Ubiquitination of Histone H3 in Elongating Spermatids of Rat Testes*

Hou Yu ChenDagger , Jian-Min SunDagger , Yun Zhang§, James R. DavieDagger , and Marvin L. Meistrich§

From the Dagger  Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0W3 Canada and the § Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Because of the potential role of histone ubiquitination in altering chromatin structure, we characterized the levels of ubiquitination of specific histones in meiotic and postmeiotic germ cells in rat testes by two-dimensional gel electrophoresis. The levels of the major ubiquitinated histone forms, mono- and poly-ubiquitinated H2A, were highest in the pachytene spermatocyte stage, declined thereafter through the round spermatid stage, and reached their lowest levels in elongating spermatids. Three additional ubiquitinated histone species, besides H2A, were detected using anti-ubiquitin antibodies specifically in the fraction enriched in elongating spermatids. Based on their electrophoretic mobilities, they corresponded to uH3, uTH3, and uH2B. Polyubiquitinated forms of these proteins were also observed. The identity of these proteins was confirmed by immunoblotting with anti-H3 antisera and by differential extraction of the proteins from the nucleus with increasing salt concentrations. This is the first report of ubiquitination of H3 in vivo. We speculate that its ubiquitination could loosen the nucleosome structure in preparation for histone removal, be a consequence of nucleosome relaxation or disruption caused by other means, or target H3 for degradation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

During spermiogenesis, which is the part of spermatogenesis involving the development of the spermatid, there are dramatic molecular and functional changes in chromatin structure. In the rat, spermiogenesis is divided into 19 steps (1). The round spermatids (steps 1-8) contain a complement of somatic plus testis-specific histones, such as H1t, TH2B, TH2A, and TH3 (2). The round spermatids are transcriptionally active, but transcription ceases at about steps 9 or 10 (3). In the elongating spermatids (steps 9 to 12), several significant events occur in the chromatin. Covalent modifications of the histones include hyperacetylation of H4 (4) and phosphorylation of H1t.1 Chromatin changes are indicated by the increased accessibility of the N terminus of TH2B to antibody binding (5) and the increased susceptibility of the DNA to thermal denaturation (6). Nearly all of the histones are subsequently displaced by the transition proteins TP1, TP2, and TP4, whose synthesis begins at steps 11 or 12 (7, 8). The displacement of the histones by the transition proteins occurs during the first phase of chromatin condensation, which begins at step 11. The transition proteins accumulate in condensing spermatids (steps 13-15) and are subsequently replaced by protamine, which is the major nuclear protein in the condensed spermatids (steps 16-19) and spermatozoa.

Although nothing is known regarding the levels of ubiquitinated histones during mammalian spermatogenesis, several studies in other vertebrate groups have established that they vary during development. In the rooster, the levels of uH2A2 increase markedly from an undetectable level in pachytene spermatocytes to the highest level in late spermatids, just before the histones are replaced by protamine (9). In contrast in trout testes, the level of uH2A remains unchanged from early to late stage germ cells, but there is a decline in uH2B and an increase in u2H2B (10).

The major role identified thus far for ubiquitination in cells is the targeting of proteins for degradation by polyubiquitination (11). In contrast, some proteins, particularly the histones H2A and H2B are often mono-ubiquitinated, which appears to be involved in processes other than degradation (12). The role of histone mono-ubiquitination is currently not known (13), but it has been shown that uH2A (14) and uH2B (15, 16) are associated with transcriptionally active chromatin. Mono- and polyubiquitinated H2A and H2B were more readily dissociated from chromatin by salt, indicating that they destabilize the nucleosome (17).

An important role for ubiquitination of proteins in mammalian spermiogenesis has been inferred from genetic and enzymatic studies. Ubiquitination of proteins is achieved by two or three enzymes, a ubiquitin-activating enzyme, E1, and a ubiquitin-conjugating (or ubiquitin-carrier) protein, E2; in some cases a ubiquitin-protein ligase, E3 is also required (11). Although both of the mouse homologues, designated mHR6A and mHR6B, of the yeast E2 enzyme RAD6 are expressed in all tissues, the elongating spermatids express predominantly mHR6B, which in spermatids is localized in the nucleus (18). Mice homozygous for the knockout of mHR6B have abnormalities in the displacement of histones during spermiogenesis and the further development of the late spermatids (19). In addition, three E2 proteins with homology to yeast UBC4/UBC5 have been identified in the rat testis; one, the 8A isoform of a 17-kDa E2, is testis-specific, reaches maximal levels in spermatids, and has an acidic pI, indicating that basic proteins might be its target (20, 21). E1 enzymes, coded by the Ube1x and Ube1y genes, have also been demonstrated to be expressed at high levels in spermatids of mouse testes (22).

