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BOR - Papers in Press, published online ahead of print December 26, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.024760
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BIOLOGY OF REPRODUCTION 70, 1239–1245 (2004)
DOI: 10.1095/biolreprod.103.024760
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Control of Mouse hils1 Gene Expression During Spermatogenesis: Identification of Regulatory Element by Transgenic Mouse

Naoko Iguchi2, Hiromitsu Tanaka2, Shuichi Yamada3, Hiromi Nishimura2, and Yoshitake Nishimune1,2

Department of Science for Laboratory Animal Experimentation,2 Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan Department of Cell Biology,3 Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histone H1-like protein in spermatids 1 (Hils1) is a testis- specific histone H1-like protein exclusively expressed in haploid spermatids and should be involved in chromatin remodeling during mouse spermatogenesis. Spatial and temporal regulation of the hils1 gene expression would be critical for the formation of functional sperm, controlled at both transcriptional and translational levels. Previously, we reported that transcripts of the hils1 gene are exclusively expressed in mouse testis from 23 days of age whereas the Hils1 protein is not detected until 28 days of age, suggesting that hils1 is a member of a class of translationally regulated genes. By analyzing transgenic mice, we could demonstrate that 318-base pair (bp) 5'-proximal region corresponding to the first 70-bp proximal TATA-less promoter, and 248 bp of 5'-untranslated region is sufficient to confer testis- and spermatid-specific transcription as well as posttranscriptional control of the mouse hils1 gene in vivo.

gamete biology, gene regulation, spermatid, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During spermiogenesis, round spermatids undergo complex morphological, biochemical, and physiological modifications resulting in the formation of mature spermatozoa. Major modifications in both nuclear and cytoplasmic structures continue throughout spermiogenesis, and stringent temporal and stage-specific gene expression is a prerequisite for the correct differentiation of round spermatids into mature spermatozoa. As spermatogenesis progresses, there is a widespread reorganization of the haploid genome followed by extensive DNA condensation [1, 2]. During this period, increasing evidence suggests that transcriptional activity is rather high in postmeiotic spermatids until the sperm genome is packed tightly into the inert DNA-protamine complex [3, 4]. Histone modifications such as acetylation, ubiquitination, methylation, and phosphorylation may lead to changes in chromatin condensation and the accessibility of transcription factors and chromatin-remodeling machineries [5]. Actually, a number of the testis-specific histones and nonhistone chromosomal proteins, such as histone H3.3, macroH2A, ssH2B, tsH2A, H1t, and tsHMG, appear to play key roles that linked to transcription, histone deposition at replication, and histone removal during spermatogenesis, in which the majority of somatic gene expression is silenced and a very unique set of genes are transcriptionally activated [6]. This transcriptional regulation is achieved by several strategies, such as extensive chromatin reorganization, the use of specialized transcription factors, and the use of alternative distinct promoters [7]. It has also been demonstrated that the mRNAs of many genes are stored as ribonucleoprotein particles in a translationally repressed state for several days and are translated during a later stage of spermatogenesis [1, 2, 8–10]. Therefore, the analyses of histone variants and nonhistone chromosomal proteins in these processes are extremely valuable tools for elucidating the mechanisms involved in controlling chromatin structure and unique transcriptional and translational regulation in postmeiotic cells.

Hils1 is a histone-H1-like protein present only in condensing spermatid nuclei, suggesting a role in the chromatin remodeling that occurs during spermiogenesis [11, 12]. As we previously reported, hils1 mRNA is expressed in steps 1–9 spermatids whereas Hils1 protein is not detected until step 9 by immunohistochemistry. Thus, hils1 appears to be both transcriptionally and translationally regulated during mouse spermiogenesis, as are other chromatin proteins in spermatids, such as protamines and transition proteins. In this study, we have examined the regions of sequence required for transcription and posttranscriptional control of the hils1 gene in vivo by analyzing transgenic mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs for Promoter Analysis

