Distinct Regions of the Mouse Cyclin A1 Gene, Ccna1, Confer Male Germ-Cell Specific

Expression and Enhancer Function1

Expression of Ccna1 Reporter Genes in Mice

cyclin, spermatogenesis, meiosis, gene regulation, testis

Karen M. Lele2 and Debra J. Wolgemuth2,3,4,5,6,7

1This research was supported by NIH grants HD34915 (D.J.W) and T32 DK07647

(K.M.L.)

2The Institute of Human Nutrition, Departments of 3Genetics and Development and

4Obstetrics and Gynecology, 5The Center for Reproductive Sciences, 6The Herbert Irving

Comprehensive Cancer Center, Columbia University Medical Center, New York, New

York 10032

7Correspondence: Dr. Debra Wolgemuth, Dept. Genetics and Development, Columbia

University Medical Center, 630 West 168th Street, Black 1613, New York, New York

10032; FAX: 212 305 6084; e-mail: djw3@columbia.edu

BOR Papers in Press. Published on June 23, 2004 as DOI:10.1095/biolreprod.104.030387

Copyright 2004 by The Society for the Study of Reproduction.

ABSTRACT 1

The gene encoding mouse cyclin A1, Ccna1, is expressed at highest levels in late 2

pachytene-diplotene spermatocytes, where it is required for meiotic cell division. To 3

begin to understand the mechanisms responsible for its highly restricted pattern of 4

expression, transgenic mouse lines carrying constructs consisting of the cyclin A1 5

regulatory region fused with the reporter gene lacZ were generated. Analysis of tissue-6

specific and testicular cell type-specific transgene expression indicated that sequences 7

within -1.3 kb of the cyclin A1 putative transcriptional start site were sufficient to direct 8

transgene expression uniquely to late spermatocytes while maintaining repression in other 9

tissues. However, sequences located between -4.8 kb and -1.3 kb of the putative 10

transcriptional start site were apparently required to transcribe the reporter at levels 11

needed for consistent X-gal staining. Comparison of the mouse, rat and human proximal 12

promoters revealed regions of high sequence conservation and consensus sequences both 13

for known transcripton factors, some of which are co-expressed with Ccna1, such as A-14

myb and Hsf2, and for elements that control expression of genes in somatic cell cycles, 15

such as CDE, CHR, and CCAAT elements. Thus, the promoter region within 1.3 kb 16

upstream of the putative Ccna1 transcriptional start can direct expression of lacZ to 17

spermatocytes, while sequences located between -4.8 kb and -1.3 kb of the putative 18

transcriptional start site may enhance expression of lacZ. 19

20

2

INTRODUCTION 20

Spermatogenesis is an ordered process during which germ cells enter successive 21

mitotic, meiotic, and post-meiotic phases. The program of gene expression required for 22

this process is tightly controlled, involving both germ cell-specific and common 23

transcription factors, whose expression is also stringently regulated (rev. in [1, 2]). This 24

regulation allows precisely ordered events to occur such as genetic recombination and 25

meiotic cell division, followed by the morphological development of spermatids. 26

Genes for several key somatic cell cycle regulators, including cyclins, cyclin-27

dependent kinases (Cdks), Cdk inhibitors, and Cdc25 family members are expressed 28

during spermatogenesis in specific patterns that suggest their function in cell cycle 29

control in the germ line as well [3, 4]. Cyclins are regulatory subunits of the Cdk 30

complexes and are expressed periodically during the cell cycle. There are at least ten 31

classes of cyclins in higher vertebrates, designated cyclins A to I and T, and multiple 32

members of the A-, B-, and D-type cyclin families (rev. in [5-7]). 33

In higher organisms, there are two A-type cyclins, cyclin A1 and cyclin A2. In 34

mice, both are expressed in male germ cells, albeit during quite different stages of 35

differentiation, suggesting that they are tightly regulated and may have distinct functions 36

