DDBJ/EMBL/GenBank Accession No. U68178, BankIt 65801
ABSTRACT
Molecular and functional studies of the LDH/C 5' upstream promoter elements were undertaken to elucidate the molecular mechanisms involved in temporal activation of LDH/C gene expression in differentiating germ cells. Ligation mediated-PCR (LM-PCR) gene walking techniques were exploited to isolate a 2.1 kb fragment of the mouse LDH/C 5' promoter region. DNA sequence analysis of this isolated genomic fragment indicated that the mouse LDH/C promoter contained TATA and CCAT boxes as well as a GC-box (Sp1-binding site) situated upstream from the transcription start site. PCR-based in vivo DNase I footprinting analysis of a 600 bp fragment of the proximal LDH/C promoter region (-524/+38) in isolated mouse pachytene spermatocytes identified a single footprint over the GC-box motif. Three DNase I hypersensitive sites were also detectable in vivo, in a region containing (CT)n(GA)n repeats upstream from the CCAT box domain. Functional characterization of the promoter region was carried outin a rat C6 glioma cell line and an SV40 transformed germ cell line (GC-1 spg)using wild-type and mutated LDH/C promoter CAT reporter constructs. These studies provide experimental evidence suggesting that transcriptional activation of the LDH/C promoter is regulated by enhancer-mediated coactivation of the Sp1 proteins bound to the GC-box motif footprinted in vivo in pachytene spermatocytes.
The lactate dehydrogenase isoenzymes [L-lactate: NAD+ oxidoreductase (LDH) EC 1.1.1.27] are tetrameric enzymes responsible for the reversible conversion of pyruvate to lactate. There are five isoforms of LDH expressed as combinations of the individual A (muscle) and B (heart) subunits that are detectable in most somatic cells: LDH-1 (B4), LDH-2 (A1B3), LDH-3 (A2B2), LDH-4 (A3B1), LDH-5 (A 4) (1 ). A sixth homotetrameric isoform, LDH-6 (C4), is found exclusively in germ cell lineages within the testicular seminiferous epithelium. The LDH/C gene encoding subunits of the LDH-6 (C4) isoenzyme is expressed in spermatocytes during the meiotic stages of spermatogenesis and in the spermatids in the later stages of germ cell differentiation (2 ). The three LDH subunits (A, B and C) are encoded by separate structural genes, each of which displays tissue-specific and developmentally regulated patterns of expression (3 ,4 ). Temporal expression of all three of the LDH subunit genes occurs only in differentiating germ cell lineages (5 ,6 ). Spermatogenesis, therefore, is an ideal experimental model system to study the molecular mechanisms involved in temporal regulation of LDH subunit gene expression. These cell-type and stage-specific patterns of LDH subunit expression observed in germ cells are most likely regulated at the level of transcription. Detailed molecular studies and functional analysis of promoter elements of the individual LDH subunit genes, such as described in this report for the LDH/C promoter, are required to confirm this hypothesis.
The mouse and rat promoter regions of the LDH/A subunit genes have been extensively characterized and studied (7 ,9 ). In somatic cells, LDH/A expression patterns have been shown to be regulated mainly by a cAMP-dependent regulatory element (CRE) present within the 5' upstream promoter motif. Recent studies, however, have demonstrated that Sp1 binding to GC-box motifs within the LDH/A promoter contributes to the coordinate regulation of the rat LDH/A promoter activity (10 ). The human LDH/C promoter does not contain the canonical TATA and CCAT boxes present in the mouse LDH/C promoter characterized in this study (11 ). These observed differences in genomic DNA sequence organization support the importance of molecular and functional analysis of LDH/C promoter structure in different species.
Seminiferous cords and tubules were prepared from Swiss Webster mice (Charles River Breeding Labs) by collagenase treatment. Monodispersed suspensions of spermatogenic cells were obtained from seminiferous cords/tubules by trypsin digestion (12 ). Pachytene spermatocytes were isolated from the testes of >= 60-day-old mice. The germ cells were separated by velocity sedimentation at unit gravity on 2-4% bovine serum albumin (BSA) gradients; their purity, as determined by cytological examination was >92% and their viability, by dye exclusion, was >98%.