The full-length mouse hils1 cDNA sequence was used to search the entire GenBank database. The genomic sequence containing the 5'-flanking region of the hils1 gene was downloaded and further analyzed through alignment with mouse hils1 cDNA using the GENETYX sequence analysis program. The 5'-flanking region of hils1 was cloned by polymerase chain reaction (PCR) screening of a C57BL/6J mouse genomic library. The primers for PCR (forward: 5'-GAGATCTGCCTACCTCACAATGGCCCCCT-3', nucleotides –70 to –46 with BglII linker sequence; reverse: 5'-CCGCTCGAGGCTGCGTATGGTGACACCAGCG- 3', nucleotides 224 to 248 with XhoI linker sequence) were based on the mouse genomic sequence registered on the GenBank (NT_039521). PCR was performed in reaction mix (50 µl) containing 1 µg of C57BL/6J mouse genomic DNA, 0.1 mM of each primer, 8 mM of deoxyribonucleotide triphosphate (dNTP) mixture, 1x Ex taq buffer, and 1 U of Ex taq (Takara, Shiga, Japan). The cycling condition was as follows: 30 cycles of denaturation at 95°C for 45 sec, annealing at 60°C for 30 sec, and extension at 72°C for 60 sec. The resulting PCR product contained 70 base pairs (bp) of 5'-upstream and 248 bp of 5'-untranslated region. The product was termed hils1 promoter and was digested with the appropriate restriction enzymes, then subcloned into the promoterless reporter vector pEGFP-1 (Clontech, Palo Alto, CA) and sequenced. Dideoxy chain-termination sequencing reactions were performed with fluorescent dye-labeled primers and thermal cycle sequencing kits purchased from Applied Biosystems (Foster City, CA). The reaction products were analyzed using an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems).

Generation of Transgenic Mice

The constructed EGFP vectors described above were digested with ApaLI and DraIII. A DNA fragment of approximately 1.9 kilobase (kb) including hils1-promoter-EGFP-SV40 poly(A) was resolved by agarose gel electrophoresis and recovered from the gel using glass beads (Qiaex, Qiagen K.K., Tokyo, Japan). The excised DNA fragment was injected into the pronuclei of BCF2 eggs. The offspring from the microinjected eggs were identified by PCR with a set of EGFP-specific primers (385 primer 5'-TGAACCGCATCGAGCTGAAGGG-3' and 386 primer 5'-TCCAGCAGGACCATGTGATCGC-3'). DNA from mouse tail was added to 15 µl of reaction mixture containing 0.1 mM of each primer, 8 mM of dNTP mixture, 1x Ex taq buffer, and 0.3 U of Ex taq (Takara). The cycling condition was as follows: 30 cycles of denaturation at 96°C for 45 sec, annealing at 60°C for 45 sec, and extension at 72°C for 45 sec. Transgenic founders were subsequently mated to C57BL/6 mice and positive male offspring from this cross were used for experiments. All the animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Research Institute for Microbial Diseases, Osaka University).

Northern Blot Analyses

Total RNAs were isolated from various tissues of PCR-positive male mice with RNA zol B (TEL-TEST Inc., Friendswood, TX) according to the manufacture's protocol and quantified by optical density measurement. Five or 10 µg of total RNA containing 2.2 M formaldehyde was separated on a 1.0% agarose gel with 0.66 M formaldehyde and transferred to a Zeta-Probe Membrane (Bio-Rad, Hercules, CA). Hybridization was performed with probes for EGFP, the entire coding regions of hils1 cDNA, and the mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, used as a control. The filter was prehybridized for 30 min and then hybridized for 16 h in PerfectHyb Hybridization Solution (Toyobo, Osaka, Japan) at 65°C with [{alpha}-32P]dCTP-labeled DNA probes. Each filter was washed at 65°C in 0.2x saline-sodium citrate buffer containing 0.1% SDS. Signals were detected using an ImageAnalyzer (Fuji Photo Film Co. Ltd., Kanagawa, Japan).

