Abstract
The gene encoding mouse cyclin A1, Ccna1, is expressed at highest levels in late pachytene-diplotene spermatocytes, where it is required for meiotic cell division. To begin to understand the mechanisms responsible for its highly restricted pattern of expression, transgenic mouse lines carrying constructs consisting of the cyclin A1 regulatory region fused with the reporter gene lacZ were generated. Analysis of tissue-specific and testicular cell-type-specific transgene expression indicated that sequences within −1.3 kilobases (kb) of the cyclin A1 putative transcriptional start site were sufficient to direct transgene expression uniquely to late spermatocytes while maintaining repression in other tissues. However, sequences located between −4.8 kb and −1.3 kb of the putative transcriptional start site were apparently required to transcribe the reporter at levels needed for consistent X-gal staining. Comparison of the mouse, rat, and human proximal promoters revealed regions of high sequence conservation and consensus sequences both for known transcription factors, some of which are coexpressed with Ccna1, such as A-myb and Hsf2, and for elements that control expression of genes in somatic cell cycles, such as CDE, CHR, and CCAAT elements. Thus, the promoter region within 1.3 kb upstream of the putative Ccna1 transcriptional start can direct expression of lacZ to spermatocytes, while sequences located between −4.8 kb and −1.3 kb of the putative transcriptional start site may enhance expression of lacZ.
Introduction
Spermatogenesis is an ordered process during which germ cells enter successive mitotic, meiotic, and postmeiotic phases. The program of gene expression required for this process is tightly controlled, involving both germ-cell-specific and common transcription factors, the expression of which is also stringently regulated (reviewed in [1, 2]). This regulation allows precisely ordered events to occur, such as genetic recombination and meiotic cell division, followed by the morphological development of spermatids.
Genes for several key somatic cell cycle regulators, including cyclins, cyclin-dependent kinases (Cdks), Cdk inhibitors, and Cdc25 family members are expressed during spermatogenesis in specific patterns that suggest their function in cell-cycle control in the germ line as well [3, 4]. Cyclins are regulatory subunits of the Cdk complexes and are expressed periodically during the cell cycle. There are at least 10 classes of cyclins in higher vertebrates, designated cyclins A to I and T, and multiple members of the A-, B-, and D-type cyclin families (reviewed in [5–7]).
In higher organisms, there are two A-type cyclins, cyclin A1 and cyclin A2. In mice, both are expressed in male germ cells, albeit during quite different stages of differentiation, suggesting that they are tightly regulated and may have distinct functions [8, 9]. The cyclin A1 gene, Ccna1, appears to be testis-specific and is expressed late in the meiotic cell cycle, just prior to chromosomal desynapsis [8, 9]. Disruption of mouse Ccna1 resulted in male infertility and complete spermatogenic arrest prior to the first meiotic division [10]. In contrast, cyclin A2 (Ccna2) is widely expressed in mouse tissues [8]. In the male germ line, Ccna2 is expressed earlier than Ccna1, in spermatogonia and preleptotene spermatocytes, and is no longer detected as germ cells enter the leptotene stage [9]. Disruption of the Ccna2 gene resulted in embryonic lethality [11]. Therefore, its role in spermatogenesis could not be discerned from a null model.
Human CCNA1 is expressed most highly in testis and, at lower levels in adult brain and in hematopoietic cells [12, 13]. It is also expressed in several human myeloid leukemia cell lines and an osteosarcoma cell line, where its level of expression appears to be cell-cycle regulated [12, 14]. In testis, human cyclin A1 is localized in late meiotic prophase spermatocytes, similar to mouse cyclin A1 [15]. A short fragment of the human CCNA1 promoter, comprised of 190 base pairs (bp) of sequence upstream and 145 bp of sequence downstream of the transcriptional start site, has been reported to activate expression of a reporter gene in HeLa cells [16] and CV-1 cells [17]. In transgenic mice, a 1.3-kilobase (kb) fragment of the human CCNA1 promoter has further been reported to direct expression of enhanced green fluorescent protein (EGFP) to male germ cells but in a much less restricted expression pattern than observed for the endogenous mouse gene [18]. That is, in addition to stage-IX to -XII spermatocytes, EGFP fluorescence was detected in spermatogonia, at earlier stages of spermatocyte development and in spermatids [18, 19].
