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Biology of Reproduction 67, 1157-1164 (2002)
© 2002 Society for the Study of Reproduction, Inc.

H1t/GC-Box and H1t/TE1 Element Are Essential for Promoter Activity of the Testis-Specific Histone H1t Gene1

Donald C. Wilkersona, Steven A. Wolfea, and Sidney R. Grimes2,a

a Research Service (151), Overton Brooks Veterans Affairs Medical Center, Shreveport, Louisiana 71101-4295 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue-specific expression of the gene is mediated primarily through elements located within the proximal promoter. Previous work in transgenic animals identified a unique 40-base pair promoter element designated H1t/TE that is essential for spermatocyte-specific expression. The H1t/TE element contains three subelements designated TE2, GC-box, and TE1 based on in vitro footprinting and electrophoretic mobility shift assays. Because GC-box is a consensus site for binding of Sp transcription-factor family members, experiments were performed demonstrating that two Sp family members, Sp1 and Sp3, were present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids. A 95- to 105-kDa form of Sp1 is most abundant in the tissues and cell lines examined, but a 60-kDa form of Sp1 is the most abundant species in spermatocytes and early spermatids. Further examination of Sp1 and Sp3 from adult testis, primary spermatocytes, and early spermatids showed that they can bind to the H1t/TE element. In order to determine the contributions of the subelements to H1t transcription, we mutated each of them in H1t promoter luciferase reporter vectors. Mutation of the GC-box and TE1 subelement reduced expression 77% and 49%, respectively, compared with the wild-type H1t promoter in transient expression assays in a testis GC-2spd cell line that was derived from germinal cells. These studies suggest that Sp transcription factors may be involved in transcription of the H1t gene and the GC-box and the TE1 subelement are required for activation of the H1t promoter.

developmental biology, gene regulation, meiosis, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The histones are a family of basic proteins involved in packaging DNA. Eight H1 subtypes have been described [1–3], with the most recently discovered being H1oo, an oocyte-specific H1 [1]. Linker histones exhibit differences in expression patterns during development and differentiation [4] and DNA binding affinity, with H1t having the weakest binding [2]. H1 histones may also function in regulation of gene transcription [5–8].

Of the eight linker histones, H1t is unique in that it is expressed exclusively in mid to late pachytene primary spermatocytes and is found in both primary spermatocytes and early spermatids [9, 10]. A knockout of the mouse H1t gene has no discernible abnormalities and the mice were fertile, but a compensatory increase in the levels of other linker histones supports the importance of H1t [11]. Knockouts of other H1 variants showed that eliminating H1° alone or in addition to a second H1 (dual H1 knockout) had no detectable effect on the animals [12].

The H1t promoter contains sequence elements conserved among H1 family members, including a TATA-box, a CCAAT-box, a GC-box, and an AC-box [13–18]. Additional regulatory elements found in the H1t promoter are likely to contribute to cell-specific expression in testis primary spermatocytes [19–21]. This cell-type specific expression may be due in part to a 40-base pair (bp) enhancer region designated TE [22] that is located within the proximal promoter of the H1t gene and in part to silencing elements within the proximal and distal promoter [22, 23]. The TE element is composed of three subelements that are present in the following linear arrangement: TE2, GC-box, and TE1 (Fig. 3). The importance of the H1t/TE element in regulating H1t transcription is supported by transgenic animal studies. Mice carrying a rat H1t transgene and promoter with either a wild-type or mutant H1t/TE element showed that this element was essential for proper tissue-specific expression [24, 25].



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  		FIG. 3. TE mutants used in expression assays and the TE probe used in EMSA. The wild-type TE element (WT) and three TE mutants (MutTE2, MutGC, MutTE1) are shown. Specific mutations in TE2, GC-box, and TE1 subelements are bold and underlined. The positions of the full-length wild-type TE2, GC-box, and TE1 subelements used in EMSA assays are indicated by heavy bars at the bottom of the figure. Boxes are drawn around the CpG dinucleotides that are unmethylated in rat primary spermatocytes but that are methylated in rat liver. The nucleotides underlined with a dashed line (CGCCTAGGGATG) in the Wt sequence correspond to TE1 sequence that is protected by bound protein in methylation interference assays. Filled triangles represent restriction sites for EcoNI (left) and AvrII (right). These sites were used by our laboratory and another laboratory to produce the various H1t/TE mutants

The TE1 subelement has been shown by electrophoretic mobility shift assays (EMSA) to bind specifically to nuclear proteins from primary spermatocytes [20, 26]. The TE2 subelement appears to bind the same or similar testis nuclear proteins although with less affinity [22]. Competitions with either TE1 or TE2 subelements eliminate binding of testis-specific proteins to a full-length TE element oligonucleotide, indicating the similarity of these two subelements.

