INTRODUCTION

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1,4-Galactosyltransferase-I (4GalT-I; EC 2.4.1.38) is a constitutively expressed, trans-Golgi resident, type II membrane-bound glycoprotein that is widely distributed in vertebrates. This enzyme catalyzes the transfer of galactose (Gal) to N-acetylglucosamine (GlcNAc) residues, forming the 4-N-acetyllactosamine (Gal4-GlcNAc) or poly-4-N-acetyllactosamine structures found in glycoconjugates (1). In mammals, 4GalT-I has been recruited for a second biosynthetic function, the tissue-specific production of lactose (Gal4-Glc), which takes place exclusively in the mammary gland during lactation (reviewed in reference 37; see also references 25 and 40). The synthesis of lactose is carried out by a heterodimer assembled from 4GalT-I and the abundant milk protein -lactalbumin, which is expressed de novo only in the mammary gland during lactation (3, 4, 16).

The organization of the 5' end of the murine 4GalT-I gene is unusual in that three transcriptional start sites are contained within an ~725-bp contiguous piece of DNA (Fig. 1). In somatic cells and tissues, the 4GalT-I gene specifies two transcripts of 4.1 and 3.9 kb. These two transcripts arise as a consequence of initiation at two different start sites separated by ~200 bp (35, 39). Since the two start sites are positioned either upstream of the first two in-frame ATGs (4.1 kb) or between these two in-frame ATGs (3.9 kb), translation of each mRNA results in the synthesis of two catalytically identical, structurally related protein isoforms that differ only in the lengths of their respective short, NH₂-terminal cytoplasmic domains. In somatic tissues, the 4.1-kb start site is used preferentially. The only exception to this pattern is found in the mammary gland during lactation, where there is a switch to the preferential use of the 3.9-kb start site. Expression from this start site is controlled by a stronger promoter (relative to the constitutive promoter governing transcription from the 4.1-kb start site) that is, in part, regulated by lactating mammary gland-restricted transcription factors (31). This results in an estimated 10-fold increase in steady-state 4GalT-I mRNA levels (14). This increase in mRNA levels, combined with the demonstration that the 3.9-kb transcript is translated in vivo three- to fivefold more efficiently than the 4.1-kb mRNA, results in the ~50-fold increase in 4GalT-I enzyme levels needed for lactose biosynthesis (7).

View larger version (8K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the 5' end of the murine 4GalT-I gene. Shown is the upstream genomic DNA (solid box), the corresponding 5'-untranslated region (727 nt) of the male germ cell-specific transcript (hatched box), the coding sequence of exon 1 (open box), and a portion of the first intron (thin line). The bent arrows denote the three 4GalT-I transcriptional start sites. The male germ cell-specific start site (Gc) is used exclusively in late pachytene spermatocytes and round spermatids. The 4.1-kb start site (4.1) is used exclusively in spermatogonia and predominantly in all somatic cells. The 3.9-kb start site (3.9) is used predominately in the mammary gland during lactation. The locations of the first two in-frame ATGs are shown. Numbering is relative to the first in-frame ATG, which is designated +1. Exon 1 is not drawn to scale. Translation of the 4.1- and 3.9-kb 4GalT-I mRNAs results in two catalytically identical, trans-Golgi resident protein isoforms with NH2-terminal cytoplasmic domains of 24 and 11 amino acids, respectively.

The third, most distal transcriptional start site (Gc in Fig. 1) is used exclusively during the later stages of spermatogenesis (15). Spermatogenesis refers to the process in which spermatogonia, through a series of mitotic and meiotic divisions, give rise to spermatozoa. In spermatogonia, only the 4.1-kb transcript can be detected and it is identical in structure to the 4.1-kb mRNA found in somatic cells. As spermatogonia develop into early pachytene spermatocytes, transcription of the 4GalT-I gene is reduced to barely detectable levels. Continued differentiation to late pachytene spermatocytes and haploid round spermatids is coincident with renewed 4GalT-I expression to levels comparable to that observed in spermatogonia. However, the 4.1-kb 4GalT-I transcript is replaced with two truncated germ cell-specific transcripts of 2.9 and 3.1 kb (15, 41). These two transcripts encode the same open reading frame as the 4.1-kb transcript; however, their respective 3'-untranslated regions are only ~0.8 or ~1.0 kb in length, due to the utilization of either one of two alternative polyadenylation signals positioned ~200 nucleotides apart within the long 3'-untranslated region (2.7 kb) of the somatic transcript. The 2.9- and 3.1-kb germ cell-specific transcripts are also distinguished from the 4.1-kb transcript by the presence of an additional ~560 nucleotides of 5'-untranslated sequence.

