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Gamete Biology |
Department of Molecular and Cell Biology,3 The University of Texas at Dallas, Richardson, Texas 75080
Department of Anatomy and Cell Biology,4 McGill University School of Medicine, Strathcona Building, Montreal, Quebec H3A 2B2, Canada
Department of Physiology,5 West China Center of Medical Sciences, Sichuan University, Chengdu 610041, Peoples Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), encodes a protein similar to the large subunit of general transcription factor TFIIA. To understand how this factor is regulated, we characterized transgenic mice that contain the ALF promoter linked to either ß-galactosidase or green fluorescent protein (GFP) reporters. The results show that as little as 133 base pairs are sufficient to drive developmentally accurate and cell-specific expression. Transgene DNA was methylated and inactive in liver, but could be reactivated in vivo by system administration of 5-aza, 2'-deoxycytidine. Fluorescence-activated cell sorting allowed the identification of male germ cells that express the GFP transgene and provides a potential method to collect cells that might be under the control of a nonsomatic transcription system. Finally, we found that transcripts from the endogenous ALF gene and derived transgenes can also be detected in whole ovary and in germinal vesicle-stage oocytes of female mice. The ALF sequence falls into a class of germ cell promoters whose features include small size, high GC content, numerous CpG dinucleotides, and an apparent TATA-like element. Overall, the results define a unique core promoter that is active in both male and female reproductive tissues, and suggest mouse ALF may have a regulatory role in male and female gametogenic gene expression programs.
gamete biology, gametogenesis, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The TFIIA-like factor ALF (TFIIA) encodes a germ cell-specific form of the large (
/ß) subunit of the general transcription factor TFIIA [5, 6]. This protein forms a heterodimeric complex with the small (
) subunit of TFIIA and stabilizes binding of the TATA-binding protein (TBP) to core promoter DNA [7]. The interaction between TFIIA, TBP, and DNA is a key event in transcription initiation in somatic cells, and the observation that TFIIA and ALF share many biochemical properties suggests that formation of this complex will also be important for transcription initiation in germ cells.
ALF is an interesting model for the study of gene regulation in germ cells because its developmentally regulated expression during the pachytene stage of meiotic prophase I in males is representative of other germ cell-specific transcription factors [8, 9]. The promoters of the mouse and human ALF genes share a short GC-rich region of homology and are active when transfected into COS-7 or 293 cells [10]. Expression of the endogenous ALF gene in male germ cells is associated with reduced methylation at promoter-proximal CpG dinucleotides, whereas silencing in somatic tissues is associated with increased methylation [10]. Recent studies have shown, interestingly, that ALF is not strictly male-specific in Xenopus, but is also expressed in immature oocytes [11].
To define the minimal region necessary to drive ALF gene expression in the testis and to determine whether this gene was also expressed in female mice, we took a transgenic approach. Using promoter constructs linked to ß-galactosidase (ß-gal) or enhanced green fluorescent protein (GFP) reporters, we show that accurate expression in male germ cells of mice requires only 133 base pairs (bp) upstream of the major initiation site. Cells isolated from transgenic testis could be separated by fluorescence-activated cell sorting (FACS) based on GFP fluorescence intensity. We also show that the endogenous ALF gene and ALF promoter-driven transgenes are expressed in rodent oocytes. Overall, the results describe a germ cell-specific gene expressed in both male and female reproductive tissues and predict a possible regulatory role in both gametogenic gene expression programs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two ALF promoter fragments, –1151 to +27 and –133 to +21, were amplified from a bacterial artificial chromosome that contained the mouse ALF gene using primers J1 (5'-CACGGTACCAGATTAGAAAGGCTGCTTGA-3'), J2 (5'-CACAGATCTGCCAGCCGCTCTGTGCCTAA-3'), mALF GFP-1 (5'-GGACTCGAGAACTTTATTTCTCCAACCCC-3'), and mALF GFP-2 (5'-GAGGGTACCGCCCGCTCTGTGCCTAACC-3'). The large fragment was cloned upstream of a ß-gal reporter in pß-Gal-Basic (Clontech, Palo Alto, CA). A KpnI and SapI fragment containing the promoter and reporter was gel-purified for microinjection into pronuclei. The short fragment was cloned upstream of an enhanced GFP reporter in pEGFP (Clontech) and excised for microinjection with XhoI and AflII. Transgenic CD-1 mice were produced at the University of Texas Health Sciences Center at Houston.