In vitro transcription assays have been performed with an H1t-promoter G-less cassette containing two different deletions within the H1t/TE element. One deletion, which removed a portion of the TE2 subelement and all sequences upstream of TE2, had little effect on transcription, while a second deletion, which removed a portion of the TE1 subelement and all sequences upstream of TE1 (including a GC-box), decreased transcription [27]. However, from these studies, it was not possible to determine the individual contributions of TE2, the GC-box, and TE1 site to transcription. Transient transfection assays using a ß-galactosidase reporter gene with an H1t/GC-box mutation showed some loss of promoter activity in nongerminal cells, but maximal loss of activity was seen only when a downstream GC-box was also mutated [28].

The Sp family of transcription factors includes Sp1, Sp2, Sp3, and Sp4. All four share similar structural features, including glutamine-rich activation domains located near the N-terminus and a highly conserved DNA binding domain composed of three zinc fingers located near the C-terminus [29]. Of the four members, Sp1, Sp3, and Sp4 bind preferentially to a GC-rich sequence [30, 31] while Sp2 prefers a GT-rich sequence [32]. Sp1 and Sp3 are ubiquitously expressed [31, 33], but Sp4 is found predominantly in the brain, epithelial tissues, testis, and developing teeth [31, 34]. Less is known regarding expression or function of Sp2. Sp1 is a potent activator of many genes [35–37], while Sp3 can serve as an activator [38, 39] or repressor [30, 40]. Sp4 may function as a transcriptional activator [41, 42] or repressor [43].

The molecular weight of Sp1 is 95 kDa [44, 45], but electrophoretic bands of 95 and 105 kDa are seen because of heterogeneity caused by glycosylation [46] and phosphorylation [47]. Sp1 species of 49- and 80-kDa molecular weights also have been reported. The smaller 49-kDa species, not present in mouse spermatogenic cells, results from alternative splicing, while the origin of the 80-kDa species is unknown [48]. An abundant and very small 2.4-kilobase (kb) Sp1 mRNA was found in mouse germinal cells and may contribute to Sp1 protein detected in mouse spermatids by immunohistochemical methods [33]. Sp3 consists of three predominant species. The largest is approximately 115 kDa [32, 45] and the two smaller ones are 60–70 kDa. The smaller species arise from two internal AUG codons [29, 45].

In this article, we show that Sp1 and Sp3 transcription factors are present in primary spermatocytes and early spermatids and they can bind to the H1t/TE element. We show that both the GC-box and the TE1 subelement within the H1t/TE element contribute to promoter activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Synthetic oligonucleotides were purchased from Genosys (The Woodlands, TX). Male Sprague-Dawley rats used for preparing testis extracts (including centrifugal elutriation to isolate enriched populations of testis cells) were from Harlan Sprague-Dawley (Madison, WI). The Sp1 and Sp3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Radioisotopes were from New England Nuclear (Boston, MA). The cell lines used were a mouse GC-2spd cell line derived from germinal cells that was kindly provided by José Luis Millán [49], a rat Leydig cell line obtained from American Type Culture Collection (ATCC), and a mouse C127I mammary cell line (ATCC no. CRL1616). The GC-2spd cells were grown at 32°C. All other cell lines were grown at 37°C. The secondary antibody, {alpha}-IgG-HRP, was obtained from an enhanced chemiluminescence (ECL) kit (Amersham Life Science, Inc., Arlington Heights, IL).