We previously reported that a 796-bp TATA-less genomic fragment that flanks the 4GalT-I male germ cell start site mediates expression of the -galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice in a manner comparable to that of the endogenous 4GalT-I gene (38). In this study, by using additional transgenic constructs in combination with DNase I footprinting and electrophoretic mobility shift assays (EMSAs), we demonstrated that a 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3'), that we have termed the TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1) motif, is essential for in vivo expression of 4GalT-I in developing male germ cells. Observations that suggest that TASS-1 is a member of the Ets family are discussed.

	MATERIALS AND METHODS

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DNA constructs. All constructs were derived from the promoterless pLacI vector, which contains the Escherichia coli lacZ gene plus an intron and a polyadenylation sequence from the mouse metallothionein II gene. The 4GalT[1270/474]LacZ construct was described in reference 38, where it was referred to as 4GT-LacZ. It contains a 796-bp PvuII-NruI fragment that includes 543 bp of the genomic sequence upstream of the 4GalT-I germ cell start site and 253 bp of the flanking downstream sequence (spanning 1270 to 474) fused to the bacterial LacZ coding sequence. The male germ cell start site is located at 727 relative to the first in-frame ATG, designated +1 (Fig. 1). The 4GalT[1085/474]LacZ construct was produced by subcloning a 611-bp Eco47III-NruI fragment, spanning 1085 to 474, into the blunt-ended SalI site of pLacI. A 651-bp PvuII-HphI fragment, spanning 1270 to 619, was isolated, blunt ended with Klenow DNA polymerase, and subcloned into the blunt-ended SalI site of pLacI to produce the 4GalT[1270/619]LacZ construct. To produce the 4GalT[1085/628]LacZ construct, two primers 5'-ATTGTCGACGCTGTGTACAACGGGCTGTTC-3' and 5'-TTTGTCGACACCACTTCCTGACGTAC-3', containing a SalI site at each end (underlined sequences) were used to amplify a 457-bp fragment spanning 1085 to 628. After digestion with SalI, the amplified fragment was subcloned into the SalI site of pLacI. To produce the 4GalT[793/707]LacZ construct, two primers, 5'-ATAGGTACCAGAGAATCCGTCCGCT-3' and 5'-AGCGTCGACTTGGGATAGTGAGAAA-3', containing KpnI and SalI sites, respectively (underlined sequences), were used to amplify an 87-bp fragment spanning 793 to 707. After digestion with KpnI and SalI, the amplified fragment was subcloned into pLacI digested with KpnI and SalI. To assemble the 4GalT[mut756/743]LacZ construct, the forward primer 5'-GCCGGTACCTGTGAGATTTTACAGGCCATTCATTCT-3' and the reverse primer 5'-ATTAGGCCTATTATTCGACGTCCCGCGAGGCCAGCG-3' were used to amplify a 531-bp fragment spanning 1270 to 757. The PCR product was then digested with KpnI and StuI (underlined sequences). Next, the forward primer 5'-CCGTTTAAAGCTCCGACCTTTCTCCACTCCATTTTC-3' and the reverse primer 5'-ATAGTCGACCGACACTTGGGGACTAAACGTGGCGCT-3' were used to amplify a 287-bp fragment spanning 742 to 474. The PCR fragment was then digested with DraI and SalI (underlined sequences). A three-way ligation of the KpnI-StuI fragment, the DraI-SalI fragment, and pLacI digested with KpnI and SalI yielded the final construct, in which the 756/743 sequence 5'-GCCGGTTTCCTAGA-3' was mutated to 5'-TAATAGGAAAGCTC-3'. The sequences of all constructs were confirmed by DNA sequencing.

