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Testis-Specific Expression of a Novel Human H3 Histone Gene
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
229, 301–306 (1996)
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Testis-Specific Expression of a Novel Human H3 Histone Gene OLAF WITT, WERNER ALBIG,
AND
DETLEF DOENECKE1
Institut fu¨r Biochemie und Molekulare Zellbiologie, Georg-August-Universita¨t Go¨ttingen, Humboldtallee 23, D-37073 Go¨ttingen, Germany
We have investigated the expression of a recently described, solitary human H3 histone gene. Using RNase protection assays, the corresponding mRNA could only be detected in RNA preparations from human testis, whereas several human cell lines and somatic tissues did not exhibit expression of this gene. In situ hybridization of sections from human testis revealed expression to be confined to primary spermatocytes. In addition to H1t, this novel H3 gene, which is located on chromosome 1, is the second tissue-specific human histone gene that has been found to be expressed solely in the testis. q 1996 Academic Press, Inc.
INTRODUCTION
Virtually all genomic DNA of eukaryotic cells is organized in nucleosomes. A nucleosomal core contains about 140 bp of DNA, wrapped around a protein octamer containing two copies each of histones H2A, H2B, H3, and H4, designated core histones [1, 2]. Additionally, histone H1 proteins are accociated with the linker DNA connecting nucleosomal cores, hence their name linker histones. All five classes of these nuclear proteins, except H4, consist of several subtypes [3]. Histone genes may be subdivided into three major groups. First, replication-dependent genes which are expressed only during S-phase of the cell cycle; second, replication-independent histone genes encoding replacement histones which are also expressed in nondividing quiescent or terminally differentiated cells; and third, tissuespecific histone isotypes. The latter group comprises the histones H1t, TH2A, TH2B, and TH3 that are exclusively expressed in the testis of different mammalian species [4–9]. These testis-specific isoforms partly replace the somatic histones during spermatogenesis and this may modulate the chromatin organization for meiotic and postmeiotic processes in spermatogenic cells [10]. Recently, our group has isolated a novel solitary histone H3 gene with some special features [11]: The gene maps to chromosome 1q42, outside the known histone gene clusters on chromosomes 6p21.3 [12] and 1q21 1 To whom reprint requests should be addressed. Fax: *49-551395960.
[13], indicating a possible replacement histone gene. On the other hand, this H3 gene shows the consensus promoter and 3* flanking portions which are typical for replication-dependent genes. The deduced protein sequence varies at four amino acid residues from the consensus mammalian H3 structure and can therefore be classified as H3.4 [14]. Based on these findings we have investigated the expression of this novel human histone H3 gene in different human cell lines and tissues. The results presented here show that detectable mRNA levels were only found in human testis with primary spermatocytes being the major cell population of expression. In addition to H1t, this gene is the second tissue-specific human histone gene expressed solely in the testis. Therefore, we propose the term H3t for this gene. MATERIAL AND METHODS Cell lines and human tissues. Human cell lines were purchased from the American Type Culture Collection. The lines were maintained in the recommended media supplemented with 10% fetal calf serum at 377C and 5% CO2 . The following human cell lines were used: HeLa/HeLaS3 (cervix carcinoma), HEK293 (embryonal kidney), U937 (histiocytic lymphoma), K562 (chronic myeloid leukemia in blast crisis), TERA2 (pluripotent embryonal carcinoma), Hep-G2 (hepatocellular carcinoma), and HL60 (acute myeloid leukemia). Tissue from human testis of a 56-year-old man with prostate cancer after orchiectomy was a generous gift from Prof. Dr. R.-H. Ringert, Department of Urology, University of Goettingen. Plasmids and probes. For RNase protection analysis and in situ hybridization experiments, DNA fragments of the 5* flanking, coding, and 3* flanking regions of the H3t gene (see Fig. 1) were subcloned into Bluescript (KS/). A 39-mer oligonucleotide (5*-TGAACGTTGCGCAACCTCTCAGGTGGCGAGATAGCCCTC-3*, purchased from Roth, Karlsruhe, Germany) of the 3* untranslated region of H3t was used in in situ hybridization experiments to avoid cross-hybridization with other histone H3 mRNAs (see Fig. 1). A H1t probe was kindly provided by Dr. B. Drabent, Department of Biochemistry, University of Goettingen [4]. RNA isolation and RNase protection analysis. Total RNA preparations of human cell lines were made using the RNeasy kit from Qiagen according to the manufacturer’s instructions. Human testicular RNA was isolated by the guanidinium isothiocyanate method [15]. Total RNAs from human tissues were a generous gift from Dr. E. J. Bellefroid, Universite´ de Lie`ge [16]. For RNase protection analysis, probes were labeled with [32 P]UTP (Amersham) by in vitro transcription using the MAXIscript kit (Ambion). After transcription, probes were purified on a denaturing 8 M urea/5% (v/v) acrylamide gel and eluted at 47C overnight. We used the RPAII kit (Ambion) for RNase protection analysis according to the standard procedure