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays (EMSA) were performed as previously described [19, 21]. Synthetic upper (U) and lower (L) oligonucleotide pairs shown in Table 1 were annealed and used for EMSA analysis. Oligonucleotides were labeled using [{alpha}-32P]dCTP by filling with the Klenow fragment of DNA polymerase or using [{gamma}-32P]ATP for 5'-end labeling with T4 polynucleotide kinase as previously described. EMSA competitions were performed as described above with an additional 15-min binding reaction with unlabeled wild-type or mutant double stranded oligonucleotides following incubation with nonspecific competitor. Unlabeled wild-type or mutant TE subelement sequences were used as competitors at a 20-fold molar excess. EMSA supershift assays were performed as described above with an additional 1 h incubation at room temperature with antibody following the probe-binding step. Gels were dried and radioactivity was detected using a Cyclone Phosphorimager (Packard Instrument Company, Meriden, CT).


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  		TABLE 1. Oligonucleotides and PCR primers

Western Blots

Nuclear proteins were prepared by the Dignam procedure [50] from unfractionated testis cells from adult and 9-day-old rats and cell populations enriched in pachytene primary spermatocytes and early spermatids (Fig. 1) prepared by centrifugal elutriation [19, 51]. For comparison, nuclear extracts from GC-2spd germinal cells, Leydig cells, and C127I cells were also used. Proteins were loaded on a 60-mm x 80-mm SDS polyacrylamide gel composed of 5% stacker and 7.5% separating gel and electrophoresed for 1.5 h at 100 V. Proteins were electroblotted to a 0.45 µM nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Western blots were performed according to the protocol provided by Santa Cruz Biotechnology. The membrane was blocked, incubated with primary antibody diluted 1:1250, washed, then incubated with the secondary antibody {alpha}-IgG-HRP (horseradish peroxidase) diluted 1:20 000. Protein bands were detected by ECL according to the manufacturer's protocols (Amersham Life Science). Chemiluminescence was visualized by brief exposure to Kodak XAR film. Bands were recorded by scanning autoradiograms on a Hewlett Packard Scanjet 6100C/T flatbed scanner, and relative band intensities were analyzed using Sigma Gel software (SPSS, Chicago, IL).



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  		FIG. 1. Western blot analysis of Sp1 and Sp3 in rat testis cells and various cell lines. Proteins from the different sources were loaded in the amounts indicated and separated by SDS-PAGE. The lanes from left to right contained nuclear proteins from adult testis, 9-day-old testis, enriched populations of primary spermatocytes (P) and early spermatids (ES), the testis GC-2spd cell line derived from germinal cells, a Leydig cell line (L), and a C127I cell line. After electrophoresis and electroblotting of the proteins, membranes were probed with either anti-Sp1 antibodies (top panel) or anti-Sp3 (middle panel) to detect proteins. The 60-, 95-, and 105-kDa bands of Sp1 and the 60-, 70-, and 115-kDa bands of Sp3 are indicated. The lower panel shows the time of appearance of germinal cells during spermatogenesis in the rat. Enriched populations of pachytene primary spermatocytes (P) and early spermatids (ES) are prepared by centrifugation

Mutagenesis

Specific mutation of the GC-box within the H1t/TE element The GC-box mutation within the TE element was performed using the plasmid pHD1 [52]. This plasmid was digested with AvrII and EcoNI, which cut at the positions indicated by two filled triangles in Figure 3, to remove the GC-box from the histone H1t promoter. The vector was purified from an agarose gel slice with a QIAEX II kit (QIAGEN, Valencia, CA). Two synthetic oligonucleotides containing a mutation in the GC-box of the TE element (MutGC Oligos, Table 1) were annealed and ligated into the previously digested and purified pHD1 vector. Upon annealing, these oligonucleotides form AvrII and EcoNI restriction site ends, which facilitated their direct cloning into pHD1.

The resulting plasmid named pCW2A was digested with Tth111I to cut at the unique restriction site located at the ATG initiation codon of the H1t promoter, and the restriction site was filled using the Klenow fragment of DNA polymerase [53]. The resulting cut and filled plasmid was digested with KpnI, which cuts at the unique restriction site located within polylinker upstream of H1t promoter to release the promoter fragment. The promoter fragment was isolated from an agarose gel slice, purified with a QIAEX II kit, and cloned into the expression vector pGL3B (Promega, Madison, WI), which was previously digested with KpnI and SmaI and purified from an agarose gel slice. The resulting plasmid pGL3B-CW2A was digested with PstI and AvrII. The PstI and AvrII restriction sites are located 141 and 64 bp, respectively, upstream from the H1t mRNA start site. This mutant PstI and AvrII promoter fragment was used to replace the wild-type PstI and AvrII fragment in pGL3B-1866 [54] so that this GC-box mutant would have the same upstream and downstream fusion points as the TE1 and TE2 mutant expression vectors and wild-type H1t promoter-driven expression vector.