Production and identification of transgenic mice. DNA inserts containing the relevant 4GalT-I genomic fragment inserted into pLacI were excised by KpnI/PstI digestion of plasmid DNA, isolated on agarose gels, purified by using an Elutip-d minicolumn (Schleicher & Schuell), and resuspended in injection buffer (10 mM Tris-HCl [pH 7.4] and 0.1 mM EDTA filtered through a 0.22-µm-pore-size membrane). Each transgene was injected into single-cell C57B6/BALBC3 embryos (at the Johns Hopkins University Transgenic Core Laboratory). Identification of transgenic founders and copy number determination were carried out essentially as previously described (38). Founders were bred to obtain heterozygous male transgenic offspring. At least three independent transgenic lines were generated for each construct. Transmission of the transgene proceeded according to Mendelian laws in the transgenic lines studied.

-Galactosidase assays and histology. -Galactosidase assays and histology were performed as previously described (38). Fresh tissues were homogenized for 20 s in 100 mM potassium phosphate (pH 7.8)-0.2% Triton X-100-1 mM dithiothreitol-5 µg of leupeptin per ml (the last component was added just before use). The homogenate was centrifuged at 12,500 × g for 10 min, and the supernatant was heated at 48°C for 60 min to inactivate the endogenous eukaryotic -galactosidase. Following centrifugation at 12,500 × g for 10 min, 10 µl of the supernatant was assayed for -galactosidase activity by using the Galacto-Light chemiluminescence assay kit (Tropix, Inc., Bedford, Mass.). Protein concentrations were estimated by using the Bio-Rad Protein Assay kit. For histology, tissues were removed from transgenic and nontransgenic mice, fixed and incubated in buffer containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal), and paraffin embedded. Sections (3 to 5 µm) were cut, mounted on glass slides, deparaffinized, counterstained for 30 s with nuclear fast red, and photographed by using a Zeiss Axiophot microscope.

Cells and culture conditions. Mouse L cells were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 100 U of penicillin per ml, and 50 µg of streptomycin per ml at 37°C in 5% CO₂.

Preparation of nuclear extracts. Pooled male germ cells were prepared by digesting adult mouse testes with collagenase and hyaluronidase (41). The final preparation contained >95% male germ cells. Within this pooled male germ cell population, ~50% of the cells are round spermatids. Nuclear extracts from mouse L cells and from pooled male germ cells were prepared by the method of Dignam et al. (10). Nuclear extracts were made from frozen, decapsulated testes of adult mice by a combination of the methods of Roy et al. (34) and Dignam et al. (10) as previously described (31). Protein concentrations were estimated by using the Bio-Rad Protein Assay kit.

DNase I footprinting analysis. DNase I footprinting was performed as previously described by using two DNA probes (31). To amplify the 396-bp region spanning 870 to 474, the forward primer 5'-TGCAGAATTCTCAATAAGCAGCTCCT-3' and the reverse primer 5'-TTTGTCGACTGTAAAACGACGGG-3' were used. To amplify the 380-bp region spanning 1150 to 770, the forward primer 5'-TGCAGAATTCGATTACAGGTGTTCA-3' and the reverse primer 5'-TTTGTCGACAGCGGACGGATTCTC-3' were used. Primers were designed so that PCR fragments contained an EcoRI site at the 5' end and a SalI site at the 3' end (underlined sequences). After digestion with EcoRI and SalI, the fragments were subcloned into the EcoRI/SalI site of pSL301 (Invitrogen). The sequences of the two inserts were confirmed by DNA sequencing. DNA fragments were excised by using EcoRI and SalI and purified as described above. To analyze the interactions of nuclear proteins with the coding (top) strand, probes were end labeled at the SalI site with [-³²P]dCTP and the remaining deoxynucleoside triphosphates by using the Klenow DNA polymerase (the EcoRI site is not labeled, since it lacks cytosine). To investigate the interactions of nuclear proteins with the noncoding (bottom) strand, probes were end labeled at the EcoRI site with [-³²P]dATP by using the Klenow DNA polymerase in the absence of additional nucleotides (thus, the SalI site is not labeled). DNase I (5-mg/ml stock solution) was diluted in 10 mM HEPES-NaOH (pH 7.6)-25 mM CaCl₂. The dilutions used were 1:4,000 for bovine serum albumin (BSA), and 1:50 for mouse L-cell and testis nuclear extracts.