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0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Restriction map of the H3t gene and flanking portions (EMBL Accession No. Z49861). For RNase protection analysis the 400-bp HindIII/NciI fragment of the 5* flanking region and the 1-kb PstI/PstI fragment of the 3* flanking region were cloned into Bluescript. For detection of H3t mRNA in in situ hybridization experiments (see Fig. 5), a 39-mer oligonucleotide corresponding to the 3 * untranslated region between the TAG stop codon and the 3 * palindromic sequence, indicated as 0 , was used. Total histone H3 mRNA in in situ hybridization experiments was detected by the 300-bp HincII/PstI fragment of the coding region. described in the manufacturer’s manual. Ten micrograms of total RNA from cells/tissues was hybridized with [32P]UTP-labeled probe and subjected to RNase digestion using the RNase A/T1 mixture provided with the kit. After precipitation, protected fragments were separated on a denaturing 8 M urea/5% (v/v) acrylamide gel and detected by exposure to a Cronex X-ray film (Du Pont). Digoxigenin labeling of probes. H3t mRNAs in tissue sections were detected by oligonucleotide in situ hybridization using a digoxigenin (DIG)-tailed oligonucleotide. Tailing of the 3*OH end was done by the terminal transferase reaction as has been described previously [17]. Average tail length was 30 bp and DIG incorporation about five molecules per tail as determined by acrylamide gel electrophoresis and staining of serial dilutions spotted on nylon membranes with an alkaline phosphatase-conjugated anti-DIG-antibody (Boehringer). For assessment of nonspecific binding in in situ hybridization experiments, we used a 39-mer oligonucleotide of the 5* untranscribed region of the H1t gene, which was tailed in the same way. For detection of total histone H3 mRNA and H1t mRNA in tissue sections, DIG-labeled RNA anti-sense and sense probes were made by in vitro transcription using the DIG RNA labeling kit (Boehringer) according to the manufacturer’s instructions. In situ hybridization. In situ hybridization was performed with modifications following standard procedures [18]. Hybridization: Sections were hybridized with 100 ml hybridization solution (50% deionized formamide, 41 SSC, 11 Denhardt’s solution, 10% dextran sulfate, 0.5 mg/ml Escherichia coli DNA, 0.25 mg/ml yeast tRNA, and 0.5 mg/ml yeast rRNA) containing 40 ng DIG-tailed oligonucleotide or 100 ng DIGlabeled RNA, respectively, at 427C overnight. Washing: After oligonucleotide hybridization, sections were washed in 21 SSC for 1 h at room temperature, in 11 SSC for 1 h at room temperature, in 0.51 SSC for 30 min at 427C, and in 0.51 SSC for 30 min at room temperature. Sections hybridized with RNA probes were washed 31 10 min in 41 SSC at 427C, incubated with 100 ml RNase A (20 mg/ml in TE containing 0.5 M NaCl) at 377C, 10 min in 21 SSC, 10 min in 0.11 SSC, and 10 min in 0.051 SSC at 427C. Immunological detection: Hybridized probes were detected with an alkaline phosphatase-conjugated anti-DIG-antibody (Boehringer), diluted 1:500. Color reaction was performed using NBT (4-nitro blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3indolyl phosphate) as substrates and reaction was monitored under microscopic control for 4–6 h at 377C in the case of hybridization with RNA probes and for 3 days at 57C with daily changes of color solution in the case of hybridization with oligonucleotide probes.
RESULTS
Expression of the H3t Gene in Human Cell Lines and Tissues The expression of the H3t gene was investigated by RNase protection analysis. Because of the highly con-
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served nature of the histone H3 coding region, we used a 400-bp probe of the 5* flanking region of the H3t gene to avoid cross-hybridizations with other H3 histone mRNAs. Figure 2a shows the analysis of total RNA preparations from eight different human cell lines. None of the investigated cell lines showed detectable histone H3t mRNA levels. In contrast, human testis revealed a relatively strong expression of this gene. The same results were obtained in three different experiments with different RNA preparations. On the other hand, strong expression was found in testicular RNA from three different individuals. We then further investigated the H3t mRNA levels in total RNA preparations from different human organs and normal cell populations (Fig. 2b). Again, none of the indicated organs and cells revealed detectable H3t expression, except for testis. Note that seminoma, a testis-derived malignant tumor, fails to express the H3t gene. Since these organs represent a broad spectrum of different human tissues and do not show any H3t transcript, we conclude that the histone H3t gene is expressed in a testis-specific manner. Mapping of the H3t 3* Termination Site The 3* end of histone mRNAs may either contain a dyad symmetry element that is essential for the processing of the replication-dependent mRNA species [19, 20] or, in the case of replacement-type histone mRNAs, be polyadenylated [21, 22]. In order to determine the 3* end of the H3t transcript, we used a 1-kb probe of the 3* flanking region in an RNase protection assay. The size of the 190-bp protected fragment shown in Fig. 3 localizes the 3* terminus of the H3t transcript immediately downstream of a consensus dyad symmetry element. No longer transcript could be detected. The sizes of the protected 5* and 3* probes allowed us to map the approximate start site, length, and termination site of the H3t transcript (Fig. 4). The TATA box is located at position 037 and the two CCAAT boxes at 059 and 0101 relative to the start site of transcription. The length of the H3t transcript is 500 bp. H3t in Situ Hybridization in Human Testis Sections Since cell-type-specific expression during spermatogenesis has been demonstrated for histone genes [6], we investigated the expression of the H3t gene in tissue sections from human testis by in situ hybridization and compared it in serial sections with the expression of total H3 and H1t. As shown in Fig. 5, H3t mRNA could indeed only be detected in distinct cell populations of spermatogenesis. High magnification demonstrates that the H3t-expressing cells are separated from the basement membrane by ‘‘pale’’ spermatogonia. As determined by hematoxylin –eosin staining of serial sections (Fig. 5), these H3t-positive cells contain large nu-
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FIG. 2. Expression of the histone H3t gene in human cell lines (a) and human tissues and normal cell populations (b). Equivalent amounts of RNA were subjected to the RNase protection assay as determined by both spectrophotometry and ethidium bromide staining of RNA (bottom). Additionally, integrity of the analyzed RNAs was monitored by using a histone H1.2 probe as a positive control (results not shown). Arrow indicates the 160-bp fragment protected by H3t mRNA. Several minor fragments õ130 bp were detected, representing degradation products of other, only partially hybridized, H3 mRNAs (not shown). PBMC, peripheral blood monocytic cells.