Western Blot Analyses

Protein samples of various tissues from PCR-positive mice were lysed with RIPA buffer (10 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 1% protease inhibitor cocktail [Sigma-Aldrich Co., St. Louis, MO]). After centrifugation, aliquots of the samples (50 µg/lane) were separated by 10% SDS- PAGE and transferred to polyvinylidene fluoride filters (Millipore, Bedford, MA) followed by blocking with TBS-T (TBS; 25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 50 mM KCl, -T; 0.05% Tween 20) containing 5% nonfat dried milk. The filters were incubated with anti-EGFP rat monoclonal antibody (x400) or polyclonal anti-Hils1 rabbit IgG (x500) diluted in TBS, followed by treatment with horseradish peroxidase-conjugated anti-rat IgG (DAKO Cytomation Norden A/S, Glostrup, Denmark) or anti- rabbit IgG (Amersham Biosciences, Tokyo, Japan), respectively. Signals were detected by development using a POD staining kit (Wako, Osaka, Japan).

Histological Analysis

Testes were collected from PCR-positive adult transgenic male mice and fixed in 4% paraformaldehyde for 16 h. After removing extra paraformaldehyde by washing with PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4), testes were dehydrated with 100% acetone for 1 h and then embedded in glycol methacrylate (Technovit 8100; Heraeus Kulzer, Hanau, Germany). Serial sections of the whole testes were prepared with a thickness of 5 µm. The sections were examined under a fluorescent microscope for expression of EGFP in germ cells at various stages of differentiation. Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI) and observed under a photomicroscope to determine the spermatogenic stage.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Genomic Construction and Characterization of the Promoter Region of hils1 Gene

Using the mouse hils1 cDNA sequence (AB022320) to search the nonredundant database of the National Center for Biotechnology Information (NCBI; Bethesda, MD), we found a mouse genomic sequence that contained the full- length hils1 gene (NT_039521). The database suggested that mouse hils1 gene consists of 1 exon located in the ninth intron of the alpha-sarcoglycan gene (sgca; NM_009161) on the long arm of chromosome 11 (NCBI locus ID: 54388). To confirm this information from the database on the genomic structure of mouse hils1, PCR amplification was performed using mouse genomic DNA. The length of the resulting PCR product was the same size as the cDNA, and DNA sequences of all the subcloned PCR products were identical, indicating hils1 is an intronless gene (Fig. 1A).



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  		FIG. 1. Nucleotide sequence of hils1 gene and surrounding genomic region. A) A schematic presentation of the hils1 gene and alpha-sarcoglycan gene (sgca). The hils1 gene is transcribed in the opposite direction of the sgca gene. The gray boxes indicate the exons of sgca and the number of the exons. Arrows indicate the positions of primers (forward, Fw; and reverse, Rv) used for PCR. B) Small and capital letters indicate the upstream sequence and hils1 gene, respectively. The transcription start site is indicated as +1. Bold letters (ATG) indicate the translation initiation sequence. White letters in black-boxes show a CRE-binding site. Y- and H-element sequences are double underlined. YRS is open boxed. The fragment used for promoter analysis in transgenic mice is shadowed

