From The testicular isoform of hormone-sensitive
lipase (HSLtes) is encoded by a testis-specific exon
and 9 exons common to the testis and adipocyte isoforms. In mouse,
HSLtes mRNA appeared during spermiogenesis in round
spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb)
of the human HSLtes gene 5'-flanking region cloned upstream
of the chloramphenicol acetyltransferase gene were microinjected into
mouse oocytes. Analyses of enzyme activity in male and female
transgenic mice showed that 0.5 kb of the HSLtes promoter
was sufficient to direct expression only in testis. Cell transfection
experiments showed that CREM Hormone-sensitive lipase
(HSL)1 is a triacylglycerol
lipase and a cholesterol esterase expressed at high levels in
adipocytes, testes, and adrenals (1-3). In adipocytes, HSL catalyzes
the rate-limiting step in the hydrolysis of triglycerides into fatty acids and glycerol (4). HSL activation is mediated through phosphorylation by the cAMP-dependent protein kinase (5).
In rat testis, HSL mRNA and protein are expressed in the
seminiferous tubuli and not in interstitial cells with a
stage-dependent pattern corresponding to the appearance of
haploid germ cells (3, 6). Several isoforms of HSL produced by a single
gene have been characterized (2, 7, 8). Human adipose tissue expresses
a 2.8-kb mRNA that encodes an 88-kDa protein (7, 9). The mRNA
and protein species expressed in testis are larger, 3.9 kb and 120 kDa,
respectively (3). Analysis of coding sequences revealed that human
adipocyte and testis HSL (HSLtes) are 775 and 1076 amino
acids long, respectively. HSLtes differs from the adipocyte
form by a unique NH2-terminal region. Elucidation of the
HSL gene organization showed that nine coding exons are common to both
forms. The additional sequence in HSLtes is encoded by a
1.2-kb-long testis-specific exon (3, 7). When a gene is expressed in
somatic tissues and in germ cells, tissue-specific expression often
results from alternate promoter use (10, 11). The promoter of the
adipocyte form of HSL (9) is located 13 kb downstream of the
HSLtes 5'-flanking region suggesting that the expression of
the different forms of HSL is controlled by several tissue-specific promoters.
During spermatogenesis, specialized transcriptional mechanisms ensure
stage-specific gene expression in the germ cells. The factors
controlling gene expression in post-meiotic germ cells are beginning to
be elucidated. Several germ cell-specific putative transcription
factors have been cloned, but target genes have been identified only
for a few of them. CREM In this study, we investigated the molecular mechanisms that control
the testis-specific expression of HSLtes. During
spermatogenesis, HSLtes mRNA was expressed in haploid
germ cells concomitantly with protamine 1 mRNA. We show that 0.5 kb
of the HSLtes promoter was sufficient to drive
testis-specific expression in transgenic mice. In cell transfection
experiments, transactivation of the HSLtes promoter was
independent of the cAMP signaling pathway. Four regions bound nuclear
proteins present in testis and not in liver. One of the region bound
Sox proteins expressed in post-meiotic germ cells raising the
possibility that Sox proteins are involved in the transactivation of
the HSLtes promoter.
Northern Blot Analyses--
A mouse HSL DNA probe (477 bp) was
generated by PCR on mouse genomic DNA with primers located in the first
adipocyte coding exon 5'-ATG GAT TTA CGC ACG ATG ACA CAG-3' and 5'-TAG
CGT GAC ATA CTC TTG CAG GAA-3'. Proenkephalin (227 bp) and protamine 1 (207 bp) cDNA probes were generated by reverse transcription-PCR on
mouse testis total RNA using 5'-GAC AGC AGC AAA CAG GAT GA-3' and
5'-TTC AGC AGA TCG GAG GAG TT-3', and 5'-AGC AAA AGC AGG AGC AGA TG-3'
and 5'-AGA TGT GGC GAG ATG CTC TT-3' primers, respectively. PCR
reactions were performed using the proofreading pfu DNA
polymerase (Stratagene). PCR products were cloned into pBluescript
(Stratagene) using the TA cloning procedure (17). Identity of the
amplicon sequences to published sequences was checked by automatic DNA sequencing (Applied Biosystems).