clei, indicative of primary spermatocytes, whereas adjacent spermatogonia and round spermatids did not exhibit expression of the H3t gene. Since it is well established that H1t mRNA is synthesized by pachytene spermatocytes [4–6, 23], we also performed in situ hybridization experiments with a human histone H1t probe (Fig. 5). Comparison of the H3t and H1t results reveals almost identical expression patterns of the two histone genes in serial sections. Therefore, we conclude
that the human H3t gene is expressed in primary spermatocytes, being transcribed by the same cell population as the H1t gene. In order to compare the H3t expression with the total histone H3 transcription during spermatogenesis, we included experiments using a probe of the H3t coding region which should crosshybridize also with any other histone H3 mRNAs due to the highly conserved nature of the H3 coding regions. Hybridization with this general H3 probe was, however, also confined to primary spermatocytes (Fig. 5). This restriction of H3t expression to the same cells which express the H1t gene is not the result of a selective accessibility of these cells to in situ hybridization probes. This is excluded by hybridization of a histone H17 probe with spermatogonia and of a protamine probe with spermatids under the same hybridization conditions (data not shown) in agreement with established patterns of expression of these genes [6, 24]. In addition to its hybridization with H3t-specific mRNA, the total H3 probe may there also react with H3.3 mRNA which has been described in testicular cells [25]. A cross-hybridization with other, i.e., S-phase-dependent, H3 mRNA, can be ruled out, since the hybridizing cells correspond to postmitotic stages. Only rarely, weak staining of spermatogonia in some tubuli was observed (results not shown). Moreover, we carried out in situ hybridizations with sections of a
FIG. 3. Mapping of the H3t termination of transcription by RNase protection analysis. Arrow indicates 190-bp fragment protected by testis mRNA.
FIG. 4. Start site and termination of transcription of the H3t gene and relative positions of the TATA and CCAAT boxes. 0 , dyad symmetry element of the sequence GGCTCTTTTAAGAGCC.
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FIG. 5. In situ hybridization of serial sections from human adult testis. A series of four sections was stained with hematoxylin –eosin (H.E.) and hybridized with probes for the indicated histone mRNAs, respectively. Magnification 4001; detailed aspects 10001.