The promoter regions of somatic and testis-specific histone H1 variants contain many regulatory elements, including TATA-, CAAT-, and GC-boxes, and the H1-specific AC motif within 100 bp of the cap site [13, 14]. In contrast, the 70-bp 5' upstream region of the hils1 gene has some transcriptional factor-binding motifs, i.e., v-ErbA (–48 to –33), AML-1a (–45 to –40), cAMP response element (CRE) (–45 to –38), CF1 (–44 to –36), and GATA-1, -2, and -3 (–33 to –24), but absence of TATA-, CAAT-, or GC-boxes, an SP1 binding site, or Inr (Fig. 1B). Thus, the transcriptional regulation mechanism of the hils1 gene must be different from those of histone H1s. Specific expression of hils1 in differentiated haploid germ cells and the silencing of the hils1 gene in somatic cells [11, 12] could be caused by its unique promoter region, which lacks the general transcriptional regulatory elements, and because the CRE sequence is also common for haploid germ cell-specific gene expression [7]. To estimate the promoter activity of the 5'-upstream region of hils1 and to assess whether the putative YRS [15], H, and Y elements [16] present in 5'-untranslated region (UTR) of hils1 (Fig. 1B) support posttranscriptional regulation in vivo, we generated a transgenic construct with the EGFP reporter gene containing the CRE, YRS, H, and Y elements in the 5'UTR of hils1 (–70 to +248) (Fig. 1). We established 13 founder transgenic lines carrying the construct. Northern blotting of RNAs from various organs of transgenic mice showed the EGFP and hils1 transcripts (about 1.5 kb and 0.9 kb, respectively) were detected exclusively in the testis (Fig. 2A). To investigate the developmental changes of the gene expression in the mouse testis, total testicular RNAs were isolated at the ages of 15, 21, and 24 days and from an adult older than 2 mo. The mRNA transcript of the EGFP reporter gene was detected first at the age of 21 days, the same as endogenous hils1 gene expression (Fig. 2B). These results indicate that the mRNA expression of the reporter gene was under the control of the hils1 promoter used in the present study (Fig. 1) and hence mimicked endogenous hils1 expression.



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  		FIG. 2. RNA expression analyses of the transgenic mice. Total RNAs (5 µg) from various organs of adult transgenic mice older than 2 mo (A) or total RNAs (10 µg) from 15-day-, 21-day-, and 24-day-old and adult transgenic mice testes and adult liver (B) were electrophoresed, transferred to nylon membranes, and hybridized with the EGFP (upper panel) and hils1 (middle panel) cDNA probe. The filters were rehybridized with mouse GAPDH cDNA as a control (lower panel). The positions of 28S and 18S ribosomal RNAs are indicated at the left margin

Posttranscriptional Regulation in Transgenic Mice

As we previously reported, Hils1 protein was first detected in postmeiotic male germ cells at 28 days, whereas hils1 transcript was detectable at 21 days of age [11]. Thus, translation was delayed for at least several days, suggesting that the expression of the hils1 gene is also regulated by translational control. Translational silencing of a number of mRNAs is a prominent phenomena during spermatogenesis, which is known to be controlled by RNA-binding proteins for the 3'UTR. Some cis-acting sequences and RNA- binding proteins have been previously identified, which are necessary for translational repression [15–18]. Some of these are also located in the 5'UTR as well as the 3'UTR. Sequence analysis showed that there are two Y- and one H-element-like sequences and also one YRS-like site in 5'UTR of hils1 gene (Fig. 1B). Although Northern blot analysis showed that the EGFP mRNA has already been expressed in the 21-day-old testis, the same as endogenous hils1 mRNA (Fig. 2B), Western blot analysis showed that the EGFP protein was exclusively detected in the testis older than 24 days of age (Fig. 3B). EGFP protein was minimally detected in the 24-day-old testis, the same as the Hils1 protein (Fig. 3B). These results clearly demonstrated that reporter EGFP expression was both transcriptionally and posttranscriptionally regulated, as was endogenous hils1 gene expression during spermiogenesis. That is, 318 bp of 5'UTR sequence contained the hils1-promoter region, which could control the haploid germ cell-specific expression of hils1 gene at both the transcriptional and translational levels.