Specific mutation of the TE1 and TE2 subelements within the H1t/TE element The plasmid pGL3B1866 served as a template for generating the specific TE1 mutation within the H1t/TE element in the polymerase chain reaction (PCR). The specific mutation in the TE1 subelement was achieved with the MutTE1 primers (Table 1). The reverse primer was designed to replace the wild-type TE1 subelement (5'-CCTAGG-3') with an EcoRI restriction site (underlined in the reverse primer). Linear amplification using the reverse primer was conducted for 30 cycles using 4 µg of pGL3B1866 plasmid DNA as template with heating at 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. Following linear PCR, the forward primer was added and amplification continued for an additional 30 cycles using the same cycle parameters. The resulting PCR product containing the mutant TE1 subelement was digested with PstI and BssHII, and the released mutant promoter fragment was purified from an agarose gel slice using a QIAEX II kit and cloned into pGL3B1866, which was previously digested with PstI and BssHII and purified. The PstI and BssHII restriction sites are located 141 and 43 bp upstream from the H1t mRNA start site, respectively.

The TE2 subelement was mutated using the same general method that was used to mutate the TE1 subelement. The forward primer used in the TE1 subelement mutation was used with the MutTE2 reverse primer (Table 1). The amplified mutant promoter fragment was digested using PstI and AvrII restriction enzymes and used for subcloning. Dideoxy sequencing of constructs confirmed their sequences and the relevant mutations are shown in Figure 3.

Transient transfection assays Transient transfections were performed as previously described [34] except that each subconfluent dish of GC-2spd cells was transfected using 20 µg of Lipofectamine (Invitrogen, Carlsbad, CA) and 2 µg of expression vector. Transfections were performed in triplicate to correct for slight variations in transfection efficiency and cell density.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sp1 and Sp3 Are Present in Primary Spermatocytes> and Early Spermatids

Previous work in our laboratory demonstrated the importance of the H1t/TE element for tissue-specific expression of the histone H1t gene [22]. Studies presented in this article were designed to examine the importance of the GC-box and TE1 and TE2 subelements for H1t gene expression. The GC-box within the H1t/TE element has a consensus sequence for binding members of the Sp family of transcription factors. In preliminary studies, Sp4 was not detected in primary spermatocytes; therefore, the focus of this article was on the Sp1 and Sp3 transcription factors.

If Sp1 or Sp3 are involved in H1t gene transcription, then these factors should be present in pachytene primary spermatocytes where the H1t gene is expressed (Fig. 1, bottom panel). Western blot analyses allowed detection of Sp1 and Sp3 in nuclear extract from whole testis and enriched populations of pachytene primary spermatocytes and early spermatids (Fig. 1). For comparison, nuclear extracts from testes of sexually immature 9-day-old rats, the testis GC-2spd germinal cell line, a rat Leydig cell line, and a mouse C127I cell line were examined. The blots revealed that the predominant 95- and 105-kDa Sp1 bands were present in all nuclear extracts assayed, although the levels varied (Fig. 1, upper panel). The three predominant 60-, 70-, and 115-kDa Sp3 bands were also present in all extracts assayed (Fig. 1, middle panel). Although visible in nuclear proteins from cells enriched in primary spermatocytes (P) and early spermatids (ES), Sp1 and Sp3 levels were low. During spermatogenesis, there is a drop in intensities of the 95- and 105-kDa Sp1 bands and 60-, 70-, and 115-kDa Sp3 bands during the transition from the early germinal cells present in testes of 9-day-old rats (9DO) to the primary spermatocyte stage (P) present only in more mature rats. A second drop in band intensities is observed during the transition from early spermatids to late spermatids (data not shown).