carcinoma of the kidney in order to rule out cross-hybridizations between the H3t probe used with other H3 histone mRNAs. In these experiments, we found that the total H3 probe, but not the H3t-specific probe, showed staining of the tumor cells, indicating no cross-hybridization of the two probes (data not shown). DISCUSSION
During spermatogenesis a rearrangement of the histone composition of spermatogenic cells takes place. In
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addition to the histone isoforms, which are also found in somatic tissues, testis-specific histones are synthesized during the process of sperm development, indicating special functions of these isoforms in modulating the chromatin structure during spermatogenesis [10]. In this paper, we have shown testis-specific expression of a novel human histone H3 gene, designated H3t, in primary spermatocytes. Other testis-specific histone variants that have been described are H1t, the testisspecific isoform of histone H1 in human, rat, and mouse
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[4–6, 26], and TH2A and TH2B, the testis-specific isoforms of histones H2A and H2B in rats [7, 9, 27]. Furthermore, Trostle-Weige et al. have described a germ cell-specific H3 histone variant (TH3) in rat testis based on electrophoretic mobility and protein analysis [8]. As yet, no TH3 gene has been identified in any mammalian genome. However, the histone H3t gene reported here appears to encode a different histone protein. First, the experimentally determined amino acid composition of the rat histone TH3 varies from the human H3t gene deduced amino acid sequence in its methionine, cysteine, and serine composition. Second, TH3 has been shown to be synthesized mainly in type A and B spermatogonia, whereas spermatocytes did not show synthesis of TH3, based on centrifugal elutriation following intratesticular injection of tritiated arginine [8]. In contrast, our in situ hybridization data demonstrate H3t expression to be absent in spermatogonia. H3t mRNA was only detectable in primary spermatocytes, being transcribed in the same cells as H1t which is predominantly expressed in pachytene spermatocytes [4 –6, 23]. The differences between TH3 and H3t might partly be explained by the different experimental approaches and different species studied. However, our results provide the first evidence for testis-specific expression of a core histone variant in man. The H3t gene shares some common features with the testis-specific H1t, TH2A, and TH2B genes. First, the pattern of H3t expression is similar to that of H1t, TH2A, and TH2B, which are exclusively expressed in primary spermatocytes of the pachytene stage [6, 7, 27]. Second, as we have shown for the H3t mRNA, these testis-specific histone isoforms contain a dyad symmetry element at their 3* end [7, 9, 26, 27]. This highly conserved stem–loop is found in all replication-dependent histone genes and has been implicated in transcription termination, efficiency of mRNA processing, mRNA stability, and the coupling of histone mRNA levels to the cell-cycle [28], whereas the replacement histone variants contain a polyadenylation signal. On the other hand, the H3t gene is expressed in meiotic cells, where no DNA synthesis takes place. Since it has been shown that H1t, TH2A, and TH2B replace about 60% of the somatic isoforms in pachytene spermatocytes [29, 30], a high synthesis rate of the testis-specific histones is required in these cells. Therefore the stem – loop of the testis-specific histone isoforms might be necessary for efficient mRNA processing or high mRNA stability in spermatocytes. Replacement of the stem – loop by a polyadenylation signal has been found to lead to a 30-fold reduction of expression of histone H4 [31]. The extent to which H3t replaces the somatic H3 histones during spermatogenesis is not yet clear, but our data show that the bulk of total H3 gene transcription as well as the H3t transcription takes place in primary spermatocytes. It could well be that the total H3 tran-
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script in these cells mainly consists of the H3t isoform, since these post-mitotic cells are not expected to transcribe replication-dependent H3 histones, but we cannot exclude that part of the hybridization obtained with the total H3 probe may be due to expression of the replacement variant genes H3.3A and H3.3B. Since spermatogonia in S-phase are rare in a given tissuesection compared with primary spermatocytes, we only occasionally detected spermatogonia reacting with the total H3 probe. An obvious difference between H3t and the other testis-specific isoforms is their genomic organization. Whereas H1t is located within the major histone gene cluster on chromosome 6 [12], the H3t gene is solitarily located on chromosome 1 in the human genome [11]. TH2A and TH2B, which appear to exist only in the rat genome, similarly have been mapped to a cluster on rat chromosome 17, which is syntenic with human 6p21.3 and carries H1t and other histone genes [32]. The function of the singular location of the H3t gene is not clear, but it may play a role in an independently regulated transcription as has been suggested for the solitarily located replacement histone genes H17 and H3.3B [12, 33]. The testis-specific expression of the H3t gene raises questions about the mechanism of its tissue-specific regulation. Several promoter elements have been implicated in testis-specific expression of the H1t gene and the testis-specific protamine genes. However, the H3t promoter does not contain any of these sequences including the conserved H1t promoter elements TE1 and TE2 or the mouse protamine 1 sequence TGACTTCATAA binding TET-1 [34, 35]. Queralt and Oliva have found a conserved protamine 1 promoter element, designated as Prot1C, at position 064 to 053 relative to the start site of transcription by comparing protamine 1 promoters of 10 different mammals [36]. The H3t promoter contains a sequence matching this consensus element by 100%, but its position is located at 0180. Further studies of the H3t promoter are needed to define the mechanism of testis-specific expression of this gene. The function of the testis-specific histone isoforms is still not known, but tissue specificity as well as expression in a distinct cell population, namely pachytene spermatocytes, where crossing-over of homologous chromosomes takes place, points to a functional role in chromatin morphology during spermatogenesis and in particular the process of meiosis. H1t has been shown to have a lower condensing capacity than the other H1 subtypes and may thus help to decondense the chromatin structure for the specific needs of the meiotic and haploid stages of germ cell development [37]. The amino acid sequence of H3t varies at four positions from the mammalian H3 consensus sequence. At position 98, the H3t protein contains serine instead of ala-
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nine. This additional potential phosphorylation site might be of functional relevance, since phosphorylation of H1t in elongating spermatids and a complex pattern of H2A.X phosphorylation have been described in murine testicular cells [25, 38]. It remains to be shown whether other mammalian genomes contain homologues of the human H3t gene. Isolation and characterization of a murine H3t gene would allow functional studies in vitro or by artificially deleting the H3t gene. This work was supported by a research grant from the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie. O.W. gratefully acknowledges a fellowship from the Deutsche Forschungsgemeinschaft.