[8, 9]. The cyclin A1 gene, Ccna1, appears to be testis-specific and is expressed late in 37

the meiotic cell cycle, just prior to chromosomal desynapsis [8, 9]. Disruption of mouse 38

Ccna1 resulted in male infertility and complete spermatogenic arrest prior to the first 39

meiotic division [10]. In contrast, cyclin A2 (Ccna2) is widely expressed in mouse 40

tissues [8]. In the male germ line, Ccna2 is expressed earlier than Ccna1, in 41

spermatogonia and preleptotene spermatocytes, and is no longer detected as germ cells 42

3

enter the leptotene stage [9]. Disruption of the Ccna2 gene resulted in embryonic 43

lethality [11]. Therefore, its role in spermatogenesis could not be discerned from a null 44

model. 45

Human CCNA1 is expressed most highly in testis, and at lower levels in adult 46

brain and in hematopoietic cells [12, 13]. It is also expressed in several human myeloid 47

leukemia cell lines and an osteosarcoma cell line, where its level of expression appears to 48

be cell cycle-regulated [12, 14]. In testis, human cyclin A1 is localized in late meiotic 49

prophase spermatocytes, similarly to mouse cyclin A1 [15]. A short fragment of the 50

human CCNA1 promoter, comprised of 190 bp of sequence upstream and 145 bp of 51

sequence downstream of the transcriptional start site, has been reported to activate 52

expression of a reporter gene in Hela cells [16] and CV-1 cells [17]. In transgenic mice, 53

a 1.3 kb fragment of the human CCNA1 promoter has further been reported to direct 54

expression of EGFP to male germ cells but in a much less restricted expression pattern 55

than observed for the endogenous mouse gene [18]. That is, in addition to stage IX to 56

XII spermatocytes, EGFP fluorescence was detected in spermatogonia, at earlier stages of 57

spermatocyte development and in spermatids [18, 19]. 58

The DNA regulatory elements required for mouse Ccna1 gene expression in male 59

germ cells have not been identified. We hypothesize that there will be regulatory 60

elements unique to Ccna1, not present in the Ccna2 promoter, reflecting their distinct 61

expression patterns, and further that such elements might be evolutionarily conserved. 62

To begin to define the location of these elements and, thus, begin to understand 63

mechanisms that control the stage-specific expression of Ccna1 and repression at other 64

stages of male germ cell development, we have tested expression of Ccna1 65

4

promoter/reporter constructs in mice. This in vivo approach was chosen to define 66

promoter function in male germ cells since procedures for transfection of these cells in 67

culture have not been established. In the present study, the regulatory function of 68

genomic fragments spanning –8.2 kb to +0.8 kb from the putative transcriptional start site 69

of mouse Ccna1 was examined in transgenic mice, using the reporter lacZ. The patterns 70

of transgene expression revealed that the genomic fragment that lies between –1.3 kb to 71

+0.8 kb of the putative Ccna1 transcriptional start site could fully recapitulate the correct 72

developmental expression of the endogenous Ccna1 gene in male germ cells with no 73

ectopic expression observed, unlike the human CCNA1 reporter constructs. However, 74

sequences between –4.8 to –1.3 of the putative transcriptional start appeared to be 75

necessary for fully penetrant expression of the gene in spermatocytes. Within this 76

genomic region, consensus binding sequences for several transcription factors were 77

identified. They include sequences known to bind factors that are co-expressed with 78

Ccna1 and also sequences that control cell cycle regulated gene expression in somatic 79

cell cycles. Alignment of the proximal promoters of the mouse, rat, and human genes for 80

cyclin A1 revealed regions of high homology; however, the human proximal promoter 81

also contained inserted sequences that were not present in the mouse and rat promoters. 82

83

MATERIALS AND METHODS 84

Transgene constructs and transgenic mice 85

A 5.6 kb fragment that extends 5’ from the BamHI site in exon 2 of Ccna1 to an 86