Potential founders were screened using polymerase chain reaction (PCR) primers spanning the reporter gene (ß-gal or GFP) and verified by Southern blot analysis. Six founders for ALF-TG-1151 (5
, 22
, 37, 40, 41, and 42) and four for ALF-TG-133 (9, 12, 26, and 32) were generated. Initial experiments showed correct expression of the ALF-TG-1151 transgene in founders 22, 40, 41, and 42 or their F1 offspring produced by matings with wild-type CD-1 mice. Subsequent experiments were performed primarily using the 41 and 42 lines. Initial experiments on the ALF-TG-133 transgene were performed with founders 9, 12, and 26 and their F1 offspring. Animals derived from founder 9 expressed the transgene in tissues other than testis, so subsequent experiments were performed with the 12 and 26 lines. We did not extensively characterize the 5, 37, or 32 lines. Tissues were typically prepared from 7- to 10-wk-old animals. These studies were approved by the Institutional Animal Care and Use Committee.
Reverse Transcription-PCR and Western Blot Analysis
RNA was prepared from samples using the Trizol reagent (Gibco-BRL, Invitrogen, Carlsbad, CA). Reverse transcription (RT)-PCR was performed with primers for ß-gal (pßGAL-1 [5'-ATGGTACCGGATCGAAAGAGCCTGCTAAAG-3'] and pßGAL-2 [5'-ATGGTACCGCACAGATGAAACGCCGAG-3']), enhanced GFP (egfp-1 [5'-GGTGAGCAAGGGCGAGGAGCTGTT-3'] and egfp-2 [5'-TGTTGTGGCGGATCTTGAAGTTCACC-3']), ALF (mALF-8 [5'-ATCGAGGAGCAGGTGTTGAAAGACCTG-3'] and mALF-1 [5'-GAGTTTGTAAAGGTGACCAGATG-3']), glyceraldehyde 3-phosphate dehydrogenase (5'-G3PDH [5'-TCCACCACCCTGTTGCTGTAG-3'] and 3'-G3PDH [5'-GACCACAGTCCATGCCATCACT-3']), actin (mACTN-11 [5'-AGGACTCCTATGTGGGTGACG-3'] and mACTN-12 [5'-CTCATCGTACTCCTGCTTGCT-3']), TFIIA/ß (mTFIIAL-1 [5'-GTGTTGTGTGTGGAAATGGCGAAC-3'] and mTFIIAL-4 [5'-TCCAGGAAGTGGGGCTAAGA-3']), TFIIA (mTFIIA-1 [5'-ATGGCCTATCAGTTATACAGA-3'] and mTFIIA-2 [5'-TCATTCCGTAGTTAACGAGCC-3']), and zona pellucida 2 (mZP2-1 [5'-ATCTGTAAGCTCTCCGTGC-3'] and mZP2-2 [5'-GTACTATGGCATCCTTCAAGG-3']). To show that RT-PCR signals were not due to contamination with genomic DNA, control reactions were performed in the absence of reverse transcriptase.
Developmental expression of the transgenes was determined by RT-PCR analysis using RNA samples taken from the testis of animals at various days after birth. Cell type-specific expression was determined by RT-PCR analysis of RNA from male germ cells separated at unit gravity over a 1%–4% gradient of BSA [12, 13]. Fractions that contained pachytene, round, and elongated spermatocytes (70%–95% pure as judged by light microscopy) were collected by centrifugation.