The DNA regulatory elements required for mouse Ccna1 gene expression in male germ cells have not been identified. We hypothesize that there will be regulatory elements unique to Ccna1, not present in the Ccna2 promoter, reflecting their distinct expression patterns, and further that such elements might be evolutionarily conserved. To begin to define the location of these elements and, thus, begin to understand mechanisms that control the stage-specific expression of Ccna1 and repression at other stages of male germ cell development, we have tested expression of Ccna1 promoter/reporter constructs in mice. This in vivo approach was chosen to define promoter function in male germ cells because procedures for transfection of these cells in culture have not been established. In the present study, the regulatory function of genomic fragments spanning −8.2 kb to +0.8 kb from the putative transcriptional start site of mouse Ccna1 was examined in transgenic mice, using the reporter lacZ. The patterns of transgene expression revealed that the genomic fragment that lies between −1.3 kb to +0.8 kb of the putative Ccna1 transcriptional start site could fully recapitulate the correct developmental expression of the endogenous Ccna1 gene in male germ cells with no ectopic expression observed, unlike the human CCNA1 reporter constructs. However, sequences between −4.8 and −1.3 of the putative transcriptional start appeared to be necessary for fully penetrant expression of the gene in spermatocytes. Within this genomic region, consensus binding sequences for several transcription factors were identified. They include sequences known to bind factors that are coexpressed with Ccna1 and also sequences that control cell-cycle-regulated gene expression in somatic cell cycles. Alignment of the proximal promoters of the mouse, rat, and human genes for cyclin A1 revealed regions of high homology; however, the human proximal promoter also contained inserted sequences that were not present in the mouse and rat promoters.
Materials and Methods
Transgene Constructs and Transgenic Mice
A 5.6-kb fragment that extends 5′ from the BamHI site in exon 2 of Ccna1 to an EcoRI site in the 5′ flanking region was inserted into a vector containing a cassette composed of lacZ and intron 1 and the polyadenylation signal of mouse protamine-1 to yield construct 4.8cyA1lacZ (Fig. 1). The reporter cassette had been used previously to produce a Hoxa4 promoter-reporter construct that was expressed in male germ cells [20]. 8.2cyA1lacZ was generated from 4.8cyA1lacZ by inserting a SalI-EcoRI fragment 5′ of the EcoRI site. 1.3cyA1lacZ was produced from 4.8cyA1lacZ by digestion with XhoI and religation. All constructs were verified by sequencing. The fusion gene product is expected to have the first 33 amino acids of the cyclin A1 protein linked to β-galactosidase (β-gal). The production of transgenic mice was carried out as previously described [20, 21]. All procedures were performed in accord with guidelines of the Institutional Animal Care and Use Committee of the Columbia University Medical Center. Transgenic animals were identified by Southern blot analysis of tail DNA digested with EcoRI, using as a probe a 2-kb EcoRV-EcoRI fragment of lacZ (Fig. 1) labeled with [γ-32P]dCTP by random priming, according to our standard procedures [20]. The probe detects a 3-kb band corresponding with full length lacZ cDNA.
Diagram of constructs for generating transgenic mice. The 8.2 kb, 4.8 kb, and 1.3 kb of Ccna1 flanking region and 0.8 kb of Ccna1 structural gene were fused to lacZ and the polyadenylation signal and intron 1 of protamine1. Solid black lines represent Ccna1 flanking sequence or intron 1. Open boxes represent Ccna1 exon sequence. Exon 2 is fused with lacZ, which is represented by the boxes with black dots. Boxes with vertical lines represent the Prm1 polyadenylation signal and intron 1. The boxes with downward diagonal stripes and with upward diagonal stripes, depicted below the cartoon of the constructs, represent the regions of lacZ sequence used as probe templates for genotyping or for Northern and in situ hybridization analysis, respectively. S, SalI; R, EcoRI; X, XhoI; B, BamHI; V, EcoRV
Tissue Staining and Histology