EcoRI site in the 5’ flanking region was inserted into a vector containing a cassette 87

composed of lacZ and intron 1 and the polyadenylation signal of mouse protamine-1 to 88

5

yield construct 4.8cyA1lacZ (Fig. 1). The reporter cassette had been used previously to 89

produce a Hoxa4 promoter-reporter construct that was expressed in male germ cells [20]. 90

8.2cyA1lacZ was generated from 4.8cyA1lacZ by inserting a SalI-EcoRI fragment 5’ of 91

the EcoRI site. 1.3cyA1lacZ was produced from 4.8cyA1lacZ by digestion with XhoI 92

and religation. All constructs were verified by sequencing. The fusion gene product is 93

expected to have the first 33 amino acids of the cyclin A1 protein linked to β-94

galactosidase (β-gal). The production of transgenic mice was carried out as previously 95

described [20, 21]. All procedures were performed in accord with guidelines of the 96

Institutional Animal Care and Use Committee of the Columbia University Medical 97

Center. Transgenic animals were identified by Southern blot analysis of tail DNA 98

digested with EcoRI, using as a probe a 2 kb EcoRV-EcoRI fragment of lacZ (Fig. 1) 99

labeled with [γ-32P]dCTP by random priming, according to our standard procedures [20]. 100

The probe detects a 3 kb band corresponding to full length lacZ cDNA. 101

102 Tissue staining and histology 103

The X-gal staining procedure was similar to that of Behringer et al. [20]. Briefly, 104

mice were sacrificed by cervical dislocation or CO2 asphyxiation and perfused 105

transcardially with fixative (0.2% glutaraldehyde, 2% formaldehyde, 5 mM EGTA, 0.1% 106

deoxycholic acid, 0.2% Nonidet P-40, 2 mM MgCl2, and 0.1 M phosphate buffer pH 107

7.3). The tissues were dissected out immediately (the testes were decapsulated and the 108

tubules were gently teased apart), and soaked in fixative for 1 hr at room temperature. 109

After thorough washing in rinse buffer (0.1 M Phosphate buffer pH 7.3, 0.1% 110

deoxycholic acid), the tissues were stained overnight at 30°C in X-gal solution (5 mM 111

potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2). After staining, the 112

6

tissues were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.3. 113

Intact tubules were viewed and photographed with a Wild MPS 51 Dissecting 114

Microscope (Wild Heerbrugg Ltd., Heerbrugg, Switzerland). For histological study, the 115

stained tissues were dehydrated and embedded in paraffin. Five-µm-thick sections were 116

deparaffinized with Histo-Clear (National Diagnostics, Atlanta, GA) and counterstained 117

with neutral red. The sections were photographed using a Nikon Eclipse 800 microscope 118

(Nikon Instrument Group, Melville, NY) with a SPOT digital camera (Diagnostic 119

Instruments, Inc., Sterling Heights, MI). 120

121

Northern blot hybridization analysis 122

Total RNA was isolated from tissues dissected from euthanized adult mice using 123

TriReagent (Molecular Research Center, Inc., Cincinnati, OH) or an RNeasy kit (Qiagen, 124

Chatsworth, CA) following the manufacturer’s instructions. Twenty µg samples of 125

denatured total RNA were resolved on a 0.8% denaturing agarose gel according to our 126

published procedures [10]. Ethidium bromide staining of the 18S and 28S rRNA bands 127

was used to determine equal sample loading. The gels were blotted onto nitrocellulose 128

membranes. The 32P-labeled antisense cRNA probe was 1 kb and corresponded to the 129

