Vol. 265, No. 33, Issue of November 25, pp. 20662%20666,199O Printed in U.S. A.

Isolation of Four Core Histones from Human Sperm Chromatin Representing a Minor Subset of Somatic Histones*

(Received for publication, May 16, 1990)

Joe M. Gatewood@llII , Gary R. Cook& Rod Balhorn **, Carl W. Schmid$, and E. Morton Bradburyeli From the *Department of Chemistry and the §Department of Biological Chemistry, School of Medicine, University of California, Davis. California 95616. the (Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and the **Lawrence Livermore National Liboratory, Livermore, California 94550

Using high performance liquid chromatography we have successfully purified four core histones from ma- ture human sperm chromatin. The H2A variants pres- ent in sperm (H2A.X and limited H2A.Z) have been shown previously to be minor variants in somatic chro- matin. The histones are highly modified as evidenced by extensive acetylation and an as yet uncharacterized multicharge modification of H2B. Based on our data, we conclude that histone proteins are a minor compo- nent of each mature spermatozoa. Given the unique nature of the histone variants present in sperm, we propose that this chromatin component has a specific function and may possibly facilitate the programming of genes which will be active in early development.

During the final stages of mammalian spermatogenesis, the histones of spermatid chromatin are replaced by transition proteins and finally by protamines (Balhorn et al., 1984; Mayer et al., 1981). Due to the extremely high arginine content of protamine, the DNA is condensed into an insoluble complex which is transcriptionally inactive (Kierszenbaum and Tres, 1975). In humans approximately 15% of the DNA in the spermatozoa is not condensed by protamine and remains associated with histone (Tanphaichitr et al., 1978). Early studies of the histone component identified one major histone, THSB (Tanphaichitr et al., 1978, 1982) or group of histones, SpH (Sobhon et al., 1982, 1984; Puwaravutipanich and Pan- yim, 1975). A more recent report described the presence of H3 and H4 in addition to THBB (Gusse et al., 1986). We have shown previously that human sperm also contain H2A (Gate- wood et al., 1987). In this report we describe the purification and characterization of four core histones from human sperm. In addition the basis for the differing number of observed histones in the various studies is identified.

* This work was supported by Nuclear Science Fund and Biotech- nology Research grants from the University of California, by the College of Letters and Science, University of California, Davis, and by the Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed: Mail Stop M880, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545. Tel.: 505-665-2906; Fax: 505-665-3024.

MATERIALS AND METHODS’

RESULTS

In a 17.5% SDS* gel (Fig. L4) the sperm core histones (lane I) migrate as three bands with mobilities identical to HeLa H3 and H4 and intermediate to H2A and H2B (lane 2). The inclusion of Triton X-100 in the Triton/acid/urea gel system separates the more hydrophobic H2A from H2B (Fig. lB, updated Fig. 1B from Gatewood et al., 1987). Analysis of sperm histones in this gel system clearly demonstrates the presence of H2A (labeled H2A.X, lane I). All four acetylated forms of H4 are present as well as unacetylated H4. H3.1 and H3.3 are present as multiple bands which is characteristic of acetylation. The 2B variant prominent in sperm (denoted H2B.0) migrates as one major band and three minor bands. The nature of the modification responsible for this mobility variation is unknown. A minor amount of protein with mo- bility identical to H2B.0 is detected in HeLa histones (lane 2).

The two-dimensional analysis of the sperm histones facili- tates the band assignment in the Triton/acid/urea first di- mension (Fig. 1B). H3.1, H3.3, highly acetylated H4, four forms of H2B.0, H2A.X. (West and Bonner, 1980) and trace amounts of H2A.Z (West and Bonner, 1980) are detected (Fig. 2A). The H2A.X and H2B.0 which were resolved in the Triton/acid/urea gel have identical mobilities in the SDS dimension. The HeLa histone pattern is shown for compari- son in Fig. 2B. A limited amount of protein is detected in HeLa cells with mobilities similar to H2B.O present in sperm.

