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a Medical Research Service (151), Overton Brooks Veterans Administration Medical Center, Shreveport, Louisiana 71101-4295
b Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Promoter comparisons reveal that the proximal promoter of the testis-specific histone H1t gene contains all of the consensus elements common to the somatic H1 promoters including an AC box, a GC box, a CCAAT box, and a TATA box [9–14]. Two elements, the AC box and the CCAAT box, have been examined in detail and have been shown to have a role in cell cycle control of enhanced histone H1 gene transcription during the S-phase of the mitotic cell cycle [12, 15–18]. The proximal promoter of the testis-specific histone H1t contains an additional enhancer element designated TE that binds factors responsible for activation of transcription in the testis [19–23]. The H1t promoter also contains a poly C element termed the GC box 2 [24, 25] that contributes to repression of H1t gene transcription in somatic cells. Since there are common elements within the proximal promoters of the 7 H1 histone genes, it has been assumed that factors that bind to elements farther upstream may modulate histone H1t gene transcription. This may be accomplished by interaction of transcriptional factors that are bound to the upstream region with transcriptional factors that are bound to the proximal promoter to repress transcription in actively dividing somatic cells.
Studies with transgenic animals have revealed regions of DNA that are needed for proper testis-specific transcription. All sequences necessary and sufficient for proper developmental and spermatocyte-specific transcription of the H1t gene are present within a genomic fragment containing 2.5 kilobases (kb) of upstream and 3.8 kb of downstream flanking DNA [26]. Results of experiments with transgenic mice containing 141 nucleotides of the rat H1t promoter fused to a lacZ reporter gene show that the proximal promoter containing the TE enhancer element can activate transcription within the testis [27]. However, transcription of the H1t gene has been examined primarily in adult animals within tissues that are not actively proliferating and undergoing mitosis [26, 27]. The 141-bp H1t promoter fusion has not been examined in immature animals or in actively proliferating tissues.
Studies with proliferating cell lines have revealed several upstream elements that may contribute to control of testis-specific histone H1t gene transcription [1, 28]. H1t transcription as detected with reporter constructs driven by the H1t promoter is low compared to transcription from somatic H1 constructs in cells undergoing mitosis [28]. Low transcriptional activity seen with the H1t promoter may be indicative of the presence of silencer elements and of significant differences within the proximal promoter that render H1t less efficient in interacting with transcriptional factors utilized by the somatic variants. Prior work suggested that regulation of H1t gene transcription during the mitotic cell cycle may be influenced by sequences upstream from the H1t proximal promoter [1, 28]. A key question that needs to be addressed is whether there are active mechanisms that silence transcription of the H1t gene or maintain its transcription at a low level in somatic cells. Therefore, examination of DNA upstream and downstream from the H1t gene as well as within the structural gene may lead to identification of elements that are involved in the silencing of H1t gene transcription in nonexpressing cells.
To determine the contribution of the 5' flanking region of the proximal promoter to transcriptional silencing of the H1t gene, we prepared a set of reporter vectors to examine H1t promoter activity in transfected somatic cells. In results presented here, a region of the H1t promoter located between 948 to 875 nucleotides upstream from the H1t transcriptional start site acted as a silencer of H1t gene transcription in mouse C127I cells. When this element was placed upstream from the rat somatic H1d proximal promoter in a luciferase reporter plasmid construct, transcription was reduced by approximately 50%.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methods for construction of recombinant DNA followed standard procedures [29]. Plasmid DNA was harvested from cultures of Escherichia coli strain HB101 using alkaline lysis [30] followed by passage through a Wizard DNA clean-up minicolumn (Promega, Madison, WI). Plasmid DNA was quantitated by reading absorbance at 260 nm on a Beckman DU 64 spectrophotometer (Beckman Instruments, Palo Alto, CA).