To further elucidate the histone modifications that occur just prior to histone displacement and to identify possible targets for ubiquitination in elongating spermatids, we investigated the modifications of histones in three populations of cells from rat testes, including one enriched in elongating spermatids. We report that the histones showing elevated levels of ubiquitination in this cell type are variants of H3.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Animals-- Adult male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN), at least 10 weeks of age, were used in all experiments.

Separation of Cells-- Spermatogenic cells were separated into enriched subpopulations by centrifugal elutriation (23) under published conditions (24, 25). The cellular composition of the fractions is given in Table I. Nuclei were prepared from the separated cells by lysis in hypotonic sodium phosphate buffer (5 mM, pH 6.5) containing 5 mM MgCl2, 0.25% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.025% soybean trypsin inhibitor, 5 mM naphthol disulfonic acid, and 1 mM p-chloromercuriphenyl sulfonic acid (26, 27) with the addition of iodoacetamide to 10 mM to inhibit isopeptidase. The nuclei were washed in sodium phosphate/MgCl2 containing 0.22 M NaCl (28) and 10 mM iodoacetamide. Basic proteins from the isolated nuclei were extracted with 0.25 N HCl. In one experiment, the nuclei were sequentially extracted with 0.3, 0.9, 1.2, and 1.5 M NaCl. Proteins were precipitated with either 4 or 5% trichloroacetic acid (TCA) to increase the proportion of core histones, or with 25% TCA, which precipitates all histones (26).

                              
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Table I
Cellular composition of enriched fractions of testicular cells obtained by centrifugal elutriation
The percentages of each cell type were adjusted based on their contribution of histones to the pool of cells in the fraction.

Gel Electrophoresis-- One-dimensional AUT gel electrophoresis and two dimensional AUT into SDS gel electrophoresis were performed using a minislab (10 × 8 cm) system (10, 29). Gels were stained with either Coomassie Blue or silver or transferred to nitrocellulose for immunochemical detection (30). First antibodies used included anti-ubiquitin (10), anti-H3 antisera (a kind gift from Dr. C. D. Allis), and two antibodies that react with TH2B (5, 31). The reaction product was detected either with alkaline phosphatase-conjugated second antibodies and a chromogenic substrate (30) or with horseradish peroxidase-conjugated second antibodies and the enhanced chemiluminescence system (Amersham ECL Western blotting system).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Histones from different fractions of testicular cells were resolved by two-dimensional gel electrophoresis (Fig. 1). The patterns of spots corresponding to the core histones were similar in all three fractions at the level of resolution afforded by the minislab gels used in this analysis. In addition, a pair of spots, which were particularly prominent in pachytene spermatocytes (Fig. 1B), were observed at the position corresponding to uH2A. One of the distinctive features of the ubiquitinated histones is that they usually migrate as doublets in the second-dimension SDS gel (16). The levels of uH2A, relative to the total histone amounts, were highest in the pachytene spermatocytes (Fig. 1B) and lowest in the elongating spermatids (Fig. 1D).


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Fig. 1.   Histones (25%-TCA precipitate) from enriched cell populations of rat testis separated on AUT 15% polyacrylamide and two-dimensional (AUT into SDS 15% polyacrylamide) gels. Each gel was loaded with 20 µg of protein as measured by a TCA turbidity assay and was stained with Coomassie Blue. Proteins from an unfractionated testis cell suspension (A) and from fractions enriched in pachytene spermatocytes (B), round spermatids (C), and elongating and elongated spermatids (D) are shown. The spot marked H2A consists of three unresolved spots (from slow to fast mobility in the AUT dimension) corresponding to TH2A, H2A.1, and H2A.2.

