 shows a typical two-dimensional gel of acid-soluble proteins of Tetrahymena macronuclei. lthough a complex group of nonhistone proteins is seen, histones are the major constituents. The histone assignments in Fig. 1 were established by electrophoresis of individual purified macronuclear histones. The separation achieved is produced in part by detergent binding which decreases the mobility of H2 and H3 in the first dimension (12). Molecular weight heterogeneity of H2 [two species (15)] and H1 (at least three species) is also resolved in the second dimension. When acid extracts were prepared from micronuclei by our previous methods and analyzed on these gels, two faint species Ir U) VD co z Proc. Natl. cad. Sci. US 76 (1979) a Triton acid urea-- H3 H2 _ H2n F 4 _ H4 H2 Sq_ p- H3s - H2 _ F _ H4 C 6 9* FIG. 1. ( and ) Two-dimensional gels (Triton/acid/urea; Na- DodSO4) of the acid-soluble material extracted from macronuclei () and micronuclei () by the new histone extraction procedure. (C) Histone region of a gel of micronuclear acid-soluble proteins extracted according to previous procedures. The arrow in C points to two faint species that are selectively lost by these procedures. These species correspond to the micronuclear H3 species shown in. The firstdimension gels used in and were equilibrated in buffer without NaDodSO4; the first-dimension gel used in C was equilibrated in buffer with 2.3% NaDodSO4. H4 smears badly when equilibrated under these conditions. The arrow in points to a species that is present in acid extracts from macronuclei and is also present in roughly the same proportion (relative to the other histones) in mononucleosomes isolated from macronuclei. were observed in the vicinity of H3 (Fig. 1C). The H3-like mobility of these two species suggests that they might represent the missing micronuclear H3. ecause the staining intensity of these species was greatly decreased relative to that of the other histones, we considered the possibility that these species were selectively lost during preparation. Systematic examination of each step in our previous procedures for preparing histones has shown that both of these species and several other prominent micronuclear-specific acid-soluble proteins (see below) are selectively lost as the result of three principal causes: (i) sticking to glass surfaces, (ii) inefficient precipitation from the acid extract, and (iii) inclusion of urea in the acid extract, which appears to trap significant amounts of material at the top of the first-dimension gel. The first two of these problems become more severe as the concentration of

3 protein in the acid extract is decreased. Sticking of histones to glass can be largely prevented by using either plast-ts liconized glassware. Histone precipitation is improved by using trichloroacetic acid rather than ethanol to precipitate proteins from the acid extract. Finally, we no longer include urea in the acid extraction mixture. Fig. 1 shows a typical two-dimensional gel of acid soluble micronuclear proteins prepared by our new procedures. Two prominent species were found in the vicinity of macronuclear H3. lthough the staining intensity of these species was roughly equal in some experiments (for example, see Fig. 2), the staining intensity of the faster migrating species (113 F) usually was greater than that of the slower species (113 S) in most of our preparations. The micronuclear material in the H3 region cannot be explained by macronuclear contamination. These gels also confirm earlier reports that micronuclei contain little if any H1 (5, 6). It is unlikely that the continuing absence of a macronuclear type Hi from micronuclei is due to selective loss because no antigenic determinants complementary to macronuclear HI can be detected in micronuclei by in situ immunofluorescence (6). Three other proteins (labeled a, f3, and My in Fig. 1) also were prominent in acid extracts from micronuclei and also appear to have been lost selectively when previous procedures were used. Finally, we consistently observed more of the more slowly migrating species of H2 (H2 S) in micronuclei than in macronuclei. In most organisms, H3 is the only histone that contains cysteine. Tetrahymena macronuclear H3 contains one cysteine residue per molecule (3, 4, 15). These