The mouse 1 kb LDH/A subunit promoter fragment (-950/+52) was synthesized by utilizing gene-specific PCR primers and mouse liver genomic DNA as template. This 1 kb fragment was subcloned into the pCAT-basic and pCAT enhancer vectors (Promega, Madison, WI, USA). The PCR primer pair (LDHA5, LDHA6) were designed from published genomic DNA sequence for the mouse LDH/A subunit gene:
LDH/A5: 5'-AAGGGATACAGACCAAGCG A-3';
LDH/A6: 5'-TGGCGAGGAGAAGCAGCGTG-3' (7 ).
CAT expression plasmids (pLDH/C) were constructed by subcloning a 600 bp PCR-generated fragment of the 5' upstream proximal LDH/C promoter region in the pCAT-basic and pCAT enhancer vectors (Promega, Madison, WI, USA). The 600 bp mouse LDH/C subunit promoter fragment (-524/+38) was synthesized by utilizing ligation-mediated PCR (LM-PCR) genomic walking strategies with nested PCR primers designed from 5' cDNA sequence for the mouse LDH/C subunit transcript (8 ).
Rat C6 glioma and/or GC-1 spg (13 ) cells (3 * 105) were seeded in 2 ml of Ham's F10 growth medium (Gibco-BRL) supplemented with 10% (v/v) serum. The cells were incubated at 37oC under 5% CO2 for ~20 h until they were 50-80% confluent. For each transfection, 0.4 [mu]g of pGL3 (luciferase expression vector, Promega, Madison, WI) and 0.8 [mu]g of each pCAT DNA construct were added to each transfection reaction using LiptofectAmine reagent according to the manufacturer's recommended procedures (Gibco-BRL). The pGL3 plasmid (Promega, Madison, WI, USA) containing the luciferase gene driven by the Syrian Virus 40 promoter was used as an internal control to determine transfection efficiency.
The growth medium was removed and the transfected cells washed 3 times with PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaHPO4. 7 H2O, 1.4 mM KH2PO4 , pH 7.1). Aliquots (200 [mu]l) of 1* Reporter Lysis Buffer (Promega, Madison, WI, USA) were added to each plate containing the transfected cells. The reactions were incubated at room temperature for 15 min, the cell lysates transferred to a microcentrifuge tube, vortexed for 15 s and 50 [mu]l aliquots of the cell extracts removed for use in luciferase assays prior to heating. The remaining extracts were heated to 60oC for 10 min, spun in a microfuge for 2 min, and the supernatants transferred to fresh tubes and stored at -20oC for use in CAT assays.
For CAT analysis, each reaction (40 [mu]l of cell extracts) contained 3.5 [mu]l of 14C-chloramphenicol (0.05 mCi/ml DuPont/NEN) and 5 [mu]l of n-butyryl coenzyme A (Sigma Chemical Co.). Distilled water was added to a final volume of 125 [mu]l and the reactions incubated at 37oC for 3 h. Aliquots (300 [mu]l ) of mixed xylenes (Aldrich Chemical Co., Inc.) were added to each tube for liquid scintillation counter (LSC) assay or 500 [mu]l aliquots of ethyl acetate were added to each tube used for thin layer chromatography (TLC) assay.
The samples for TLC analysis were vortexed and centrifuged for 3 min, and supernatants were transferred to a fresh tube and air dried. Residues were resuspended in 10 [mu]l of ethyl acetate and spotted onto a silica gel TLC plate. The silica plate chromatography was run for 1 h in a closed chamber §P vortexed and spun in the microcentrifuge for 3 min. The upper phase was removed and back-extracted twice in 100 [mu]l of fresh 0.25 M Tris-HCl, pH 8.0, and 200 [mu]l aliquots of each sample were counted in a liquid scintillation counter. All measurements were carried out within the linear range of the CAT activity assay, and the data which were initially expressed as the percentage of 14C chloramphenicol converted to butyrylated derivatives (% butyrylation/[mu]g protein), were normalized to luciferase activity (expressed as U/[mu]g protein) to give a final value of % butyrylation/unit luciferase.
For luciferase assays, the 50 [mu]l aliquots of the transfected cell extracts, removed previously, were spun in a microfuge for 5 s and 1-5 [mu]l aliquots of the supernatant mixed with 100 [mu]l of room temperature equilibrated Luciferase Assay Reagent as indicated in protocols supplied by the manufacturer (Promega, Madison, WI). Luciferase activity was measured in a liquid scintillation counter (LSC). Total protein concentrations in the extracts were determined using the BioRad Protein Assay Kit I (BioRad Laboratories, Hercules, CA, USA).