The levels of the 95- and 105-kDa Sp1 proteins in spermatocytes are 33% and 49% of levels in adult testis, and the levels in early spermatids are 74% and 76% of the levels in adult testis. In GC-2spd cells, these bands are approximately 201% and 241% of the levels in adult testis. Interestingly, the testes from 9-day-old rats had very high Sp1 levels. The levels of the two testis Sp1 bands in 9-day-old rats are 11.5-fold and 10-fold higher than the levels in adult testis. These sexually immature rats do not contain spermatocytes and H1t mRNA is not present [26]. The levels of the 60-, 70-, and 115-kDa Sp3 proteins follow the same general pattern. These bands in spermatocytes are 26%, 39%, and 33% of the levels in adult testis, and the levels in early spermatids are 29%, 46%, and 42% of the levels in adult testis. These bands in GC-2spd cells are 125%, 165%, and 124% of the levels in adult testis. In testes of 9-day-old rats, they are 11-, 12-, and 7-fold higher than in testes from adults. The Sp1 electrophoretic band intensities change from the prominent 95- and 105-kDa bands in testes of 9-day-old and adult rats to a predominant band of approximately 60 kDa in pachytene spermatocytes and early spermatids (Fig. 1, top panel). The ratios of the 60-kDa Sp1 band to the 105-kDa band in spermatocytes and early spermatids are 1.86 and 1.6, respectively, while the ratio in testes from 9-day-old rats is 0.82. The 60-kDa band was seen in preparations from spermatocytes, early spermatids, testes from immature and adult rats, and at low levels in the cell lines examined. It is likely that this band in adult testis is due largely to contributions from spermatocytes and early spermatids.

Testis Sp1 and Sp3 Are Functional and Can Bind> to the Histone H1t GC-Box

After demonstrating that Sp1 and Sp3 were present in nuclear extracts from rat testis primary spermatocytes and early spermatids, we conducted experiments to test whether they could bind to the GC-box within the H1t/TE element (Fig. 2). Initially, we examined the binding of testis Sp1 and Sp3 to the Santa Cruz Sp consensus sequence using antibodies specific to these factors to supershift EMSA bands (Fig. 2A). In the first lane showing a binding reaction with testis nuclear extracts in the absence of antibodies, two low-mobility bands are formed corresponding to Sp1 and Sp3. Lanes 2 and 3 show that antibodies against Sp1 or Sp3 gave supershifted bands (labeled Sp1ss and Sp3ss), indicating that both the Sp1 and Sp3 factors found in these extracts bind to the probe and that commercial antibodies recognized these factors. We also wanted to confirm the binding of Sp1 and Sp3 found in the GC-2spd cells to the same probe. In Figure 2B, lane 1 shows binding in the absence of antibodies. Lanes 2 and 3 confirm that the antibodies specifically supershifted the Sp1 and Sp3 bands.



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  		FIG. 2. Electrophoretic mobility shift assays (EMSA) to detect binding of Sp factors. A) EMSA supershift showing the binding of 15 µg of nuclear extract prepared from adult testis cells to an Sp consensus probe (Table 1) in the absence and presence of Sp antibodies. Lane 1 shows binding without antibody, lane 2 shows binding with 6 µg of anti-Sp1 antibody, and lane 3 shows binding with 6 µg of anti-Sp3 antibody. The bands corresponding to Sp1 and Sp3 binding and the supershifted bands (labeled Sp1ss and Sp3ss) are indicated. B) Binding with 10 µg of nuclear extract prepared from the GC-2spd cell line and the Santa Cruz Sp consensus probe. C) Binding using the 40-bp wild-type H1t/TE element (WT) shown in Figure 3 and Table 1 and nuclear extracts prepared from enriched populations of early spermatids (ES) (lanes 1–3, 27 µg/lane), pachytene primary spermatocytes (P) (lanes 4–6, 27 µg/lane), and adult testis (lanes 7–8, 25 µg/lane). D) This panel shows protein binding using the GC-box probe (Table 1) and 10 µg of GC-2spd cell nuclear extracts

Because the H1t gene is expressed in pachytene primary spermatocytes, supershift assays were performed using nuclear extracts prepared from different testis cell types and a 40-bp H1t/TE probe. This TE sequence (Table 1 and Fig. 3) is found within the proximal promoter of the H1t gene and contains an Sp binding site (GC-box). Nuclear extracts prepared from cell populations enriched in early spermatids and in pachytene primary spermatocytes and total testis cells all contain Sp1 and Sp3 that can bind to the GC-box within the H1t/TE element (Fig. 2C). Lanes 1 and 4 show a low-mobility band (labeled Sp1 and Sp3) that is formed with this probe and extracts from early spermatids (ES) and pachytene primary spermatocytes (P). Antibodies against Sp1 or Sp3 bind and produce supershifted bands. We have previously shown that sequences flanking the GC-box within the TE element are bound by testis-specific proteins, and it was possible that these proteins might exclude Sp factor binding. However, both Sp1 and Sp3 bind to the GC-box. Figure 2D shows that Sp1 and Sp3 in nuclear extracts from the GC-2spd cell line can bind to the shorter GC-box subelement (within the 40-bp H1t/TE element) and can be supershifted by Sp1 and Sp3 antibodies.