With the inclusion of Triton in the horizontal gradient gel system, the single prominent band observed when sperm histones are electrophoresed in an acid-urea gel is resolved into HBA.X, H2B.0, H3.1, and H3.3 (Fig. 3A). H4 is highly acetylated and migrates as five bands in both gel systems making visualization relatively difficult. HeLa histones are shown for comparison in Fig. 3B.

In the initial attempts to purify the histones from sperm, the sample was injected in 0.1% trifluoroacetic acid, 0% acetonitrile to insure binding of all protein components to the column. Using the gradient profile shown in Table IA, a peak (pk 27) eluted -27 min into the gradient that contained a complex (aggregate) of all the histones (Fig. 4A). In contrast HeLa histones injected and chromatographed under identical conditions are resolved (Fig. 4B). By modifying the gradient

’ Portions of this paper (including “Materials and Methods” and Figs. 1, 5, and 6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

2 The abbreviations used are: SDS, sodium dodecyl sulfate: HPLC, high performance liquid chromatography.

20662

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Active Chromatin-associated Histone Variants in Human Sperm 20663

H3.1 H3.2 Hud!l, .!

0

r I I ‘9 H2A.Z H2B.0 H2B H4

FIG. 2. Triton/acid/urea-SDS two-dimensional electropho- retie analysis of sperm histones. The Triton/acid/urea (TAU) first dimensions shown in Fig. 1B are separated in an SDS second dimension. Histones from sperm are shown in A. The major H3 variants in sperm are H3.1 and H3.3. A limited amount of H2A.Z is present in addition to H2A.X. H2B.0 is present in one major form and three minor forms. The nature of the charge modification result- ing in the multiple forms of H2B.0 is unknown. HeLa histones are shown for comparison in B.

A. B.

FIG. 3. Acid/urea-Triton/acid/urea horizontal gradient electrophoretic analysis of sperm histones. Sperm histones (A) and HeLa histones (B) were separated on gels containing a horizontal gradient of Triton X-100 ranging from 0 to 0.5% (Zweidler, 1978). The single prominent band which appears in an acid/urea gel is shown to be a trimer of H2A, H2B, and H3. As the Triton concentra- tion increases from left to ri&t, the mobilities of the more hydropho- bic H2A and H3 are retarded due to interaction with the detergent.

to include injection in an acetonitrile concentration above the value at which pk 27 eluted (Table IB), the complex was disrupted and bound to (eluted from) the column as individual proteins (Fig. 4C). A major 2B peak (H2B.O), a single 2A peak (H2A.X), an H4 peak, and two H3 peaks (H3.1, H3.3) are detected in sperm.

The sperm histone HPLC peak fractions were analyzed on SDS and Triton/acid/urea gels. H2A.X and H2B.0 had iden- tical mobilities in the SDS gel system (Fig. 5A, lanes 4 and 1). The mobilities of H3.3, H3.1, and H4 (Fig. 5A, lanes 5, 6, and 2) were identical to somatic H3 and H4. In the TAU gel system the purified H3.3, H3.1, and H4 (Fig. 5B, lanes 6, 7, and 3) have mobilities which correspond to acetylated H3.3, H3.1, and H4 from somatic tissue. H2A.X migrated slightly

TABLE I HPLC reverse phase gradient profiles

A, histones were injected in 0.1% trifluoroacetic acid and eluted with a Hz0 (solvent A)/acetonitrile (solvent B) gradient containing 0.1% trifluoroacetic acid. All gradient changes were linear. The flow rates were 0.7 ml/min (analytical) and 2.4 ml/min (semipreparative). B, to eliminate complexation, the sperm histones were injected in 25% acetonitrile, 0.1% trifluoroacetic acid, the proteins were eluted with the gradient as shown.