When blots from similar gels were immunoreacted with anti-ubiquitin antibodies, uH2A was most prominent (Fig. 2). We could not determine at the resolution level of these gels whether the spots corresponded to ubiquitinated forms of TH2A, H2A.1, or H2A.2 or all of these proteins. Spots corresponding to uH2A.X and uH2A.Z were observed, respectively, slightly slower than uH2A in the SDS dimension and faster than uH2A in the AUT dimension. In addition, polyubiquitinated forms of H2A (32) were observed prominently in the pachytene spermatocytes (Fig. 2B). The round spermatids had appreciable levels of u2H2A, but little polyubiquitinated forms (Fig. 2C). Elongating spermatids lacked the forms of histones with two or three ubiquitins attached but contained a diffuse distribution of highly ubiquitinated forms (Fig. 2D).


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Fig. 2.   Immunoblots of gels from the same preparations as Fig. 1 (20-µg loads), obtained using anti-ubiquitin antibodies. Note that there is nonspecific staining of TH3 (major spot).

In addition to uH2A, uH2A.X, and uH2A.Z, three additional spots can be seen in the regions corresponding to mono-ubiquitinated forms. Based on the mobilities of these histones relative to the H2As (see Fig. 1), two of the spots were assigned as uH3 (either uH3.2 or uH3.3) and uTH3. These spots moved slower than the uH2A spots in the SDS dimension; uTH3 migrated between uH2A and uH2A.Z, while uH3 electrophoresed faster than H2A.Z in the AUT dimension. The third spot (more prominent in Fig. 3B), which migrated just slightly faster than uH3 in the AUT dimension and slightly slower than the uH2As in the SDS dimension, was assigned to uH2B. This assignment has been confirmed by alignment with ubiquitinated forms of rat liver histones (not shown). None of the spots in the ubiquitinated region showed immunoreaction with antibodies to TH2B (not shown). The spot corresponding to uH3 was also observed in unseparated testicular cell suspensions (not shown) but never in the pachytene or round spermatid fractions.


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Fig. 3.   Two-dimensional electrophoresis separation of core histones (4% TCA precipitate) from elongating spermatid fraction. A, silver-stained gel (7.5-µg load). Some of the core histones are indicated for orientation, as are uH2A and uH2A.Z, which are clearly resolved. B, immunoblot (15-µg load) with anti-ubiquitin antibodies clearly showing mono- and polyubiquitinated forms of histones. Note the minimal nonspecific staining in this gel.

The ubiquitinated forms of the histones in the elongating spermatid fraction were better resolved and detected in Fig. 3, in part because the 4%-TCA precipitate was used, enriching the core histones. In the silver-stained gel (Fig. 3A), uH2A and uH2A.Z were clearly observed; it was not possible to identify with certainty the ubiquitinated forms of H3 and H2B because of numerous spots in those regions. However, the corresponding immunoblot (Fig. 3B) clearly shows the presence of uH3, uTH3, and uH2B. In addition, there were extensive polyubiquitinated forms. The polyubiquitinated forms of different histones can be resolved by their positions in the diagonals that extend from the upper left corner of the gel in an arc to the mono-ubiquitinated form (Fig. 3B). It is apparent that both H3 and H2A were extensively polyubiquitinated. This is in contrast to the ubiquitinated histones of pachytene spermatocytes (Fig. 2B), where H2A was the major polyubiquitinated histone species.

Two approaches were taken to support the identification of the spots labeled uH3 and uTH3. The first involved the use of anti-H3 antibodies (Fig. 4). The limitation of this approach was the low levels of the uH3 and some nonspecific reaction of the antibody. Nevertheless, the spot designated uH3 was detected by the antibody. This observation was repeated on additional blots of gels from this and another preparation (not shown); nonspecific reactions were always present.


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Fig. 4.   Two-dimensional gel electrophoretic separation of histones (25% TCA precipitate) from elongating spermatid fraction. A, silver stain. B, immunoblot with anti-ubiquitin antibodies. The nonspecific reaction appears to be with TH3 and H3 (H3.2/H3.3). C, immunoblot with anti-H3 antibodies. Note the intense immunoreaction with all nonubiquitinated isoforms of H3. Immunoreaction with uH3 is observed. The row of bands above uH3 is consistent with the position of H3 dimers. In addition, some nonspecific reaction with other spots is observed.