Total testis RNA was prepared from prepuberal and sexually mature mice
by a single-step guanidinium thiocyanate phenol/chloroform extraction
(18). RNA samples (25 µg) were separated on a 1% agarose, 2.2 M formaldehyde gel, transferred, and UV cross-linked to a
nylon membrane (Nytran, Schleicher & Schuell). Equal loading of the
different lanes was checked by ethidium bromide staining of the gel and
by hybridization with a rat Plasmid Constructs--
A 1.6-kb
HindIII/BglII human DNA genomic restriction
fragment was isolated from a cosmid clone containing the entire human HSL gene (3). The fragment was subcloned into the HindIII
and BamHI sites of pBluescript and sequenced by automatic
DNA sequencing (Applied Biosystems). It contained 1.4 kb of the
5'-flanking region upstream of the testis-specific exon. The construct
was digested with HindIII and XbaI, and the
1.6-kb fragment was ligated upstream of the chloramphenicol
acetyltransferase (CAT) gene into the promoterless pCAT-basic vector
(Promega) (p1.4HSLtesCAT). The 1.6-kb
HindIII/BglII DNA genomic restriction fragment
was also cloned upstream of the luciferase gene into the promoterless
pGL3-basic vector (Promega) (p1.4HSLtesLUC). About 900 bp of
p1.4HSLtesLUC 5'-flanking sequence was deleted by digestion with
SmaI to produce p0.5HSLtesLUC. The SmaI site used
to generate p0.5HSLtesLUC and the AvaI site used to generate
the microinjected fragment 0.5HSLtesCAT (see below) are overlapping.
The complete 1-kb CREM Transgenic Mice--
The two transgenes were prepared by
digesting p1.4HSLtesCAT with HindIII and BamHI or
with AvaI and BamHI to give, respectively, 1.4HSLtesCAT and 0.5HSLtesCAT. These two fragments were isolated on
agarose gel by electroelution and purified using an elutip-d column
(Schleicher & Schuell). Transgenic mice were produced by microinjection
of the transgenes into the pronuclei of fertilized B6D2/F1 mouse eggs
(19). Microinjected embryos were transferred to pseudo-pregnant
B6-CBA/F1 female mice and carried to term. Screening of the positive
transgenic animals was performed with DNA prepared from tail samples
using Southern blot or PCR using as sense primer an oligonucleotide
located in the human HSLtes 5'-flanking sequence and as
antisense primer an oligonucleotide located in the CAT gene. Subsequent
generation of heterozygous mice were produced by mating transgenic mice
with wild type B6-CBA/F1 mice. The transmission of the transgene was
~50% in the progeny of all founders indicating Mendelian
transmission. Protein extracts for CAT assays were prepared from
hemizygous transgenic mice. Briefly, tissues were rapidly frozen in
liquid nitrogen and homogenized in 0.5 ml of 250 mM Tris,
pH 7.6, 5 mM EDTA, and 1 mM DTT. Homogenates were heated 7.5 min at 65 °C and centrifuged at 4 °C for 15 min at 13,000 rpm. Supernatants were kept for CAT and protein analyses (17,
20).
RNase H Mapping--
Ninety µg of RNA from transgenic sexually
mature mouse testis or 1 µg of human testis poly(A)+ RNA
(CLONTECH) were lyophilized and resuspended in 10 µl of RNase H buffer (20 mM Tris, pH 7.5, 10 mM MgCl2, 100 mM KCl, 0.1 mM DTT, 5% sucrose) containing 10 pmol of the
human-specific single strand antisense oligonucleotide 5'-GTA GAG TAA
CTA AGG AGT TG-3' (nt 197 to 179 downstream of the transcriptional
start site). After 10 min at 70 °C, hybridization was performed for
30 min at 37 °C. Then, 40 µl of RNase H buffer containing 7 units
of RNase H (Amersham Pharmacia Biotech) were added, and digestion was
carried out for 45 min (21). The digestion products were separated on a
polyacrylamide-urea gel after ethanol precipitation. The gel was washed
twice in 7% formaldehyde, 9 mM Tris borate, 0.2 mM EDTA, and RNA was passively transferred onto a nylon
membrane. Hybridization was performed as described above using a
32P-labeled probe corresponding to the 197 bp located
downstream of the transcription start site.
Cell Transfection Experiments--
JEG3 cells grown in
28-cm2 plates were transfected using Fugene-6 (Boehringer
Mannheim) with 700 ng of p0.5HSLtesLUC or pCRE-LUC (Stratagene), 700 ng
of CREM Preparation of Liver and Testis Nuclear Extracts--
Total
nuclei extracts were performed as described by Howard et al.
