hvl is an evolutionarily conserved h2a variant that is preferentially

8
(3) 1986 by The American Soclety of Bioiogxcal Chemists, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY ,261. , No. 4. Issue of February 5, pp. 1941-1948, 1986 Printed in U.S.A. hvl Is an Evolutionarily Conserved H2A Variant That Is Preferentially Associated with Active Genes* (Received for publication, April 12, 1985) C. David Allis$$, Ronald Richman$, Martin A. Gorovskyli, Yvonne S. Zieglerli, Billy Touchstonell, William A. Bradley11 , and Richard G. Cook** From the $Verna and Marrs McLean Department of Biochemistry, [[Department of Medicine, **Howard Hughes Medical Institute and the Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Teras 77030 and the llDepartment of Biology, University of Rochester, Rochester, New York 14627 Polyclonal antibodies to the Tetrahymena macronu- clear-specific histone variant hvl cross-react with his- tone-like molecules from yeast, wheat, and mouse. A novel purification scheme has allowed isolation of suf- ficient h v l to enable determination of the sequence of 61 amino-terminal residues as well as 27 additional internal residues. These data clearly demonstrate that hvl shares a number of conserved sequence elements with the H2A family of histones. Comparison of hvl with H2A.F (=H2A.Z=M1), another evolutionarily conserved H2A variant whose sequence is known, re- veals that they share an unblocked amino-terminal alanine (instead of acetylserine) and a distinctive structure in a “variant box” region that distinguishes them from major H2As. In addition, 10 residues have been identified which are identical (or highly similar) in hvl and H2A.F, but are different from residues conserved in the major H2As. Therefore, in many ways h v l resembles chick H2A.F more than the major Tet- rahymena H2A. The sites of acetylation of hvl also differ from those of the major Tetrahymena H2As. In spite of their similarities, hvl and H2A.Z differ significantly in their amino termini, and antibodies against hvl do not react with H2A.Z. Interestingly, the nucleolar staining pattern reported with anti-hv1 serum is similar to that reported for an antiserum to another H2A variant, mouse testes-enriched H2A.X. Since both H2A.Z and hvl appear to be enriched in transcriptionally active chromatin, these results sug- gest that there may be a number of different, function- ally distinct, nonallelic variants in the H2A family of histones and that hvl is a hybrid H2A variant with properties of both vertebrate H2A.Z and H2A.X. DNA of eukaryotes is packaged into the fundamental nu- cleosomal unit of chromatin through its interaction with a small group of core histones (H2A, H2B, H3, and H4), (for a review see Igo-Kemenes et al., 1982). While the overall struc- ture of the nucleosome core particle has been maintained throughout evolution, microheterogeneity within nucleosomes could provide a means for regulating the structural and func- tional state of chromatin. In addition to secondary modifica- tions and association with nonhistone proteins (see Reeves *This research was supported by Grant HD16259 (to C. D. A.) and Grant GM21793 (to M. A. G.) from the National Institutes of Health. The costsof publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom reprint requests should be addressed. 1984 for review), another factor that could contribute to heterogeneity is the existence of histone variants. Nonallelic histone variants or subtypes differing in primary sequence have now been reported in a wide variety of organisms and have been described for all the inner histones (see Allis et al., 1980b; Zweidler, 1984; Grunstein et al., 1984; von Holt et al., 1984; Harvey and Wells, 1984; Marzluff and Graves, 1984; Wu et al., 1984; and Hayashi et al., 1984). Despite ample documentation for the existence of histone subtypes, the role (if any) that these proteins play in modu- lating chromatin structure/function is not clear. Three pos- sible functions canbe proposed for the existence of nonallelic histone variants: 1) dosage repetition, 2) regulatory repeti- tion, or 3) functional variation. In the first instance, nonal- lelic variants are simply genesthat have duplicated (either by chance or to fulfilldosage requirements) and accumulated neutral mutations. In this case, the nonallelic variants per- form the same function. This view of histonevariantsis supported by recentelegantstudies of Grunstein and his collaborators on yeast H2A and H2B genes (see Grunstein et al., 1984), which argue strongly that the two nonallelic var- iants for each of these genes are completely interchangeable. In the case of regulatory repetition, the gene products them- selves again are similar or identical in function, but multiple genes are required for hypothesized mechanisms of gene reg- ulation to ensure expression at the right time and in the right amount. Histone H3.3 of vertebrates may be such a case since it appears to differ only slightly in sequence from the major H3s (H3.1 and H3.2), but differs markedly in its expression in that its synthesis is uncoupled from DNA replication (see Zweidler, 1984; Wu et al., 1984). Functional variation of nonallelic histone variants is ex- tremely difficult to demonstrate in the absence of precise assays for histone function. In an attempt to gain insights into histone function, we have previously carried out detailed analyses of the histone composition of Tetrahymena macro- and micronuclei (see Bannon andGorovsky, 1984 for a recent review). These nuclei originate from daughter products of a single mitotic division during the sexual phase of the life cycle (conjugation), and therefore contain closely related genetic information (see Nanney, 1953; Gorovsky, 1973). However, during vegetative growth, large structural and functional dif- ferences exist between them. Macronuclei are transcription- ally active anddivide amitotically; micronuclei are transcrip- tionally inactive and divide mitotically (see Gorovsky, 1973 and Gorovsky et al. 1978). Thus, thesenuclei serve as a model system for studying how similar genetic information is main- tained in different structural and functional states. One of the more striking differences in histone composition between macro- and micronuclei is the existence of two quan- 1941

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Page 1: hvl Is an Evolutionarily Conserved H2A Variant That Is Preferentially

(3) 1986 by The American Soclety of Bioiogxcal Chemists, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY ,261. , No. 4. Issue of February 5, pp. 1941-1948, 1986

Printed in U.S.A.

hvl Is an Evolutionarily Conserved H2A Variant That Is Preferentially Associated with Active Genes*