Construction of Histone H1t-Promoted Luciferase Reporter Plasmids
The plasmid pPS5 served as the source for the histone H1t promoter fragments used in the construction of the luciferase-based reporter vectors. The plasmid pPS5 was constructed by digesting the plasmid pPS3 [31] with EcoRI and HindIII to release a 2.77-kb EcoRI-HindIII fragment that contained approximately one half of the rat H1t structural gene and 2.4 kb of DNA upstream from the initiation codon. This fragment was isolated and cloned into pUC 9 (EcoRI and HindIII sites) to yield plasmid pPS5. Initially, pPS5 was digested with Tth111 I, which cuts 5' of the "A" of the H1t initiation codon; this was followed by a filling reaction with the Klenow fragment of DNA polymerase to generate a blunt end. A KpnI digest was performed to release a 1543-bp fragment that was subcloned into the KpnI-SmaI-digested pGL3 Basic luciferase plasmid (Promega) to generate pGL3B KpnI. Starting with the plasmid pGL3B KpnI, several more H1t-promoted luciferase reporter plasmids were isolated using convenient restriction sites (StuI, PvuII, AccI, XbaI, and PstI) within the H1t promoter and a HindIII located downstream (3') of the Tth111I/SmaI fusion site within pGL3B (Fig. 1). Exonuclease III digestion of pGL3B KpnI (digested with KpnI and XbaI) followed by S1 nuclease digestion was used to generate pGL3B 1866. The plasmid pGL3B EcoRI was generated by a two-step procedure. First, in order to obtain a region upstream from the KpnI site, a 1568-bp KpnI-HindIII restriction fragment, obtained from pGL3B KpnI, was subcloned into KpnI-HindIII-digested pPS5 to generate plasmid pUC9 EcoRI-Tth111I/SmaI. Second, pUC9 EcoRI-Tth111I/SmaI was digested with EcoRI, filled with Klenow, and digested with HindIII to release a 2480-bp fragment that was subcloned into the SmaI and HindIII sites of pGL3 Basic to generate pGL3B EcoRI. Construction of all H1t-promoted reporter vectors was performed in the above fashion to maintain identical fusion points to the luciferase reporter.
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Construction of the Somatic Histone H1d-Promoted Luciferase Reporter Plasmid
The rat H1d-promoted luciferase reporter vector was constructed using the pGL3 Basic luciferase plasmid and a polymerase chain reaction (PCR)-generated fragment using the known rat H1d sequence [32]. A pair of primers was selected using the Oligo program (National Biosciences, Inc., Plymouth, MN), which would amplify a 576-bp DNA fragment that started 233 nucleotides upstream from the ATG and extended 336 nucleotides into the coding region of the H1d gene. The upstream primer 5'-GACGCGTACTGGGCAATTCTATGTGGGGAAT-3' was designed so that it contained a MluI site (ACGCGT) at the 5' end to facilitate cloning, and the downstream primer sequence was 5'-AGCCGCCTTCTTGTTGAGTTTGAA-3'. Amplification was conducted for 30 cycles using 1 µg of rat testis genomic DNA, with heating at 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The resulting PCR product was digested with MluI (engineered into 5' primer) and XhoI (positioned in the untranslated H1d leader region 27 bp 3' of the transcriptional initiation site) and cloned into the same sites in the pGL3 Basic plasmid.
To have a convenient source of DNA for generating plasmids and probes from the silencer/enhancer region, two plasmids containing a 285-bp MaeIII fragment were constructed. Briefly, pPS5 was digested with StuI and PstI to release a 955-bp fragment that was recovered from low-melting temperature (LMT) agarose using an ELUTIP-D column (Schleicher & Schuell, Keene, NH). The 955-bp StuI-PstI fragment was digested with MaeIII and treated with Klenow to fill the ends of the DNA fragments. The DNA was electrophoresed through a 1% LMT agarose gel, and a 285-bp fragment was recovered using hot phenol extraction [6]. The 285-bp blunt-ended fragment was cloned into the SmaI site of pUC 19 to generate pM3F (pUC 19/MaeIII forward orientation) and pM3R (pUC 19/MaeIII reverse orientation). The plasmid pGL3B H1d/Silencer was generated by digesting pM3R with AccI and then treatment with Klenow to fill the 5' overhangs. After electrophoresis through 1% LMT agarose, the 163-bp blunt-ended fragment was recovered using hot phenol and cloned into the MluI site (Klenow-filled) of pGL3B H1d (5' of the H1d proximal promoter). All of the preceding plasmid constructs were sequenced to confirm orientation and proper fusion points.
Mammalian Cell Lines
Mouse C127I mammary cells and HeLa cells, obtained from American Type Culture Collection, were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified incubator at 37°C with an atmosphere of 5% CO2.