(22) with modifications. Four adult rat testis and 10-15 mg of adult
rat liver (perfused with 0.9% NaCl to wash out blood) were washed in
ice-cold saline containing 0.1 mM PMSF, decapsulated, and
minced with scissors in 40 ml of homogenization buffer (10 mM HEPES, pH 8, 1 mM EDTA, 25 mM
KCl, 0.5 mM spermidine, 0.15 mM spermine, 10%
glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, and sucrose (1.85 M for testis, 2 M for liver)). Tissues were then homogenized in a glass
tissue grinder with a motor-driven Teflon pestle until cells were
broken. The homogenate was then completed to 80 (testis) and 100 ml
(liver) with homogenization buffer, and 28-ml aliquots were layered
over 10-ml cushions of the same buffer in SW28 tubes. The tubes were
centrifuged at 27,000 rpm for 1 h at 4 °C. The supernatants
were carefully removed, and the tube walls were washed with water and
dried. Nuclei pellets were resuspended in 3 (testis) or 5 ml (liver) of
buffer A (20 mM HEPES, pH 8, 100 mM EDTA, 8.8 mM MgCl2, 25% glycerol, 0.5 mM
spermidine, 0.15 mM spermine, 0.14 M NaCl)
using an all-glass Dounce homogenizer (pestle B). An aliquot was
diluted 100 times in 0.5% SDS, and the absorbance at 260 nm was
measured. The nuclear suspension was diluted at 40 A260 units per ml. An equal volume of buffer B
(20 mM HEPES, pH 8, 100 mM EDTA, 8.8 mM MgCl2, 25% glycerol, 0.5 mM
spermidine, 0.15 mM spermine, 0.7 M NaCl) was
added dropwise, and the extract was gently shaken for 45 min. The
viscous lysate was then centrifuged at 35,000 rpm for 1.5 h at
4 °C to pellet the chromatin. Solid
(NH4)2SO4 was progressively added
(0.4 g/ml) to the supernatant and dissolved by gentle mixing. After
incubation 45 min on ice, the precipitated proteins were centrifuged in
an SW60 rotor at 37,000 rpm for 30 min at 4 °C. The pellets were
resuspended in 200 µl of dialysis buffer (20 mM HEPES, pH
8, 1.2 mM EDTA, 60 mM KCl, 25% glycerol, 1 mM DTT, 0.5 mM PMSF) for 40 A260 units of nuclear lysate (see above). The
protein extract was dialyzed twice for 2 h against 200 ml of the
dialysis buffer without DTT and PMSF. The precipitate, formed during
dialysis, was discarded by a 10-min centrifugation at 10,000 rpm at
4 °C. The protein extract was frozen in small aliquots in liquid
nitrogen and stored at Gel Retardation Assays--
Single strand oligonucleotides (35 bp) covering 0.5 kb of the testis HSL promoter were gel-purified. Other
oligonucleotides used were as follows: mSRY/Sox, 5'-GTA GGG CAC CCA TTG
TTC TCT-3' (23); signal transducer and activator of transcription,
5'-CTG ATT TCC CCG AAA TGA CGG-3' (24); HNF3, 5'-CTA GAA CAA ACA AGT CCT GCG T-3' (25); C/EBP, 5'-GAT CCG CGT TGC GCC ACG ATG-3' (26). 100 ng of single strand oligonucleotides were 5'-end-labeled using T4
polynucleotide kinase (Eurogentec) and [ Circular Permutation Assay--
Annealed synthetic
oligonucleotides containing the TSBR4 region (Fig. 4) were cloned into
the XbaI site of the circular permutation vector pBend2
(29). Circularly permuted DNA fragments were made by cleavage with the
restriction enzymes indicated in Fig. 8A, dephosphorylated
using calf intestine phosphatase (Eurogentec), and gel-purified. The
DNA fragments were 5'-end-labeled using T4 polynucleotide kinase and
[ HSLtes Expression in Germ Cells--
The developmental
expression of HSLtes mRNA was examined by Northern blot
analysis of testis total RNA from mice at different ages. In rodents,
the time at which a transcript appears during the first wave of
spermatogenesis in prepuberal animal can be used to identify the
spermatogenic cell type in which transcription initiates (31). The
levels of proenkephalin and protamine 1 mRNA were therefore
determined. In rodents, somatic and spermatogenic cells expressed a
1.4- and a 1.7-kb proenkephalin mRNA, respectively. The
testis-specific proenkephalin mRNA is expressed at high levels in
late pachytene spermatocytes, and protamine 1 mRNA expression appears in round spermatids (32-34). The proenkephalin germ cell form
was detected from day 21 on (Fig. 1).
HSLtes and protamine 1 mRNAs appeared on day 24. Densitometric analyses of the bands showed that the kinetics of
HSLtes and protamine mRNA expression were very similar
(data not shown), suggesting an expression of both genes in haploid
round spermatids.