(Received for publication, April 12, 1985)

C. David Allis$$, Ronald Richman$, Martin A. Gorovskyli, Yvonne S. Zieglerli, Billy Touchstonell, William A. Bradley11 , and Richard G . Cook** From the $Verna and Marrs McLean Department of Biochemistry, [[Department of Medicine, **Howard Hughes Medical Institute and the Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Teras 77030 and the llDepartment of Biology, University of Rochester, Rochester, New York 14627

Polyclonal antibodies to the Tetrahymena macronu- clear-specific histone variant h v l cross-react with his- tone-like molecules from yeast, wheat, and mouse. A novel purification scheme has allowed isolation of suf- ficient h v l to enable determination of the sequence of 61 amino-terminal residues as well as 2 7 additional internal residues. These data clearly demonstrate that hvl shares a number of conserved sequence elements with the H2A family of histones. Comparison of h v l with H2A.F (=H2A.Z=M1), another evolutionarily conserved H2A variant whose sequence is known, re- veals that they share an unblocked amino-terminal alanine (instead of acetylserine) and a distinctive structure in a “variant box” region that distinguishes them from major H2As. In addition, 10 residues have been identified which are identical (or highly similar) in hvl and H2A.F, but are different from residues conserved in the major H2As. Therefore, in many ways h v l resembles chick H2A.F more than the major Tet- rahymena H2A. The sites of acetylation of h v l also differ from those of the major Tetrahymena H2As.

In spite of their similarities, hvl and H2A.Z differ significantly in their amino termini, and antibodies against h v l do not react with H2A.Z. Interestingly, the nucleolar staining pattern reported with anti-hv1 serum is similar to that reported for an antiserum to another H2A variant, mouse testes-enriched H2A.X. Since both H2A.Z and hvl appear to be enriched in transcriptionally active chromatin, these results sug- gest that there may be a number of different, function- ally distinct, nonallelic variants in the H2A family of histones and that hvl is a hybrid H2A variant with properties of both vertebrate H2A.Z and H2A.X.

DNA of eukaryotes is packaged into the fundamental nu- cleosomal unit of chromatin through its interaction with a small group of core histones (H2A, H2B, H3, and H4), (for a review see Igo-Kemenes et al., 1982). While the overall struc- ture of the nucleosome core particle has been maintained throughout evolution, microheterogeneity within nucleosomes could provide a means for regulating the structural and func- tional state of chromatin. In addition to secondary modifica- tions and association with nonhistone proteins (see Reeves

*This research was supported by Grant HD16259 (to C. D. A.) and Grant GM21793 (to M. A. G.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom reprint requests should be addressed.

1984 for review), another factor that could contribute to heterogeneity is the existence of histone variants. Nonallelic histone variants or subtypes differing in primary sequence have now been reported in a wide variety of organisms and have been described for all the inner histones (see Allis et al., 1980b; Zweidler, 1984; Grunstein et al., 1984; von Holt et al., 1984; Harvey and Wells, 1984; Marzluff and Graves, 1984; Wu et al., 1984; and Hayashi et al., 1984).

Despite ample documentation for the existence of histone subtypes, the role (if any) that these proteins play in modu- lating chromatin structure/function is not clear. Three pos- sible functions can be proposed for the existence of nonallelic histone variants: 1) dosage repetition, 2) regulatory repeti- tion, or 3) functional variation. In the first instance, nonal- lelic variants are simply genes that have duplicated (either by chance or to fulfill dosage requirements) and accumulated neutral mutations. In this case, the nonallelic variants per- form the same function. This view of histone variants is supported by recent elegant studies of Grunstein and his collaborators on yeast H2A and H2B genes (see Grunstein et al., 1984), which argue strongly that the two nonallelic var- iants for each of these genes are completely interchangeable. In the case of regulatory repetition, the gene products them- selves again are similar or identical in function, but multiple genes are required for hypothesized mechanisms of gene reg- ulation to ensure expression at the right time and in the right amount. Histone H3.3 of vertebrates may be such a case since it appears to differ only slightly in sequence from the major H3s (H3.1 and H3.2), but differs markedly in its expression in that its synthesis is uncoupled from DNA replication (see Zweidler, 1984; Wu et al., 1984).

Functional variation of nonallelic histone variants is ex- tremely difficult to demonstrate in the absence of precise assays for histone function. In an attempt to gain insights into histone function, we have previously carried out detailed analyses of the histone composition of Tetrahymena macro- and micronuclei (see Bannon and Gorovsky, 1984 for a recent review). These nuclei originate from daughter products of a single mitotic division during the sexual phase of the life cycle (conjugation), and therefore contain closely related genetic information (see Nanney, 1953; Gorovsky, 1973). However, during vegetative growth, large structural and functional dif- ferences exist between them. Macronuclei are transcription- ally active and divide amitotically; micronuclei are transcrip- tionally inactive and divide mitotically (see Gorovsky, 1973 and Gorovsky et al. 1978). Thus, these nuclei serve as a model system for studying how similar genetic information is main- tained in different structural and functional states.

One of the more striking differences in histone composition between macro- and micronuclei is the existence of two quan-

1941

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1942 Conserved H2A Variant in Active Chromatin

titatively minor macronuclear-specific histone variants, hvl and hv2 (Allis et al., 1980b). While hv2 has been positively identified as a variant of H3, the exact identity of hvl has remained obscure. Preliminary characterization of hvl sug- gested that it is a moderately lysine-rich histone with a peptide map significantly different from that of either H2A or H2B (Allis et al., 1980b). Consistent with this result is the more recent finding that polyclonal antiserum raised against hvl reacts strongly with the immunogen following immunoblot- ting, but fails to cross-react with any other major macronu- clear histone (including H2A or H2B, see Allis et al., 1982). Thus, while several indirect lines of evidence (its pattern of secondary modification, its detergent-binding properties, and its methionineless nature) suggested that hvl is more like H2A than any other histone (Allis et al., 1980b), its primary structure has diverged sufficiently from other major Tetru- hymena histones to preclude a definitive identification, In fact, in the absence of compositional or sequence data and a functional assay for a histone, it was possible even to question hvls identity as such.