To detect GFP protein, testes from ALF-TG-133 animals (
100 mg) were ground in a Dounce homogenizer (Wheaton Science Products, Millville, NJ) in 200 µl PBS supplemented with 0.2% SDS. The homogenate was centrifuged in a clinical centrifuge for 5 min and the supernatant was collected. Approximately 100 µg total protein was separated by SDS-PAGE, transferred to nitrocellulose, blotted with an anti-GFP antibody (Clontech), and detected with the SuperSignal West Pico chemiluminescent system (Pierce, Rockford, IL).
ß-Galactosidase and GFP Activity
Testis from adult wild-type or transgenic animals analyzed for ß-gal activity [14–16] were incubated with 5 ml of fixative solution (100 mM sodium phosphate buffer pH 7.3, 2% paraformaldehyde, 0.2% glutaraldehyde, 0.01% sodium deoxycholate, and 0.02% NP-40) at 4°C for 2 h, washed three times (100 mM sodium phosphate pH 7.3, 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% NP-40), and incubated overnight at room temperature in staining solution (100 mM sodium phosphate pH 7.3, 1.3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, with 1 mg/ml X-gal).
Isolated cells or tubules from testis were examined for GFP fluorescence using an Olympus BX60 microscope (Olympus, Tokyo, Japan) equipped with a GFP filter. For sections, testis were transferred into a mold filled with optimal temperature cutting compound, sectioned into 15-µm slices with an HM500OM cryostat, fixed for 2 min with 100% methanol, and stained with hematoxylin-eosin (Fisher, Hampton, NH). To detect GFP expression in germinal vesicle (GV)-stage oocytes, untreated cells were placed in a Petri dish containing warm Dulbecco modified Eagle medium (DMEM) media and examined by phase contrast and fluorescence microscopy.
Mouse Oocyte Preparation and Growth of SIGC Cells
Isolation of GV oocytes and mature oocytes was performed essentially as described [17]. Briefly, adult mice were injected with 20 IU of eCG (Sigma, St. Louis, MO). After 48 h, ovaries were isolated and immature GV-stage oocytes were removed by microdissection under an Olympus SZ40 stereomicroscope. Mature oocytes were isolated from animals that were treated with 20 IU eCG for 48 h followed by 15 IU hCG for 16 h. Oviducts were isolated, oocytes were removed by microdissection, and then incubated for 3 min in DMEM with hyaluronidase (100 µg/ml) to remove cumulus cells.
SIGC cells were grown in 5% CO2 in DMEM/F12 media (Gibco/BRL) with 5% fetal bovine serum to 70%–80% confluence. Cells were detached from plates with 2x trypsin/EDTA (Gibco/BRL) at 37°C for 5 min, and used for RNA preparation.
Methylation Analysis
Genomic DNA from liver or testis of an ALF-TG-133 animal was digested with HpaII, MspI, and HindIII. Digested DNA (15 µg) was separated on an agarose gel, transferred to nitrocellulose, and probed with a GFP-specific probe prepared by PCR using primers LiEGFP-1 (5'-CAACTACAACAGCCACAAC-3') and LiEGFP-2 (5'-GCGGCGGTCACGAACTCCAG-3'). Animals treated with 5-aza, 2'-deoxycytidine (azaC) were given two i.p. injections of azaC (2.5 µg/g) 3 days apart [18], and tissues were harvested 2 days later for sectioning and microscopy.