Analysis of Tissue-specific Expression in Transgenic Mice--
To
investigate whether the 5'-flanking region of the human
HSLtes specific exon contained cis-acting sequences
involved in tissue-specific expression, we generated transgenic mice
with 1.4HSLtesCAT and 0.5HSLtesCAT constructs. Three (A, B, and C) and
two (D and E) lines were generated from the large and small constructs,
respectively. High levels of CAT activity were detected in testis and
epididymis from sexually mature mice (between 60 and 90 days old) for
the two transgenes (Table I). The
activity in epididymis was ascribed to sperm since, when mature sperm
is washed from the epididymis, CAT activity was between 200 and 800 cpm/min/mg protein in the collected fluid. HSL enzymatic activity and
protein was also detected in the collected fraction indicating the
expression of HSL in sperm after spermiation (data not shown). No
apparent variation in CAT activity was observed in the offspring of the
founders and subsequent generations (data not shown). These data
provided evidence that 0.5 kb of the 5'-flanking region are sufficient
to drive expression of the CAT gene in testis. Next, we sought to
determine if the 5'-flanking regions conferred tissue-specific expression. In males, CAT activity levels were very low in all non-gonadal tissues. In females, the low level of CAT activity seen in
all tissues was comparable to the level detected in tissues of
non-transgenic male and female mice (data not shown). Therefore, the
sequences present in the first 0.5 kb of the human HSLtes promoter are critical for specific expression in testis. CAT activity was also determined in testis of 25- and 60-day-old mice from lines A
and D. Four animals were analyzed per line at both ages. In young mice,
the levels of CAT activity were 21 ± 3 cpm/min/mg protein for
line A and 18 ± 1 cpm/min/mg protein for line D. In older mice,
CAT activity levels were 818 ± 36 and 862 ± 43 cpm/min/mg protein, respectively. The marked increase in CAT activity showed that
the transgenes were expressed in post-meiotic germ cells.
In order to check if the transcriptional start site of the chimeric
genes expressed in transgenic mice and of the endogenous human
HSLtes gene were identical, we performed RNase H mapping analyses with human-specific oligonucleotides on RNAs from human and
transgenic mouse testis (Fig. 2). In both
tissues, a band of ~175 nucleotides was detected. The data show that
the human HSLtes promoter in transgenic mice used the same
initiation site as the endogenous human promoter. Moreover, the length
of the 5'-noncoding region deduced from RNase H mapping corresponded to
the size (277 nucleotides) found using 5'-rapid amplification of
cDNA ends PCR (3).
HSLtes Promoter Activation Is cAMP- and
CREM Testis Nuclear Protein-binding Sites within the Human
HSLtes Promoter--
Transgenic analyses demonstrated that
the 0.5-kb region located upstream of the transcriptional start site
was sufficient to confer testis-specific expression. To assess directly
whether sequences within the human HSLtes promoter bound
nuclear proteins present in testis, a series of in vitro DNA
binding studies was performed. The strategy used consisted in designing
20 overlapping double strand oligonucleotides spanning the entire
region (Fig. 4). Each of the 20 oligonucleotides was used to map
interaction sites for factors present in nuclear extracts prepared
either from rat testis or from rat liver, an organ that does not
express HSL. Four probes bound nuclear proteins expressed in testis but not in liver (Fig. 5). Analysis of the sequences of three
testis-specific binding regions (TSBR) revealed no binding motifs for
known testis transcription factors. TSBR4 contained a sequence AACAAAG
(Fig. 4) that has been shown to bind members of the SRY/Sox protein family (37). The testis-specific binding on TSBR4 (Fig. 6) was competed
by mSRY/Sox oligonucleotide but not by signal transducer and activator
of transcription and HNF3 oligonucleotides. The mSRY/Sox
oligonucleotide contains an AACAAT sequence with high affinity for
mouse SRY, Sox5, and Sox6 (27, 28, 38-40). An efficient competition
was observed that was maximal with a 30-fold excess of mSRY/Sox
oligonucleotide. Binding of the testis-specific nuclear proteins to
TSBR4 was increased when poly(dG-dC) was added as nonspecific
competitor (Fig. 6), a feature suggesting interaction of HMG domain
proteins such as Sox proteins with A-T pairs in the minor groove of the
DNA helix (41, 42). A short form of Sox5 and Sox6 is expressed in mice
in post-meiotic germ cells (27, 38). Binding of recombinant Sox5 HMG
box peptide and in vitro translated Sox6 protein on TSBR4
was studied using linker scan mutagenesis (Fig. 7). The HMG domain of
Sox5 bound the mSRY/Sox probe containing the AACAAT sequence and TSBR4
but not a C/EBP recognition motif (Fig. 7B). Mutation of the
AACAAAG sequence of TSBR4 strongly decreased the binding. Because Sox6
homodimers do not bind DNA (27), a truncated form of Sox6 deleted of
the leucine zipper region was produced by in vitro
translation. As previously reported (27), incubation of the
unprogrammed lysate (data not shown) and of the programmed lysate of
the empty vector pRc/CMV with labeled double strand oligonucleotides
resulted in retardation of the probe (Fig. 7C). The binding
appeared to be due to endogenous DNA binding factor of the reticulocyte
lysate. When the pCMV/Sox-LZ(D105-356) vector was used, an additional binding complex was detected. This binding was abolished by mutation of
the AACAAAG sequence. These data show that testis Sox proteins produced
in vitro and from nuclear extracts bind TSBR4.