Recent interest in hvl stems from several different lines of evidence, suggesting that it is associated with transcription- ally active (or potentially active) chromatin. First, hvl is found only in transcriptionally active macronuclei and is missing from transcriptionally inactive micronuclei (Allis et ul., 1980b). Second, immunocytological data indicate that hvl antibodies stain subregions of nucleoli in several mammalian cells (Allis et al., 1982). Thus, hvl may be enriched in (al- though not exclusively associated with) ribosomal gene-con- taining chromatin. Third, this antiserum stains develop- mentally active loci on polytene chromosomes of Drosophila.’ Fourth, hvl antiserum can partially inhibit run-on transcrip- tion in isolated Tetrahymena macronuclei.’ Fifth, appearance of hvl in developing new macronuclei in conjugating Tetra- hymena has been shown to coincide closely with the onset of RNA synthesis in these nuclei (Wenkert and Allis, 1984). These data suggest that hvl (or an hvl-related polypeptide) plays a fundamental role in the differentiation of active and inactive chromatin.

To proceed with studies on the function of hvl and to determine whether primary sequence variants of histones can actually serve different evolutionarily conserved functional roles, we have examined the distribution of hvl in diverse species and have devised a novel purification scheme that has allowed compositional analysis and partial sequencing. These data enable us to identify hvl as a member of the H2A family. Close comparison of the hvl sequence to one other evolution- arily conserved, quantitatively minor H2A variant (H2A.F of Harvey et al., 1983; H2A.Z of Ball et al., 1983; or M1 of Urban et al., 1979) has also enabled us to identify regions within the H2A molecule which may distinguish these variants from their respective major H2As. However, antibodies to hvl do not cross-react with H2A.Z, and the amino termini of these two proteins are sufficiently different that it is not possible at this time to equate hvl and H2A.Z unequivocally. Recent studies (Bhatnagar et al., 1984) also indicate that another mammalian H2A variant, H2A.X (also known as protein A or MI or Zz), has a nucleolar-specific immunocytochemical staining pattern similar to that of hvl. Coupled with the observations that both hvl (Allis et al., 1980b; Allis et al., 1982)’ and H2A.Z (Gabrielli et al., 1981) appear to be enriched in transcriptionally active chromatin, these studies argue that the H2A family of histones consists of a number of functional

Elgin, S. C. R., Dietrich, V., Steiner, E., Olmsted, J. B., Allis, C. D., and Gorovsky, M. A. (1985) submitted for publication.

K. Shupe and M. Gorovsky, unpublished Observations.

variants and that Tetrahymena hvl may be a “hybrid” exhib- iting properties of more than one mammalian H2A variant.

EXPERIMENTAL PROCEDURES

Cell Culture and Isolation of Macronuclei Tetrahymena thermophila were cultured axenically in enriched

proteose peptone as previously described (Gorovsky et al., 1975). Cells were harvested at densities not exceeding 800,000 cells/ml. Macro- nuclei were isolated according to published procedures (Gorovsky et al., 1975) and stored frozen (-80 “C) as pellets under medium A (Gorovsky et al., 1975) until needed.

Labeling Studies Growing cells (200,000-400,000 cells/ml) were labeled with [3H]

lysine, [3H]sodium acetate, or [32P]orthophosphate as described pre- viously (see Allis et al., 1980a, 1980b; Allis and Gorovsky, 1981; and Vavra et al., 1982 for specific details).

In Vitro Deacetylation of Macronuclear Histone Macronuclei were thawed, washed once with 0.01% spermidine

(Gorovsky et al., 1975), and resuspended in deacetylation buffer (10 mM Tris, pH 8.0; 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, see Vavra et al., 1982) at a concentration of 1 X lo7 macronuclei/ml. Nuclei were held in deacetylation buffer at 4 “C overnight before being pelleted for histone extraction.

Extraction of Histones Histones were prepared as previously described (Allis et al., 1979),

utilizing all precautions to avoid artifactual protein losses. In some cases, macronuclear HI was removed from the preparation of histones by selective precipitation of the inner histones with perchloric acid (Glover et al., 1981).

Gel Electrophoresis First- (Triton/acid/urea or acid/urea) and second-dimension

(SDS3) gels used in this report have been described in detail previously (Allis et al., 1979; Allis et al., 1980a, 1980b). In experiments where hvl was being purified preparatively, gels were briefly stained ( 4 5 min) with Coomassie Blue (Allis et al., 1979).

Elution and Recovery of Protein from Stained Gels Individual stained protein bands (or spots) were cut from one- or

two-dimensional gels after being thoroughly equilibrated with 62.5 mM Tris, pH 6.8. In some cases, gel bands were stored at (-20 “c) in the above solution containing 10% glycerol. Gel pieces were minced into small slices and incubated with continuous agitation at 37 “C for 6-8 h in Laemmli SDS electrophoresis buffer (Laemmli, 1970). Ap- proximately 1 ml of elution buffer was used per 1.4 cm2 of gel (0.75 cm thick) being extracted. Usually gel pieces were re-extracted a second time for 2 h at 37 ”C with approximately one-fifth of the initial eluting volume. The two supernatants from both elution steps were pooled, dialyzed exhaustively against deionized water, and con- centrated with a speed-vacuum concentrator (Savant). Dried material was resolubilized in a small amount of water, and the protein was recovered as a 20% trichloroacetic acid precipitate. This was then acetone-washed and dried as described previously (Allis et al., 1979). Experiments with radiolabeled histones indicated that 50-90% recov- ery of the input radioactivity can be recovered by these methods.