The X-gal staining procedure was similar to that of Behringer et al. [20]. Briefly, mice were killed by cervical dislocation or CO2 asphyxiation and perfused transcardially with fixative (0.2% glutaraldehyde, 2% formaldehyde, 5 mM EGTA, 0.1% deoxycholic acid, 0.2% Nonidet P-40, 2 mM MgCl2, and 0.1 M phosphate buffer, pH 7.3). The tissues were dissected out immediately (the testes were decapsulated and the tubules were gently teased apart) and soaked in fixative for 1 h at room temperature. After thorough washing in rinse buffer (0.1 M phosphate buffer, pH 7.3, 0.1% deoxycholic acid), the tissues were stained overnight at 30°C in X-gal solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2). After staining, the tissues were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. Intact tubules were viewed and photographed with a Wild MPS 51 Dissecting Microscope (Wild Heerbrugg Ltd., Heerbrugg, Switzerland). For histological study, the stained tissues were dehydrated and embedded in paraffin. Five-micrometer-thick sections were deparaffinized with Histo-Clear (National Diagnostics, Atlanta, GA) and counterstained with neutral red. The sections were photographed using a Nikon Eclipse 800 microscope (Nikon Instrument Group, Melville, NY) with a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Northern Blot Hybridization Analysis
Total RNA was isolated from tissues dissected from euthanized adult mice using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) or an RNeasy kit (Qiagen, Chatsworth, CA) following the manufacturer’s instructions. Twenty-microgram samples of denatured total RNA were resolved on a 0.8% denaturing agarose gel according to our published procedures [10]. Ethidium bromide staining of the 18S and 28S rRNA bands was used to determine equal sample loading. The gels were blotted onto nitrocellulose membranes. The 32P-labeled antisense cRNA probe was 1 kb and corresponded to the 5′ end of the lacZ cDNA (Fig. 1). The membranes were hybridized at 65°C overnight using 107 cpm/ml of lacZ cRNA in hybridization solution (5× SSC, 20 mM sodium phosphate buffer [pH 7], 60% formamide, 1% SDS, 5× Denhardt solution, 100 μg/ml salmon sperm RNA, and 7% dextran sulfate). The membranes were washed with increasing stringency in solutions of 0.2× SSC and 1% SDS. The hybridization signal was detected using XAR 5 film (Eastman Kodak Co., Rochester, NY).
In Situ Hybridization Analysis
Testes and selected other tissues were dissected and fixed in 4% paraformaldehyde in PBS overnight at 4°C and then dehydrated prior to paraffin embedding. Six-micrometer sections were cut. Paraffin was removed using Histo-Clear and the sections were rehydrated. Sense and antisense cRNA probes were transcribed from template consisting of the 5′ 1 kb of lacZ cDNA (Fig. 1) using [35S]UTP as radiolabel. Hybridization was carried out overnight at 65°C in 50% formamide, 0.3 M NaCl, 20 mM Tris pH 8.0, 5 mM EDTA, 10 mM sodium phosphate, pH 8.0, 10% dextran sulfate, 1× Denhardt solution, 500 μg/ml yeast tRNA, and 1 × 104 cpm/ml probe. After hybridization, the slides were washed in 5× SSC for 10 min at 50°C and then in 80% formamide and 2× SSC for 20 min at 65°C. Single-stranded RNA was digested in 0.3 M NaCl, 10 mM Tris, pH 8.0, 5 mM EDTA, and 50 μg/ml RNase A for 30 min at 37°C. The RNase-treated slides were washed with 50% formamide and 2× SSC for 20 min at 65°C and further rinsed with 2× SSC and with 0.1× SSC each for 15 min at room temperature. The slides were dehydrated, coated with NTB-2 emulsion (Eastman Kodak Co.) and exposed for 2 wk at 4°C. The slides were developed and stained with hematoxylin and eosin. Photomicrographs were taken with a DIALUX 20 microscope (Leica Microsystems, Wetzlar, Germany) using epifluorescence optics and a SPOT digital camera.
Results
Generation of cyA1lacZ Transgenic Mice
To begin to identify sequences responsible for the highly restricted pattern of mouse Ccna1 expression, constructs composed of varying lengths of Ccna1 5′ genomic sequence linked to lacZ cDNA were prepared and used to generate transgenic mice. The transgenes were constructed with mouse genomic fragments consisting of 8.2, 4.8, or 1.3 kb of Ccna1 5′ flanking sequence and the 5′ end of the Ccna1 structural gene to exon 2 fused in-frame to a reporter cassette consisting of lacZ and the 3′ UTR and intron 1 of the testis-specific gene protamine1, yielding 8.2cyA1lacZ, 4.8cyA1lacZ, and 1.3cyA1lacZ, respectively (Fig. 1). Two permanent mouse lines carrying 8.2cyA1lacZ, two permanent mouse lines carrying 1.3cyA1lacZ, and one permanent mouse line carrying 4.8cyA1lacZ were established. Each of the five lines was characterized further.