Nuclear Protein Binding to the Mutant H1t/TE Subelement Sequences Is Reduced or Altered

The 40-bp TE element of the H1t proximal promoter can be divided into three subelements: TE2 subelement, GC-box, and TE1 subelement. To determine the individual contributions of each of these subelements to promoter activity, they were individually mutated as described in Materials and Methods, and the effects of these mutations were assayed by gel shift assays and transient transfection. The wild-type 40-bp TE sequence of the H1t proximal promoter (labeled Wt) is shown along with mutTE2, mutGC, and mutTE1 (Fig. 3). The underlined sequences indicate where wild-type promoter sequence was replaced with an EcoRI restriction site. Once the mutations were confirmed by sequencing, they were blasted against a number of transcription factor databases and assayed by EMSA competitions. Analysis, using TESS (Transcription Element Search Software on the worldwide web, URL: http://www.cbil.upenn.edu/tess), showed that no transcriptional repressor binding sites were created in the mutant subelements.

To confirm the loss or alteration of protein binding to the mutant sequences, EMSA competitions were performed (Fig. 4). Both wild-type and mutant TE2 sequences bind proteins from GC-2spd cells, but competitions show that the proteins that bind to the mutant sequence differ from proteins that bind to the wild-type sequence (Fig. 4A, both panels). Lanes are marked for probe alone (P), no competitor (NC), competition with self (S), and competition with nonself (NS). When wild-type TE2 sequence is used as a probe (Fig. 4A, left panel), nonself competition is with unlabeled mutant sequence. When mutant TE2 sequence is used as a probe (Fig. 4A, right panel), non-self-competition is with unlabeled wild-type sequence. The mutant TE2 binds testis nuclear extracts very weakly compared with the wild-type sequence (Fig. 4D, both panels). This suggests that different proteins are binding to the wild-type and mutant TE2 probes.



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  		FIG. 4. EMSA competitions to examine protein binding to TE subelement mutants. Gel shift assays were conducted using mutant and wild-type TE subelement probes and nuclear extracts from GC-2spd cells (10 µg/lane) and adult testis (15 µg/lane). Lanes are labeled P (probe alone), NC (no competitor), S (self competitor), and NS (non-self-competitor). Oligonucleotides used as probes and competitors are shown in Table 1. Nuclear protein binding to wild-type and mutant TE2 probes are shown (A, GC-2spd cells; D, testis). Competitions were performed with a 20-fold molar excess of nonlabeled double-stranded wild-type TE2 or mutant TE2 DNA fragments. Binding of nuclear proteins to wild-type and mutant GC-box probes are shown (B, GC-2spd cells; E, testis). Note that the mutant GC-box does not bind Sp1 or Sp3. Binding of nuclear proteins to wild-type and mutant TE1 probes are shown (C, GC-2spd cells; F, testis). The mobilities of bands formed with GC2 extracts using TE2 and TE1 probes that are marked with an open triangle (A and C) are similar to mobilities of bands formed with testis extracts marked with an open rectangle (D and F)

We next assayed the mutant GC-box probe and found that neither Sp1 or Sp3 from either GC-2spd cells or testis bound to this mutant (Fig. 4, B and E). Sp1 and Sp3 only bound to the wild-type GC-box probe. Both the wild-type and mutant GC-box probes produced a high mobility nonspecific band.

Wild-type and mutant TE1 probes also bind proteins from GC-2spd cells (Fig. 4C, right panel), but competitions show that the proteins that bind to the mutant sequence differ from proteins that bind to the wild-type sequence (Fig. 4C, both panels). The mutant probe binds poorly to testis proteins and forms only the higher mobility nonspecific band (Fig. 4F, right panel). The mutant does not form the testis-specific low-mobility band marked with a filled triangle.