Amino Acid Analyses Protein hydrolysis (24 h, 5.7 N hydrochloric acid) and amino acid

analyses were performed by Sequemat, Inc. (Watertown, MA). Tryp- tophan, cysteine, and cystine were not determined.

Automated Sequencing Procedures Radiolabeled hul-In separate sequencing runs, [3H]lysine-, 13H]

sodium acetate-, or [32P]orthophosphate-labeled hvl (see “Labeling Studies”) was mixed with unlabeled sperm whale myoglobin (4 nm) and subjected to automated sequencing procedures described previ- ously (Allis et al., 1980a). Automated sequence analysis of [3H]lysine-

The abbreviations used are: SDS, sodium dodecyl sulfate; PTH, phenylthiohydantoin.

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Conserved H2A Variant in Active Chromatin 1943

labeled hvl was performed on a Beckman Model 120B (updated) while that of [3H]acetate- and [32P]orthophosphate-labeled hvl was performed on the instrument described below. For obvious reasons, [3H]acetate- (and [32P]orthophosphate)-labeled hvl was purified by two-dimensional gel electrophoresis (Triton/acid/urea by SDS) using histone preparations which were not taken through the deacetylation reaction.

Amino Acid Sequence Determination-Approximately 750 pmol of “deacetylated” hvl protein was sequenced on the Applied Biosystems, Inc. model 470A protein sequencer using the OZnrun program with aqueous trifluoroacetic acid conversion to the phenylthiohydantoin (PTH) derivatives. For some analyses, similar amounts of hvl were first cleaved with chymotrypsin or Staphylococcus aureus V8 protease as described previously (Allis et al., 1984). Peptides produced were fractionated by the technique of Cleveland et al. (19771, recovered from stained gels as described above, and subjected to automated sequencing. PTH derivatives were identified by high performance liquid chromatography on a Waters Nova Pak C-18 column using a Waters high performance liquid chromatography system (Waters Associates, Milford, MA). Solvent A was 84% sodium acetate (33 mM, pH 5.0) and 16% acetonitrile; solvent B was 60% isopropanol and 40% water. The column was maintained at 40°C and PTH derivatives were eluted at a flow rate of 1 ml/min using the following complex gradient: 0-0.5 min, 0% B; 0.5-3.5 min, 35% B, Waters curve number 4. All PTH derivatives were eluted from the column by 12 min at 35% B. PTH derivatives were monitored at both 254 and 313 nm. Repetitive yield, calculated on the lysine residues at positions 4 and 16, was 96%.

RESULTS AND DISCUSSION

Purification of hv l Previous attempts to obtain enough hvl for biochemical

analyses were frustrated by the fact that only two-dimensional gel electrophoresis (Triton/acid/urea X SDS) could separate hvl from the other core histones. However, attempts to use these gels preparatively with larger protein loads frequently gave low yields and incomplete resolution of hvl. During our initial studies with hvl, the specificity of 32P-labeling of hvl (hv2 and H3 are not phosphorylated) was used to study electrophoretic properties of this variant in long acid/urea gels (Allis et al., 1980b). I t was concluded that hvl co-migrates largely with H3 and its acetylated subspecies in this gel. However, it seemed that the unmodified form of hvl (hvl is both acetylated and phosphorylated, Allis et al., 1980b) mi- grates l unit charge faster than unmodified H3 (see the star in Fig. 1A). In this report, we verified this fact and have exploited it along with a potent endogenous deacetylase activ- ity in isolated macronuclei (Vavra e t al., 1982) to generate large quantities of essentially pure unmodified hvl from pre- parative acid/urea gels. Fig. lA shows a typical profile of macronuclear acid-soluble proteins fractionated on a long acid/urea gel. Since unmodified hvl co-migrates with mono- acetylated H2B in this gel system (see star), resolution of hvl from H2B requires SDS second-dimension electrophoresis (see Fig. IC, top profile). When this is done, one observes unmodified hvl migrating 1 unit charge ahead of unmodified H3. We reasoned that if monoacetylated H2B could be quan- titatively converted to unmodified H2B by the deacetylation reaction, unmodified hvl could be purified by preparative electrophoresis on acid/urea gels. Two-dimensional gel anal- ysis of deacetylated histones (Fig. IC, bottom profile) demon- strates that this is indeed the case. Since a small amount of monoacetylated H2B can escape the deacetylation reaction, we routinely completed the purification of hvl by preparative second-dimension electrophoresiss in SDS gels. Fig. 1B shows an SDS gel of hvl which has been purified by these electro- phoretic procedures. We estimate that hvl is greater than 95% pure in these samples. Finally, immunoblotting with hvl

antibodies has been used to verify that the band shown in Fig. 1 is in fact hvl (data not shown).

Amino Acid Composition of hvl Peptide mapping, isotopic compositional analyses, and pat-

terns of postsynthetic modification (see Introduction and Allis et aL, 1980b) have provided only suggestive evidence that hvl is a histone of the H2A type. Shown in Table I is the amino acid composition of hvl. For comparison, the composition of Tetrahymena H2A (Fusauchi and Iwai, 1983) and H2B (Nom- oto et al., 1982) is presented as well as that of the H2A variants, rat X2 (Trostle-Weige et al., 1982), chicken M2 (Urban et al., 1979), Drosophila D2 (Palmer et al., 1980), and calf thymus H2A.Z (Ball et al., 1983). In agreement with earlier isotopic studies (Allis et al., 1980b), the lysine to arginine ratio of hvl is more similar to that of other H2As than to Tetrahymena H2B. Also consistent with hvl being HPA-like is its high glycine content relative to its own H2B. It is also worth noting that hvl and these quantitatively minor H2A variants are particularly rich in glycine when compared to major H2As (see below). Thus, we conclude that hvl has an amino acid composition similar to other H2As and especially to H2A-related variants.