Sector Time Solvent A

min %

A. Equilibrate 1 2 3 4 5 6

B. Equilibrate 1 2 3 4 5

15 5

10 45 40 45 10 10

15 15 25 25 10 10

100.0 100.0

84.0 62.0 59.5 50.0 45.0

100.0

75.0 62.0 58.5 35.0 75.0 75.0

slower than H2A.l (he 4). H2B.0 (lane 1) migrates as one major band and three minor bands which correspond to the four H2B.0 spots observed in the Triton/acid/urea-SDS 2- dimensional gel system (Fig. 2A).

The results of the amino acid analysis are shown in Table II. The known amino acid compositions of other histone variants are included for comparison.

The H3 antibody reacted with isolated sperm chromatin, and individual chromatin particles were easily detected (Fig. 6B). There was little evidence of crossreactivity above back- ground in the histone depleted chromatin (Fig. 6D). The corresponding phase contrast photomicrographs are shown in Fig. 6, A and C.

DISCUSSION

Several differing reports concerning the existence of his- tones in human sperm have been published. Calvin (1976) considered the nonprotamine component of sperm chromo- somal proteins to be contaminating nonchromosomal proteins or proteins of somatic origin. Several reports described the presence of a single major histone species, THPB (Tanphaich- itr et al., 1978, 1981) or group of histones, SpH (Puwaravuti- panich and Panyim, 1975; Sobhon et al., 1982, 1984; Tan- phaichitr et al., 1982) based on acid-urea gel electrophoresis. Gusse et al. (1986) identified H3 and H4 as components of human sperm in addition to THZB based on SDS gel electro- phoresis. We have demonstrated previously the existence of four core histones in 0.6 M NaCl extractions of human sperm chromatin based on Triton-acid-urea gel electrophoresis (Gatewood et al., 1987). Based on the data presented here, all these results can be reconciled.

The Triton X-100 horizontal gradient system demonstrates unequivocally the inability of the acid/urea gel system used in earlier studies to resolve sperm histones. We suspect the H2A, H2B, and H3 variants present in human sperm simply have similar mobilities in the acid-urea gel system although some type of complexation between the histones is a possibil- ity. The inclusion of Triton in the acid-urea gel readily resolves the histones based on their varying hydrophobicity. The SDS gel system is also unable to fractionate all the sperm

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Active Chromatin-associated Histone Variants in Human Sperm

20 40 80 80 1M 120

Retention Time (min)

O=d 0.7

A230

0.1

FIG. 4. C4 reverse phase HPLC of sperm histones. A, sperm histones in 0.1% trifluoroacetic acid were separated on a C4 column using the gradient shown in Table I. A major peak eluted at approx- imately 27 min @k27) which contained all four histones. A prominent H2B peak (H2B.0) is present along with a single H2A species (H2A.X), H3.3, H3.1, oxidized H3, and H4. B, HeLa histones ex- tracted from nuclei with 0.6 M NaCl were fractionated using the same gradient as used in A. The major early eluting peaks are Hls. All the core histones are present to a limited extent in the 0.6 M NaCl extraction and are successfully fractionated using this gradient. C, sperm histones are separated using the gradient profile shown in Table II. The early eluting peak is eliminated by injecting the histones in 25% acetonitrile, and the four core histones are successfully frac- tionated.

histones. As demonstrated by the Triton/acid/urea-SDS two- dimensional gel, H2A.X and H2B.0 have identical mobilities in the SDS gel system.

Somatic histones can be injected in 0.1% trifluoroacetic acid onto a C4 reverse-phase column and successfully frac- tionated with an elution gradient of acetonitrile (Fig. 4B) (Lindner et al., 1986). Under the same conditions sperm histones elute primarily as a single peak (Fig. 4A). Similar behavior has been observed when attempts were made to fractionate sperm chromosomal proteins on sizing columns (Tanphaichitr et al., 1978 and Puwaravutipanich and Panyim,

1975). In our own attempts to crudely size-fractionate sperm histones using a Superose-12 fast protein liquid chromatog- raphy column, the sperm histones also eluted as a single peak.3 The chromatography data suggest that sperm histones are capable of forming a complex under conditions in which their somatic counterparts dissociate. The failure of earlier chromatographic fractionations of sperm chromosomal pro- teins to identify multiple histone species may be due to this unusual behavior. The nature and extent of the interactions responsible for this complexation are unknown. By disrupting the complex in 25% acetonitrile prior to injection, the sperm histones can be successfully fractionated using reverse-phase chromatography (Fig. 4C).