EMSAs. Single-stranded oligomers were synthesized by the Johns Hopkins DNA Synthesis Facility, and complementary strands were annealed before use. Each double-stranded oligomer contained a recessed 3' end which was filled in with either [-³²P]dATP or [-³²P]dCTP and the remaining deoxynucleoside triphosphates by using the Klenow DNA polymerase. The ³²P-labeled probes were separated from the unincorporated nucleotides by chromatography on Sephadex G-50. EMSAs were performed essentially as previously described, by using 10 µg of nuclear extract (31). For competition experiments, a 100-fold molar excess of an unlabeled, double-stranded probe was incubated with 10 µg of nuclear extract for 20 min prior to the addition of the labeled probe. For the sequences of the double-stranded oligomers, see Table 2.

	RESULTS

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Experimental approach. One major limitation in studying promoters regulating the expression of genes during murine spermatogenesis is the lack of established male germ cell lines. To circumvent this deficiency, investigators have relied on in vitro transcription assays (6, 46) or transgenic mice (22, 30, 33, 52). We have chosen the latter approach, since the functional significance of putative promoter elements can be established in vivo.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Schematic representation of the LacZ-based constructs used to generate the first three transgenic lines. The original 4GalT[1270/474]LacZ construct contains a 796-bp fragment that includes 543 bp of the genomic sequence upstream of the male germ cell start site (Gc) and 253 bp of the flanking downstream sequence (38). In the 4GalT[1085/474]LacZ construct, the sequence spanning 1270 to 1086 was deleted, while in the 4GalT[1270/619]LacZ construct, the sequence spanning 618 to 474 was deleted. The 4GalT[1085/628]LacZ construct combines both 5' and 3' deletions. The solid, hatched, and open boxes represent the upstream genomic DNA, the 5'-untranslated region of the male germ cell transcript, and the LacZ coding sequence, respectively.

Promoter activity is retained in vivo after removal of 185 bp from the 5' end, or 145 bp from the 3' end, of the 796-bp fragment. DNA fragments derived from the 4GalT[1085/474]LacZ and 4GalT[1270/619]LacZ constructs were used to generate transgenic mice. A total of four founders were identified for 4GalT[1085/474]LacZ, while a total of three founders were identified for 4GalT[1270/619]LacZ (Table 1, groups B and C, respectively). Subsequent breeding with wild-type mice produced heterozygous F₁ and F₂ progeny for analysis. In order to assess if expression of the reporter gene was observed exclusively in the testes of the transgenic mice, -galactosidase activity was measured in extracts from various tissues (brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis) by using a chemiluminescence assay (Tropix Inc.). Levels of -galactosidase enzymatic activity in the tissues tested were found to be comparable to background levels in nontransgenic control mice (data not shown), except for the testis tissue, which exhibited high levels of enzyme activity. As seen in Table 1, the enzyme levels in the testes of mice from groups B and C were similar to that of the previously characterized transgenic line in group A, which contained the entire 796-bp fragment. It can also be seen that levels of activity were independent of the transgene copy number. The testes of mice from transgenic lines 310, 915, and 930 exhibited the same level of -galactosidase activity as found in nontransgenic controls (Table 1, group E), presumably due to the insertion of the transgene at a chromosomal location that suppresses expression.

View this table: [in this window] [in a new window]   TABLE 1.   -Galactosidase activity in testes of transgenic mice

View larger version (66K): [in this window] [in a new window]   FIG. 3.   Expression of the -galactosidase reporter gene in the testes of transgenic mice. Testes from a nontransgenic control and the indicated transgenic lines were paraformaldehyde fixed, incubated in X-Gal for 48 h, and visualized directly (parts 1 to 4). To establish which male germ cell populations expressed the -galactosidase reporter gene, the fixed and stained testes were paraffin embedded, sectioned, counterstained with nuclear fast red, and inspected under a microscope. (A) Parts: 1, a testis from a nontransgenic mouse; 2, a testis from a 4GalT[1085/474]LacZ-346 mouse; 3, a testis from a 4GalT[793/707]LacZ-320 mouse; 4, a testis from a 4GalT[mut756/743]LacZ-374 mouse. Note that the blue staining, indicative of the expression of the -galactosidase reporter gene (LacZ) is concentrated in the seminiferous tubules. (B) Section of a seminiferous tubule obtained from the testis of a 4GalT[1085/474]LacZ-346 mouse. The purple arrows indicate the pachytene spermatocytes, the yellow arrows indicate the round spermatids, and the black arrows indicate the elongated spermatids (final magnification, ×625). (C) Section of a seminiferous tubule obtained from the testis of a 4GalT[793/707]LacZ-320 mouse (final magnification, ×625). Note that this 87-bp genomic fragment which contains the TASS-1 regulatory element is sufficient to drive male germ cell-specific expression of the reporter gene in a pattern that is comparable to that of the endogenous 4GalT-I gene.