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Received August 1, 1996 Revised version received September 16, 1996
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0375
Testis-Specific Expression of a Novel Human H3 Histone Gene OLAF WITT, WERNER ALBIG,
AND
DETLEF DOENECKE1
Institut fu¨r Biochemie und Molekulare Zellbiologie, Georg-August-Universita¨t Go¨ttingen, Humboldtallee 23, D-37073 Go¨ttingen, Germany
We have investigated the expression of a recently described, solitary human H3 histone gene. Using RNase protection assays, the corresponding mRNA could only be detected in RNA preparations from human testis, whereas several human cell lines and somatic tissues did not exhibit expression of this gene. In situ hybridization of sections from human testis revealed expression to be confined to primary spermatocytes. In addition to H1t, this novel H3 gene, which is located on chromosome 1, is the second tissue-specific human histone gene that has been found to be expressed solely in the testis. q 1996 Academic Press, Inc.
INTRODUCTION
Virtually all genomic DNA of eukaryotic cells is organized in nucleosomes. A nucleosomal core contains about 140 bp of DNA, wrapped around a protein octamer containing two copies each of histones H2A, H2B, H3, and H4, designated core histones [1, 2]. Additionally, histone H1 proteins are accociated with the linker DNA connecting nucleosomal cores, hence their name linker histones. All five classes of these nuclear proteins, except H4, consist of several subtypes [3]. Histone genes may be subdivided into three major groups. First, replication-dependent genes which are expressed only during S-phase of the cell cycle; second, replication-independent histone genes encoding replacement histones which are also expressed in nondividing quiescent or terminally differentiated cells; and third, tissuespecific histone isotypes. The latter group comprises the histones H1t, TH2A, TH2B, and TH3 that are exclusively expressed in the testis of different mammalian species [4–9]. These testis-specific isoforms partly replace the somatic histones during spermatogenesis and this may modulate the chromatin organization for meiotic and postmeiotic processes in spermatogenic cells [10]. Recently, our group has isolated a novel solitary histone H3 gene with some special features [11]: The gene maps to chromosome 1q42, outside the known histone gene clusters on chromosomes 6p21.3 [12] and 1q21 1 To whom reprint requests should be addressed. Fax: *49-551395960.
[13], indicating a possible replacement histone gene. On the other hand, this H3 gene shows the consensus promoter and 3* flanking portions which are typical for replication-dependent genes. The deduced protein sequence varies at four amino acid residues from the consensus mammalian H3 structure and can therefore be classified as H3.4 [14]. Based on these findings we have investigated the expression of this novel human histone H3 gene in different human cell lines and tissues. The results presented here show that detectable mRNA levels were only found in human testis with primary spermatocytes being the major cell population of expression. In addition to H1t, this gene is the second tissue-specific human histone gene expressed solely in the testis. Therefore, we propose the term H3t for this gene. MATERIAL AND METHODS Cell lines and human tissues. Human cell lines were purchased from the American Type Culture Collection. The lines were maintained in the recommended media supplemented with 10% fetal calf serum at 377C and 5% CO2 . The following human cell lines were used: HeLa/HeLaS3 (cervix carcinoma), HEK293 (embryonal kidney), U937 (histiocytic lymphoma), K562 (chronic myeloid leukemia in blast crisis), TERA2 (pluripotent embryonal carcinoma), Hep-G2 (hepatocellular carcinoma), and HL60 (acute myeloid leukemia). Tissue from human testis of a 56-year-old man with prostate cancer after orchiectomy was a generous gift from Prof. Dr. R.-H. Ringert, Department of Urology, University of Goettingen. Plasmids and probes. For RNase protection analysis and in situ hybridization experiments, DNA fragments of the 5* flanking, coding, and 3* flanking regions of the H3t gene (see Fig. 1) were subcloned into Bluescript (KS/). A 39-mer oligonucleotide (5*-TGAACGTTGCGCAACCTCTCAGGTGGCGAGATAGCCCTC-3*, purchased from Roth, Karlsruhe, Germany) of the 3* untranslated region of H3t was used in in situ hybridization experiments to avoid cross-hybridization with other histone H3 mRNAs (see Fig. 1). A H1t probe was kindly provided by Dr. B. Drabent, Department of Biochemistry, University of Goettingen [4]. RNA isolation and RNase protection analysis. Total RNA preparations of human cell lines were made using the RNeasy kit from Qiagen according to the manufacturer’s instructions. Human testicular RNA was isolated by the guanidinium isothiocyanate method [15]. Total RNAs from human tissues were a generous gift from Dr. E. J. Bellefroid, Universite´ de Lie`ge [16]. For RNase protection analysis, probes were labeled with [32 P]UTP (Amersham) by in vitro transcription using the MAXIscript kit (Ambion). After transcription, probes were purified on a denaturing 8 M urea/5% (v/v) acrylamide gel and eluted at 47C overnight. We used the RPAII kit (Ambion) for RNase protection analysis according to the standard procedure