The experimentally determined and unadjusted amino acid compositions of sperm H2A.X, H3.1, and H3.3 are in reason- able agreement with the known amino acid composition of somatic H2A.l and M2 (H2A.X), H3.1, and H3.3 (Table II). The amount of glycine in H4 isolated from sperm (13.9 mol W) is lower than the corresponding level in somatic H4 (16.7 mol X) and may be due to an H4 sequence variant. H2B.0 has an amino acid composition which is in closer agreement with H2B.2 than with either H2B.l or the published compo- sition of THBB. We interpret the differences in the levels of glutamic acid, proline, glycine, and isoleucine in H2B.0 and H2B.2 as evidence for a H2B sequence variant as opposed to a modified H2B.2. This interpretation is further supported by the differences in reverse-phase retention times of H2B and H2B.0. Although we have compared the amino acid compo- sition of H2B.0 and THBB directly, the published amino acid composition of human TH2B is based on the analysis of a sizing column peak fraction and may represent an average of the unresolved histones in the peak.

Based on gel mobility and amino acid analysis we have identified the major H2A variant in human sperm as H2A.X. Although limited amounts of H2A.Z were detected in the two- dimensional gels of sperm histones, we have not succeeded in purifying H2A.Z from sperm. The 2B variant isolated from sperm was designated 2B.0 based on the relative mobility of 2B.0 and 2B.l in the Triton/acid/urea gel system and reverse- phase retention times. We have chosen to avoid the “TH” nomenclature because there is no evidence that these histone variants are unique to testis. A minor amount of protein is detected in the HeLa histone Triton/acid/urea/SDS gel in the same position as H2B.0 indicating H2B.0 is a minor component of somatic tissue.

The results of the immunocytochemistry confirm in part an observation by Samuel et al. (1976) who detected H2A, H2B, and H3 in swollen human sperm chromatin. Samuel et al. (1976) considered the H3 result contradictory to the pro- tein analysis since H3 had not been biochemically detected in human sperm. Given the identification of H3 by Gusse et al. (1986) and Gatewood et al. (1987) and the results presented here, this discrepancy is resolved.

The highly modified character of the sperm histones, par- ticularly the acetylation levels, resembles the situation ob- served in both active chromatin (Johnson et al., 1978; Prior et al., 1983) and chromatin during late spermatogenesis (Christensen and Dixon, 1982). If the H2A and H2B variants present in human sperm prove to be minor components of somatic tissue as the data presented here indicate, then a unique role for the histones in sperm is indicated.

We have previously published DNA hybridization data which indicates the DNA in human sperm is packaged in a sequence-specific manner (Gatewood et al., 1987). Several

3 J. M. Gatewood, G. R. Cook, R. Balhorn, and E. M. Bradbury, unpublished data.

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20665 Active Chromatin-associated Histone Variants in Human Sperm

TABLE II Comparison of human sperm histone amino acid analysis with published histone variant amino acid compositions

The sperm histone amino acid composition data (bold type) were obtained from a single point 24-h acid hydrolysis and performic acid oxidation analyses. No adjustments were made for hydrolytic losses, Trp was not determined, and the Cys values were adjusted for background levels of cysteine. The sources of the reference protein composition data are shown as Footnotes a-j.