Distribution of cyA1lacZ Transgene Expression in Adult Tissues
To establish the tissue specificity of expression of the transgenes, RNA was isolated from a variety of tissues from adult transgenic mice and analyzed by Northern blot hybridization analysis. For all lines, a lacZ transcript was detected in RNA from testis only (Fig. 2, A–C; and data not shown), similar to endogenous Ccna1. This result was confirmed by in situ hybridization analysis of selected tissues from each line using a lacZ probe (data not shown). These results suggested that the testis specificity of expression of Ccna1 is conferred by sequences within 1.3 kb upstream of its putative transcriptional start.
Northern blot hybridization analysis of adult tissues from transgenic mice. Total RNA from 8.2cyA1lacZ, 4.8cyA1lacZ, or 1.3cyA1lacZ transgenic tissues was hybridized with a radiolabeled lacZ probe. The top lanes in each panel represent the pattern of the lacZ hybridization signal, while the bottom lanes show the 18s rRNA ethidium bromide staining pattern to assess equal sample loading. Results from (A) 8.2cyA1lacZ, (B) 4.8cyA1lacZ, and (C) 1.3cyA1lacZ transgenic lines are shown
Pattern of β-Galactosidase Activity in cyA1lacZ Testes
To determine the cellular specificity of expression of the cyA1lacZ transgenes, testes were stained with X-gal as whole mounts and then sectioned. Visualization of X-gal staining in intact tubules of 8.2cyA1lacZ testis (Fig. 3A) and 4.8cyA1lacZ testis (Fig. 3B) were identical and revealed dark blue staining except at the edges of the tubules, which appeared unstained. This suggested that the cells closest to the basement membrane of the tubules, the spermatogonia and early meiotic spermatocytes, did not express the transgenes. In contrast, tubules from wild-type testis were unstained, although nonspecific staining appeared in interstitial cells, which are located between tubules (inset of Fig. 3A). Histological sections of the X-gal-stained tubules from 8.2cyA1lacZ testis (Fig. 3, D, G, and J) and 4.8cyA1lacZ testis (Fig. 3, E, H, and K) confirmed this observation. No staining was observed in spermatogonia or in spermatocytes through stage VIII (Fig. 3, D and E). Rather, the onset of expression appeared in stage-IX spermatocytes (Fig. 3, G and H), similar to the appearance of endogenous Ccna1 mRNA and protein [8]. X-gal staining was present in spermatocytes at later stages, including stage XII (Fig. 3, J and K), and persisted in all round and elongating spermatids (Fig. 3, D, E, G, H, J, and K), cells known not to express cyclin A1 (see Results). This staining pattern suggested that the transgenes contained sequences required to direct the onset of expression of lacZ to stage-IX spermatocytes.
Expression of the lacZ reporter gene in representative testis tubules and histological sections from transgenic mice. X-gal-stained cells appear blue. A, D, G, J) 8.2cyA1lacZ testis, (B, E, H, K) 4.8cyA1lacZ, (C, F, I, L) 1.3cyA1lacZ testis, and (the inset in A) wild-type testis. A–C) Whole mount at ×12 magnification. D–F) Stage-VIII tubule at ×40. G–I) Stage-IX tubule at ×40. J–L) Stage-XII tubule at ×40
Examination of stained, intact testicular tubules from the 1.3cyA1lacZ mice revealed a less uniform X-gal staining pattern (Fig. 3C) as compared with that of 8.2cyA1lacZ (Fig. 3A) or 4.8cyA1lacZ mice (Fig. 3B). Unlike the consistent appearance of X-gal staining in late spermatocytes and spermatids in 8.2cyA1lacZ and 4.8cyA1lacZ testis, the staining of spermatocytes and spermatids within tubules of 1.3cyA1lacZ testis was sometimes absent or was of varying intensity (Fig. 3, C, F, and L). However, the cellular specificity of expression exhibited was similar to 8.2cyA1lacZ and 4.8cyA1lacZ lines. First, the onset of expression of the transgenes was similar. Examination of sectioned tubules revealed that staining was absent in spermatogonia and in spermatocytes through stage VIII (Fig. 3F), and β-gal activity first appeared in stage-IX spermatocytes (Fig. 3I). Also, β-gal activity persisted in the spermatids in the 1.3cyA1lacZ transgenic testis (Fig. 3, F, I, and L). These observations suggest that the Ccna1 genomic fragment within −1.3 kb to +0.8 kb from its transcriptional start contains sequences needed to direct the onset of expression to stage-IX spermatocytes, but that additional upstream sequences from −4.8 kb and −1.3 kb of the Ccna1 transcriptional start site were needed to direct detectable expression of 1.3cyA1lacZ in all spermatocytes. In support of these observations, Northern blot analysis of total RNA from adult testis of mice carrying each construct shows that 1.3cyA1lacZ is expressed at lower levels than 4.8cyA1lacZ or 8.2cyA1lacZ (Fig. 4).