hvl Is an H2A Preliminary experiments monitoring [3H]lysine release dur-

ing an automated sequence run (see “Experimental Proce- dures’’) demonstrated that hvl had an unblocked amino ter- minus, allowing direct sequencing of its amino terminus. hvl is similar in this respect to H2A.Z (Ball et al., 1983) and differs from all other sequenced H2As which have an amino- terminal acetylated serine. Shown in Fig. 2a is the sequence of the first 61 amino acids of hvl and, for comparison, that of another H2A variant (chicken H2A.F which differs in only 1 residue out of 30 from the amino terminus of calf H2A.Z; Harvey et al., 1983; Ball et al., 1983), and each of their respective major H2As are given. The first point to emerge from this analysis is that hvl contains several regions (see closed boxes) which have been highly conserved among these (and other) H2As. Perhaps most striking is the existence in hvl (and all H2A family members which have been examined to date) of the conserved sequence S(T)-R-S separated by 1 residue from a highly conserved region which yields a unique phenylalanine-containing tryptic peptide (hvl residues 33- 41, see West and Bonner, 1980; Isenberg, 1979). Shown in Fig. 26 is the sequence of 27 residues of an internal peptide produced by cleavage of hvl with V8 protease. As in Fig. 2a, closed boxes surround regions of hvl which have been highly conserved in H2As. From these data, we conclude that hvl is an H2A.

The “Variant Box” A second issue is whether hvl shares any primary structural

features which are found in other H2A variants, but are missing from major H2As. Such variant-specific regions would argue for a related function for these molecules. When the sequence of hvl is closely compared to that of chicken H2A.F and each of these is then compared to their respective H2As and to other HZAs, one particularly informative region has been identified (see dashed box in Fig. 2 ) . In this region (see Fig. 3) both hvl and H2A.F share the following properties which differ from most other H2As. 1) Two positively charged residues are observed at the extreme left-hand end of the dashed box in each protein. In both of the variants (hvl and H2A.F) these residues are separated by one uncharged residue; in all nonvariant HZAs, these two positively charged residues

Page 4: hvl Is an Evolutionarily Conserved H2A Variant That Is Preferentially

1944

FIG. 1. Electrophoretic profile and purification strategy of hvl. A, one-dimensional gel analysis (acid/urea) of macronuclear histones from total (lane I), total minus H1 (lane 2), and deacetylated histone ( l a n e 3) prepara- tions. The star between lanes 1 and 2 denotes the position at which unmodi- fied hvl and monoacetylated H2B co- migrate in this gel system. For reference, a lane of purified hvl (lane 4) is also shown. B, one-dimensional gel analysis (SDS) of total acid-soluble macronuclear histone minus H1 (lane I) and purified hvl (lane 2). The bracket beside lane I denotes the position of core histones (H2A, H2B, H3, H4, hvl, and hv2) in this gel system. C, two-dimensional gel analysis (acid/urea by SDS) of macro- nuclear histone from either normal (top profile) or deacetylated (bottom profile) preparations. Note that deacetylation causes hvl to migrate between unmodi- fied H3 and H2B as an essentially pure protein.

Conserved H2A Variant in Active Chromatin

A acid-urea

L 2 E

H1

H4

I * 4

1 2

are not separated. 2) In both of the variant H2As, another positively charged residue is observed 1 residue removed from the right-hand edge of the dashed box; in each of the respec- tive major H2As, this residue is a negatively charged glutamic acid. In all other H2As, except sea urchin (early embryo), this residue is either negatively charged or neutral (Sures et al., 1978; Schaffner et al., 1978). 3) Finally, in both variant H2As an insertion of 1 residue must be imposed at the extreme right-hand edge of the dashed box to maintain proper align- ment of the neighboring conserved residues; in all the “non- variant” H2As, however, this insertion is not required.

It is worth noting that this putative variant box occurs in a region that is highly conserved both in length and in sequence in other H2As. For example, if all published major H2A sequences are aligned at the S(T)-R-S box (Fig. 2), they are co-linear without any deletions or insertions for 102 residues; only hvl and H2A.F require the indicated insertion in the variant box to maintain alignment. It is also clear that hvl and H2A.F are the only H2As whose sequence differs markedly from that of the consensus sequence in this region. Since the variant box falls within one of the two known regions of histone-histone interaction between H2A and H2B (see Zweidler, 1984), it seems likely that differences here have functional significance.

C

S D S

1

3 4

acid-urea-

U H2B

1 2

deacetyl.

Other Variant-specific Features

Close analyses of the data presented in Fig. 2, a and b, reveal other regions besides the variant box where hvl is remarkably similar to H2A.F. Ten residues have been identi- fied (see small arrows over residues of hvl and H2A.F se- quence) where the sequence of hvl and H2A.F is identical (or highly similar, assuming S=T and K=R=H) and is different from the major H2As. Remarkably, at all of these positions the main H2A sequences of chicken and Tetrahymena are identical (9 positions) or very similar (1 position). Three of these residues occur within the variant box (Fig. 3); the rest are scattered between blocks of highly conserved residues in the H2A sequence. These data suggest that hvl and H2A.F share similar features of structure between a region which begins with the variant box (Fig. 3) and extends through the next 70 amino acids (roughly residues 50-120 in the hvl sequence). However, we note that residues have been identi- fied where hvl and H2A.F differ (for example, residue 86 in hvl=F, L in H2A.F, and N in both of the main H2As). Whether or not differences such as this bring about significant differences in the structure and/or function of these variants is not yet known.