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FIG. 1. Restriction map of the H3t gene and flanking portions (EMBL Accession No. Z49861). For RNase protection analysis the 400-bp HindIII/NciI fragment of the 5* flanking region and the 1-kb PstI/PstI fragment of the 3* flanking region were cloned into Bluescript. For detection of H3t mRNA in in situ hybridization experiments (see Fig. 5), a 39-mer oligonucleotide corresponding to the 3 * untranslated region between the TAG stop codon and the 3 * palindromic sequence, indicated as 0 , was used. Total histone H3 mRNA in in situ hybridization experiments was detected by the 300-bp HincII/PstI fragment of the coding region. described in the manufacturer’s manual. Ten micrograms of total RNA from cells/tissues was hybridized with [32P]UTP-labeled probe and subjected to RNase digestion using the RNase A/T1 mixture provided with the kit. After precipitation, protected fragments were separated on a denaturing 8 M urea/5% (v/v) acrylamide gel and detected by exposure to a Cronex X-ray film (Du Pont). Digoxigenin labeling of probes. H3t mRNAs in tissue sections were detected by oligonucleotide in situ hybridization using a digoxigenin (DIG)-tailed oligonucleotide. Tailing of the 3*OH end was done by the terminal transferase reaction as has been described previously [17]. Average tail length was 30 bp and DIG incorporation about five molecules per tail as determined by acrylamide gel electrophoresis and staining of serial dilutions spotted on nylon membranes with an alkaline phosphatase-conjugated anti-DIG-antibody (Boehringer). For assessment of nonspecific binding in in situ hybridization experiments, we used a 39-mer oligonucleotide of the 5* untranscribed region of the H1t gene, which was tailed in the same way. For detection of total histone H3 mRNA and H1t mRNA in tissue sections, DIG-labeled RNA anti-sense and sense probes were made by in vitro transcription using the DIG RNA labeling kit (Boehringer) according to the manufacturer’s instructions. In situ hybridization. In situ hybridization was performed with modifications following standard procedures [18]. Hybridization: Sections were hybridized with 100 ml hybridization solution (50% deionized formamide, 41 SSC, 11 Denhardt’s solution, 10% dextran sulfate, 0.5 mg/ml Escherichia coli DNA, 0.25 mg/ml yeast tRNA, and 0.5 mg/ml yeast rRNA) containing 40 ng DIG-tailed oligonucleotide or 100 ng DIGlabeled RNA, respectively, at 427C overnight. Washing: After oligonucleotide hybridization, sections were washed in 21 SSC for 1 h at room temperature, in 11 SSC for 1 h at room temperature, in 0.51 SSC for 30 min at 427C, and in 0.51 SSC for 30 min at room temperature. Sections hybridized with RNA probes were washed 31 10 min in 41 SSC at 427C, incubated with 100 ml RNase A (20 mg/ml in TE containing 0.5 M NaCl) at 377C, 10 min in 21 SSC, 10 min in 0.11 SSC, and 10 min in 0.051 SSC at 427C. Immunological detection: Hybridized probes were detected with an alkaline phosphatase-conjugated anti-DIG-antibody (Boehringer), diluted 1:500. Color reaction was performed using NBT (4-nitro blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3indolyl phosphate) as substrates and reaction was monitored under microscopic control for 4–6 h at 377C in the case of hybridization with RNA probes and for 3 days at 57C with daily changes of color solution in the case of hybridization with oligonucleotide probes.
RESULTS
Expression of the H3t Gene in Human Cell Lines and Tissues The expression of the H3t gene was investigated by RNase protection analysis. Because of the highly con-
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served nature of the histone H3 coding region, we used a 400-bp probe of the 5* flanking region of the H3t gene to avoid cross-hybridizations with other H3 histone mRNAs. Figure 2a shows the analysis of total RNA preparations from eight different human cell lines. None of the investigated cell lines showed detectable histone H3t mRNA levels. In contrast, human testis revealed a relatively strong expression of this gene. The same results were obtained in three different experiments with different RNA preparations. On the other hand, strong expression was found in testicular RNA from three different individuals. We then further investigated the H3t mRNA levels in total RNA preparations from different human organs and normal cell populations (Fig. 2b). Again, none of the indicated organs and cells revealed detectable H3t expression, except for testis. Note that seminoma, a testis-derived malignant tumor, fails to express the H3t gene. Since these organs represent a broad spectrum of different human tissues and do not show any H3t transcript, we conclude that the histone H3t gene is expressed in a testis-specific manner. Mapping of the H3t 3* Termination Site The 3* end of histone mRNAs may either contain a dyad symmetry element that is essential for the processing of the replication-dependent mRNA species [19, 20] or, in the case of replacement-type histone mRNAs, be polyadenylated [21, 22]. In order to determine the 3* end of the H3t transcript, we used a 1-kb probe of the 3* flanking region in an RNase protection assay. The size of the 190-bp protected fragment shown in Fig. 3 localizes the 3* terminus of the H3t transcript immediately downstream of a consensus dyad symmetry element. No longer transcript could be detected. The sizes of the protected 5* and 3* probes allowed us to map the approximate start site, length, and termination site of the H3t transcript (Fig. 4). The TATA box is located at position 037 and the two CCAAT boxes at 059 and 0101 relative to the start site of transcription. The length of the H3t transcript is 500 bp. H3t in Situ Hybridization in Human Testis Sections Since cell-type-specific expression during spermatogenesis has been demonstrated for histone genes [6], we investigated the expression of the H3t gene in tissue sections from human testis by in situ hybridization and compared it in serial sections with the expression of total H3 and H1t. As shown in Fig. 5, H3t mRNA could indeed only be detected in distinct cell populations of spermatogenesis. High magnification demonstrates that the H3t-expressing cells are separated from the basement membrane by ‘‘pale’’ spermatogonia. As determined by hematoxylin –eosin staining of serial sections (Fig. 5), these H3t-positive cells contain large nu-
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FIG. 2. Expression of the histone H3t gene in human cell lines (a) and human tissues and normal cell populations (b). Equivalent amounts of RNA were subjected to the RNase protection assay as determined by both spectrophotometry and ethidium bromide staining of RNA (bottom). Additionally, integrity of the analyzed RNAs was monitored by using a histone H1.2 probe as a positive control (results not shown). Arrow indicates the 160-bp fragment protected by H3t mRNA. Several minor fragments õ130 bp were detected, representing degradation products of other, only partially hybridized, H3 mRNAs (not shown). PBMC, peripheral blood monocytic cells.