Northern blot hybridization analysis of adult testes from transgenic mice. Total RNA from wild-type (wt), 1.3cyA1lacZ, 4.8cyA1lacZ, and 8.2cyA1lacZ testes was hybridized with a radiolabeled lacZ probe. The top lanes represent the pattern of the lacZ hybridization signal, while the bottom lanes show the 18s rRNA ethidium bromide staining pattern
The detection of β-gal activity in spermatids may have been due to expression of the transgenes at stages later than that of endogenous Ccna1. Alternatively, the lacZ reporter may have been expressed at the same developmental stages as Ccna1, but its gene product may have persisted in spermatids. To determine if ectopic expression of the transgene was responsible for the β-gal activity detected in spermatids in cyA1lacZ testes, lacZ expression was assayed by in situ hybridization. LacZ mRNA was detected in spermatocytes as late as stage XII (Fig. 5A), but was not detected in spermatids at stage I (Fig. 5A) or in round or elongating spermatids at later developmental stages (Fig. 5, A–5D). Each of the cyA1lacZ constructs exhibited this same pattern of expression (Fig. 5, and data not shown). The localization of lacZ mRNA only in spermatocytes suggested that the β-gal activity detected in spermatids was not produced by ectopic expression of the transgenes, but rather to stable β-gal protein. This further suggested that sequences within −1.3 kb to +0.8 kb from the mouse Ccna1 transcriptional start site, unlike its human homologue, were sufficient to control the correct timing of spermatocyte-specific expression of the transgenes in germ cells.
In situ hybridization analysis in representative histological sections from transgenic testes. Testes sections were hybridized with antisense lacZ probe (A–D) or sense lacZ probe (E, F). Green-colored speckled areas represent the positive signal of reflected fluorescent light off autoradiographic silver grains. A, B) 8.2cyA1lacZ testis sections at ×40 magnification, (C–E) 1.3cyA1lacZ testis sections at ×40, and (F) a nontransgenic testis section at ×40
A curiosity of the expression of these transgenes in the testis was noted in our in situ hybridization analysis when a sense lacZ probe was used as a control. Signal was detected in stage VI–VIII spermatocytes in transgenic testis sections for all three constructs (Fig. 5E, and data not shown). This signal was not present in sections from nontransgenic testis (Fig. 5F). This expression might result from activity of a cryptic promoter driving transcription in the opposite orientation in the protamine1 sequence placed 3′ to lacZ in each transgene. However, promoter elements that support transcriptional initiation, as assessed by TFSearch [22], were not detected in this sequence.
Consensus Elements for Transcriptional Regulators Located Between −4.8 kb and +0.8 kb of the Ccna1 Putative Transcriptional Start Site
Intron 1 and the region between −4.8 kb and the putative transcriptional start site of the Ccna1 gene were sequenced and scanned for putative transcription factor binding sites using the search engine TFSEARCH and the TRANSFAC database [22]. The locations of consensus sequences of DNA binding motifs for known transcription factors and for binding activities are listed in Table 1. Among these motifs are binding sites for transcription factors that are coexpressed in spermatocytes with Ccna1, such as A-myb [23], Hsf2 [24], and Sp1 [25]. Consensus sequences for A-myb and Hsf2 binding are located between −4.8 kb and −1.3 kb of the putative Ccna1 transcriptional start site, and A-myb and Sp1 consensus sequences located within −1.3 kb of the putative Ccna1 transcriptional start site (Table 1).