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Conserved H2A Variant in Active Chromatin 1945

TABLE I Amino acid composition of Tetrahymena hvl

The values presented represent mol % and are the average of two analyses on hvl. The values presented for Tetrahymena (Tet.) H2A and H2B are calculated from the known amino acid sequence. Drosop. Drosophila. References used rat X2, Trostle-Weige et al., 1982; chicken M2, Urban et al., 1979; Drosophila D2, Palmer et al., 1980; H2A.Z, Ball et al., 1983; Tet. H2A, Fusauchi and Iwai, 1983; Tet. H2B. Nomot,o et al.. 1982. ~~

Amino acid Tet. Rat Chicken Drosop. Calf Tet. Tet. hvl X2 M2 D2 H2A.Z H2A H2B

, ~ ~~~ ~ -~

.. . ". .~

mol 7% Asp/Asn 7.0 5.0 7.0 5.7 4.2 8.2 6.7 Thr 5.1 3.6 3.5 5.0 6.0 6.3 6.7 Ser 6.7 5.0 5.0 6.6 6.3 7.4 10.1 Glu/Gln 7.6 9.8 9.4 10.0 8.7 7.8 8.4

Pro 4.1 4.6 4.4 2.7 0 4.5 4.2 G ~ Y 16.9 11.6 10.0 11.1 11.8 9.3 2.5" Ala 9.9 13.8 13.5 13.9 14.3 11.9 10.1

CY s 0 0

Val 5.0 7.9 5.7 5.9 5.9 5.2 6.7 Met 0.5 0.4 trace 0 0 1.5 1.7 Ile 4.6 4.2 4.7 6.8 7.3 5.6 5.9 Leu 7.9 9.6 11.6 10.0 10.9 10.4 6.7

Tyr 2.4 2.6 2.3 1.6 1.6 2.2 0.8 P he 3.2 1.2 1.2 0.8 1.0 1.5 5.0

His 2.6 2.1 2.3 3.6 4.9 1.5 1.7 LY S 9.5 10.2 9.8 9.5 11.2 9.7 16.8 A X 7.2 7.8 9.1 6.8 7.9 7.1 5.9

Basic 19.3 20.1 21.2 19.9 24.0 18.3 24.4

Basic/acidic 1.3 1.4 1.3 1.3 1.9 1.1 1.6 Lys/Arg 1.3 1.3 1.1 1.4 1.4 1.4 2.8"

Hydrophobicb 23.6 25.9 25.5 25.1 26.7 26.4 26.8 a s denote places where Tetrahymena H2B amino acid composition

deviates from Tetrahymena H2A and other H2A variants. Includes Val, Met, Ile, Leu, Tyr, and Phe.

Other Features of the hul Sequence The data presented in Fig. 2a also reveal what seems to be

a somewhat unusual feature of the N-terminal domain of Tetrahymena hvl. Striking usage of the triplet Gly-Gly-Lys is observed which is repeated without interruption 5 times in hvl (see the underlined residues between 2 and 16 in hvl). While chick H2A.F (Harvey et al., 1983), calf H2A.Z (Ball et al., 1983), and other H2As (see Isenberg, 1979) contain several glycine and lysine residues in this region, they do not contain such a regular repeating subunit structure. Although both chick and calf H2A variants begin with essentially the same N-terminal 7 residues as hvl (see the horizontal boxes in Fig. 2), it is worth mentioning that this sequence also initiates Tetrahymena H4 (Glover and Gorovsky, 1979; Bannon et al., 1984; Hayashi et al., 1984). Nonetheless, it is clear that hvl (like the other H2A variant) is not blocked at its amino terminus by the conserved acetyl-Ser-Gly-Arg which begins most other H2As (Isenberg, 1979; Ball et al., 1983). I t is also obvious that hvl has a significant N-terminal extension over either Tetrahymena H2A (10-residue extension), chicken H2A.F (10 residues), and most other H2As. Most of this extension in hvl seems to be accounted for by the repeated Gly-Gly-Lys stretch which is practically nonexistent in other H2As.

As already noted (see Fig. 3), the distribution of basic and acidic residues seems to be variant-specific in the variant box. There is also a high degree of positive charge in hvl relative

to other H2As and chicken variant H2A.F. A net of 17 positively charged residues exists in the amino-terminal re- gion of hvl (Fig. 2 4 , while 14, 13, and 11 are found in chicken H2A.F, chicken H2A.1, and Tetrahymena H2A, respectively. Thus, both H2A variants have more overall positive charge than their respective H2As, and in the case of Tetrahymena this difference is quite large.

Sites of Secondary Modification of hul

Acetylation-Acetylation is a postsynthetic modification which affects primarily internal lysine residues in the anino- terminal portion of core histones (see Isenberg, 1979) and is thought to be correlated with transcriptional activity (see Mathis et al., 1980). In the case of several H2As, acetylation has been observed at the lysine in position 5 (see Isenberg, 1979; Pantazis and Bonner, 1981). Since hvl is known to be acetylated (Allis et al., 1980b; Vavra et al., 1982) and since hvl contains a unique stretch (Gly-Gly-Lys) which contains lysines at positions 4, 7, 10, 13, 16, we have asked which (if any) of these positions are acetylated in hvl. Fig. 4 shows a plot of the distribution of [3H]acetate radioactivity released a t each successive cycle of an automated sequencing run. Hayashi et al. (1984) and Glover (1979) have shown that the sites of acetylation on Tetrahymena H3 are conserved lysines at positions 9, 14, 18, and 23. Since hv2 is an H3 (Allis et al., 1980b) and is a likely contaminant of hvl that has been purified without deacetylation, the radioactive acetates re- leased at these positions are probably derived from hv2. Conversely, since hvl but not H3 has lysines a t positions 4, 7, 10, 13, 16 and 21, it also seems likely that these represent actual acetylation sites in hvl. Fusauchi and Iwai (1984) have shown that positions 5 and 12 of the major Tetrahymena H2As are acetylated. All of the acetylated sites in Tetrahy- menu H2A or hvl are lysines preceded by either glycine (usually) or alanine.