5’end of the lacZ cDNA (Fig. 1). The membranes were hybridized at 65°C overnight 130

using 107 cpm/ml of lacZ cRNA in hybridization solution (5x SSC, 20 mM sodium 131

phosphate buffer (pH 7), 60% formamide, 1% SDS, 5x Denhardt’s solution, 100 ug/ml 132

salmon sperm RNA, and 7% dextran sulfate). The membranes were washed with 133

increasing stringency in solutions of 0.2X SSC and 1% SDS. The hybridization signal 134

was detected using XAR 5 film (Eastman Kodak Co., Rochester, NY). 135

7

136 In situ hybridization analysis 137

Testes and selected other tissues were dissected and fixed in 4% 138

paraformaldehyde in PBS overnight at 4°C and then dehydrated prior to paraffin 139

embedding. Six µm sections were cut. Paraffin was removed using Histo-Clear and the 140

sections were rehydrated. Sense and antisense cRNA probes were transcribed from 141

template consisting of the 5’ 1 kb of lacZ cDNA (Fig. 1) using [35S]UTP as radiolabel. 142

Hybridization was carried out overnight at 65°C in 50% formamide, 0.3M NaCl, 20 mM 143

Tris pH 8.0, 5 mM EDTA, 10 mM sodium phosphate pH 8.0, 10% dextran sulfate, 1x 144

Denhardt’s solution, 500 µg/ml yeast tRNA and 1 x 104 cpm/ml probe. After 145

hybridization, the slides were washed in 5x SSC for 10 min. at 50°C and then in 80% 146

formamide and 2x SSC for 20 min. at 65°C. Single-stranded RNA was digested in 0.3 M 147

NaCl, 10 mM Tris pH 8.0, 5 mM EDTA and 50 µg/ml RNase A for 30 min. at 37°C. 148

The RNase-treated slides were washed with 50% formamide and 2x SSC for 20 min. at 149

65°C and further rinsed with 2x SSC and with 0.1x SSC each for 15 min. at room 150

temperature. The slides were dehydrated, coated with NTB-2 emulsion (Eastman Kodak 151

Co., Rochester, NY) and exposed for 2 weeks at 4°C. The slides were developed and 152

stained with hematoxylin and eosin. Photomicrographs were taken with a DIALUX 20 153

microscope (Leica Microsystems, Wetzlar, Germany) using epifluorescence optics and a 154

SPOT digital camera. 155

156

157 Results 158 159 Generation of cyA1lacZ transgenic mice 160

8

To begin to identify sequences responsible for the highly restricted pattern of 161

mouse Ccna1 expression, constructs composed of varying lengths of Ccna1 5’ genomic 162

sequence linked to lacZ cDNA were prepared and used to generate transgenic mice. The 163

transgenes were constructed with mouse genomic fragments consisting of 8.2 kb, 4.8 kb, 164

or 1.3 kb of Ccna1 5’ flanking sequence and the 5’ end of the Ccna1 structural gene to 165

exon 2 fused in-frame to a reporter cassette consisting of lacZ and the 3’ UTR and intron 166

1 of the testis-specific gene protamine1, yielding 8.2cyA1lacZ, 4.8cyA1lacZ, and 167

1.3cyA1lacZ, respectively (Fig. 1). Two permanent mouse lines each carrying 168

8.2cyA1lacZ and 1.3cyA1lacZ and one permanent mouse line carrying 4.8cyA1lacZ 169

were established. Each of the 5 lines was characterized further. 170

171

Distribution of cyA1lacZ transgene expression in adult tissues 172

To establish the tissue specificity of expression of the transgenes, RNA was 173

isolated from a variety of tissues from adult transgenic mice and analyzed by Northern 174

blot hybridization analysis. For all lines, a lacZ transcript was detected in RNA from 175

testis only (Fig. 2A – C; and data not shown), similarly to endogenous Ccna1. This result 176

was confirmed by in situ hybridization analysis of selected tissues from each line using a 177

lacZ probe (data not shown). These results suggested that the testis-specificity of 178

expression of Ccna1 is conferred by sequences within 1.3 kb upstream of its putative 179

transcriptional start. 180

181

Pattern of B-galactosidase activity in cyA1lacZ testes 182

9

To determine the cellular specificity of expression of the cyA1lacZ transgenes, 183

testes were stained with X-gal as whole mounts and then sectioned. Visualization of X-184

gal staining in intact tubules of 8.2cyA1lacZ testis (Fig. 3A) and 4.8cyA1lacZ testis (Fig. 185