molecules can be oxidized to form H3-H3 dimers by formation of disulfide bridges. To investigate whether the H3-like species from micronuclei contain cysteine, first-dimension gels (either Triton/acid/urea or acid/urea) were run as usual, equilibrated for 4 hr in buffer with or without 2-mercaptoethanol, and then run on NaDodSO4 second-dimension gels. When these proteins were allowed to oxidize in the first-dimension gels, a triplet was observed (arrow, Fig. 2) with the same first-dimension mobility as the "13 doublet" but with a significantly decreased mobility in the NaDodSO4 dimension. ecause it was not observed when equilibration was performed in the presence of 2-mercaptoethanol (Fig. 2), this triplet must result from the oxidation of Triton acid urea-b O cz jc iochemistry: llis et al. G FwG. 2. cid extracts from micronuclei ( and ) and macronuclei (C and D) were electrophoresed in Triton/acid/urea firstdimension gels. These gels were equilibrated for 4 hr in buffer containing 2-mercaptoethanol [reduced, ( and C)J or lacking 2-mercaptoethanol and having air bubbled through it [oxidized ( and D)l. fter equilibration, standard second-dimension NaDodSO4 gels were run. rrows in and D indicate the oxidation products of H3. D 4w -MP 4. Proc. Natl. cad. Sci. US 76 (1979) 4859 species at this mobility in the first-dimension gel. The triplet nature of these oxidation products can most easily be explained in the following manner: (i) the upper species represents the homodimer (H3 S-H3 S) of the slower migrating "H3" species; (ii) the bottom species represents the homodimer (H3 F-H3 F) of the faster migrating "H3" species; (iii) the intermediate species represents the heterodimer (H3 S-H3 F). Heterodimer formation is not unexpected because H3 F and H3 S are not completely separated in the first-dimension gel, and the proteins in an unstained and unfixed gel would be free to diffuse during the equilibration period. This oxidation-reduction experiment has also been performed with macronuclear acid-soluble material (Fig. 2 C and D). s expected, only one oxidation product (H3-H3 homodimer, arrow in Fig. 2D) is observed above the H3 region in macronuclei. Thus, each of the H3-like species found in micronuclei contains at least one cysteine residue (and probably no more than one, because we have observed no evidence of intramolecular dimers or higher oligomers), suggesting that both are, in fact, H3 molecules. To determine whether either of the micronuclear H3s has exactly the same mobility as H3 from macronuclei, we examined (on long acid/urea, first-dimension gels) the acid-soluble proteins prepared from macronuclei and from micronuclei by the new and old procedures (Fig. 3). oth of the micronuclear H3 species migrated much closer to H2 than they did in Triton-containing gels. It is clear that one of the micronuclear species (H3 S) has the same mobility as the unmodified form of H3 in macronuclei. This species is markedly decreased in extracts prepared by previous procedures. These gel scans illustrate the purity of these micronuclear preparations (little HI and modified H4 species) and support our contention that H3 S cannot be due to macronuclear contamination. It is also clear that a protein (labeled H3 F) migrates between the positions of monoacetylated and unacetylated macronuclear H2. This protein is unique to micronuclei and was also lost in previous procedures. Two-dimensional gels (acid/urea; NaDodSO4) of micronuclei indicate that this species is the fast species of the micronuclear H3 doublet (data not shown). The other micronucleus-specific species in these scans (also decreased in extracts prepared by previous procedures) correspond to the high molecular weight proteins, 3 and y (see Fig. 1). The data of Fig. 3 suggest that H3 S has a mobility remarkably similar to that of H3 from macronuclei and that H3 F is unique to micronuclei. To confirm this, acid-soluble material from macronuclei and micronuclei was mixed and coelectrophoresed on a two-dimensional gel (Triton/acid/urea; Na- DodSO4) (Fig. 4). It is apparent that H3 from macronuclei migrates at the position of H3 S and obscures it but does not obscure H3 F which is therefore unique to micronuclei. In addition, we have coelectrophoresed unlabeled macronuclear proteins with 3H-labeled