H1t/GC-Box and the TE1 Subelement Are Important> for Transcription in Testis GC-2spd Cells

In the previous section, we demonstrated that protein binding to each of the mutant subelements was eliminated or altered. We next wanted to determine whether the GC-box and/or the TE1 and TE2 subelements have a significant role in regulating transcription of the H1t gene. To examine transcription, we utilized the three H1t promoter mutants designated MutTE1, MutGC, and MutTE2 (Fig. 3 and Table 1) in transient transfection assays. The results of these assays are shown in Figure 5. There was a 77% drop in activity when comparing the wild-type promoter (Wt H1t) with the promoter containing the GC-box mutation (MutGC). There was a 49% drop in activity with the TE1 promoter mutation compared with wild-type H1t promoter, while there was little change observed with the TE2 mutant. Assays of the GC-box and TE1 mutants in other cell lines also showed a similar loss of activity (data not shown).



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  		FIG. 5. Transient expression assay of H1t promoter mutants in testis GC-2spd cells. Cells were transfected with the expression vectors shown on the x-axis. From left to right, these vectors are the promoterless pGL3 basic (Basic), the wild-type testis-specific histone H1t promoter vector (Wt H1t), the H1t promoter vector with a GC-box mutation (MutGC), the H1t promoter vector with a TE1 mutation (MutTE1), and the H1t promoter vector with a TE2 mutation (MutTE2). All vectors have the same upstream and downstream fusion points in the pGL3B expression vector. The three mutant vectors have the exact H1t promoter sequence as wild type (Wt H1t) except for those nucleotides indicated in Figure 3. The columns represent the means of triplicate samples and the error bars indicate the standard error of the mean


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The H1t gene has a GC-box located within the H1t/TE promoter element, a region known to be important for tissue-specific expression of the gene as shown in transgenic animals and for promoter activity in transient expression assays in nongerminal cell lines [22, 24, 25, 27, 28]. In a previous study, we were able to detect Sp1 in testis nuclear extracts using an Sp1 consensus probe, but we did not detect Sp1 in nuclear extracts from enriched populations of primary spermatocytes [22]. Other testis-specific genes including the LDHc have a GC-rich element that appears to be important for activity [55]. Although studies from other laboratories reported the presence of certain Sp transcription factors in testis using immunohistochemical staining [33] and EMSA supershift assays [55], the studies did not specifically address the presence of Sp factors in pachytene primary spermatocytes, the cell type where the H1t gene is transcribed.

More recently, we demonstrated hypomethylation of CpG dinucleotides located within the H1t proximal promoter in rat primary spermatocytes and hypermethylation of the same CpG dinucleotides in rat liver [56]. Two of these CpG dinucleotides are located within the H1t/TE element, one within the core of the GC-box and one within the TE1 subelement as indicated in Figure 3 by the boxed dinucleotides. These results suggest that methylation of this promoter region may contribute to silencing of the gene in nonexpressing cells. Other testis-specific genes such as LDHc [57] also display hypomethylation in testis and hypermethylation in nongerminal cells, suggesting that methylation may contribute in a more general way to silencing many testis-specific genes in nongerminal cells. If this is the case, then these results imply even greater importance for this TE region of the H1t promoter in transcriptional regulation.

The H1t/TE element was shown by our laboratory and others to be important for promoter activity, but the relative contributions of the GC-box and TE1 and TE2 subelements to promoter activity had not been determined. Filled arrow heads in Figure 3 indicate the positions of two deletion mutants that were produced to study this promoter region in another laboratory [27]. Because these sites were located within the TE2 subelement and the TE1 subelement, individual contributions of the GC-box, the TE1 subelement, or the TE2 subelement to H1t promoter activity could not be determined. This point is important because our laboratory has found that the TE1 and TE2 subelements bind testis-specific nuclear proteins [22, 26]. Furthermore, methylation interference experiments with the rat [58] and human [22] TE1 subelements revealed specific residues protected by bound testis nuclear proteins. The entire protected region is underlined with a dashed line in Figure 3, and in the forward DNA strand, specific protected G and A residues are marked with dots above the residue. Although mutagenesis of the GC-box within the TE element decreased H1t promoter activity in nongerminal cells, maximal activity loss occurred only with removal of a downstream repressor [28].