Some of the transcription factors whose consensus DNA binding motifs are found within 4.8 kb of the mouse Ccnal putative transcriptional start site or in intron 1.
| Transcription factor | Consensus sequencea | Locationb |
|---|---|---|
| SRY | AAAC(A/T)A(/C) | −4292, −4784, −4777, −4371, −3195 |
| −3156, −3125, −1 727, +583 | ||
| GATA-1 | (G/C)NNGATNNNN | −4597, −1336, −979, −574, −220, +692 |
| Sox-5 | NNAACAATNN | −3386, −301 1, −2598, +435 |
| c-Mybc | NNNAAC(G/T)G(G/C)C | −2069 |
| YY1 | NNNNNCCATNT(A/T)NNN(A/T)N | −1460 |
| HSF2 | NGAANN(A/T)C(G/T) | −1314, −913 |
| USF | NCACGTGN | −1031, −861 |
| AML-1a | TGCGGT | −319, −309, −278 |
| CDE | (G/C)GCGG | −255 |
| Sp1 | G(A/G)GGC(G/T)GGG(A/T) | −247 |
| delta EF1 | NNNCACCTNAN | −201 |
| CDE/CHR | (G/C)GCGG/TGGAA | −153/−142, −89/−79 |
| AP-1 | NTGA(C/G)TCAG | −102 |
| CHR | TGGAA | −26 |
| Transcription factor | Consensus sequencea | Locationb |
|---|---|---|
| SRY | AAAC(A/T)A(/C) | −4292, −4784, −4777, −4371, −3195 |
| −3156, −3125, −1 727, +583 | ||
| GATA-1 | (G/C)NNGATNNNN | −4597, −1336, −979, −574, −220, +692 |
| Sox-5 | NNAACAATNN | −3386, −301 1, −2598, +435 |
| c-Mybc | NNNAAC(G/T)G(G/C)C | −2069 |
| YY1 | NNNNNCCATNT(A/T)NNN(A/T)N | −1460 |
| HSF2 | NGAANN(A/T)C(G/T) | −1314, −913 |
| USF | NCACGTGN | −1031, −861 |
| AML-1a | TGCGGT | −319, −309, −278 |
| CDE | (G/C)GCGG | −255 |
| Sp1 | G(A/G)GGC(G/T)GGG(A/T) | −247 |
| delta EF1 | NNNCACCTNAN | −201 |
| CDE/CHR | (G/C)GCGG/TGGAA | −153/−142, −89/−79 |
| AP-1 | NTGA(C/G)TCAG | −102 |
| CHR | TGGAA | −26 |
Consensus sequences are those listed in the TRANSFAC database [22] or, for CDE and CHR elements, were listed in Zwicker et al. [28].
Locations of motifs are as they appear 5′ to 3′ on the sense strand, regardless of orientation, and fit the consensus sequence exactly or differ by one base.
The c-Myb consensus sequence that is listed in the database is identical to that for A-myb and B-myb.
Some of the transcription factors whose consensus DNA binding motifs are found within 4.8 kb of the mouse Ccnal putative transcriptional start site or in intron 1.
| Transcription factor | Consensus sequencea | Locationb |
|---|---|---|
| SRY | AAAC(A/T)A(/C) | −4292, −4784, −4777, −4371, −3195 |
| −3156, −3125, −1 727, +583 | ||
| GATA-1 | (G/C)NNGATNNNN | −4597, −1336, −979, −574, −220, +692 |
| Sox-5 | NNAACAATNN | −3386, −301 1, −2598, +435 |
| c-Mybc | NNNAAC(G/T)G(G/C)C | −2069 |
| YY1 | NNNNNCCATNT(A/T)NNN(A/T)N | −1460 |
| HSF2 | NGAANN(A/T)C(G/T) | −1314, −913 |
| USF | NCACGTGN | −1031, −861 |
| AML-1a | TGCGGT | −319, −309, −278 |
| CDE | (G/C)GCGG | −255 |
| Sp1 | G(A/G)GGC(G/T)GGG(A/T) | −247 |
| delta EF1 | NNNCACCTNAN | −201 |
| CDE/CHR | (G/C)GCGG/TGGAA | −153/−142, −89/−79 |
| AP-1 | NTGA(C/G)TCAG | −102 |
| CHR | TGGAA | −26 |
| Transcription factor | Consensus sequencea | Locationb |
|---|---|---|
| SRY | AAAC(A/T)A(/C) | −4292, −4784, −4777, −4371, −3195 |
| −3156, −3125, −1 727, +583 | ||
| GATA-1 | (G/C)NNGATNNNN | −4597, −1336, −979, −574, −220, +692 |
| Sox-5 | NNAACAATNN | −3386, −301 1, −2598, +435 |
| c-Mybc | NNNAAC(G/T)G(G/C)C | −2069 |
| YY1 | NNNNNCCATNT(A/T)NNN(A/T)N | −1460 |
| HSF2 | NGAANN(A/T)C(G/T) | −1314, −913 |
| USF | NCACGTGN | −1031, −861 |
| AML-1a | TGCGGT | −319, −309, −278 |
| CDE | (G/C)GCGG | −255 |
| Sp1 | G(A/G)GGC(G/T)GGG(A/T) | −247 |
| delta EF1 | NNNCACCTNAN | −201 |
| CDE/CHR | (G/C)GCGG/TGGAA | −153/−142, −89/−79 |
| AP-1 | NTGA(C/G)TCAG | −102 |
| CHR | TGGAA | −26 |
Consensus sequences are those listed in the TRANSFAC database [22] or, for CDE and CHR elements, were listed in Zwicker et al. [28].