A 457-bp core fragment spanning the 4GalT-I germ cell start site is sufficient to confer correct germ cell-specific expression in transgenic mice. Since male germ cell promoter activity was maintained in transgenic mice harboring either a 5' or a 3' deletion of the 796-bp promoter region, the third construct (4GalT[1085/628]LacZ), combining both 5' and 3' deletions, was generated and analyzed. As seen in Table 1, group D, testes of mice from two of the five lines (lines 42 and 58) showed high levels of -galactosidase enzymatic activity similar to that seen in the animals in groups A to C. Levels of -galactosidase in all of the somatic tissues analyzed were comparable to the background levels in nontransgenic control mice (data not shown). Background levels of -galactosidase activity were found in the testes of mice from the other three lines (641, 642, and 648), presumably due to the insertion of the transgene at a chromosomal location that suppresses expression.

DNase I footprinting analysis of the 457-bp 4GalT-I male germ cell promoter region. The functional analysis using transgenic mice demonstrated that a 457-bp genomic fragment spanning 1085 to 628 contains the cis elements necessary for correct male germ cell-specific expression of the transgene. Of the several motifs within this region identified by computer analysis, two putative CRE-like elements, spanning 766 to 759 and 645 to 630, had been noted (38). To directly assess whether the two CRE-like motifs and/or other sequences within the 457-bp fragment bind testis-specific nuclear proteins, DNase I footprinting assays were carried out. A restriction fragment spanning either the 1150 to 770 or the 870 to 474 sequence was end labeled on either the coding or the noncoding strand. Each probe was then incubated with 25 µg of a testis nuclear extract or an equivalent amount of BSA. Partial digestion with DNase I revealed only a single protected region spanning 756 to 734 on both the coding and noncoding strands (Fig. 4A and B, lanes 3). The sequence of the protected region is shown in Fig. 4C. To determine whether this region was also protected by nuclear proteins derived from somatic cells, probes were incubated with 25 µg of mouse L-cell nuclear extract. A footprint extending across the same sequence was observed, but only on the noncoding strand (Fig. 4B, lane 2). This observation suggests that the binding activity in the mouse L-cell nuclear extract is due to a different protein or protein complex.

View larger version (61K): [in this window] [in a new window]   FIG. 4.   Identification of a single 23-bp protected region within the 457-bp promoter region by DNase I footprinting analysis. Protected regions on the coding strand (A) and noncoding strand (B) are indicated by the black and gray bars, respectively. A DNA fragment corresponding to the region between 870 to 474 was end labeled with 32P and digested with DNase I in the presence of BSA (lanes 1) or nuclear proteins from mouse L cells (lanes 2) or testes (lanes 3). (C) Sequence of the region spanning 768 to 637. The black rectangle indicates the region protected by the testis nuclear extract, while the gray rectangle indicates the region protected by both testis and L-cell nuclear extracts. The locations of the two CRE-like motifs are shown. The male germ cell transcription start site (Gc) is located at position 727.

Neither CRE-like sequence within the 457-bp promoter region binds nuclear protein. The DNase I footprinting analysis showed that the two CRE-like motifs did not bind protein, as the region containing these motifs was not protected after incubation with either the testis (Fig. 4A and B, lanes 3) or mouse L-cell (Fig. 4A and B, lanes 2) nuclear extract. To confirm this result, two double-stranded oligomers containing either the CRE-like motif spanning 766 to 759 or 645 to 638 were synthesized (Table 2) and tested by EMSA. Both failed to bind nuclear proteins from the testis (Fig. 5, lanes 6 and 9) or mouse L cells (Fig. 5, lanes 5 and 8). As a positive control, incubation of an oligomer containing the CRE consensus sequence with either the mouse L-cell or the testis nuclear extract produced two retarded bands (Fig. 5, lanes 2 and 3). Thus, even though CRE binding factors are present in both nuclear extracts, the inability to demonstrate complex formation with either of the CRE-like motifs present in the 457-bp promoter region suggests that members of the CRE family do not play a role in the regulation of 4GalT-I expression in male germ cells.