FACS Analysis
Razor-minced testis from wild-type CD-1 (Charles River Laboratories, Wilmington, MA) and transgenic mice were incubated in PBS containing 50 U/ml collagenase (Sigma) and 2.4 U/ml Dispase (Invitrogen) for 10 min at 37°C. Undigested tissues were allowed to settle, and the cell suspension was collected and spun for 3 min at 300 x g. The cell pellet was resuspended in 5 ml PBS containing 0.5% bovine calf serum and cells. Multiple aliquots of 1 x 106 cells was diluted into 1 ml of PBS containing 0.5% bovine calf serum and incubated with 5 µg/ml Hoechst 33342 (Sigma) for 45 min at 37°C. During the incubation, tubes were gently mixed several times. Cells were then filtered through a 40-µm nylon mesh (BD Bioscience, San Jose, CA) and kept in the dark on ice until FACS analysis. A total of 1–2 x 107 cells were analyzed on a MoFlo (DakoCytomation, Glostrup, Denmark) flow cytometer. Excitation of GFP was at 488 nm, and signals were collected with a 530/40 bandpass filter. Hoechst 33342 excitation was at 360 nm and signals were collected with a 424/44 nm filter. Sorted cell populations were collected by centrifugation for 3 min at 300 x g and resuspended in 20 µl PBS. Cells were visualized by brightfield and fluorescence microscopy using an Olympus BX51. To confirm cell populations were sorted and collected properly they were reanalyzed by FACS after collection.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this report we used a transgenic approach to define sequences of the ALF gene promoter that can drive correct developmental and cell type-specific expression in an animal. One construct contained a promoter fragment from –1151 to +27 inserted upstream of a ß-gal reporter (ALF-TG-1151; total promoter size 1178 bp) (Fig. 1). Another construct contained a promoter fragment from –133 to +21 inserted upstream of a GFP reporter (ALF-TG-133; total promoter size 154 bp). The short construct contains the GC-rich region of homology between the mouse and human ALF core promoters, including a TATA-like element and 14 CpG dinucleotides. Sequences downstream of the major initiation site (to either +21 or +27) were included because this promoter initiates at multiple sites [10].
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Tissue-Specific Expression of the ALF-TG-1151 Construct in Mice
Expression of the ALF-TG-1151 construct in founder mice and their offspring was examined in heart, kidney, liver, lung, and testis (see Materials and Methods for a description of transgenic lines). Using an RT-PCR assay, mRNA from the ß-gal reporter could be detected in testis but not in any of the somatic tissues (Fig. 2A, lane 1). This pattern of expression was observed in the offspring of both male and female founders, and in the original founder males (data not shown).
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The mouse ALF gene is first expressed in the testis of mice at Day 14 postpartum, corresponding to the time at which cells in the pachytene stage of meiotic prophase I first appear [9]. To test whether the ALF-TG-1151 transgene was also developmentally regulated, RNA from testis of transgenic mice at 8, 10, 13, 16, and 19 days after birth was analyzed by RT-PCR. The results showed ß-gal mRNA transcripts to be present starting at Day 13 (Fig. 2B), consistent with earlier results [9]. In addition, pachytene spermatocytes, round spermatids, and elongated spermatids isolated by unit gravity sedimentation all contained transcripts from the endogenous ALF gene and the ALF-TG-1151 transgene (Fig. 2C).
To provide a visual demonstration of ß-gal transgene expression, testes were stained with X-gal (Fig. 2D). The results showed that whole testis and individual seminiferous tubules from transgenic animals stained strongly, whereas those from wild-type animals did not. Blue discoloration of the epididymis was observed in both types of animals and appears to be an intrinsic property of this tissue. Based on these results we conclude that sequences within 1151 bp of the initiation site contain sufficient regulatory information to drive expression identical to the endogenous ALF gene.
Tissue-Specific Expression of the ALF-TG-133 Construct in Mice
We had previously shown that a 100 bp region of homology between the mouse and human ALF gene promoters was active in several somatic cell lines [10] and we wanted to test whether this region alone might be sufficient to drive expression in transgenic mice. We therefore characterized a transgene construct, ALF-TG-133, that contained promoter proximal sequences between –133 to +21 linked to a GFP reporter (Fig. 1).
Transgenic animals that harbored the ALF-TG-133 transgene expressed the GFP reporter in testis but not in somatic tissues such as spleen, brain, kidney, liver, heart, or lung (Fig. 3A). Consistent with the RT-PCR results, the GFP protein itself could be detected by Western blot analysis in testis and not in somatic tissues (Fig. 3B). Moreover, GFP fluorescence was detected in seminiferous tubules (Fig. 3C), testis sections (Fig. 3D), and purified male germ cells separated by unit-gravity sedimentation (Fig. 3E). Together, the results demonstrate that 133 bp of the ALF promoter are sufficient to drive male germ cell-specific expression.