Plasmid DNA (2 [mu]g) pALTER-1 vector containing the 600 bp LDH/C promoter DNA fragment (-524/+38) inserted into the HindIII/XbaI cloning site was denatured for 5 min at room temperature in 20 [mu]l of 0.2 M NaOH, 0.2 mM EDTA solution. All remaining reactions and procedures were carried out in the presence of Sp1 mutagenic oligo (5'-CAGCACGCAAGCCCATACCACTGCCTGC-3') according to the instructions provided by the manufacturer of the Altered Sites II in vitro mutagenesis kit (Promega, Madison, WI, USA). Individual clones were isolated, the DNA purified and sequenced to confirm which clones had the correct mutational changes.
All long distance polymerase chain reactions (LD PCR) were carried out according to manufacturers' protocols included with the PromoterFindertm mouse genomic DNA walking kit (Clontech Lab. Inc., Palo Alto, USA). The PCR fragments of interest were purified from a 1% agarose gel and subcloned into a TA-cloning Vector (PCRtmII TA plasmid, Invitrogen, San Diego, CA, USA).
The following PCR primers were used for both LDH/C genomic walking and in vivo footprint analysis:
primer LDHC4: 5'-CTTACTGCTGACTCCGCAGCACA-3'
primer LDHC5: 5'-CTGACTCCGCAGCACAGGTAAGAAACCA-3'
primer LDHC6: 5'-CCGCAGCACAGGTAAGAAACCAGGATAACTGTT-3'
primer LDHC7: 5'-AAGCCCTGGGACCACACAGAA-3'
primer LDHC8: 5'-GACCACACAGAAGATGGCAGCCAGT-3'
primer LDHC9: 5'-AAGATGGCAGCCAGTTTGGCCAG-3'
For in vivo DNase I digestions, isolated pachytene spermatocytes (5 * 106 cells) were permeabilized by treatment for 1 min at 37oC with 0.05% lysolecithin in buffer A (150 mM sucrose, 80 mM KCl, 35 mM HEPES, pH 7.4, 5 mM K2HPO4, 5 mM MgCl2, 0.5 mM CaCl2). The cells were centrifuged, washed once with the buffer A without lysolecithin and were treated at room temperature for 5 min with DNase I (50-100 [mu]g/ml) in buffer A. The DNase I treated cells were centrifuged and lysed in 10 mM Tris-HCl, pH 8.0, 85 mM NaCl, 12.5 mM EDTA, 0.5% SDS, 300 [mu]g/ml proteinase K, 100 [mu]g/ml RNase A by incubation at 37oC for 5 h. The reaction was phenol/chloroform extracted and the DNA ethanol precipitated. For in vitro DNase I digestions, DNA (40 [mu]g) purified from pachytene spermatocytes was digested with DNase I (0.01-0.03 [mu]g/ml) at room temperature in buffer A for 10 min. The in vitro treated DNA was phenol/chloroform extracted and precipitated with ethanol. The in vivo and in vitro digested DNAs were resuspended in buffer (40 mM Tris-HCl, pH 7.7, 25 mM NaCl, 6.7 mM MgCl2), denatured at 95oC for 3 min, cooled for 1 min and the free 3'OH groups blocked by incubation of the DNA with Sequenasetm 2 (5 U) in the presence of 5 [mu]M ddNTPs for 20 min at 45oC. The reaction mixtures were heated at 94oC for 3 min, cooled and adjusted to a final concentration of 200 mM potassium cacodylate, pH 7.0, 1 mM 2-mercaptoethanol. Terminal transferase (30 U) was added to each reaction mixture and the incubation continued for an additional 30 min at 37oC. The DNA fragments were phenol/chloroform extracted and recovered by ethanol precipitation with 2 M ammonium acetate. The precipitated DNA was resuspended in TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and used in LM-PCR reactions with LDH/C gene-specific primers.