View larger version (83K): [in this window] [in a new window]   FIG. 5.   The two CRE-like sequences within the 457-bp promoter fail to bind a nuclear protein(s) when analyzed by EMSA. Double-stranded oligomers containing the CRE consensus binding site (left panel) or the CRE-like motif spanning 766 to 759 (middle panel) or 645 to 638 (right panel) were end labeled with 32P and incubated with 10 µg of testis (lanes 3, 6, and 9) or mouse L-cell (lanes 2, 5, and 8) nuclear extract. FP designates the migration position of the free probe.

Analysis of the DNase I-protected region by EMSA. By using the information from the DNase I protection assays, we synthesized a double-stranded oligomer that corresponded to the sequence of the single footprint identified within the 457-bp core promoter region. Incubation of the mouse testis nuclear extract with this 756/734 oligomer (Table 2) resulted in the formation of one very intense retarded band (Fig. 6A, lane 2). To confirm the specificity of protein binding, we also performed EMSAs by using specific and nonspecific DNA competitors. As seen in Fig. 6B, complex formation did not occur in the presence of a 100-fold molar excess of the unlabeled 756/734 oligomer (lane 3) whereas two nonspecific oligomers had no effect on complex formation (lanes 4 and 5).

View larger version (79K): [in this window] [in a new window]   FIG. 6.   Nuclear factor(s) binding to the DNA sequence spanning 756 to 734. (A) The 756/734 oligomer was end labeled with 32P using Klenow DNA polymerase and incubated with nuclear extracts from testes (lane 2), pooled male germ cells (lane 3), or mouse L cells (lane 4). Upon a lighter exposure, the band present in lane 4 can be resolved into two bands. (B) Specificity of the complex obtained with the testis nuclear extract (lane 2) was demonstrated by addition of a 100-fold molar excess of the unlabeled 756/734 oligomer (lane 3) prior to addition of the labeled probe. Incubation with a 100-fold molar excess of a nonspecific oligomer containing the CRE-like motif spanning 766 to 759 (lane 4) or the CRE consensus motif (lane 5) did not prevent complex formation. FP indicates the migration position of the free probe.

The 756/734 oligomer binds a protein factor(s) present in a male germ cell nuclear extract. Since the testis is composed of both germ and somatic cell types, an unequivocal confirmation that the gel shift pattern seen with the testis nuclear extract is due to proteins in male germ cells can be established by preparing nuclear extracts from isolated, pooled male germ cells, in which round spermatids comprise ~50% of the cell population. Incubation of the male germ cell nuclear extract with the 756/734 oligomer gave rise to the identical band observed when the testis nuclear extract was used (Fig. 6A, compare lane 3 with lane 2). This result clearly demonstrates that the testis nuclear protein(s) that recognizes the binding site in the 756/734 oligomer is present in male germ cells.

Mutation analysis defines a 14-bp DNA binding site. The DNA binding site within the 756 to 734 sequence was determined by using a series of oligomers containing overlapping groups of 5- or 6-bp mutations (Fig. 7A). Complex formation equivalent to that seen with the 756/734 oligomer (Fig. 7B, lane 1) was only observed when oligomers mut 6 and mut 7 were used (Fig. 7B, lanes 7 and 8). This shows that the 13 bp at the 3' end of the sequence are not critical for protein binding. The other oligomers tested either abolished (mut 2 and mut 3) or greatly reduced (mut 1, mut 4, and mut 5) complex formation (Fig. 7B, lanes 2 to 6), showing that the 14-bp sequence (5'-GCCGGTTTCCTAGA-3') at the 5' end of the sequence is involved in protein binding. As anticipated, an oligomer (mut 8) in which the 14 bp were mutated by transversion also abrogated complex formation (Fig. 7B, lane 9).

View larger version (46K): [in this window] [in a new window]   FIG. 7.   Effect of mutations within the 756 to 743 region on complex formation. (A) Sequences of the wild-type and mutated (mut 1 to mut 8) double-stranded oligomers used for EMSAs. The mutated bases are in boldface. (B) Eight mutated double-stranded oligomers (lanes 2 to 9) labeled with 32P at their respective 5' ends and incubated with 10 µg of testis nuclear extract. Complex formation observed with each mutated oligomer was compared with that observed with the 756/734 oligomer (lane 1). FP designates the migration position of the free probe.