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  		FIG. 3. Protein expression analyses of the transgenic mice. Protein samples (50 µg/lane) prepared from various adult (older than 2 mo) mouse organs (A) and testes of 15-day-, 21-day-, and 24-day-old and adult mice (B) were examined with antibodies against EGFP (upper panels) or Hils1 (lower panels). Arrowheads indicate the positions of each protein. The molecular marker positions (Mr x 10–3) were indicated at the left margin (A)

Histological Analysis of Transgenic Mouse Testis

EGFP fluorescence was observed in the testes of the transgenic mice, but not in other organs examined, consistent with the results of Northern and Western blot analysis (Figs. 2A, 3A). Histological observation showed that EGFP fluorescence was limited to the cytoplasm of haploid germ cells (Fig. 4), except for the natural fluorescence in Leydig cells observed even in nontransgenic littermates (data not shown). EGFP expression was first observed in elongating spermatids at step 9, consistent with the expression of endogenous Hils1 protein [11]. Although the endogenous Hils1 protein became undetectable in step 13 elongated spermatids by immunohistochemistry, EGFP persisted in the cytoplasm of spermatids until the end of spermiogenesis, although spermatozoa recovered from epididymis were no longer fluorescent positive. This was most likely the result of the stability of the EGFP protein, which would have been discarded in the residual body at the end of sperm morphogenesis. The expression pattern of the EGFP reporter gene was the same in all eight independently raised transgenic mouse lines. These results showed that the hils1 promoter demonstrated here could confer haploid germ cell-specific transcription and translational delay of mRNA, which mimics endogenous hils1 gene expression.



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  		FIG. 4. EGFP expression in adult transgenic mice. EGFP expression in cross sections of transgenic mouse testis (upper panel). Leydig cells show endogenous natural fluorescence. Nuclei were stained with DAPI (central panel). The lower panel is the merged image. The left panels show the higher magnification of merged images. Greek numerals indicate the stages of seminiferous tubules. Bar = 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During spermiogenesis, stringent temporal and stage- specific gene expression is a prerequisite for the proper differentiation of male germ cells into functional sperm. Gene expression of spermatid nucleoproteins, such as protamines and transition proteins, is well documented to be regulated at both the transcriptional and translational level [2, 19– 21]. Hils1 is a DNA-binding nuclear protein specifically expressed in haploid germ cells in the testis during the time chromatin is under reorganization, suggesting that it may be an important factor for sperm chromatin remodeling. We have shown previously that the hils1 gene is regulated at both the transcriptional and posttranscriptional level like other spermatid nucleoproteins [11].

In this study, we investigated the genomic structure and determined the regulatory element of hils1 expression by raising transgenic mice. The intronless mouse hils1 gene is located in the ninth intron of the alpha-sarcoglycan gene (sgca) (Fig. 1A), which is the gene responsible for muscular dystrophy [22] and is mapped to chromosome 11D. The hils1 gene seems to have been generated by retroposition and integrated into an area capable of supporting transcription. Although retroposition has frequently occurred during mammalian evolution, most retroposons have degenerated into nonexpressed pseudogenes [23], whereas expression of functional retroposons is a rather common phenomenon in meiotic and haploid spermatogenic cells [24–28].

Regulation of gene expression in postmeiotic male germ cells follows a number of specialized rules, distinct from somatic cells. In this context, transcriptional control mediated by the activator cAMP-responsive element modulator (CREM) is one of common occurrence [29]. In contrast with somatic cells, CREM-mediated regulation of postmeiotic genes is performed by activator of CREM in testis (ACT), independent of CRE-binding protein (CREB)-binding protein (CBP) and phosphorylation [7, 30]. The CREM- {tau} protein is expressed only in haploid spermatids [31] and regulates a number of haploid genes involved in the process of spermatogenesis [32–35]. CREM-{tau} has an essential role in spermiogenesis, as demonstrated by its targeted mutation in the mouse [36, 37]. In the present study, transgenic mice revealed that a relatively short 5'-flanking region (70 bp) containing one CRE site is sufficient to drive haploid-specific gene expression in vivo. It suggests that CREM-{tau} may play an important role in the transcriptional regulation of hils1 in mouse haploid germ cells.