For the reasons described above, we felt that it was important to examine Sp1 and Sp3 in testis cells and to measure their binding activities. We also wanted to examine H1t promoter activity using specific mutants of the TE2 subelement, the GC-box, and the TE1 subelement. Our data clearly show for the first time by Western blot analysis the presence of Sp1 and Sp3 in primary spermatocytes and early spermatids as well as in other tissues and cell lines examined. We also show for the first time a shift from the 95- to 105-kDa Sp1 species to a 60-kDa species in spermatocytes and early spermatids. The peptide forming this low molecular weight species needs further characterization. It is possible that a specific Sp1 mRNA such as the abundant small 2.4-kb Sp1 mRNA found in germinal cells encodes this small Sp1 species [33].

We were able to show that Sp1 and Sp3 from adult testis, primary spermatocytes, and early spermatids bind to the H1t/TE probe (Fig. 2C). Nuclear proteins, some probably unique to spermatocytes and early spermatids and that have been shown to bind to the TE2 and TE1 subelements that flank the GC-box, do not block Sp factor binding even though Sp1 and Sp3 levels are low in these cell types. There are also other GC-box and GT-box binding proteins such as BTEB1 (basic transcription element binding protein) [59] and TIEG1 and TIEG2 (TGFß-inducible early protein genes 1 and 2) [60, 61] that share characteristics with Sp transcription factors and have nearly identical binding properties [61, 62]. It is possible that germinal cell-specific Sp isoforms or similar Sp-like proteins function during spermatogenesis. To determine whether such proteins regulate H1t or other gene transcription during spermatogenesis requires further experimentation.

Relative contributions of the H1t/TE subelements to H1t promoter activity were explored by conducting mutagenesis experiments. Specific mutations in the TE2 subelement, the GC-box, and the TE1 subelement were constructed and tested by EMSA for protein binding affinity and in transient expression assays in the testis GC-2spd cell line for promoter activity (Figs. 4 and 5). We show for the first time that the GC-box is critical for H1t promoter activity in a testis cell line (Fig. 5). The loss in promoter activity is correlated with a loss in binding of Sp1 or Sp3 to the mutant GC-box. An equally important novel observation is that the TE1 subelement is critical for promoter activity (Fig. 5). The loss in promoter activity is correlated with a change in proteins that bind the TE1 element (Fig. 4), and it is likely that a transcriptional activator fails to bind to the TE1 mutant. It is also possible, although unlikely, that a repressor binds to the mutant.

Although a cell line derived from germinal cells was used for these transient expression assays, there are important differences in this cell line and normal germinal cells. For example, we have not been able to see H1t gene expression in the GC-2spd line, although basal expression below the level of detection by Northern blots is possible. Nevertheless, this GC-2spd cell line and several nongerminal cell lines have proven to be useful for transient expression assays to examine H1t promoter elements that enhance or silence transcription.

In summary, Sp1 and Sp3 are present in primary spermatocytes as well as other germinal cell types and nongerminal cells. The levels of Sp1 and Sp3 are low in primary spermatocytes, but we have shown that they can bind to the H1t GC-box located within the TE element. We have also shown that both the GC-box and the TE1 subelement are important for enhanced H1t promoter activity in transient expression assays. Therefore, it is possible that ubiquitous transcription factors such as Sp1 and Sp3 contribute to transcriptional regulation of the H1t gene, and it seems very likely that other factors such as the TE binding proteins are involved in directing tissue-specific transcription of this gene. A more definitive answer to the question about the importance of the H1t promoter elements in activating transcription of the H1t gene in spermatocytes and silencing of the gene in early spermatids and in nongerminal cells may require use of transgenic mice bearing transgenes with specific mutations within these promoter sites.


   ACKNOWLEDGMENTS
 
We thank José Luis Millán for the kind gift of GC-2spd cells and Jane vanWert for reading the manuscript.


   FOOTNOTES
 
1 This research was supported by grant HD29381 from the National Institutes of Health and a Merit Review grant from the Department of Veterans Affairs to S.R.G. Back

2 Correspondence: Sidney R. Grimes, Medical Research Service (151), Overton Brooks Veterans Affairs Medical Center, 510 E. Stoner Avenue, Shreveport, LA 71101-4295; FAX: 318 429 5734; srgrimes{at}prysm.net Back

Received: 22 January 2002.

First decision: 12 February 2002.

Accepted: 6 May 2002.


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 MATERIALS AND METHODS
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 DISCUSSION
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