An 87-bp fragment containing the TASS-1 binding motif is sufficient to direct correct germ cell-specific 4GalT-I expression in transgenic mice. The in vitro DNA-protein binding assays established that only one DNA binding site, located 16 bp upstream of the transcriptional start site, was recognized by a protein(s) present in the male germ cell nuclear extract. If the TASS-1 binding motif is the only regulatory element required for male germ cell-specific expression of 4GalT-I, the following predictions can be made. (i) Mice harboring a transgenic construct containing this motif within a short genomic fragment should express -galactosidase in late pachytene spermatocytes and round spermatids. (ii) Mice harboring a transgenic construct in which the TASS-1 binding motif has been mutated, within the context of the original 796-bp fragment, should not express -galactosidase in these cells.

View larger version (17K): [in this window] [in a new window]   FIG. 8.   Schematic representation of the 4GalT[793/707]LacZ and 4GalT[mut756/743]LacZ constructs. The original 4GalT[1270/474]LacZ construct is shown for comparison. In the 4GalT[793/707]LacZ construct, an 87-bp fragment containing 67 bp of the genomic sequence upstream from the 4GalT-I male germ cell start site and 20 bp of the flanking downstream sequence was fused to the bacterial LacZ coding sequence. In the 4GalT[mut756/743]LacZ construct, the nucleotides within the 14-bp TASS-1 motif (spanning 756 to 743) were mutated by transversion. The positions and sequences of the original and mutated sites are shown. The solid, hatched, and open boxes represent the upstream genomic DNA, the 5'-untranslated region of the male germ cell transcript, and the LacZ coding sequence, respectively.

View this table: [in this window] [in a new window]   TABLE 3.   -Galactosidase activity in testes of transgenic mice

The TASS-1 binding motif is essential for transgene expression in late pachytene spermatocytes and round spermatids in vivo. The second prediction was tested by generating the 4GalT[mut756/743]LacZ construct (Fig. 8), in which the TASS-1 binding site was mutated by transversion within the context of the original 796-bp sequence. Seven transgenic founders were identified (Table 3, group G), and in all of the lines analyzed, -galactosidase activity was found to be 12- to 35-fold lower than that measured in the testes of mice from group A.

	DISCUSSION

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One of the most intriguing results from this study is the finding that a short 87-bp genomic fragment that flanks the 4GalT-I male germ cell-specific start site and contains a single 14-bp regulatory element (termed the TASS-1 motif) drives the in vivo expression of a reporter gene in the later stages of spermatogenesis. It is instructive to compare the relative sizes of the respective promoter regions that govern the expression of 4GalT-I in male germ cells or somatic cells. As summarized in the Introduction, DNase I footprinting and EMSAs have been used to elucidate the DNA binding motifs in an ~1.3-kb genomic fragment positioned upstream of the two somatic transcriptional start sites (Fig. 1; see references 14 and 31). When this genomic fragment was evaluated for the ability to drive somatic cell expression of a reporter gene in transgenic mice, we failed to detect expression in six different lines (unpublished data). Thus, in contrast to the 87-bp male germ cell promoter, the 4GalT-I somatic cell promoter region extends beyond the 1.3-kb genomic fragment analyzed.

While we find the compactness of the 4GalT-I male germ cell promoter remarkable, this appears to be an emerging general rule and not the exception, based on the limited subset of male germ cell promoters that have been analyzed in vivo. In addition to 4GalT-I, other examples of short, compact promoter regions that regulate male germ cell-specific gene expression include the 91-bp angiotensin-converting enzyme promoter (17), the 330-bp calmegin promoter (50), the 100-bp lactate dehydrogenase c promoter (23), the 187-bp promoter of the E1 subunit of the pyruvate dehydrogenase complex (18), the 328-bp phosphoglycerate kinase 2 (Pgk-2) promoter (33), the 116-bp proenkephalin promoter (24), and the 113-bp protamine 1 promoter (53).