Locations of motifs are as they appear 5′ to 3′ on the sense strand, regardless of orientation, and fit the consensus sequence exactly or differ by one base.
The c-Myb consensus sequence that is listed in the database is identical to that for A-myb and B-myb.
The proximal promoter of Ccna1 was aligned with the corresponding rat and human promoters. A region of high homology was found from −511 to −448 of the putative Ccna1 start site (Fig. 6A), although this region lacked consensus sequences for transcription factor binding sites listed in the TRANSFAC database [22]. However, highly conserved consensus sequences for Sp1, GATA-1, Ap-1, and delta EF1 binding sites, and a CCAAT box were identified (Table 1; Fig. 6B) [22]. Downstream of these consensus sequences and 153 bp upstream of the putative transcriptional start site of the Ccna1 gene, the first of two bipartite cell-cycle dependent element/cell-cycle genes homology region (CDE/CHR) elements were found (Table 1; Fig. 6B). In the mouse and rat promoters, there are two conserved sets of putative CDE/CHR elements. In the human promoter, the 5′ set and only the CDE sequence of the 3′ set are found; that is, the human promoter lacks the CHR sequence of the 3′ CDE/CHR present in the mouse and rat promoters. In addition, unpaired CDE and CHR elements are present, which are conserved among all three cyclin A1 genes (Fig. 6B).
Alignment of the nucleotide sequence of the 5′ flanking regions of mouse Ccna1 (mCcna1), rat Ccna1 (rCcna1), and human CCNA1 (hCCNA1). The proximal promoter sequence of mCcna1 located (A) from −517 to −438 and (B) from −266 to +53 of the putative transcriptional start site is compared with the corresponding regions in rCcna1 and hCCNA1. Numbers indicate the position of the nucleotides of mCcna1 sequence relative to the putative transcription start site, which is indicated by an upward bending arrow. Nucleotides identical to mouse sequence are dark shaded. Nucleotide differences relative to mouse sequence are light shaded, and insertions or deletions are unshaded. The region between the solid arrowheads is a highly conserved sequence. Consensus sequences for regulatory elements in mCcna1 are labeled above the sequence. GC boxes previously identified in the human sequence are underlined
In the human proximal promoter, there are sequences that are poorly conserved or not conserved at all with mouse and rat, for example, several GC boxes (Fig. 6B). These sequences, in the context of a human CCNA1 reporter construct, have been suggested to contribute to transcriptional activation in HeLa cells [16].
Discussion
In mice, the Ccna1 gene is expressed specifically in testis in stage-IX to -XII spermatocytes [8]. Human CCNA1 is reported to be expressed at highest levels in male germ cells [15] and at very low levels in brain and hematopoietic cells of normal tissues [12, 13, 26]. Other sites of expression of mouse Ccna1 are yet to be confirmed. The tissue specificity of Ccna1 expression contrasts with that of Ccna2, which is expressed in a variety of tissues [8, 27]. Ccna1 and Ccna2 also have nonoverlapping expression patterns during male germ cell development, as Ccna2 expression is downregulated early in the meiotic cell cycle before Ccna1 is expressed [8, 9]. We therefore hypothesize that there will be specific regulatory elements unique to each A-type cyclin as well. The essential role of Ccna1 in male germ cell development and the concurrent expression of cyclin A1 message and protein suggested the importance of understanding mechanisms controlling transcription of the Ccna1 gene. The lack of cell lines derived from male germ cells made it necessary to analyze the Ccna1 promoter in these cells in vivo in transgenic mice. The use of serial deletions of Ccna1 upstream sequence has allowed us to define several functional segments of the promoter.