Transient Transfections
Cells were transfected using LipofectAMINE (Gibco BRL, Gaithersburg, MD) as described by the supplier's protocol. Mouse C127I cells from a single cell suspension were plated on 60-mm dishes and grown to a density of 40–60% before transfection. Transfections were performed in triplicate to help control for variation in cell number and culture conditions. Cells in serum-free medium were co-transfected using 2 µg of a specific plasmid construct plus 200 ng of pRL TK complexed with 20 µl of LipofectAMINE per dish. The LipofectAMINE/DNA complexes were incubated with cells 5 h before feeding with an equal volume of DMEM containing 20% fetal bovine serum. Lysates were made from transfected cells 48 h after feeding.
Luciferase Assays
Cell lysates were prepared and Luciferase assays were performed using slight modifications of the Dual-Luciferase reporter assay system protocols manual as supplied by Promega. After transfection, the growth medium was removed, and 4 ml of PBS was added to each 60-mm dish of cells. The dishes were gently swirled to wash the cell surfaces, the rinse solution was removed, and 400 µl of single-strength passive lysis buffer (Promega) was added to each dish. The dishes were incubated for 15 min at room temperature before the cell lysates were harvested by scraping of the bottom of the dishes with disposable plastic scrapers. Each lysate was pipetted several times to obtain a homogenous solution and was transferred to a microfuge tube. The lysates were then cleared by centrifugation in the microfuge at 4°C for 1–2 min. The lysates were transferred to fresh tubes and stored at -70°C.
Dual luciferase assays were performed in a Beckman LS6000SC scintillation counter with the coincidence counter disabled. The firefly luciferase and the Renilla luciferase assays were performed manually in one reaction tube. The firefly luciferase activity and Renilla luciferase activity were sequentially measured for 2 min each essentially as directed by the protocol set forth for manual luminometers in the Dual-Luciferase reporter assay system technical manual. Protein determinations were made spectrophotometrically using a program contained in a Soft-Pac module on a Beckman DU series 64 spectrophotometer based upon the Warburg and Christian coefficients [33].
Probes
Radiolabeled DNA for DNase I footprint analysis was produced by PCR amplification from a plasmid template. Two synthetic oligonucleotides were purchased from Genosys Biotechnologies, Inc. (The Woodlands, TX) and used for amplification, a 17-mer for the upper strand (5'-CAAGTTGCCACCATGCC-3') corresponding to nucleotides 967–951 upstream from the rat H1t transcription initiation site and a 21-mer for the lower strand (5'-CCACATTACAACAGCTTCCAA-3') corresponding to nucleotides 761–741 upstream from the rat transcription initiation site. Before PCR amplification, the oligonucleotide corresponding to the strand to be footprinted was end-labeled with [
-32P]ATP using T4 polynucleotide kinase [29]. PCR amplification was performed with AmpliTaq DNA polymerase and 200 ng of template as described [26] for 30 cycles at 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min. The resulting 227-bp product was purified by electrophoresis through a 0.7% LMT agarose gel and recovered using a hot phenol procedure [6].
DNase I Footprint Analysis
Nuclear extracts were prepared from crude nuclei derived from mouse C127I cells as previously described [23,34]. DNase I footprinting reactions were carried out in a total volume of 20 µl generally following published procedures [35]. C127I nuclear extract containing 25–30 µg protein was incubated with nonspecific competitor DNA (2 µg poly dI-dC [Pharmacia Biotech, Piscataway, NJ]) and buffer D (20 mM Hepes, pH 7.9, 100 mM KCl, 0.2 mM EDTA, and 20% glycerol) in a total volume of 16 µl at 4°C for 15 min. After incubation, 2–6 ng of probe end-labeled on only one strand was added along with 2 µl of 10-strength footprinting buffer (250 mM HEPES, pH 7.6, 50 mM MgCl2, and 340 mM KCl), bringing the volume to 19 µl. Binding was carried out for 30 min at 4°C followed by DNase I digestion for 1 min using 0.5–2 Kunitz units of DNase I at the same temperature. The amount of DNase I was adjusted to achieve a ladder for each extract. Reactions were stopped by adding of 2 µl of 100 mM EDTA followed by two phenol:chloroform (1:1, v:v) extractions. The DNA in each sample was precipitated by addition of 2.5 volumes of 95% ethanol and incubation at -20°C for 1 h. DNA was recovered by centrifugation at 16 000 x g at 4°C for 30 min. The resulting DNA was dried, resuspended in 3 µl of loading solution (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanole), and heated to 90°C for 3 min before loading on a denaturing 6% polyacrylamide sequencing gel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies using transient transfection of cell lines performed with histone H1t-promoted reporters showed that both proximal and distal elements contribute to control of transcription [1, 28]. Transcription of reporter genes driven by the H1t proximal promoter in transiently transfected cells was comparatively low with respect to somatic histone H1 variant transcription [28] and exhibited a cyclic pattern of transcription similar to the pattern of somatic histone H1 transcription [36]. Regions upstream from the proximal promoter have been reported to enhance transcription [1,28] or reduce transcription [1, 28]. One upstream element, a TG box located between 521 and 305 bp upstream from the mouse H1t transcriptional initiation site, has been reported to elevate H1t transcription in transiently transfected mouse testicular cells [1].