micronuclear protein. nalysis by staining and fluorography has also shown that the faster species is unique to micronuclei (data not shown). Finally, we have investigated whether the micronuclear H3 species are present in purified nucleosomes. fter digestion with micrococcal nuclease, solubilized micronuclear chromatin was sedimented in a 5-2% sucrose gradient. Two-dimensional gels (Triton/acid/urea; NaDodSO4) of the acid-soluble proteins extracted from mononucleosomes and oligomers are shown in Fig. 5. In addition to H2, H2, and H4, both H3 F and H3 S were major constituents of mononucleosomes and oligomers and together were present in amounts similar to those of the other inner histones. The proportion of each species in nucleosomes is similar to that in extracts from whole nuclei. lthough the four inner histones are the only major proteins detected in extracts from mononucleosomes, we have consistently observed

4 486 iochemistry: llis et al. lh2.1 H3 mi I I -T-l Proc. Natl. cad. Sci. US 76 (1979) Triton acid urea- CO) T CZ I S F H2 S -F H3 H2 H4 cid/urea d FIG. 3. Densitometer tracings of gels containing acid-soluble material prepared from macronuclei () or from micronuclei extracted by new () or previous (C) method. In this experiment, long (25. cm) acid/urea gels were used, and only the histone region of the gels is shown. Gels were stained with fast green (14). the three micronucleus-specific high molecular weight species (y, (3, and y in extracts from oligomers. This suggests that these proteins may be associated with linker regions between nucleosomes in a manner analogous to HI (16). We conclude from these data that nucleosomes in micronuclei contain the four inner histones H2, H2, H3, and H4 and therefore are similar in histone composition to nucleosomes from macronuclei and from other eukaryotes. CU) _ I riton/acid/urea 4 z C FIG. 4. cid-soluble material from macronuclei () and micronuclei (C) were electrophoresed either singly or mixed () on three parallel two-dimension gels. Only the H3 region of the gels is shown. The H3 region of this micronuclear preparation (C) was unusually contaminated with nonhistone proteins. FIG. 5. cid-soluble material from mononucleosomes () and oligomers () was electrophoresed on two-dimensional gels. The proteins extracted from mononucleosomes are more streaked in the first dimension than usual, presumably because of the presence of small pieces of digested DN that were inefficiently precipitated with.2 M H2SO4 during the acid extraction but were precipitated with trichloroacetic acid. This nucleic acid would contaminate the preparation of acid-soluble proteins and may have caused the streaking. The amount of the high molecular weight protein y extracted from isolated oligomers () is decreased relative to amounts normally seen in extracts from whole nuclei (see Fig. 1). DISCUSSION Contrary to earlier reports (3, 4), the data presented here demonstrate that micronuclei from Tetrahymena contain histone H3. Interestingly, our data suggest that these nuclei contain significant amounts of two types of H3. One of these (the slower migrating species on all gel systems used, H3 S) is remarkably similar if not identical to H3 contained in macronuclei. The other species (faster migrating, H3 F) is a unique form of H3 that is not found in macronuclei. Two types of H3 have been reported in other organisms. In these cases, however, the second species is usually present in minor amounts (17, 18) Ṡeveral lines of evidence suggest that both of these species are in fact H3 molecules: (i) together they are present in amounts similar to those of the other inner histones (Fig. 3) in acid extracts from whole nuclei; (ii) these species display mobilities similar or identical to those of macronuclear H3 on three different gel systems (acid/urea, Triton/acid/urea, and Na- DodSO4), suggesting that their size, charge, and detergentbinding properties are roughly the same; (iii) like macronuclear H3, each of these species appears to contain a single cysteine residue; and (iv) each of these species is present in significant