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FACS Analysis of Cells that Express the ALF-TG-133 Transgene
We next examined whether cells isolated from the testis of transgenic animals could be separated based on expression of the GFP reporter. Cells were washed away from surrounding connective tissue, stained with Hoescht dye, and separated by FACS based on DNA content and GFP fluorescence intensity (Fig. 4A). A subset of cells separated at a position along the X-axis indicative of high GFP reporter gene expression. To verify this result, these cells were collected and shown to re-sort at the same position as in the original run (data not shown). Moreover, fluorescence microscopy demonstrated that 2N cells were in fact GFP positive (Fig. 4A). In contrast, cells obtained from the testis of wild-type animals were negative for GFP expression (Fig. 4B).
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Methylation of the ALF-TG-133 Transgene
Because the ALF-TG-133 transgene was properly expressed, we wanted to know whether it was subject to the same pattern of tissue-specific methylation observed for the endogenous ALF gene [10]. To address this issue, we digested genomic DNA from liver and testis with the methylation-sensitive HpaII or methylation-insensitive MspI enzymes and hybridized with a GFP-specific probe. Digestion of both liver and testis DNA with MspI gave a 600 bp hybridization product (Fig. 5A, lanes 3 and 6). This band was also observed in testis DNA that had been digested with HpaII (Fig. 5A, lane 5), but did not appear in HpaII-digested liver DNA (Fig. 5A, lane 2).
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To determine whether silencing of the ALF-TG-133 transgene in liver was associated with DNA methylation, male mice were treated with the methylcytosine analog azaC and examined for GFP fluorescence in liver. The results showed that azaC treatment upregulates GFP activity compared to untreated controls (Fig. 5B) and did not affect expression in testis where the promoter is already demethylated and active.
Expression of ALF and ALF Transgenes in Female Reproductive Tissues
To determine whether ALF might have a role in regulating gene expression in female germ cells, we tested whether it was expressed in ovary. To test this possibility, RNA from liver, testis, and ovary was an analyzed by RT-PCR using primers for ALF, ß-gal, and GFP, as well as actin and ZP2 controls. Remarkably, the results showed that the endogenous ALF gene (Fig. 6A, lane 3) and both ALF-TG-1151 and ALF-TG-133 transgenes (Fig. 6A, lane 6) could be reliably detected at low levels in ovary.
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To provide a direct demonstration that ALF mRNA was present in ovary we performed Northern blot analysis. The results showed two ALF transcripts from control, eCG-treated, and eCG/hCG-treated animals (Fig. 6B, lanes 3–5). Although present at low levels, the sizes of these bands are similar to the abundant testis transcript (Fig. 6B, lane 1) and substantiate the earlier RT-PCR analysis. Examination of additional somatic tissues or staged mouse embryos did not reveal expression elsewhere (Fig. 6C).
We next examined whether ALF transcripts were present in GV oocytes themselves. To test this possibility, oocytes were isolated by microdissection of ovaries from eCG-treated animals and used to prepare RNA. RT-PCR analysis shows that mRNAs for ALF, and for the somatic factors TFIIA/ß and TFIIA, were present in these cells (Fig. 7A, lane 3). We also showed that ALF transcripts could not be detected in the SIGC granulosa cell line [19] (Fig. 7B, lane 1).