The second approach was to enrich for different histone fractions by differential salt extraction. H1 is the most readily salt-extractable form, followed by H2A and H2B; H3 and H4 require higher salt concentrations for extraction (17). The one-dimensional gel (Fig. 5A, lane 1) shows that the most prominent histones in the 0.3-0.9 M NaCl fraction were H1 (some was carried down in the 5% precipitate), H2A, and H2B; H3 and H4 were at low levels. The immunoblot with anti-ubiquitin (Fig. 5B) shows intense immunoreaction with the uH2A histones and some immunoreaction with uH2B, but no reaction in the region of the uH3 histones. The 0.9-1.2 M fraction contained the four core histones (Fig. 5A, lane 2). The corresponding immunoblot (Fig. 5C) showed immunoreactions with uH3, uTH3, and u2H3, in addition to the ubiquitinated forms of H2A and H2B. Finally, the 1.2-1.5 M fraction was most enriched in H3 and H4 (Fig. 5A, lane 3). Although the left part of the gel for the immunoblot (Fig. 5D) did not run properly, the right part of the gel showed that the intensity of uH3 relative to uH2B was higher in this fraction than the previous one.


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Fig. 5.   Separation of core histones (5% TCA precipitate) extracted at different salt concentrations from the elongating spermatid fraction. Load on all gels was 25 µg. A, Coomassie Blue-stained one-dimensional AUT gel. Lane 1, 0.3-0.9 M NaCl extract; lane 2, 0.9-1.2 M extract; and lane 3, 1.2-1.5 M extract. Immunoblots of two-dimensional gels from 0.3-0.9 M NaCl extract (B), 0.9-1.2 M extract (C), and 1.2-1.5 M extract (D). Note the left side of the two-dimensional gel in panel D did not run well.

    DISCUSSION
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Procedures
Results
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References

This manuscript represents the first report of ubiquitination of H3. Although the ubiquitination is apparently specific to the elongating spermatids of the testis, in reaching this conclusion it is necessary to take into consideration the purity of the fractions, the contaminating cell types, and the histones that are present in these cells. Methods of further purifying these cell types are available (7, 24), but they would reduce yields and we did not feel greater purity was essential in this study.

The pachytene spermatocytes were only 79% pure; the contamination was largely by earlier stage germ cells (12%) and by round spermatids (6%). Since the round spermatid fraction, which also contained some spermatogonia and early primary spermatocytes, had low levels of the polyubiquitinated forms, most polyubiquitinated H2A histones in the pachytene fraction must indeed be derived from the pachytene spermatocytes.

The purity of the round spermatid fraction was 73%, with early primary spermatocytes and interstitial somatic cells being the major contaminants. Only 4% were elongating spermatids, explaining the absence of bands corresponding to the uH3 histones. The uH2A and u2H2A species observed were most likely present in the round spermatids.

Purification of elongating spermatids is extremely difficult (7), and here we have obtained a fraction that is only somewhat enriched. The elongating spermatid fraction contains many cytoplasmic fragments but these would not contribute to the nuclear protein complement of the cells. The nuclei in this fraction are derived from three main classes of cells, elongating spermatids, condensing spermatids, and somatic cells. The condensing spermatid (steps 13-15) nuclei cannot contribute ubiquitinated histones to this fraction because they contain largely transition proteins and not histones. It is possible that the somatic cells might contribute some of the uH2B and uH3 histones, but they cannot contribute the uTH3 since that protein is exclusively in germ cells (27). Further, uH3 has never been detected in rat, chicken, or human somatic cells (16, 33, 34). Since one of the uH3 isoforms (uTH3) must be restricted to elongating spermatids and since ubiquitination of H3 is a rare event, we conclude that the other forms (uH3 and polyubiquitinated H3) are also in the elongating spermatids.

In contrast to the increase in ubiquitination of H3 in elongating spermatids, the levels of ubiquitinated H2A declined in these cells. Thus the rat differs from the rooster, in which uH2A increases during spermiogenesis (9), and the trout, in which it remains constant (10).

Ubiquitination of H2A (35) and H2B (36) have been described previously and have been extensively characterized. In many cell types, about 10% of the nucleosomal H2A is in the form of uH2A and 1% of H2B exists as uH2B. There are no reports of ubiquitination of H3 in vivo. However, in vitro H3 is as good a substrate as H2A for a variety of mammalian ubiquitin-conjugating enzymes (37) and is very effectively ubiquitinated by the yeast RAD6 E2 protein (38).