The HMG Box of Sox5 Bends a Testis-specific Binding
Region--
Sox proteins as other proteins containing HMG domains
induce a marked bend within the DNA (23, 37). Therefore, we
investigated whether the HMG box of Sox5 was able to modify TSBR4 DNA
curvature using a circular permutation assay. TSBR4 DNA was cloned into the pBend2 vector (29). Digestion of the resulting plasmid with various
restriction endonucleases gave DNA probes of almost identical sizes and
base composition but with TSBR4 at variable distances from the end of
the probe (Fig. 8A). Fig. 8B shows the result of
a gel retardation assay with recombinant Sox5 HMG box peptide and the
different DNA probes. The retarded complexes migrated with a mobility
that was inversely correlated to the distance between the binding site
and the end of the probe, a relationship characteristic of proteins
that bend their target DNA (43). The ratio between the fastest and the
slowest migrating species was used to estimate the extent of DNA
distortion (Fig. 8C). The center of the bending mapped to
the AACAAAG motif of TSBR4. Using the empirical equation proposed by
Thompson and Landy (30), the angle of DNA bending was estimated between
65 and 70°.
In this paper, we show that the proximal 5'-flanking region of the
HSL gene functions as a testis-specific promoter and binds testis
nuclear proteins. HSLtes mRNA appears in round
spermatids concomitantly to protamine 1 mRNA (Fig. 1). This result
is in agreement with in situ hybridization data obtained in
rat which showed that HSLtes mRNA was detected in
stages X-XIV of spermatogenesis (6). As shown for many genes expressed
during spermatogenesis, the HSLtes protein accumulation is
delayed to stages XIII-VIII corresponding to late spermatids (3, 44).
The similar stage-specific expression pattern observed for
HSLtes and protamine 1 mRNAs and other transcripts
suggests the presence of common regulatory mechanisms. Since CREM The lack of appropriate male haploid germ cell line led us to use
transgenic mice to investigate the transcriptional regulation of
HSLtes. We demonstrate here that 0.5 kb of the region
flanking the HSLtes-specific exon govern testis expression
in transgenic mice (Table I). Analysis of a large number of tissues in
male and female transgenic mice showed the strict testis expression of
the transgene. The testis form of HSL that is characterized by larger
mRNA and protein species than the other isoforms has only been
detected in testis (1-3). Moreover, the 25-day-old transgenic mice
showed very little CAT activity compared with the 60-day-old animals.
The data in transgenic mice are therefore in agreement with the pattern
and timing of expression of HSLtes.
In order to determine testis-specific DNA-protein interactions on the
HSLtes promoter 0.5-kb region, gel retardation assays were
performed using overlapping double strand oligonucleotides (Fig.
4). Four regions were shown to bind
testis nuclear proteins absent in liver nuclear extracts (Fig.
5). One of them, TSBR4, contained a DNA
sequence motif AACAAAG recognized by the HMG domain of SRY/Sox proteins
(23, 39). Competition experiments revealed that TSBR4 bound a testis
nuclear protein that shows properties of a Sox protein (Fig.
6). Two members of the family, a short form of Sox5 and Sox6, are expressed in male germ cells at the round
spermatid stage (27, 28, 38, 46). The role of these proteins in
spermatogenesis has not been documented. The HMG domain of Sox5 and a
leucine zipper region-deleted Sox6 was shown to bind TSBR4 (Fig.
7). This observation raises the
possibility that the short form Sox5 and/or Sox6 may directly or
indirectly participate to the transactivation of the HSLtes
promoter. The HSL gene would therefore represent the first target
gene of these proteins.
INSERM Unit 317,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a testis-specific transcriptional
activator, does not transactivate the HSLtes promoter.
Using gel retardation assays, four testis-specific binding regions
(TSBR) were identified using testis and liver nuclear extracts. The
testis-specific protein binding on TSBR4 was selectively competed by a
probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6
which are expressed in post-meiotic germ cells bound TSBR4. Mutation of
the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of
the high mobility group domain of Sox5 induced a bend within TSBR4.
Together, our results showed that 0.5 kb of the human
HSLtes promoter bind Sox proteins and contain cis-acting
elements essential for the testis specificity of HSL.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a product of the CREM gene that acts as a
transcriptional activator responsive to the cAMP signaling pathway
(12). Several target genes for CREM
-mediated activation have been
identified in haploid germ cells, most notably the gene encoding
protamine 1, a nucleoprotein that replaces histones and promotes
nuclear condensation (13, 14). Moreover, targeted disruption of the
CREM gene results in a complete block of germ cell differentiation at
the first steps of spermiogenesis (15, 16). Thus, CREM
may govern a coordinated regulation of gene expression in post-meiotic germ cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-actin probe. Membranes were
pre-hybridized for 1 h in hybridization buffer (500 mM
Na2HPO4, 1 mM EDTA, 7% SDS, 1%
bovine serum albumin) and then hybridized overnight in 10 ml of the
same buffer containing 1.5 106 cpm/ml HSL and proenkephalin
cDNA probes and 106 cpm/ml protamine cDNA probe.