DNase I treated genomic DNA (2 [mu]g in 5 [mu]l TE Buffer) was mixed with 43 [mu]l first-strand synthesis mix, (40 mM NaCl, 10 mM Tris-HCl, pH 8.9, 5 mM MgSO4, 0.01% gelatin, 0.3 pmol gene-specific primer 1 (LDH C4), 0.2 mM dNTPs mix, Vent DNA polymerase (0.5 U). The reactions were denatured for 5 min at 95oC, annealed for 30 min at 60oC, and extended for 10 min at 76oC. Each reaction was mixed with 20 [mu]l of ligase dilution buffer (10 mM Tris-HCl, pH 7.5, 17.5 mM MgCl2, 50 mM DTT, 125 [mu]g/ml BSA) and 25 [mu]l of ligase mix (10 mM MgCl2, 20 mM DTT, 3 mM ATP, 50 [mu]g/ml BSA, 4 [mu]M linkers (LMPCR1: 5'-GCGGTGACCCGGGAGATCTGAATTC-3', LMPCR2: 5'-GAATTCAGATC-3'). T4 ligase (3 U) was added and the reactions incubated overnight at 17oC. The DNA samples were ethanol precipitated after adding 9.4 ml ice-cold precipitation salt mix (0.3 M NaOAc, pH 7.0, 1 mg/ml yeast tRNA). After centrifugation, the precipitated DNA was dissolved in 70 [mu]l of H2O and mixed with 30 [mu]l of 1* amplification mix (40 mM NaC1, 20 mM Tris-HCl, pH 8.9, 5 mM MgSO4 , 0.01% gelatin, 0.5% Triton X-100), 10 pmol gene-specific primer 2 (LDH C5), 10 pmol linker primer, 0.7 mM dNTPs and Vent DNA polymerase (10 U). The PCR reactions were performed for 18 cycles: the first denaturation was 4 min at 95oC, and subsequent ones for 1 min; annealing was 2 min at 2oC above the calculated Tms, extension was 3 min at 76oC. For each cycle, an extra 5 s was added to the extension step. The final extension was allowed to proceed for 10 min. The secondary PCR reactions were performed by adding 5 [mu]l of end-labeling mix (1* amplification buffer, 2.3 pmol end-labeled gene-specific primer 3 (LDH C6), 2 mM dNTPs, Vent DNA polymerase (1 U) using two rounds of PCR (1 cycle: 95oC/4 min, annealing temperature 2 min, 76oC/10 min; 1 cycle: 95oC/1 min, annealing temperature 2 min, 76oC/10 min). Each reaction was terminated by adding 295 [mu]l stop solution (260 mM NaOAc, pH 7.0, 10 mM Tris-HCl, pH 7.5, 4 mM EDTA, pH 8.0, 68 [mu]g/[mu]l yeast tRNA). Samples were phenol/chloroform extracted and ethanol precipitated. Precipitated DNA was dissolved in 28 [mu]l of gel loading buffer [80% deionized formamide in 45 mM boric acid, 45 mM Tris base, 1 mM EDTA, 0.05% (w/v) bromophenol blue, 0.05% (w/v) xylene cyanol] and an aliquot (7 [mu]l) of each sample denatured by heating and loaded onto the denaturing sequencing gel.
The genomic DNA sequence of the 2.1 kb upstream promoter region of the mouse LDH/C subunit gene isolated by LM-PCR genomic walking is shown in Figure 1 . A series of overlapping PCR oligonucleotide primers designed from the published 5' untranslated region of the mouse LDH/C cDNA (primers LDHC4, LDHC5, and LDHC6) were used to initiate genomic walking into the promoter region (8 ). DNA sequence analysis indicated that a TATA box -30/-27 and CCAT box -117/-114 are present in the PCR fragment isolated from the mouse LDH/C promoter region. An Sp1 binding GC-box motif -78/-71 is also clearly recognizable upstream from the TATA box.
Results of LM-PCR DNase I in vivo footprint analysis of the LDH/C promoter in isolated mouse pachytene spermatocytes using nested LDH-specific PCR primers C4 and C5 are shown in Figure 2 . Only a single footprint was observed over the GC-box motifs with no obvious footprints appearing over the TATA and CCAT boxes. Additional footprinting studies with nested LDH/C specific PCR primers C7 and C8, situated further upstream, gave no additional distinctive footprints. However, several hypersensitive sites (enhancements) were observed consistently at positions -233/-231, -252/-250 and -261/-262 (Fig. 3 ). Interestingly, these hypersensitive sites occur within a region of the LDH/C promoter containing several (CT)n(GA)n repeats. Recent experimental evidence has implicated (CT)n(GA)n repeats in promoter domains with structural changes occurring at the level of chromatin to facilitate activation of temporarily expressed genes (33 -38 ).