To localize and study regulatory elements positioned upstream from the histone proximal promoter, a set of eight luciferase-based transcription expression vectors was constructed. This set of reporter vectors contained from 141 bp to 2385 bp of DNA upstream from the rat histone H1t transcriptional initiation site (Fig. 1). These reporter constructs were used in transient transfections of mouse C127I cells to test for any upstream sequences that would silence or enhance H1t-promoted transcription in nongerminal cells. Additionally, a 173-bp promoter from the rat histone H1d gene, a somatic histone variant, was used to construct a luciferase reporter vector as a positive control and for comparative purposes (Fig. 1).
Results from the transient transfections of the C127I cell line can be seen in Figure 2. A general trend observed in these experiments was that stepwise deletion of the histone H1t promoter led to progressively higher levels of expression (Fig. 2). The longest construct, pGL3B EcoRI, which contains 2385 nucleotides of sequence upstream from the H1t transcriptional start site, exhibited low luciferase activity that was indistinguishable from the promoterless pGL3 Basic plasmid. Removal of DNA to the KpnI site (at -1472) increased expression 4.7-fold (Fig. 2B). A 2.3-fold increase was seen with the deletion of the next 377 bp of DNA to the StuI site at -1095. No change in expression was seen with the removal of DNA to the PvuII site at -948. Maximal expression was detected with the pGL3B AccI construct, which is 875 nucleotides upstream from the H1t initiation site (Figs. 1, 2A, and 3). Expression observed with the pGL3B AccI construct was 44-fold the basal activity observed with the pGL3B EcoRI vector (Fig. 2B).
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Deletion of the next 95 nucleotides downstream from the AccI site at -875 (as observed with pGL3B XbaI) reduced the level of expression 4-fold (Figs. 2 and 4). The level of expression seen with pGL3B XbaI was similar to that seen with the constructs pGL3 StuI at -1095 and pGL3 PvuII at -948. Stepwise deletions from -780 (pGL3B XbaI) to -520 (pGL3B 1866) and to -141 (pGL3B PstI) led to 2.2- and 1.3-fold increases in expression, respectively. Expression from the shortest promoter fragment tested (pGL3B PstI) was 71% of the maximum level observed with pGL3B AccI and 31-fold higher than the pGL3B EcoRI construct, which has the longest H1t promoter fragment.
Upstream Silencer Element Is Conserved in Mouse and Rat H1t Promoters
To identify any sequence motifs that could potentially be the silencer, a sequence alignment was performed comparing the 172-bp region from PvuII (-948) to XbaI (-780) of the rat to the mouse promoter (Fig. 3). Alignment was performed using a software program named ALIGN [37]. The 172-bp region from the PvuII site to the XbaI site of the rat sequence exhibited 82.6% nucleotide identity. Alignment of the 73-bp silencer region from PvuII (-948) to the AccI (-875) revealed that rat and mouse have 87.7% nucleotide identity within this region.
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H1t Upstream Silencer Represses Transcription by the Somatic H1d Promoter
Since a 3.9-fold increase to maximal levels in expression was observed upon deletion of DNA between -948 and -875 (Figs. 2 and 3), experiments were undertaken to determine whether the 73-bp region could repress activity of another histone promoter. A 163-bp AccI fragment from pM3R containing the 73-bp putative silencer region (Figs. 1 and 3) was subcloned adjacent to and upstream from the H1d proximal promoter in pGL3B H1d to generate pH1d/silencer (Fig. 1). The wild-type H1d promoter was found to drive luciferase expression to a level more than 3-fold higher than the maximal level observed with the H1t promoter (Fig. 2).