3B) were identical and revealed dark blue staining except at the edges of the tubules, 186

which appeared unstained. This suggested that the cells closest to the basement 187

membrane of the tubules, the spermatogonia and early meiotic spermatocytes, did not 188

express the transgenes. In contrast, tubules from wild type testis were unstained, 189

although non-specific staining appeared in interstitial cells, which are located between 190

tubules (inset of Fig. 3A). Histological sections of the X-gal stained tubules from 191

8.2cyA1lacZ testis (Figs. 3D, 3G, and 3J) and 4.8cyA1lacZ testis (Figs. 3E, 3H, and 3K) 192

confirmed this observation. No staining was observed in spermatogonia or in 193

spermatocytes through stage VIII (Fig. 3D and 3E). Rather, the onset of expression 194

appeared in stage IX spermatocytes (Fig. 3G and 3H), similar to the appearance of 195

endogenous Ccna1 mRNA and protein [8]. X-gal staining was present in spermatocytes 196

at later stages, including stage XII (Fig. 3J and 3K), and persisted in all round and 197

elongating spermatids (Figs. 3D, 3E, 3G, 3H, 3J, 3K), cells known not to express cyclin 198

A1 (see results below). This staining pattern suggested that the transgenes contained 199

sequences required to direct the onset of expression of lacZ to stage IX spermatocytes. 200

Examination of stained, intact testicular tubules from the 1.3cyA1lacZ mice 201

revealed a less uniform X-gal staining pattern (Fig. 3C) as compared to that of 202

8.2cyA1lacZ (Fig. 3A) or 4.8cyA1lacZ mice (Fig. 3B). Unlike the consistent appearance 203

of X-gal staining in late spermatocytes and spermatids in 8.2cyA1lacZ and 4.8cyA1lacZ 204

testis, the staining of spermatocytes and spermatids within tubules of 1.3cyA1lacZ testis 205

10

was sometimes absent or was of varying intensity (Figs. 3C, 3F, and 3L). However, the 206

cellular specificity of expression exhibited was similar to 8.2cyA1lacZ and 4.8cyA1lacZ 207

lines. First, the onset of expression of the transgenes was similar. Examination of 208

sectioned tubules revealed that staining was absent in spermatogonia and in 209

spermatocytes through stage VIII (Fig. 3F), and β-gal activity first appeared in stage IX 210

spermatocytes (Fig. 3I). Also, β-gal activity persisted in the spermatids in the 211

1.3cyA1lacZ transgenic testis (Figs. 3F, 3I, and 3L). These observations suggest that the 212

Ccna1 genomic fragment within -1.3 kb to +0.8 kb from its transcriptional start contains 213

sequences needed to direct the onset of expression to stage IX spermatocytes, but that 214

additional upstream sequences from -4.8 kb and -1.3 kb of the Ccna1 transcriptional start 215

site were needed to direct detectable expression of 1.3cyA1lacZ in all spermatocytes. In 216

support of these observations, Northern blot analysis of total RNA from adult testis of 217

mice carrying each construct shows that 1.3cyA1lacZ is expressed at lower levels than 218

4.8cyA1lacZ or 8.2cyA1lacZ (Fig. 4). 219

The detection of β-gal activity in spermatids may have been due to expression of 220

the transgenes at stages later than that of endogenous Ccna1. Alternatively, the lacZ 221

reporter may have been expressed at the same developmental stages as Ccna1, but its 222

gene product may have persisted in spermatids. To determine if ectopic expression of the 223

transgene was responsible for the β-gal activity detected in spermatids in cyA1lacZ 224

testes, lacZ expression was assayed by in situ hybridization. LacZ mRNA was detected 225

in spermatocytes as late as stage XII (Fig. 5A), but was not detected in spermatids at 226

stage I (Fig. 5A) or in round or elongating spermatids at later developmental stages (Figs. 227