Phosphorylation-Earlier characterization also demon- strated that hvl is phosphorylated (Allis et al., 1980b; Allis and Gorovsky, 1981). Interestingly, this modification is not a constant feature of H2A variants. hvl (Allis et al., 1980b; Allis and Gorovsky, 1981) and H2A.X (West and Bonner, 1980; Pantazis and Bonner, 1981) are phosphoryIated while H2A.Z (West and Bonner, 1980; Pantazis and Bonner, 1981; Ball et aL., 1983) is not. Most H2As contain an N-terminal sequence acetyl-Ser-Gly-Arg whose serine is often utilized as a major (or exclusive) site of phosphorylation (Pantazis and Bonner, 1981). Ball et al. (1983) have suggested that the failure of H2A.Z to be phosphorylated may stem from the fact that it lacks this amino-terminal sequence. However, our data dem- onstrate that hvl also lacks this amino-terminal end and yet it is phosphorylated. To determine where in the amino-ter- minal domain of hvl phosphorylation site(s) might exist, we have examined the release of 32P radioactivity during succes- sive cycles of an automated sequencing run. No significant peaks of 32P radioactivity were observed in the first 67 residues of hvl (the internal myoglobin reference protein was sequenc- ing at 96% repetitive yield; data not shown). Furthermore, preliminary peptide maps with V8 protease suggest that the 32P site(s) in hvl is confined to the C-terminal "half' of hv1.4 The major Tetrahymena H2As are also phosphorylated in the carboxy-terminal portion of the molecule (Fusauchi and Jwai, 1984).

* C. D. Allis and R. Richman, unpublished observations.

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1946 Conserved H2A Variant in Active Chromatin

v v v , r------ v v

V v v v V

Tmt h v l

L V8

FIG. 2. Comparison of the primary sequences of Tetrahymena hvl and chicken H2A.F and each of their respective major forms of H2A. Shown in a is the N-terminal amino acid sequence of Tetrahymena hvl along with the major form of macronuclear H2A (Fusauchi and Iwai, 1983). This region of hvl was sequenced by analyses which utilized intact hvl (residues 1-59) as well as a peptide produced by chymotrypsin digestion (residues 32-61). For comparison, the sequence of a chicken H2A variant (H2A.F) and H2A (H2A.1) (see Harvey et al., 1983) are also shown. The first 30 residues of chicken H2A.F are identical with those of calf H2A.Z, except for the substitution of threonine for alanine at position 14 (see Ball et al., 1983). Shown in b is the primary sequence of an internal hvl peptide (generated by treatment with V8 protease) which most likely begins a t residue 78 in the hvl sequence (assuming no insertions or deletions occur between residues 61 and 78). The sequence of Tetrahymena hvl is given in the one-letter amino acid code. Parentheses in the hvl sequence indicate that only a tentative identification of these amino acids has been made. A blank in the hvl sequence indicates positions where no identification could be made. Alignment of sequences starting at the boxed SRS region allows alignment of the next 102 residues in all H2As examined to date. Amino acids which are identical to those in hvl have been given an asterisk; different amino acids are indicated. Closed boxes indicate regions where three of the four sequences have identical residues. These regions can be considered as indicative of conserved H2A regions. The dashed larger box denotes a region which seems to distinguish members of the H2A family as being more like a major H2A or an H2A variant (see Fig. 3). Arrows above individual residues of hvl and H2A.F in a and b denote positions where the sequences of hvl and H2A.F are identical (or highly similar, assuming S=T and K=R=H) and differ from highly conserved residues in the main H2As. In both Tetrahymena H2A and chicken H2A.1, one deletion (-) has been included to maintain the alignment shown. See text for further details. Ac, acetyl.

Cow, rat. human, chick consensus H2A

urchin (embryo) trout

urchin (gonad) Drosophila

wheat yeast

Tetrahymena

HPA.F * * H * K [ T R T T s n G I

FIG. 3. The variant box. Regions that are highly conserved in all H2As flank a segment that is highly conserved in major H2As, but is divergent in both H2A.F and hvl. Note that the distribution of positive charges and one extra residue in this box are common to the two variants and differ from all major H2As. Sequences are from the NBRF protein sequence data bank and the Genbank nucleic acid sequence data bank. Asterisks indicate that the residue is identical to that in the consensus sequence. X indicates no consensus, although both positions so marked show a clear preference for a general class of residues in the major H2As. The consistent positive charge distri- butions for major H2As and the variants are indicated above and below the sequences, respectively. See text for details.

hvl * ' F + K ' G R V S * K N;: : : : + + +

L """""_ J + ' +

Cross-reactivity of hul Antibodies Antibodies against hvl were found to stain small subregions

in nucleoli of several mammalian cell lines (Allis et al., 1982), suggesting that hvl has been highly conserved in evolution. Using the same antiserum, we have investigated the cross- reactivity of these antibodies further by reacting immunoblots

containing Tetrahymena, mouse, wheat, and yeast histone preparations with immune (and preimmune) hvl serum. Fig. 5 demonstrates that a cross-reacting species with mobility similar to hvl is observed in each of these histone prepara- tions, although the reaction is fainter (as might be expected) than that observed with homologous protein. No reaction is observed with preimmune serum. These results indicate that a histone-like peptide containing an hvl-related antigenic determinant exists in all four eukaryotic kingdoms. Thus, it seems likely that hvl (or an hvl-related proteins(s)) has been conserved throughout evolution, suggesting that it plays a fundamental role in chromatin structure/function. Since anti- hvl serum stains molecules with the electrophoretic mobility of unacetylated, unphosphorylated hvl, these modifications cannot be responsible for the conserved determinant. It seems Iess likely, therefore, although stiII possible, that an hvl- specific secondary modification is the conserved determinant.

Given the above results, we have asked whether the poly- clonal antibodies raised against Tetrahymena hvl react with other known H2A variants. Immunoblots with purified calf H2A.Z (generously provided by W. Garrard) and Drosophila D2 (generously provided by M. Blumenfeld) yielded negative results when reacted with hvl antibodies (data not shown). Furthermore, polyclonal antibodies specific for Drosophila D2 (courtesy of M. Blumenfeld) did not react with hvl on im- munoblot.6 H2A.Z (from calf) is presumably identical to

' C. D. Allis, R. Richman, P. Donahue, and M. Blumenfeld, prelim- inary experiments.