Although the present study was not designed to quantify the amount of uH3 in the elongating spermatids, it is important to make a rough estimate. The ratio of uH2A to H2A estimated from Fig. 1 is about 1%. The ratio of uH3 + uTH3 to uH2A + uH2A.Z from the immunoblots is an average of 0.78 (range 0.25-2.7). If we assume that the uH3 histones are only in elongating spermatids, which constitute 42% of the histone-containing cells of this fraction, the percentage of the H3 histones that are ubiquitinated in the elongating spermatids is within an order of magnitude of 2%.

The linkage of ubiquitin to H3 is not known. In the cases of H2A and H2B, the ubiquitin C-terminal glycine is linked via an isopeptide bond to epsilon -amino groups of lysine at the 11th and the 5th amino acid, respectively, from the C terminus. In H3, the only lysines in the C-terminal region are at the 14th and 21st amino acid from the terminus (39). However, both of these sites are located in the histone fold regions of H3 and would likely be inaccessible to ubiquitinating enzymes (40). In contrast, there are several lysines in the N-terminal tail of H3 and in exposed internal regions of the nucleosome (41). Since ubiquitination of H3 in these regions would be expected to occur in somatic cells too, which is not observed, the only explanation for the specificity of uH3 in elongating spermatids would be a specific ubiquitinating enzyme in spermatids (21). Alternatively, lysines located within the histone fold regions of H3 might be the target of the ubiquitinating enzymes, and these would become accessible only after partial disruption of the nucleosome, which might precede the ubiquitination of H3 in the elongating spermatids. The possibility that the ubiquitination of H3 occurs in free H3 is unlikely because more than 0.9 M NaCl is required to extract uH3 from the nucleus.

The role of histone ubiquitination in elongating spermatids is not clear but some hypotheses appear reasonable. The increased levels of polyubiquitination of H2A and H3 in elongating, as opposed to round spermatids, indicate that they may be targeted for degradation to permit the binding of transition proteins. Why only some of the histones are processed in this manner is not known; perhaps there are other mechanisms for the removal of the others from the chromatin.

Another possibility is that H3 ubiquitination has a role in chromatin restructuring. The localization of mono-ubiquitinated histones in regions of actively transcribed chromatin (14-16) and their absence in metaphase chromosomes (42), indicates that ubiquitination might aid in the relaxation of compact chromatin and nucleosomal structures to increase accessibility of the transcription machinery. Chromatin restructuring, possibly involving ubiquitination of H3, occurs in elongating spermatids; however, it cannot be related to transcriptional activity, because these cells are already transcriptionally inactive. One alternative possibility is that H3 ubiquitination is involved in the relaxation of the nucleosomal structure, facilitating either direct displacement of histones by the transition proteins or their accessibility to chaperones or other proteins involved in histone removal. In this model, H3 ubiquitination may act synergistically with the other histone modifications, H1 phosphorylation and H4 hyperacetylation, observed at this stage of spermiogenesis to prepare the nucleosome for removal. Another possibility is that H3 ubiquitination is a consequence of the relaxation or disruption of the nucleosome that had already occurred as a result of these other histone modifications or transition protein deposition. However, H3 ubiquitination may still serve to prevent nucleosome reformation and thereby promote histone displacement.

    FOOTNOTES

* This work was supported by grants from the National Institutes of Health (HD-16843 and core grant CA-16672 to M. L. M) and the Medical Research Council of Canada (MT-9186 to J. R. D). The award of a Medical Research Council of Canada Senior Scientist Award (to J. R. D.) is gratefully acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 1-713-792-3424; Fax: 1-713-794-4858; E-mail: meistrich{at}odin.mdacc.tmc.edu.

1 I. Zalenskaya and M. L. Meistrich, in preparation.

2 The abbreviations used are: uH2A, uH2B, uH3, mono-ubiquitinated forms of histones H2A, H2B, and H3, respectively; u2H2A, u2H2B, u2H3, diubiquitinated forms of H2A, H2B, and H3, respectively; AUT, acid-urea-Triton X-100; TCA, trichloroacetic acid.

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Abstract
Introduction
Procedures
Results
Discussion
References

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