5A – 5D). Each of the cyA1lacZ constructs exhibited this same pattern of expression 228

11

(Fig. 5 and data not shown). The localization of lacZ mRNA only in spermatocytes 229

suggested that the β-gal activity detected in spermatids was not produced by ectopic 230

expression of the transgenes, but rather to stable β-gal protein. This further suggested 231

that sequences within -1.3 kb to +0.8 kb from the mouse Ccna1 transcriptional start site, 232

unlike its human homologue, were sufficient to control the correct timing of 233

spermatocyte-specific expression of the transgenes in germ cells. 234

A curiosity of the expression of these transgenes in the testis was noted in our in 235

situ hybridization analysis when a sense lacZ probe was used as a control. Signal was 236

detected in stage VI to VIII spermatocytes in transgenic testis sections for all three 237

constructs (Fig. 5E and data not shown). This signal was not present in sections from 238

non-transgenic testis (Fig. 5F). This expression might result from activity of a cryptic 239

promoter driving transcription in the opposite orientation in the protamine1 sequence 240

placed 3’ to lacZ in each transgene. However, promoter elements that support 241

transcriptional initiation, as assessed by TFSearch [22], were not detected in this 242

sequence. 243

244

Consensus elements for transcriptional regulators located between -4.8 kb and +0.8 kb 245

of the Ccna1 putative transcriptional start site 246

Intron 1 and the region between -4.8 kb and the putative transcriptional start site 247

of the Ccna1 gene were sequenced and scanned for putative transcription factor binding 248

sites using the search engine TFSEARCH and the TRANSFAC database [22]. The 249

locations of consensus sequences of DNA binding motifs for known transcription factors 250

and for binding activities are listed in Table 1. Among these motifs are binding sites for 251

12

transcription factors that are co-expressed in spermatocytes with Ccna1, such as A-myb 252

[23], Hsf2 [24], and Sp1 [25]. Consensus sequences for A-myb and Hsf2 binding are 253

located between -4.8 kb and -1.3 kb of the putative Ccna1 transcriptional start site, and 254

A-myb and Sp1 consensus sequences located within -1.3 kb of the putative Ccna1 255

transcriptional start site (Table 1). 256

The proximal promoter of Ccna1 was aligned with the corresponding rat and 257

human promoters. A region of high homology was found from -511 to -448 of the 258

putative Ccna1 start site (Fig. 6A), although this region lacked consensus sequences for 259

transcription factor binding sites listed in the TRANSFAC database [22]. However, 260

highly conserved consensus sequences for Sp1, GATA-1, Ap-1, and delta EF1 binding 261

sites, and a CCAAT box were identified (Table 1 and Fig. 6B) [22]. Downstream of 262

these consensus sequences and 153 bp upstream of the putative transcriptional start site 263

of the Ccna1 gene, the first of two bipartite ‘cell-cycle dependent element’/’cell-cycle 264

genes homology region’ (CDE/CHR) elements were found (Table 1 and Fig. 6B). In the 265

mouse and rat promoters, there are two conserved sets of putative CDE/CHR elements. 266

In the human promoter, the 5’ set and only the CDE sequence of the 3’ set are found; that 267

is, the human promoter lacks the CHR sequence of the 3’ CDE/CHR present in the mouse 268

and rat promoters. In addition, unpaired CDE and CHR elements are present, which are 269

conserved among all three cyclin A1 genes (Fig. 6B). 270

In the human proximal promoter, there are sequences that are poorly conserved or 271

not conserved at all with mouse and rat, for example, several GC boxes (Fig. 6B). These 272

sequences, in the context of a human CCNA1 reporter construct, have been suggested to 273

contribute to transcriptional activation in Hela cells [16]. 274

13

275

Discussion 276

In mice, the Ccna1 gene is expressed specifically in testis in stage IX to XII 277

spermatocytes [8]. Human CCNA1 is reported to be expressed at highest levels in male 278

germ cells [15], and at very low levels in brain and hematopoietic cells of normal tissues 279