clei, indicative of primary spermatocytes, whereas adjacent spermatogonia and round spermatids did not exhibit expression of the H3t gene. Since it is well established that H1t mRNA is synthesized by pachytene spermatocytes [4–6, 23], we also performed in situ hybridization experiments with a human histone H1t probe (Fig. 5). Comparison of the H3t and H1t results reveals almost identical expression patterns of the two histone genes in serial sections. Therefore, we conclude
that the human H3t gene is expressed in primary spermatocytes, being transcribed by the same cell population as the H1t gene. In order to compare the H3t expression with the total histone H3 transcription during spermatogenesis, we included experiments using a probe of the H3t coding region which should crosshybridize also with any other histone H3 mRNAs due to the highly conserved nature of the H3 coding regions. Hybridization with this general H3 probe was, however, also confined to primary spermatocytes (Fig. 5). This restriction of H3t expression to the same cells which express the H1t gene is not the result of a selective accessibility of these cells to in situ hybridization probes. This is excluded by hybridization of a histone H17 probe with spermatogonia and of a protamine probe with spermatids under the same hybridization conditions (data not shown) in agreement with established patterns of expression of these genes [6, 24]. In addition to its hybridization with H3t-specific mRNA, the total H3 probe may there also react with H3.3 mRNA which has been described in testicular cells [25]. A cross-hybridization with other, i.e., S-phase-dependent, H3 mRNA, can be ruled out, since the hybridizing cells correspond to postmitotic stages. Only rarely, weak staining of spermatogonia in some tubuli was observed (results not shown). Moreover, we carried out in situ hybridizations with sections of a
FIG. 3. Mapping of the H3t termination of transcription by RNase protection analysis. Arrow indicates 190-bp fragment protected by testis mRNA.
FIG. 4. Start site and termination of transcription of the H3t gene and relative positions of the TATA and CCAAT boxes. 0 , dyad symmetry element of the sequence GGCTCTTTTAAGAGCC.
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FIG. 5. In situ hybridization of serial sections from human adult testis. A series of four sections was stained with hematoxylin –eosin (H.E.) and hybridized with probes for the indicated histone mRNAs, respectively. Magnification 4001; detailed aspects 10001.