Page 7: hvl Is an Evolutionarily Conserved H2A Variant That Is Preferentially

Conserved H2A Variant in Active Chromatin

PRE- IMMUNE

1947 17

10 N

E X

2 0

5

0

\ G G K G G K G G K G G K G G K V G G A K N K K T P ~ S R S Y K A G L * * * * * * * * *

CYCLElRESlDUE

FIG. 4. Sites of acetylation in the N-terminal domain of hvl. hvl (from [3H]acetate-labeled cells) was purified by two-dimensional electrophoresis (Triton/acid/urea by SDS), eluted, mixed with unla- beled myoglobin, and subjected to automated sequencing procedures. Shown is the distribution of [3H]acetate counts released at each step of the sequencing run. Asterisks denote the positions of lysine residues in the primary sequence of hvl given on the abscissa in one-letter amino code. Open bars indicate positions of lysine in the sequence of hvl which contain significant amounts of acetate (residues 4, 7, 10, 13, 16, and 21). Three other positions are observed where significant amounts of acetate counts were released (9, 14, and 18). We suspect that acetate a t these positions results from contamination of hvl with histone variant hv2 which is incompletely resolved from hvl in this gel system. Downward pointing arrows over the bars at 9, 14, 18, and 23 denote positions of acetylation in macronuclear H3 (Glover, 1979).

chicken H2A.F (see the legend to Fig. 2) and mouse H2A.Z (West and Bonner, 1980; Albright et al., 1979). H2A.Z (like hvl, this study) has also been shown to be highly conserved in evolution (Wu et al., 1982). Thus, while some variant features may be shared between H2A.F (=H2A.Z) and hvl (see Table I and Figs. 2 and 3), these immunological results suggest that differences also exist between.the variants so far tested in this fashion (hvl, D2, and H2A.Z). In this regard, we point out that hvl antibodies react strongly with the C - terminal half of hvl (cut at the V8 site indicated in Fig. 2b) and react poorly with its N-terminal half.4 Thus, it is possible that we have not yet sequenced the region of hvl that is being recognized by our antiserum. This could explain why H2A.F (which = H2A.Z) is not recognized by hvl antibodies even though it shares some strong sequence homology to it (Figs. 2 and 3).

The protein species that ,cross-reacts with anti-hvl anti- bodies in diverse organisms remain to be positively identified. A recent report makes H2A.X (=M2, X2, or protein A) a possible candidate. Bhatnagar et al. (1984) reported that an antibody against the mouse testis-enriched protein “A,” pref- erentially stains nucleolar chromatin in a variety of mouse cell types. Since antiserum against hvl gives a similar staining pattern with mammalian cells (Allis et al., 1982), it seems likely that these proteins are related. Unfortunately, H2A.X is present in most tissues in small amounts and appears to have a blocked amino terminus (Pantazis and Bonner, 1981), so additional biochemical characterization will be difficult. If, as seems likely, hvl and H2A.X share antigenic determinants, then we must consider the possibility that hvl is actually related to two mammalian H2A variants. H2A.Z and hvl are similar in having diverged markedly from their corresponding major H2As (Wu et al., 1982; Allis et al., 1980b), having an unblocked amino terminus and in having several regions of homology including the variant box; they differ notably in the

TET MOUSE TET WHEAT TkT YEAST

IMMUNE

I TET MOUSE TET WHEAT TET YEAST

FIG. 5. Blots of SDS gels of Tetrahymena, mouse, wheat, and yeast histones probed with anti-hvl antibody. Mouse liver nuclei were isolated by the method of Yasmineh and Yunis (1970) and extracted as for Tetrahymena (Tet.) macronuclear histone (Allis et al., 1979). Wheat embryo histone was kindly provided by Dr. Steven Spiker and yeast histone was kindly provided by Dr. James R. Davie. Protein loads were 10 pg/lane. Gels were run and blotted onto nitrocellulose and treated with anti-hvl antibody as described previ- ously (Allis et al., 1982). Blots of mouse liver and wheat embryo histone (and their corresponding Tetrahymena controls) were probed with ’251-protein A (New England Nuclear). Yeast histone (and its control) were probed with peroxidase-conjugated goat anti-rabbit serum and reacted with 0-dianisidine. See Allis et al. (1982) for experimental details and references. Immunoblots were also per- formed with these samples fractionated on one-dimensional Triton/ acid/urea gels. However, a definitive identification of which histone variant might be reacting (for example, mouse H2A.Z uersus H2A.X) was not possible due to difficulties in precise alignment of blots with corresponding gels and subtle differences in mobility between these variants.

sequence of their amino termini, in phosphorylation (Pantazis and Bonner, 1981; Allis and Gorovsky, 1981) and in lack of immunological cross-reactivity. H2A.X and hvl differ in the extent to which they resemble their respective H2As (Wu et al., 1982; Allis et al., 1980b) and in their amino-terminal residues, but are similar in that they are phosphorylated and share conserved antigenic determinant(s) that are enriched in mammalian nucleoli. Thus, there may be more than one sub-family of functionally distinct, evolutionarily conserved nonallelic H2A variants in mammals, and hvl may have properties of more than one sub-family. While the precise role of these H2A variants in transcriptional activation remains to be determined, these findings argue strongly that functional variation of the H2A family of histones plays an important role in chromatin structure and function.

Acknowledgments-We are grateful to Sequemat, Inc. for carrying out the amino acid analyses of hvl. We thank Drs. W. T. Garrard, S. Spiker, J. Davie, and M. Blumenfeld for providing us with protein samples. We also acknowledge Dr. Blumenfeld and P. Donahue for providing us with D2-specific antibodies.

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