Amino acid composition (mol%) Amino acid

H2A.X A” M2b H2A.Z’ H2A.ld H2B.0 THZB H2B.l’ H2B.p H3.1 H3.1h H3.3 H3.3’ H4 H4’

Asx 6.9 5.2 7.0 3.9 6.3 4.0 3.2 4.8 4.8 5.3 3.7 5.5 3.7 6.0 4.9 Thr 3.4 3.5 3.5 6.3 3.9 6.8 9.5 6.5 6.4 1.3 7.5 7.1 7.4 6.6 6.9 Ser 4.6 2.6 5.0 7.1 3.1 10.4 11.1 10.5 11.2 3.5 3.7 4.1 4.4 2.5 2.0

Glx 10.5 10.8 9.4 7.8 9.3 10.3 9.5 8.1 8.0 11.9 11.2 11.8 11.1 7.5 5.9 Pro 4.1 5.5 4.4 2.4 3.9 3.0 2.4 4.8 4.8 5.6 4.5 5.5 4.4 1.7 1.0 GUY 9.5 8.5 10.0 11.8 10.9 4.8 5.6 5.7 5.6 5.3 5.2 5.8 5.9 13.9 16.7

Ala 11.0 12.3 13.5 12.6 13.2 8.1 7.9 12.9 10.4 12.1 13.4 11.7 13.3 7.2 6.9 Val 5.2 7.1 5.7 6.3 6.2 6.8 6.3 6.5 7.2 5.1 4.5 4.7 3.7 8.0 8.8 Met 0.0 0.0 Trace 0.0 0.0 1.6 1.6 1.6 1.6 1.5 1.5 0.9 0.7 1.0 1.0

Ile 4.7 4.8 4.7 7.1 4.7 6.8 7.1 5.6 4.8 5.0 5.2 5.4 5.9 5.5 5.9 Leu 13.1 11.7 11.6 9.4 12.4 5.9 4.8 4.8 4.8 9.7 9.0 9.6 8.9 8.6 7.8 Tyr 2.5 2.9 2.3 1.6 2.3 4.0 4.0 4.0 4.0 2.2 2.2 2.3 2.2 4.0 3.9 Phe 0.7 1.5 1.2 0.8 0.8 2.2 2.4 1.6 1.6 3.0 3.0 3.0 3.0 1.8 2.0

His 3.2 2.5 2.3 4.7 3.1 2.5 2.4 3.2 2.4 1.6 1.5 1.7 1.5 2.1 2.0 LYS 11.3 10.0 9.8 11.0 10.9 16.4 14.3 14.5 16.0 8.8 9.7 8.9 9.6 11.3 10.8 Aw 9.3 10.6 9.1 7.1 9.3 6.5 7.1 4.8 6.4 12.0 12.7 11.8 13.3 12.3 13.7 CYS 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 1.3 1.5 0.9 0.7 0.0 0.0

’ Bhatnagar et al., 1985. *Urban et al., 1979. ’ Hatch and Bonner, 1988. ’ Hayashi et al., 1980. ’ Tannhaichitr et al.. 1978. ‘Zhong et al.. 1983. y Ohe et al., 1979. h Marachi et al., 1986. ’ Welis and Kedes, 1’985. ’ Sierra ej al., 1983.

lines of evidence already exist that point to paternal-specific gene expression in the mouse (McGrath and Solter, 1984; Cattanach and Kirk, 1985; Surani et al., 1986) and human (Jacobs et al., 1980). If our interpretation of the DNA hybrid- ization data is correct, the histones in human sperm may be part of a sequence-specific subset of the genome which is programmed for expression in early development. The sperm nucleohistone may represent chromatin which is poised for early replication and which contains genes programmed for expression due to their association with minor histone var- iants and specific transcription factors.

Acknowledgment-The anti-H3 serum was generously provided by Dr. Michael Bustin.

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J M Gatewood, G R Cook, R Balhorn, C W Schmid and E M Bradburysubset of somatic histones.

Isolation of four core histones from human sperm chromatin representing a minor

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