Although the tissue-specific differential methylation of CpG dinucleotides is regarded as a key mechanism in the silencing of genes [38], in the case of hils1, the CpG dinucleotides are absent in 500 bp of the 5'-flanking region (data not shown). Thus, the methylation would be unlikely to cause the repression of hils1 in nonhaploid germinal cells. Rather, the TATA-less promoter of hils1 should be sufficient for its haploid germ cell-specific expression. Indeed, increasing evidence shows that a number of postmeiotic CREM-target genes lack the TATA-box [39]. It has been demonstrated that the testis-specific isozyme of the angiotensin converting enzyme (ACE-{tau}) gene could not be activated by CREM-{tau} in cultured somatic cell lines unless the TATA-like element in the promoter mutated to the authentic TATA-box [40]. It has also been proposed that TBP- like Factor, highly expressed in the testis, substitutes TBP in initiation complexes associated to TATA-less promoters [41]. Taken together, CREM in spermatogenic cells would not need a TATA-element in the target gene promoter for transcriptional activation. Thus, the repression of hils1 in nonexpressed organs is probably achieved by its TATA- less, CRE-containing promoter.

There are numerous examples of translation repression of mRNA and subsequent activation of translation at specific stages of spermiogenesis [8, 22, 42–45]. Translational repression is essential for spermatid differentiation as premature translation can lead to an arrest in spermatid differentiation and cause dominant male sterility [46]. Posttranscriptional control can be mediated by sequences in the 5' and 3' UTRs of mRNAs, and in some cases separate elements may regulate translational repression and activation [1, 13, 47]. Transgenic experiments indicate that the first 248 nucleotides of the 5'UTR confer hils1-like translational control on a reporter gene, suggesting that the 5'UTR of hils1 plays a role in hils1 translational control and protection of mRNA from degradation during spermiogenesis. The 5'UTR used for transgenic analysis contains the sequences similar to YRS, Y, and H elements, which are known to be involved in posttranscriptional regulation. Several RNA-binding proteins, which interact with these elements, i.e., testis-brain RNA-binding protein (TB-RBP), Y- box proteins [15, 48–50], may play an important role in the control of hils1 mRNA translation. The YRS-like element is not the original consensus YRS, 5'-UACCACAUCCACU- 3', but is reported to be a mutation that eliminates MSY2 and MSY4 binding [51]. TB-RBP binds to conserved Y and H elements of many testis and brain mRNAs, including those for protamines 1 and 2, glyceraldehyde3-phosphate dehydrogenase-S (gapds), myelin basic protein, and tau protein, and is implicated in the movement, stability, and/ or translational suppression of specific mRNAs in male germ cells [15, 42, 51]. Recently, Yang et al. reported that posttranscriptional regulation of gapds gene expression during spermiogenesis might be regulated by the H element in its 5'UTR via TB-RBP function [45]. This suggests that the Y and H elements in the 5'UTR are mediated by the binding of TB-RBP rather than MSY2 and MSY4, leading to the posttranscriptional control of hils1 mRNA. Although the hils1 3'UTR may also play a cooperative role in translational control, the 5'UTR in our present experiment is enough to demonstrate the specific control of hils1 gene expression. Further studies are required to determine more specific control elements of transcription and translation together with the binding factors responsible for hils1 gene expression.


   ACKNOWLEDGMENTS
 
We appreciate Ms. Amy Herlihy (Monash Institute of Reproduction and Development, Australia) for critical reading of the manuscript, Mr. Kouichi Kitamura for discussion, and Ms. Yoko Shigemi and Ms. Kahori Numazawa for technical assistance.


   FOOTNOTES
 
1 Correspondence: Yoshitake Nishimune, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp Back

Received: 28 October 2003.

First decision: 23 November 2003.

Accepted: 12 December 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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