[12, 13, 26]. Other sites of expression of mouse Ccna1 are yet to be confirmed. The 280

tissue-specificity of Ccna1 expression contrasts with that of Ccna2, which is expressed in 281

a variety of tissues [8, 27]. Ccna1 and Ccna2 also have non-overlapping expression 282

patterns during male germ cell development, as Ccna2 expression is downregulated early 283

in the meiotic cell cycle before Ccna1 is expressed [8, 9]. We therefore hypothesize that 284

there will be specific regulatory elements unique to each A-type cyclin as well. The 285

essential role of Ccna1 in male germ cell development and the concurrent expression of 286

cyclin A1 message and protein suggested the importance of understanding mechanisms 287

controlling transcription of the Ccna1 gene. The lack of cell lines derived from male 288

germ cells made it necessary to analyze the Ccna1 promoter in these cells in vivo, in 289

transgenic mice. The use of serial deletions of Ccna1 upstream sequence has allowed us 290

to define several functional segments of the promoter. 291

Analysis of transgene expression in the testis, using a combination of X-gal 292

staining and in situ hybridization, revealed their expression specifically in spermatocytes 293

at stages IX to XII of the cycle of the seminiferous epithelium, and not in earlier stages. 294

The endogenous mouse Ccna1 has been shown to be expressed only during this same 295

narrow window of spermatogenesis. This suggests that the genomic fragment of mouse 296

Ccna1 spanning -1.3 kb to +0.8 kb of the putative transcriptional start site contains 297

14

sequences necessary for the stage-specific expression of Ccna1 in the meiotic cell cycle. 298

In contrast, 1.3 kb of human CCNA1 promoter directs expression of EGFP in a much less 299

restrictive pattern in male germ cells [19], reflecting differences in the mouse and human 300

promoters despite their sharing highly conserved regions. 301

Within this fragment are consensus sequences for two sets of paired CDE/CHR 302

elements. These elements were first discovered in the proximal promoter of genes for 303

human cyclin A2, Cdc2, and Cdc25C, which are expressed in S and G2 phases of the 304

mitotic cell cycle [28]. Several lines of evidence suggest that the CDE/CHR elements 305

control the timing of expression of these genes during the cell cycle. In vivo footprinting 306

of the human CCNA2 proximal promoter revealed that the bipartite element was occupied 307

at stages that the gene was not transcribed [28]. Mutation of the CDE/CHR element in 308

the context of CCNA2 or CDK1 promoter/reporter genes caused derepression of the 309

reporters in G1 phase [28]. These elements have now been shown to be present and 310

involved in controlling the timing of expression of other cell cycle-regulated genes, 311

including the genes for cyclin B2 [29], rabkinesin6 [30], polo-like kinase [31], p130 [32], 312

m-survivin [33], and aurora A [34]. Also, the CDE/CHR element appears to 313

downregulate expression of the CDK1 gene in response to TPA-induced differentiation of 314

U937 cells [35] or in response to p53-dependent DNA damage [36]. The promoters of 315

the genes for mouse and rat cyclin A1 are unique in that they contain two sets of 316

CDE/CHR elements, and like the human gene, also have unpaired CDE and CHR 317

consensus sequences. There have been no previous reports of unpaired CDE elements, 318

but the promoters of the genes for human cyclin B2 ([37]) and mouse Cdc25C ([38]) are 319

regulated by CHR elements that are not paired with functional CDE elements. Testing of 320