5 iochemistry: llis et al. Proc. Natl. cad. Sci. US 76 (1979) 4861 amounts in isolated nucleosomes. ecause nucleosomes are well-defined structural units whose principal protein constituents are histones H2, H2, H3, and H4, it is likely that these micronuclear proteins are H3 molecules. Several factors probably explain why micronuclear H3 was not detected previously. H3 F is poorly resolved from the unmodified form of H2 on the acid/urea gels previously used for the analysis of Tetrahymena histones (9, 1). lthough H3 S would have been resolved on these gels, its decreased amounts made it difficult to distinguish from macronuclear or nonhistone contamination. Finally, although several tests made on micronuclear chromatin suggested that it was not histone deficient (3-5), the loss of H3 occurs after these proteins are extracted from the DN, so that this loss would not have been detected by these methods. The identical mobilities of H3 S and the unmodified form of macronuclear H3 on three different gel systems suggest that these species are identical. Furthermore, selective loss of macronuclear H3 has also been observed when our original procedures for histone extraction were used and the concentration of protein in the acid extract was sufficiently low. Thus, we have no evidence that H3 from macronuclei behaves any differently from either of the two micronuclear H3 species with respect to its ability to be lost. The existence of a micronucleus-specific H3 molecule raises questions concerning its biological function. The biological significance of the variation in the relative amounts of H3 S and H3 F in different micronuclear preparations is unclear at present. However, preliminary results on the synthesis and retention of the two forms (pulse-chase experiments) make it extremely unlikely that H3 F is simply derived from H3 S by artifactual proteolytic cleavage. The data presented here demonstrate that micronuclei as well as macronuclei of T. thermophila contain all four inner histones (H2, H2, H3, and H4). The discovery of H3 in micronuclei yields a consistent picture of nucleosome structure in Tetrahymena. In conclusion, major alterations in nucleosome structure (4) or histone composition do not accompany the large structural and functional differences between macronuclei and micronuclei (7). However, it is likely that there are subtle differences between macronuclear and micronuclear chromatin which are related to differences in their histone complements. The precise structural and functional consequences of differences in histone modification (4, 9), of differences in H1 (5, 6), and of the presence of a unique species of H3 remain to be elucidated. We are grateful to Ms. J. owen for assistance in preparing micronuclei, Ms. K. Vavra for the samples of [3H]thymidine-labeled micronuclei, and to Dr. C. P. Giri for instruction and assistance with the micrococcal nuclease digestions. This research was supported by U.S. Public Health Service Research Grant GM C.D.. is a recipient of a National Institutes of Health Postdoctoral Fellowship; C.V.C.G. is a recipient of a National Institutes of Health Predoctoral Traineeship; and M..G. is a recipient of a U.S. Public Health Service Research Career Development ward. 1. Gorovsky, M.. (1973) J. Protozool. 2, Glover, C. V. C. & Gorovsky, M.. (1978) iochemistry 17, Gorovsky, M.. & Keevert, J.. (1975) Proc. Natl. cad. Sci. US 72, Gorovsky, M.., Glover, C., Johmann, C.., Keevert, J.., Mathis, D. J. & Samuelson, M. (1978) Cold Spring Harbor Symp. Quant. iol. 42, Gorovsky, M.. & Keevert, J.. (1975) Proc. Natl. cad. Sci. US 72, Johmann, C.. & Gorovsky, M.. (1976) J. Cell iol. 71, Kornberg, R. D. (1977) nnu. Rev. iochem. 46, Gorovsky, M.., Yao, M. C., Keevert, J.. & Pleger, G. L. (1975) in Methods in Cell iology, ed. Prescott, D. M. (cademic, New York), Vol. 9, pp Gorovsky, M.., Pleger, G. L., Keevert, J.. & Johmann, C.. (1973) J. Cell iol. 57, Gorovsky, M.., Keevert, J.. & Pleger, G. L. (1974) J. Cell iol. 61, Panyim, S. & Chalkley, R. (1969) rch. iochem. iophys. cta 16, lfageme, C. R., Zweidler,., Mahowald,. & Cohen, L. H. (1974) J. iol. Chem. 249, Laemmli, U. K. (197) Nature (London) 227, Gorovsky, M.., Carlson, K. & Rosenbaum, J. L. (197) nal. iochem. 35, Johmann, C.. & Gorovsky, M.. (1976) iochemistry 15, Varshavsky,. J., akayev, V. V. & Georgiev, G. P. (1976) Nucleic cids Res. 3, Marzluff, W. F., Sanders, L.., Miller, D. M. & McCarty, K. S. (1972) J. iol. Cheri. 247, Garrard, W. T. (1976) FES Lett. 64,