After hybridization, membranes were washed twice with 0.3 M
NaCl, 30 mM tri-sodium citrate, 0.1% SDS 20 min at room
temperature and once with 30 mM NaCl, 3 mM
tri-sodium citrate, 0.1% SDS for 30 min at 65 °C. Membranes were
subjected to digital imaging (Molecular Dynamics).
cDNA (12) was subcloned into the
expression vector pSVSport (Life Technologies, Inc.).
-pSVSport or pSVSport, 700 ng of pFC-PKA, an expression
vector encoding the catalytic subunit of the cAMP-dependent
protein kinase (Stratagene) or pFC-DBD, the negative control plasmid
(Stratagene) and 50 ng of pRL-CMV vector (Promega). The pRL-CMV vector
encoding Renila luciferase was used to normalize
transfection efficiency. Cells were treated 44 h post-transfection
with 1 mM dibutyryl cAMP (Sigma) when specified. Cells were
harvested 48 h post-transfection for Firefly and
Renilla luciferase activity determinations according to the
manufacturer's instructions (Promega).
80 °C. Protein concentrations ranged
between 5 and 10 mg/ml.
-32P]ATP
(>4000 Ci/mmol). After heat inactivation of the kinase, the labeled
oligonucleotides were annealed to 300 ng of the complementary strand
oligonucleotides. Labeled double strand oligonucleotides were purified
with the QIAquick nucleotide removal kit (Qiagen). 32P-Labeled DNA (1 ng at approximately 100,000 cpm/ng) was
incubated on ice for 30 min with testis (10 µg) or liver (8 µg)
nuclear extracts in a total reaction buffer volume of 25 µl
containing 10 mM HEPES, pH 7.9, 60 mM KCl, 0.1 mM EDTA, 1 mM DTT, 4 mM spermidine, 5 mM MgCl2, 12% glycerol, and 1 µg of
poly(dI-dC) (Amersham Pharmacia Biotech) or 0.5 µg of poly(dI-dC) and
0.5 µg of poly(dG-dC) (Amersham Pharmacia Biotech). DNA-protein
complexes were resolved on 6% nondenaturing polyacrylamide gels at 10 V/cm for 3 h in a 23 mM Tris borate, pH 8, 0.5 mM EDTA migration buffer. Polyacrylamide gels were dried
under vacuum and subjected to digital imaging (Molecular Dynamics). Gel
retardation assays were also performed with Sox proteins. A truncated
form of the trout orthologue of Sox6 deleted of the leucine zipper
region was produced in a reticulocyte lysate-coupled
transcription-translation system (Promega) using the
pCMV/Sox-LZ(D105-356) vector and the empty pRc/CMV vector (27). Six
µl of the reaction mixture were used in gel retardation assays. A
peptide containing the high mobility group (HMG) box of mouse Sox5 (28)
was produced in Escherichia coli as a glutathione S-transferase fusion protein and purified using
glutathione-Sepharose beads and bovine thrombin. The purity of the
peptide was checked on SDS-polyacrylamide gel electrophoresis. Fifty ng
of purified protein and 0.5 ng of 32P-labeled double strand
oligonucleotide were used in gel retardation assays.
-32P]ATP, and gel-purified. Binding reactions were
performed for 20 min at room temperature with 10 ng of Sox5 peptide, 50 000 cpm of DNA in a reaction buffer containing 10 mM HEPES,
pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM
DTT, and 12% glycerol. The reactions were electrophoresed on 8%
nondenaturing polyacrylamide gels at 10 V/cm for 4-5 h. Bend
parameters were calculated according to Thompson and Landy (30).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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View larger version (32K):
[in a new window]
Fig. 1.
HSLtes mRNA expression during
development in mice. RNA blots were prepared with 25 µg of
testis total RNA from 14-, 17-, 21-, 24-, 28-, 35-, and 56-day-old
mice. The blot was hybridized with HSL, proenkephalin, and protamine 1 cDNA probes. gProenk, germ cell proenkephalin;
sProenk, somatic proenkephalin.
Chloramphenicol acetyltransferase (CAT) activity of 1.4HSLtesCAT and of
0.5HSLtesCAT in transgenic mice
View larger version (62K):
[in a new window]
Fig. 2.
Analysis of 5'-untranslated region of
HSLtes mRNA in human and transgenic mice testes.
Total RNA (90 µg) from sexually mature transgenic mouse testis and 1 µg of poly(A)+ RNA from human testis were digested
by RNase H in the presence of an antisense oligonucleotide
complementary to the 5'-untranslated region of human HSLtes
mRNA. The reaction mixture was denatured and electrophoresed on a
polyacrylamide-urea gel. The resulting blot was hybridized with a
32P-labeled DNA probe located 5' of the antisense
oligonucleotide on human HSLtes cDNA. RNA size
markers were produced by in vitro transcription and labeled
by incorporating [32P]UTP into the reaction
mixture.