15

mutated CDE/CHR elements in transgenic mice will determine the role, if any, of these 321

elements in controlling the expression of Ccna1 in the meiotic cell cycle. Factors that 322

bind CDE/CHR [39] or CHR [40, 41] have been detected in various cultured cell lines, 323

but have not been identified. 324

The less efficient X-gal staining in male germ cells from both lines of 325

1.3cyA1lacZ transgenic mice, as compared to germ cells from 8.2cyA1lacZ and 326

4.8cyA1lacZ transgenic mice, suggests that sequences that lie between 4.8 kb and 1.3 kb 327

upstream of the putative Ccna1 transcriptional start contains enhancer elements needed 328

for consistent expression of lacZ. Within this region, consensus elements for several 329

regulatory factors expressed in spermatocytes have been identified (Table 1). Hsf2 [24] 330

and A-myb [23] are reported to be expressed in mid- to late-stage spermatocytes, raising 331

the possibility that they may be involved in the regulation of expression of Ccna1. 332

Interestingly, disruption of A-myb in mice causes spermatogenic arrest during mid-333

prophase and male sterility [42]. Developmentally, arrest in A-myb-/- spermatocytes 334

occurs slightly earlier than the arrest in Ccna1-/- spermatocytes. 335

In summary, our results suggest that the genomic fragment -1.3 kb to +0.8 kb 336

from the putative Ccna1 transcriptional start contains sequences that mediate the correct 337

developmental expression of Ccna1 in male germ cells, as compared to this region in the 338

human CCNA1 gene. Further, sequences located between -4.8 kb and -1.3 kb from the 339

putative Ccna1 transcriptional start site appear to contribute to enhanced expression at 340

theses stages. Finally, sequences that lie between -1.3 kb and +0.8 kb from the Ccna1 341

transcriptional start appear to be sufficient to maintain repression of Ccna1 in the correct 342

adult tissues and stages of spermatogenic differentiation. Additional deletions in the -1.3 343

16

to +0.8 region may identify the location of elements that repress transcription of Ccna1 in 344

other cell types while mutation of known transcriptional regulatory elements and/or the 345

CDE-CHR elements may shed light on enhancer and cell-cycle regulatory roles, 346

respectively. Currently, the Ccna1 promoter we have characterized will provide a useful 347

tool to direct the expression of genes specifically to late meiotic spermatocytes in vivo. 348

349

Acknowledgements 350

We are very grateful to Dr. Xiangyuan Wang for performing microinjection and 351

producing histological sections and to Ms. Stacey Baptiste for staining 4.8cyA1lacZ 352

testes. 353

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Figure Legends

Fig. 1. 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.

Fig. 2. 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.

Fig. 3. 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

5

A, wild type testis. A, B, C, whole mount at 12x magnification. D, E, F, stage VIII

tubule at 40x. G, H, I, stage IX tubule at 40x. J, K, L, stage XII tubule at 40x.

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 lane represents the pattern of the lacZ

hybridization signal, while the bottom lane shows the 18s rRNA ethidium bromide

staining pattern.

Fig. 5. 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 40x magnification, C, D, E, 1.3cyA1lacZ testis sections at 40x, and F, a non-

transgenic testis section at 40x.

Fig. 6. 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 to 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

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to mouse sequence are light-shaded, and insertions or deletions are unshaded. The region

between the solid arrowheads is 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.

Table 1: Some of the transcription factors whose consensus DNA binding motifs are found within 4.8 kb of the mouse Ccna1 putative transcriptional start site or in intron 1 Transcription factor Consensus sequence

a Location

b

_______________________________________________________________________________________ SRY AAAC(A/T)A(/C) -4292, -4784, -4777, -4371, -3195 -3156, -3125, -1727, +583 GATA-1 (G/C)NNGATNNNN -4597, -1336, -979, -574, -220, +692 Sox-5 NNAACAATNN -3386, -3011, -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 aConsensus sequences are those listed in the TRANSFAC database [22] or, for CDE and CHR elements, were listed in Zwicker et al. [28]. bLocations 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. cThe c-Myb consensus sequence that is listed in the database is identical to that for A-myb and B-myb.