Analysis of transgene expression in the testis, using a combination of X-gal staining and in situ hybridization, revealed their expression specifically in spermatocytes at stages IX–XII of the cycle of the seminiferous epithelium, and not in earlier stages. The endogenous mouse Ccna1 has been shown to be expressed only during this same narrow window of spermatogenesis. This suggests that the genomic fragment of mouse Ccna1 spanning −1.3 kb to +0.8 kb of the putative transcriptional start site contains sequences necessary for the stage-specific expression of Ccna1 in the meiotic cell cycle. In contrast, 1.3 kb of human CCNA1 promoter directs expression of EGFP in a much less restrictive pattern in male germ cells [19], reflecting differences in the mouse and human promoters despite their sharing highly conserved regions.
Within this fragment are consensus sequences for two sets of paired CDE/CHR elements. These elements were first discovered in the proximal promoter of genes for human cyclin A2, Cdc2, and Cdc25C, which are expressed in S and G2 phases of the mitotic cell cycle [28]. Several lines of evidence suggest that the CDE/CHR elements control the timing of expression of these genes during the cell cycle. In vivo footprinting of the human CCNA2 proximal promoter revealed that the bipartite element was occupied at stages that the gene was not transcribed [28]. Mutation of the CDE/CHR element in the context of CCNA2 or CDK1 promoter/reporter genes caused derepression of the reporters in G1 phase [28]. These elements have now been shown to be present and involved in controlling the timing of expression of other cell-cycle-regulated genes, including the genes for cyclin B2 [29], rabkinesin6 [30], pololike kinase [31], p130 [32], m-survivin [33], and aurora A [34]. Also, the CDE/CHR element appears to downregulate expression of the CDK1 gene in response to TPA-induced differentiation of U937 cells [35] or in response to p53-dependent DNA damage [36]. The promoters of the genes for mouse and rat cyclin A1 are unique in that they contain two sets of CDE/CHR elements, and like the human gene, also have unpaired CDE and CHR consensus sequences. There have been no previous reports of unpaired CDE elements, but the promoters of the genes for human cyclin B2 [37] and mouse Cdc25C [38] are regulated by CHR elements that are not paired with functional CDE elements. Testing of mutated CDE/CHR elements in transgenic mice will determine the role, if any, of these elements in controlling the expression of Ccna1 in the meiotic cell cycle. Factors that bind CDE/CHR [39] or CHR [40, 41] have been detected in various cultured cell lines, but have not been identified.
The less efficient X-gal staining in male germ cells from both lines of 1.3cyA1lacZ transgenic mice, as compared with germ cells from 8.2cyA1lacZ and 4.8cyA1lacZ transgenic mice, suggests that sequences that lie between 4.8 kb and 1.3 kb upstream of the putative Ccna1 transcriptional start contains enhancer elements needed for consistent expression of lacZ. Within this region, consensus elements for several regulatory factors expressed in spermatocytes have been identified (Table 1). Hsf2 [24] and A-myb [23] are reported to be expressed in mid- to late-stage spermatocytes, raising the possibility that they may be involved in the regulation of expression of Ccna1. Interestingly, disruption of A-myb in mice causes spermatogenic arrest during midprophase and male sterility [42]. Developmentally, arrest in A-myb−/− spermatocytes occurs slightly earlier than the arrest in Ccna1−/− spermatocytes.
In summary, our results suggest that the genomic fragment −1.3 kb to +0.8 kb from the putative Ccna1 transcriptional start contains sequences that mediate the correct developmental expression of Ccna1 in male germ cells, as compared with this region in the human CCNA1 gene. Further, sequences located between −4.8 kb and −1.3 kb from the putative Ccna1 transcriptional start site appear to contribute to enhanced expression at theses stages. Finally, sequences that lie between −1.3 kb and +0.8 kb from the Ccna1 transcriptional start appear to be sufficient to maintain repression of Ccna1 in the correct adult tissues and stages of spermatogenic differentiation. Additional deletions in the −1.3 to +0.8 region may identify the location of elements that repress transcription of Ccna1 in other cell types while mutation of known transcriptional regulatory elements and/or the CDE-CHR elements may shed light on enhancer and cell-cycle regulatory roles, respectively. Currently, the Ccna1 promoter we have characterized will provide a useful tool to direct the expression of genes specific to late meiotic spermatocytes in vivo.
Acknowledgments
We are very grateful to Dr. Xiangyuan Wang for performing microinjection and producing histological sections and to Ms. Stacey Baptiste for staining 4.8cyA1lacZ testes.
References
Author notes
Supported by NIH grants HD34915 (D.J.W) and T32 DK07647 (K.M.L.).