Comparison of the normal H1d-promoted construct pGL3B H1d to the H1d construct containing the potential silencing sequence is presented in Figure 4. Upon transient transfection of C127I cells, a 57% decrease in activity was observed when the normal H1d luciferase construct was compared to the construct containing the silencer region from the H1t promoter. Although overall luciferase activity in HeLa cells was 6-fold lower than in C127I cells, the silencer construct also reduced luciferase transcription 43% in this human cell line (Fig. 4).
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Protein-DNA Binding Determined by In Vitro DNase I Footprinting
Mouse C127I nuclear proteins were bound to a radiolabeled probe that covered the silencer region from -967 to -741 (data not shown). DNase I digestion was performed in order to determine sites of protein binding to DNA within this potential silencer. In vitro DNase I footprinting experiments performed with a moderate amount of nonspecific competitor DNA (0.5 µg poly dI-dC) revealed four protected elements represented by lines in Figure 3 and shown by dotted lines in Figure 5. Two footprints were observed within the regions from -955 to -945 and -935 to -919. Two additional large footprints were observed within the regions from -919 to -890 and -885 to -835. It should be noted that the AccI cut site used to prepare the truncated promoter that removed the silencer region is located within the fourth footprint (-885 to -835) (Figs. 3 and 5).
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When DNase I footprinting assays were performed under more stringent conditions (using 2 µg of nonspecific competitor), the two footprints in the region from -955 to -919 were lost, and the two large footprints were reduced to the regions -903 to -887 and -855 to -835 as shown in Figures 3 and 5.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stepwise deletion of the H1t promoter revealed several potential regulatory regions. Our results suggest that most if not all of the elements necessary for total silencing of H1t promoter activity in somatic cells are located within 2.4 kb of the H1t transcriptional start site. These results (Figs. 1 and 2) are in agreement with the known transcription pattern of this testis-specific histone gene and are consistent with the results of transgenic mouse studies in our laboratory and in other laboratories [26, 27].
Our data show that removal of DNA from -948 to -875 resulted in the maximal level of luciferase activity observed with any H1t-promoted reporter vector (Fig. 2). Loss of this 74-bp region resulted in a 3.9-fold increase in expression. The total or partial elimination of a repressor's cognate DNA binding site is likely to disrupt the normal binding of the repressor, causing an increase in transcription. Interestingly, a decrease in activity was observed with the removal of the next 95 bp of downstream sequence from -875 to -780 (pGL3B AccI to pGL3B XbaI). This decrease may be due to the removal or disruption of a downstream enhancer element between AccI and XbaI sites.
It is noteworthy that deletion of sequences from -780 to -520 (pGL3B XbaI to pGL3B 1866) led to a 2.2-fold increase in transcription, and deletion of sequences from -520 to -141 led to a further 1.3-fold increase in luciferase activity. A portion of this region from -693 to -174 has been reported to contain sequences that increase H1t transcription at least 2-fold in synchronized mouse L cells [28]. Our results indicate that the presence of these two regions contribute to transcriptional repression. Since the transfected C127I cultures we used were not synchronized, it is likely that we detected the contribution of factors that may influence transcription during all phases of the cell cycle. It should be mentioned that the region from -520 to -141 contains a TG box that has been reported to increase transcription of H1t in mouse testicular cells [1]. Our results indicate that this region acts to repress transcription in somatic cells.
Support for the ability of the region of the H1t promoter from -948 to -875 to silence transcription was observed by placing the putative H1t silencer upstream from the somatic histone H1d proximal promoter. Apparently, changing the position of the element did not eliminate its ability to repress transcription, but the strength of the silencer appeared to be reduced. To test the possibility that this silencer element may be present in other mammals, we transfected a human cell line. Transient transfection of HeLa cells with the H1d/silencer construct also showed a decrease in transcription when compared to the H1d construct (Fig. 4).
We are currently attempting to identify factors that bind to the silencer. Potential binding sites have been identified with TRANSFAC, a program to aid in identification of consensus binding elements [38]. Sites identified in the region from -903 to -887 include C/EBP beta [39] (positive strand) and AR [40] and HiNF-A [13] (negative strand). Sites identified in the region from -855 to -835 include Oct-1 [41], YY1 [42], C/EBP1 [39], and SRF [43] (positive strand) and c-Myc [44] (negative strand). The Oct-1 binding site spans the AccI restriction site used to delete the silencer region. It is possible that the silencer region actually extends through the fourth footprint region (downstream from the AccI site) and that repressor proteins bind in the region of third and fourth footprints (Fig. 5). Alignment of the rat promoter with the mouse promoter within these regions yielded a high degree of nucleotide identity. The potential binding of these factors to these silencer regions is being tested currently in our laboratory [52].