In vivo studies designed to functionally define the LDH/C promoter domain sequences necessary for initiating transcriptional activity were carried out by using CAT reporter expression analyses. These studies were undertaken by transfecting LDH/C CAT reporter constructs in two cell lines: (i) an SV40 immortalized mouse germ cell line (GC-1 spg) (13 ); and (ii) a rat C6 glioma somatic cell line. Mouse LDH/A CAT reporter constructs were used as positive controls for these experiments based on published LDH/A CAT expression studies that were carried out in the rat C6 glioma cell line (9 ).
The CAT reporter transcriptional activities directed by both LDH/C and LDH/A promoter constructs were assayed by TLC analysis of extracts from the rat C6 glioma transfected cells (Fig. 4 A). Strong CAT expression (14C butyrylated chloramphenicol derivatives) was observed from extracts of rat C6 glioma cells transfected with pCAT LDH/A basic and enhancer constructs, while a weaker signal was observed from extracts of cells transfected with pCAT LDH/C enhancer constructs. CAT expression was not detectable in extracts assayed from cells transfected with the promoterless pCAT basic and enhancer vectors, the pCAT LDH/C basic, and pCAT LDH/C Sp1 in vitro mutagenized constructs.
In this study, we used in vivo footprinting analysis in mouse pachytene spermatocytes to show that the Sp1 binding GC-box motif plays a major functional role in regulating mouse LDH/C promoter activity during spermatogenesis. The functional significance of this Sp1-binding GC motif for LDH/C promoter activity was further examined using a mutagenized GC-box promoter construct in CAT reporter expression studies in both rat C6 glioma cells and an SV40 transformed mouse germ cell line (GC-1 spg). LDH/C promoter directed transcriptional activity was significantly decreased in both cell lines transfected with mutated GC-box constructs. As a result of these site-directed mutational studies on the LDH/C promoter GC-box, the experimental observations made in this report strongly support the hypothesis that activation of LDH/C transcription in vivo is mediated through this Sp1 binding site within the LDH/C promoter.
GC-box motifs which bind the ubiquitous transcription factor Sp1 are found in a number of eukaryotic promoters including the mouse and rat LDH/A promoters (7 ,10 ). The regions surrounding most eukaryotic GC-box motifs are highly heterogeneous and appear to be active binding sites for other regulatory transcription factors. Several recent molecular and functional studies on GC-box containing eukaryotic promoters illustrated that unique interactions typically occur between bound Sp1 protein and bound neighboring transcription factors to coordinately modulate the transcriptional activity of individual genes (18 -23 ). Direct interactions are also known to occur between coactivator proteins bound to upstream enhancer sequences and Sp1 transcription factors bound to GC-boxes upstream from the initiation start site. These synergistic interactions increase the level of complexity of gene regulatory activity due to cooperativity of factors at these sites (17 -22 ).
Binding interactions of the ubiquitous transcription factor Sp1 with the GC-box in the LDH/C promoter explain why transcriptional activation of this promoter was observed in the cell lines examined (rat C6 glioma and GC-1 spg), although the LDH/C subunit gene is not normally expressed in either of these cell lines. These studies also demonstrated that the LDH/C promoter directed transcriptional activation observed in both cell lines was absolutely dependent on the presence of a strong enhancer. Functional studies of other eukaryotic promoters have indicated that enhancer domains can interact with the Sp1 protein complexes bound to GC-boxes to modulate cell-type specific gene transcriptional activity (24 -27 ). Lack of the availability of an appropriate mouse germ-cell line that expresses the LDH/C subunit gene has precluded the utilization of deletional promoter constructs in transfection studies to identify and characterize the functional germ-cell specific enhancer region of the LDH/C gene.
Differential Sp1 expression patterns have been observed in several mouse tissues including the testis (28 ). It is possible that developmentally modulated changes in Sp1 expression could occur in the differentiating germ cells during spermatogenesis. To further complicate this issue, multiple Sp1 proteins have been identified and characterized with both transcriptional activator and repressor activities (29 -31 ). Changes in the expression patterns of these Sp1 proteins or post-translation modifications, such as phosphorylation and differential glycosylation (17 ,32 ), during spermatogenesis could also contribute toward modulation of the LDH/C promoter activity in differentiating germ cells.