-independent--
It has been shown that CREM
binds to
cAMP-responsive elements (CREs) and stimulates transcription of several
germ cell-specific genes (12, 13). CREM
functions as a
transcriptional activator after phosphorylation by
cAMP-dependent protein kinase. Computer-based (35) and
visual analyses did not reveal apparent consensus sequences for CREs in
the HSLtes promoter. Since functional CRE-like sites can
substantially diverge from the palindromic sequence TGACGTCA (36), we
wished to determine whether CREM
and cAMP had an effect on
HSLtes transcriptional activity. Because of the lack of
haploid germ cell lines, cotransfection experiments were performed in JEG3, a human choriocarcinoma cell line bearing an efficient
cAMP-dependent transduction pathway (13). To ensure that
our transfection system was valid to study cAMP-dependent
transactivation, we used a control CRE-LUC vector containing four
copies of CREs upstream of a minimal promoter. This reporter construct
was strongly cAMP-inducible whether the cells were treated with
dibutyryl cAMP, a stable and permeable analogue of cAMP, or
cotransfected with an expression vector for the catalytic subunit of
the cAMP-dependent protein kinase. This result was
predictable because JEG3 cells contain endogenous CREB. CREM
coexpression resulted in a further increase of cAMP-induced luciferase
activity. The results obtained with 0.5HSLtesLUC were strikingly
different. No significant increase in luciferase activity was observed
showing that the HSLtes promoter does not represent a
cellular target of CREM/CREB transregulatory function.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plays an important role during the first steps of spermiogenesis as a
transcriptional activator, we checked whether this transcription factor
transactivates the HSLtes promoter. Cell transfection
experiments (Fig. 3) similar to the ones
performed with CREM
-activated promoters (11, 13, 45) do not support a direct role for CREM
and members of the CREB family in
HSLtes promoter transactivation. An indirect role of
CREM
that is essential for a complete differentiation of haploid
germ cells (15, 16) cannot, however, be ruled out, e.g.
through the control of expression of a transcription factor activating
the HSLtes promoter.
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Fig. 3.
Effect of CREM ,
cAMP-dependent protein kinase, and cAMP on
p0.5HSLtesLUC vector in JEG3 cells. The p0.5HSLtesLUC vector
contains 0.5 kb of the HSLtes promoter in the promoterless
luciferase vector (pGL3basic). The pCRE-LUC reporter construct contains
four copies of cAMP-responsive elements upstream of a minimal promoter
linked to the luciferase gene. The reporter constructs were
cotransfected with expression vectors encoding the catalytic subunit of
the cAMP-dependent protein kinase (PKA) and
CREM
. Cells were treated for 4 h with 1 mM
dibutyryl cAMP (db cAMP), a stable and permeable analogue of
cAMP, as indicated. Results are expressed relative to the activity of
pGL3basic treated under the same conditions. Data represent means ± S.D. of three experiments performed in duplicate.
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Fig. 4.
Sequence of 0.5 kb of the HSLtes
promoter and positions (overlined) of the 20 probes
used in gel retardation analysis. A putative TATA box is indicated
in bold. A SRY/Sox recognition motif is
underlined. The positions of the four testis-specific
binding regions (TSBR) are boldface.
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Fig. 5.
Binding of testis and liver nuclear extracts
on four oligonucleotide probes corresponding to the testis-specific
binding regions (TSBR). Lane 1,
nuclear extracts; lane 2, nuclear extracts in the presence
of 100-fold excess of unlabeled TSBR oligonucleotide; and lane
3, nuclear extracts in the presence of 100-fold excess of
unrelated unlabeled oligonucleotide. Arrowheads show
testis-specific nuclear protein binding.
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Fig. 6.
Binding of testis and liver nuclear extracts
on the testis-specific binding region TSBR4. Lane 1,
liver nuclear extracts; lanes 2-11, testis nuclear
extracts. Binding reaction buffer contained 1 µg of poly(dI-dC)
(lane 2) or 0.5 µg of poly(dI-dC) and 0.5 µg of
poly(dG-dC) (lane 1 and lanes 3-11). A 100-fold
excess of TSBR4 oligonucleotide (lane 4), of unrelated
oligonucleotide from the 0.5-kb HSLtes promoter (lane
5), and of signal transducer and activator of transcription 1 (lane 10) and HNF3 (lane 11) oligonucleotides
were added in competition experiments. Competition experiments were
also performed in the presence of 3- (lane 6), 10- (lane 7), 30- (lane 8), and 100-fold (lane
9) of mSRY/Sox oligonucleotide that contains the AACAAT sequence.
A testis-specific DNA complex is shown with an
arrowhead.
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Fig. 7.