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To further support the conclusion that ALF is expressed in female mice, we examined GFP mRNA and GFP activity in seven different animals. RT-PCR demonstrated that one ovary from each animal contained similar, relative levels of ALF and GFP transcripts (Fig. 7C). The second ovary was used to isolate GV-stage oocytes for phase contrast and fluorescence microscopy. Five animals showed GFP-positive oocytes (Fig. 7D), substantiating the conclusion that the ALF promoter is expressed in female germ cells. For reasons that are not yet understood, GFP signals were absent in the other two animals. Overall, however, multiple lines of evidence indicate that the ALF gene is expressed in reproductive tissues of male and female mice (see Discussion).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was first reported as a testis-specific counterpart of the large (/ß) subunit of TFIIA [5, 6]. In the current study, we use a transgenic approach to characterize expression of the ALF transcription factor in somatic and germ line tissues. To eliminate effects on expression that might be due to the reporter or vector rather than the promoter itself, the two constructs, ALF-TG-1151 and ALF-TG-133, were engineered into different vectors with different reporters and distinct splicing patterns. The results show that both constructs can drive similar patterns of cell type-specific expression in mice. The ALF-TG-133 transgene is one of the smallest germ cell-specific promoters characterized to date. The fact that this short construct can drive accurate expression demonstrates that the core promoter region alone is sufficient for germ cell expression and that distal enhancers are not required. When aligned with other germ cell-specific genes that have been tested in transgenic assays (20–40; Fig. 8), we observe that approximately half contain a TATA-like element positioned at about –30 relative to the start site. However, experiments with the ALF and ACE promoters suggest that this element may not be essential for activity, and its role is still uncertain [10, 41].
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The aligned promoters display an average of nearly 60% GC content in the first 100 nucleotides upstream of the initiation site, and many are subject to differential CpG methylation in somatic and germ line tissues (Fig. 5 and [10, 42]). In fact, methylation of the short transgenic ALF promoter and its reactivation by azaC (Fig. 5 and [10]) suggests that the ALF core promoter is recognized as a substrate by a methyltransferase, either directly or through recruitment by another DNA-binding transcription factor. Because the transgene was germ cell-specific in founder animals, it appears that methylation was established during embryogenesis and did not require passage through the germ line. This situation is distinct from methylation of imprinted genes, which require transit through the germ cell differentiation program for methylation to be correctly set [43]. The identification of the regulatory factors that bind to these genes and control methylation and expression in vivo will be an important goal.
In this study we also demonstrate that the ALF gene and derived transgenes are expressed in ovary. This conclusion was unanticipated from earlier studies of this gene in humans and rodents [5, 6, 9], but is supported by several lines of evidence. First, RT-PCR analysis detected transcripts from the endogenous ALF gene in whole ovary and in GV-stage oocytes isolated by microdissection. Second, transcripts from the endogenous ALF gene were detected by Northern blot analysis using whole ovary RNA. The size of larger transcript matches the one observed in testis, suggesting that it may initiate from the same promoter. Third, transcripts from the ß-gal and GFP reporters in ALF-TG-1151 and ALF-TG-133 could be detected in ovary using RT-PCR. Moreover, GFP fluorescence could be visually detected in oocytes isolated from female mice. The fact that multiple bands were observed by Northern blot analysis of RNA from ovary suggests that more than one promoter might be active for ALF transcription, as occurs for the Dnmt1 gene [44]. Together with results on ALF expression in Xenopus laevis [11], the results predict an evolutionarily conserved role for ALF in both male and female germ cell differentiation programs. The results suggest that both types of germ cells could possess similar regulatory factors, or they could impose similar chromatin packaging constraints (or both) that result in shared patterns of gene expression. The results also raise the possibility that other male-specific genes are also expressed at low levels in female reproductive tissues.
Another germ cell-specific transcription factor, TAFII-105, is expressed in male germ cells of the testis and in granulosa cells of the ovary [45]. Surprisingly, deletion of this gene resulted in female sterility but had no effect on male germ cell development [45]. These observations emphasize the need to examine cell-type specificity of germ cell-specific transcription factors on a case by case basis. Ultimately, genetic studies will be required to evaluate whether these factors are essential for gametogenesis in both sexes or, as observed for TLF/TRF2 and TAFII105, whether their effects are male- or female-specific [45–47].