Most of these male germ cell-specific promoters, including 4GalT-I, do not have a TATA binding site immediately upstream of their respective start sites. This suggests that binding of RNA polymerase II may occur via an initiator (Inr) element (YCANTYY) (19). Inspection of the sequence surrounding the 4GalT-I male germ cell start site (5'-CACTC⁺¹CATTTT-3') does reveal a potential Inr consensus motif (underlined sequence), with the requisite A nucleotide positioned near the start site (C⁺¹). Thus, the Inr and TASS-1 binding sites may be all that is required for 4GalT-I expression in late pachytene spermatocytes and round spermatids.

Does TASS-1 associate with a cofactor(s) to activate transcription of the 4GalT-I gene in male germ cells? While it is proposed that only the TASS-1 and Inr binding motifs are required for expression of 4GalT-I in male germ cells, it is possible that an additional protein(s) is needed to mediate communication between TASS-1 and the basal transcription machinery (positioned at the Inr site). In fact, the presence of a cofactor interacting directly with TASS-1 could explain the differing electrophoretic mobilities of the DNA-protein complexes formed by using the various mutated oligomers (Fig. 7). In the schematic in Fig. 9A, we propose that binding of TASS-1 to the 14-bp motif leads to the recruitment of a cofactor (Co-F), which gives rise to the intense band seen in Fig. 7B, lane 1. While mutation at the 5' end of the binding motif (Fig. 9B, mut 1) results in reduced TASS-1 binding, Co-F can still bind the complex; thus, the migration position of the complex seen with mut 1 is identical to that seen with the 14-bp sequence (Fig. 7, compare lanes 1 and 2). Mutation at the 3' end of the binding motif (Fig. 9C, mut 4 and mut 5) also results in reduced TASS-1 binding, but in this case, Co-F cannot bind the DNA-TASS-1 complex. Therefore, the migration pattern seen in Fig. 7, lanes 5 and 6, reflects the binding of TASS-1 alone. Finally, mutation of the core sequence (Fig. 9D, mut 2 and mut 3) or of the 14-bp motif (Fig. 9D, mut 8) abrogates the binding of TASS-1 and Co-F (Fig. 7, lanes 3, 4, and 9).

View larger version (21K): [in this window] [in a new window]   FIG. 9.   Model depicting the binding of TASS-1 and a putative cofactor (Co-F) to the 14-bp regulatory element. (A) High-affinity binding of TASS-1 to the 14-bp motif (open rectangle) is shown by the solid vertical lines. This binding leads to the recruitment of a putative Co-F which interacts with TASS-1 via protein-protein interactions. (B) TASS-1 binds weakly, as shown by the dashed vertical lines, to oligomer mut 1, in which the 5' end of the binding motif has been mutated (solid box). Co-F binding to DNA-bound TASS-1 is unaffected, but the complex can easily dissociate; thus, the steady-state level of the complex is greatly reduced. (C) TASS-1 binds weakly to oligomers mut 4 and mut 5, in which the 3' end of the binding motif has been mutated (solid box); however, Co-F cannot bind to the DNA-TASS-1 complex. (D) TASS-1 is unable to bind an oligomer in which the core sequence is mutated (mut 2 or mut 3) or the entire 14-bp motif is mutated (mut 8).

Transcriptional activation of the 4GalT-I male germ cell promoter is CREM independent. Recent studies have begun to provide insight into the factors responsible for the transcriptional activation of genes expressed in a stage-specific manner during spermatogenesis. The genes studied can be divided into two categories, i.e., those that are activated by CREM and those that are not. The genes regulated by CREM include those for the angiotensin-converting enzyme (17, 20, 55), calspermin (43), RT7 (9), transition protein 1 (21), and protamines 1 and 2 (13, 44, 53). As anticipated, none of these genes are expressed in the testes of CREM knockout mice (2, 28).

Is TASS-1 an Ets protein? The presence of the Ets core motif in the TASS-1 binding site suggested that TASS-1 is an Ets family member. To date, ~30 different Ets proteins have been identified; each interacts specifically with its respective cis element via a conserved DNA binding domain ~85 amino acids in length (reviewed in references 42 and 49). The 5-bp flanking sequence on either side of the 5'-GGAA-3' core appears to determine the affinity of a specific Ets protein for its target sequence, although it has been shown that different Ets proteins can bind with comparable affinity to the same target sequence in vitro (5).