carcinoma of the kidney in order to rule out cross-hybridizations between the H3t probe used with other H3 histone mRNAs. In these experiments, we found that the total H3 probe, but not the H3t-specific probe, showed staining of the tumor cells, indicating no cross-hybridization of the two probes (data not shown). DISCUSSION
During spermatogenesis a rearrangement of the histone composition of spermatogenic cells takes place. In
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addition to the histone isoforms, which are also found in somatic tissues, testis-specific histones are synthesized during the process of sperm development, indicating special functions of these isoforms in modulating the chromatin structure during spermatogenesis [10]. In this paper, we have shown testis-specific expression of a novel human histone H3 gene, designated H3t, in primary spermatocytes. Other testis-specific histone variants that have been described are H1t, the testisspecific isoform of histone H1 in human, rat, and mouse
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[4–6, 26], and TH2A and TH2B, the testis-specific isoforms of histones H2A and H2B in rats [7, 9, 27]. Furthermore, Trostle-Weige et al. have described a germ cell-specific H3 histone variant (TH3) in rat testis based on electrophoretic mobility and protein analysis [8]. As yet, no TH3 gene has been identified in any mammalian genome. However, the histone H3t gene reported here appears to encode a different histone protein. First, the experimentally determined amino acid composition of the rat histone TH3 varies from the human H3t gene deduced amino acid sequence in its methionine, cysteine, and serine composition. Second, TH3 has been shown to be synthesized mainly in type A and B spermatogonia, whereas spermatocytes did not show synthesis of TH3, based on centrifugal elutriation following intratesticular injection of tritiated arginine [8]. In contrast, our in situ hybridization data demonstrate H3t expression to be absent in spermatogonia. H3t mRNA was only detectable in primary spermatocytes, being transcribed in the same cells as H1t which is predominantly expressed in pachytene spermatocytes [4 –6, 23]. The differences between TH3 and H3t might partly be explained by the different experimental approaches and different species studied. However, our results provide the first evidence for testis-specific expression of a core histone variant in man. The H3t gene shares some common features with the testis-specific H1t, TH2A, and TH2B genes. First, the pattern of H3t expression is similar to that of H1t, TH2A, and TH2B, which are exclusively expressed in primary spermatocytes of the pachytene stage [6, 7, 27]. Second, as we have shown for the H3t mRNA, these testis-specific histone isoforms contain a dyad symmetry element at their 3* end [7, 9, 26, 27]. This highly conserved stem–loop is found in all replication-dependent histone genes and has been implicated in transcription termination, efficiency of mRNA processing, mRNA stability, and the coupling of histone mRNA levels to the cell-cycle [28], whereas the replacement histone variants contain a polyadenylation signal. On the other hand, the H3t gene is expressed in meiotic cells, where no DNA synthesis takes place. Since it has been shown that H1t, TH2A, and TH2B replace about 60% of the somatic isoforms in pachytene spermatocytes [29, 30], a high synthesis rate of the testis-specific histones is required in these cells. Therefore the stem – loop of the testis-specific histone isoforms might be necessary for efficient mRNA processing or high mRNA stability in spermatocytes. Replacement of the stem – loop by a polyadenylation signal has been found to lead to a 30-fold reduction of expression of histone H4 [31]. The extent to which H3t replaces the somatic H3 histones during spermatogenesis is not yet clear, but our data show that the bulk of total H3 gene transcription as well as the H3t transcription takes place in primary spermatocytes. It could well be that the total H3 tran-
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script in these cells mainly consists of the H3t isoform, since these post-mitotic cells are not expected to transcribe replication-dependent H3 histones, but we cannot exclude that part of the hybridization obtained with the total H3 probe may be due to expression of the replacement variant genes H3.3A and H3.3B. Since spermatogonia in S-phase are rare in a given tissuesection compared with primary spermatocytes, we only occasionally detected spermatogonia reacting with the total H3 probe. An obvious difference between H3t and the other testis-specific isoforms is their genomic organization. Whereas H1t is located within the major histone gene cluster on chromosome 6 [12], the H3t gene is solitarily located on chromosome 1 in the human genome [11]. TH2A and TH2B, which appear to exist only in the rat genome, similarly have been mapped to a cluster on rat chromosome 17, which is syntenic with human 6p21.3 and carries H1t and other histone genes [32]. The function of the singular location of the H3t gene is not clear, but it may play a role in an independently regulated transcription as has been suggested for the solitarily located replacement histone genes H17 and H3.3B [12, 33]. The testis-specific expression of the H3t gene raises questions about the mechanism of its tissue-specific regulation. Several promoter elements have been implicated in testis-specific expression of the H1t gene and the testis-specific protamine genes. However, the H3t promoter does not contain any of these sequences including the conserved H1t promoter elements TE1 and TE2 or the mouse protamine 1 sequence TGACTTCATAA binding TET-1 [34, 35]. Queralt and Oliva have found a conserved protamine 1 promoter element, designated as Prot1C, at position 064 to 053 relative to the start site of transcription by comparing protamine 1 promoters of 10 different mammals [36]. The H3t promoter contains a sequence matching this consensus element by 100%, but its position is located at 0180. Further studies of the H3t promoter are needed to define the mechanism of testis-specific expression of this gene. The function of the testis-specific histone isoforms is still not known, but tissue specificity as well as expression in a distinct cell population, namely pachytene spermatocytes, where crossing-over of homologous chromosomes takes place, points to a functional role in chromatin morphology during spermatogenesis and in particular the process of meiosis. H1t has been shown to have a lower condensing capacity than the other H1 subtypes and may thus help to decondense the chromatin structure for the specific needs of the meiotic and haploid stages of germ cell development [37]. The amino acid sequence of H3t varies at four positions from the mammalian H3 consensus sequence. At position 98, the H3t protein contains serine instead of ala-
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nine. This additional potential phosphorylation site might be of functional relevance, since phosphorylation of H1t in elongating spermatids and a complex pattern of H2A.X phosphorylation have been described in murine testicular cells [25, 38]. It remains to be shown whether other mammalian genomes contain homologues of the human H3t gene. Isolation and characterization of a murine H3t gene would allow functional studies in vitro or by artificially deleting the H3t gene. This work was supported by a research grant from the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie. O.W. gratefully acknowledges a fellowship from the Deutsche Forschungsgemeinschaft.
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Received August 1, 1996 Revised version received September 16, 1996
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