The production of ALF and other transcription factors during the pachytene stage of meiotic prophase I [9] and their predicted role as transcriptional regulators raise interesting questions about meiotic gene expression. In particular, how are genes controlled at various stages of gametogenesis and what role do germ cell transcription factors play in regulating those genes? In consideration of these questions, we suggest that gene regulation during gametogenesis might occur through a defined sequence of events. One of the first events in this progression would be to revive genes from their silent somatic state. For the ALF and PGK2 genes, this involves changes in CpG methylation status and chromatin packaging [10, 48]. Genes that become accessible at the very earliest stages of meiosis are presumably recognized and transcribed by the preexisting somatic transcription machinery. Genes expressed at intermediate stages may also be controlled by this machinery, or by a combination of somatic and newly synthesized germ cell factors. It is interesting that the somatic core promoter factor TFIIA does interact with a germ cell-specific activator, CREM [49]. Later, when all the components of a new germ cell-specific transcription system are present, they will presumably act in concert to direct downstream (late) patterns of gene expression. In support of this idea, male mice that lack the TLF/TRF2 gene are able to produce normal spermatogonia and early spermatocytes but do not complete the spermatogenic cycle. This suggests that TLF/TRF2 is not necessary to initiate the meiotic program, but its loss disrupts mid/late gene expression events needed for its completion [46]. Furthermore, ALF completely replaces the somatic TFIIA factor for all RNA polymerase II-dependent gene expression in stage I-VI oocytes in Xenopus [11]. The ability to tag and collect germ cells that express an ALF transgene (Fig. 4A) may allow an examination of gene regulation in cells that are under the control of an alternate germ cell transcription system.
The suggestion that mammalian gametogenesis proceeds through an ordered transcription program is reminiscent of the situation in yeast, in which discrete sets of meiotic genes (early, middle, late) are expressed sequentially under the control of transcription factors produced in preceding stages [50]. This hypothesis also predicts that the short sequences of germ cell promoters such as ALF and others listed in Figure 8 might fall into groups with shared core promoter architectures diagnostic for their cell-type specificity or time of expression.
In summary, the present study demonstrates that a short proximal promoter region of the ALF gene is expressed during meiotic prophase I in the testis and in mouse ovary as well. This pattern of expression predicts a regulatory role for ALF in both male and female germ cell differentiation and, together with data from other germ cell-specific transcription factors, suggests a framework of regulatory events that occur during these gene expression programs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jeff DeJong, The University of Texas at Dallas, Department of Molecular and Cell Biology, 2601 N. Floyd Road, Richardson, TX 75080. FAX: 972 883 2409; dejong{at}utdallas.edu
Received: 29 March 2004.
First decision: 14 April 2004.
Accepted: 7 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß homolog selectively expressed in testis. J Biol Chem 1999 274:18040-18048) stimulates TATA-binding protein-DNA binding and transcription activation. J Biol Chem 2000 275:122-128/ß-like factor is encoded by a germ cell-specific gene whose expression is upregulated with other general transcription factors during spermatogenesis in the mouse. Biol Reprod 2001 64:507-517This article has been cited by other articles:
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  S. M. Kehoe, M. Oka, K. E. Hankowski, N. Reichert, S. Garcia, J. R. McCarrey, S. Gaubatz, and N. Terada A Conserved E2F6-Binding Element in Murine Meiosis-Specific Gene Promoters Biol Reprod, November 1, 2008; 79(5): 921 - 930. [Abstract] [Full Text] [PDF]
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K. Tokuhiro, Y. Miyagawa, S. Yamada, M. Hirose, H. Ohta, Y. Nishimune, and H. Tanaka The 193-Base Pair Gsg2 (Haspin) Promoter Region Regulates Germ Cell-Specific Expression Bidirectionally and Synchronously Biol Reprod, March 1, 2007; 76(3): 407 - 414. [Abstract] [Full Text] [PDF]
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 K. Kitamura, H. Nishimura, Y. Nishimune, and H. Tanaka Identification of Human HAPRIN Potentially Involved in the Acrosome Reaction J Androl, July 1, 2005; 26(4): 511 - 518. [Abstract] [Full Text] [PDF]
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