LDH/C subunit gene transcriptional activation does not normally occur in somatic cells despite the observed requirement for Sp1 modulated activation of this promoter as demonstrated in this study. It is apparent, therefore, that other regulatory events are likely to be involved in addition to enhancer/Sp1 directed LDH/C gene activation. These include chromatin remodeling which may play an important role in conferring active conformation to the LDH/C promoter in these cells. Hypersensitive sites such as those identified by our DNase I in vivo footprinting studies, as indicated in Figure 3 , are likely candidates for chromatin-specified regulatory regions. It is biologically significant that these hypersensitive sites occur in a region of the LDH/C promoter containing several (CT)n(GA)n repeats.
Studies on the Drosophila hsp26 promoter have demonstrated that similar hypersensitive sites found in this promoter play an important role in regulation of the transcriptional activation of the hsp26 gene (33 ). In fact, experimental evidence exists showing that (CT)n(GA)n repeat elements bind coactivator transcription factors that synergistically bind to, and facilitate stabilization of, TFIID at the preinitiation complex forming at the site of the TATA box (34 -38 ). The (CT)n(GA)n repeats in the LDH/C promoter were footprinted in vivo in pachytene spermatocytes and, therefore, are implicated in determining the functional status of the LDH/C gene in germ cells. Since it is unlikely that chromatin mediated regulatory mechanisms will occur on the episomal DNA constructs transfected into cells, further in vivo functional studies are required to elucidate the details of the complex molecular mechanisms involved in repression of LDH/C promoter activity in somatic cells and its activation in differentiating germ cells. These studies are now in progress and will require extensive transgenic analysis of deletional and mutagenized LDH/C promoter reporter fragments stably integrated into the mouse genomic chromosomal background.
In vitro transcriptional and 5' deletional analyses indicated that a 60 bp core mouse LDH/C promoter sequence was sufficient to direct testis-specific transcriptional activity (39 ). These in vitro studies confirmed that 5' deletions of the LDH/C promoter, some of which corresponded to (CT)n(GA)n repeats and the GC-box regions footprinted in vivo, resulted in significant reductions of LDH/C transcriptional activity. The authors concluded from these studies that multiple upstream cis-regulatory elements are involved in mediating LDH/C tissue and cell-type specific gene expression in the testis. Using electrophoretic mobility shift assays (EMSAs), these investigators detected protein binding activity to a 30 bp palindromic oligonucleotide sequence overlapping the TATA box and transcriptional start site of the mouse LDH/C promoter. Two DNA-binding proteins, a 105 kDa testes-specific protein and a 65 kDa liver-specific protein were identified by Southwestern blotting with the labeled oligonucleotide. The authors speculate that tissue-specific transcription of the mouse LDH/C gene may be associated with differential binding activity of these proteins to this palindromic sequence in expressing and nonexpressing of tissues. However, mutagenesis studies were not carried out to confirm the functional significance of these observations. Due to the high sensitivity of EMSA assays, a number of DNA-protein interactions are detectable in vitro that did not normally occur in vivo. It has been demonstrated that DNA-protein interactions can be restricted in vivo by the structural conformation of chromatin occurring in these regions (33 -38 ). These conditions are difficult to duplicate in vitro using cellular extracts and oligonucleotide probes.
In vivo footprints were not detectable over the CCAT or TATA box regions of the mouse LDH/C promoter in pachytene spermatocytes despite the increased resolution and sensitivity of PCR-based DNase I in vivo footprinting strategies utilized in this study. These observations are functionally significant in light of the finding that DNA sequence analysis has clearly indicated that these domains are structurally absent from the human LDH/C promoter (11 ). However, the Sp1-binding GC-box domain footprinted in vivo in the mouse LDH/C promoter is conserved in the human LDH/C promoter. Data presented in this study have demonstrated the functional significance of this Sp1-binding GC box domain using in vivo footprinting analysis in pachytene spermatocytes. The importance of this Sp1-binding domain for LDH/C transcriptional activity was confirmed by CAT-reporter expression studies in a germ-cell derived cell line, GC1-spg, and in a C6 glioma somatic cell line using wild type and mutated promoter constructs.
We thank Mr Marsena Riley for technical assistance in isolating the purified pachytene spermatocytes and maintaining the cell lines used in this study; Dr José Millan for providing the GC-I spg cell line; Drs Deborah Lyn, Craig Bond and Peter MacLeish for critique and helpful suggestions, and Mrs Doris Pitts for typing and editing assistance. This work was supported by NICHD (HD30323) and NIH/MBRS (S06GM08248) grant awards to K.H.T. and NIH/RCMI (RRAI03034) support of the MSM Molecular Genetics Core in which the DNA sequencing studies were conducted.
REFERENCES