In summary, we have identified a distal region upstream from the rat H1t gene that contains potential sites for binding of transcriptional regulators. Eukaryotic repressors can work in several ways to reduce transcription [45]. Repressors can directly compete for specific activator binding sites on DNA and interfere with activator binding [46, 47]. Repressors can co-occupy sites on DNA with activators and interfere with the activity of the DNA-bound activator (quenching) as seen with cytokine transcriptional control [48] and insulin gene regulation [49]. Repressors can also interfere with the general transcriptional machinery and affect transcription [50, 51]. Future experiments will be designed to determine which of these mechanisms may lead to repression of transcription of the testis-specific histone H1t gene in cell types other than primary spermatocytes.
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FOOTNOTES |
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2 Correspondence: Sidney R. Grimes, Medical Research Service (151), Overton Brooks Veterans Administration Medical Center, 510 E. Stoner Ave., Shreveport, LA 71101-4295. FAX: 318 429 5747; srgrimes{at}prysm.net
Accepted: May 20, 1999.
Received: April 6, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B by activated glucocorticoid receptors. Mol Cell Biol 1995; 15:943–953.[Abstract]This article has been cited by other articles:
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  E. Shang, H. D. Nickerson, D. Wen, X. Wang, and D. J. Wolgemuth The first bromodomain of Brdt, a testis-specific member of the BET sub-family of double-bromodomain-containing proteins, is essential for male germ cell differentiation Development, October 1, 2007; 134(19): 3507 - 3515. [Abstract] [Full Text] [PDF]
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 M. Siep, E. Sleddens-Linkels, S. Mulders, H. van Eenennaam, E. Wassenaar, W. A. Van Cappellen, J. Hoogerbrugge, J. A. Grootegoed, and W. M. Baarends Basic helix-loop-helix transcription factor Tcfl5 interacts with the Calmegin gene promoter in mouse spermatogenesis Nucleic Acids Res., December 7, 2004; 32(21): 6425 - 6436. [Abstract] [Full Text] [PDF]
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 G. C. Horvath, W. S. Kistler, and M. K. Kistler RFX2 Is a Potential Transcriptional Regulatory Factor for Histone H1t and Other Genes Expressed During the Meiotic Phase of Spermatogenesis Biol Reprod, November 1, 2004; 71(5): 1551 - 1559. [Abstract] [Full Text] [PDF]
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S. A. Wolfe and S. R. Grimes Specific Binding of Nuclear Proteins to a Bifunctional Promoter Element Upstream of the H1/AC Box of the Testis-Specific Histone H1t Gene Biol Reprod, June 1, 2003; 68(6): 2267 - 2273. [Abstract] [Full Text] [PDF]
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D. C. Wilkerson, S. A. Wolfe, and S. R. Grimes H1t/GC-Box and H1t/TE1 Element Are Essential for Promoter Activity of the Testis-Specific Histone H1t Gene Biol Reprod, October 1, 2002; 67(4): 1157 - 1164. [Abstract] [Full Text] [PDF]
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G. C. Horvath, S. E. Clare, M. K. Kistler, and W. Stephen Kistler Characterization of the H1t Promoter: Role of Conserved Histone 1 AC and TG Elements and Dominance of the Cap-Proximal Silencer Biol Reprod, October 1, 2001; 65(4): 1074 - 1081. [Abstract] [Full Text] [PDF]
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R. Singal, J. vanWert, M. Bashambu, S. A. Wolfe, D. C. Wilkerson, and S. R. Grimes Testis-Specific Histone H1t Gene Is Hypermethylated in Nongerminal Cells in the Mouse Biol Reprod, November 1, 2000; 63(5): 1237 - 1244. [Abstract] [Full Text]
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J. G. Bartell, D. A. Fantz, T. Davis, M. J. Dewey, M. K. Kistler, and W. S. Kistler Elimination of Male Germ Cells in Transgenic Mice by the Diphtheria Toxin A Chain Gene Directed by the Histone H1t Promoter Biol Reprod, August 1, 2000; 63(2): 409 - 416. [Abstract] [Full Text]
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