Linker scan mutagenesis of the
testis-specific binding region TSBR4. A, sequences of
the oligonucleotide probes and positions of the mutation are shown in
bold. The SRY/Sox recognition motif is
underlined. B, DNA binding of the purified high
mobility group domain of Sox5. mSRY/Sox and C/EBP oligonucleotides were
used as positive and negative controls, respectively. Binding reactions
were performed in the absence of unlabeled oligonucleotide (lane
1), in the presence of 100-fold excess of unlabeled specific
oligonucleotide (lane 2), and of unlabeled unrelated
oligonucleotide (lane 3). C, DNA binding of trout
Sox6 deleted of the leucine zipper region. In vitro
transcription/translations were performed using the
pCMV/Sox-LZ(D105-356) vector (+) and the empty pRc/CMV vector ( ).
The position of Sox6 DNA binding is indicated by an
arrowhead.
Cooperation of several Sox proteins and other transcription factors is often necessary to promote target gene expression (41, 46-49). In teratocarcinoma cells, Sox2 and the POU domain transcription factor octamer 3 bind adjacent sites and participate together to the transactivation of the fibroblast growth factor 4 gene through protein-protein interaction (41, 49, 50). Either factor alone is ineffective. Three different Sox proteins, a long form of Sox5, Sox6, and Sox9, are coexpressed in chondrocytes and cooperatively activate the chondrocyte-specific enhancer of the type II collagen gene (46). The activation is facilitated by the dimerization of the long form of Sox5 and Sox6. Sox6 contains a leucine zipper motif that allows dimerization of the protein, and homodimers fail to bind DNA (27). These data suggest that, in testis, Sox6 may bind to another protein as heterodimers to show transactivation properties. The short form of Sox5 expressed in testis does not contain the coiled-coil domain present in the long form. This domain is necessary for homo- and heterodimerization (46). In HeLa cells, expression of the short form Sox5 alone or coexpression of the short form Sox5 and Sox6 did not activate the HSLtes promoter (data not shown). Other uncharacterized testis Sox proteins might be involved in the activation of HSLtes. In addition, the cooperation between Sox proteins and other transcription factors might be necessary. The identification of the interacting partners will require extensive investigation since, except Sox binding to TSBR4, the other TSBRs do not show significant sequence homology with consensus binding motifs for known testis transcription factors.
Sox proteins could indirectly modulate transcription activity by
organizing local chromatin structure. Binding of Sox proteins occurs in
the minor groove and results in a bend within the DNA (37). It has been
reported that Sox5 HMG box induces a bend to the AACAAT motif with an
estimated angle of 74° (28). The nature of the recognition sequence
and of the flanking nucleotides influence the angle of the bend (51).
Here, we show that the HMG domain of Sox5 induces an estimated flexure
of 65-70° through binding to the AACAAAG sequence of TSBR4 (Fig.
8). The data demonstrate that Sox5 can
induce a strong bend in DNA in the context of a natural testis-specific
promoter. Testis Sox proteins may act through an alteration of local
chromatin structure around the AACAAAG site in TSBR4 to facilitate the
interaction of distant enhancer nucleoprotein complexes
(e.g. on the other TSBRs) with the basal transcription
machinery.
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To conclude, we have identified a testis-specific promoter that
contains four regions binding testicular nuclear proteins. The
HSLtes promoter provides a molecular basis to characterize new cis-acting elements and transcription factors responsible for the
transactivation of genes in post-meiotic germ cells.
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ACKNOWLEDGEMENTS |
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We are grateful to Ghislaine Hamard
(INSERM U380, Paris, France) for help with oocyte microinjection; Dr.
Michel Raymondjean (INSERM U129, Paris, France) for critical reading of
the manuscript; Prof. Alan Ashworth (The Institute of Cancer Research,
London, UK) for short form Sox5 vector; Dr. Véronique Lefebvre
(University of Texas M. D. Anderson Cancer Center, Houston, TX) for
Sox6 vector; Dr. Nobuhiko Takamatsu (Kitasato University, Kitasato,
Japan) for Sox-LZ vector, Dr. Sankar Adhia (National Institutes of
Health, Bethesda, MD) for pBend2 vector, and Dr. Paolo Sassone-Corsi
(IGBMC, Strasbourg, France) for CREM vector.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ132272.
§ Present address: Laboratoire de Nutrition, Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation, Dijon, France.
To whom correspondence should be addressed: INSERM U317,
Institut Louis Bugnard, Bâtiment L3, CHU Rangueil, F-31403
Toulouse Cedex 4, France. Tel.: (33) 5 62172950; Fax: (33) 5 61331721; E-mail: langin{at}rangueil.inserm.fr.
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ABBREVIATIONS |
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The abbreviations used are: HSL, hormone-sensitive lipase; HSLtes, testicular hormone-sensitive lipase; CAT, chloramphenicol acetyltransferase; C/EBP, CCAAT-enhancer binding protein; CRE, cAMP-responsive element; CREB, cAMP-responsive element binding protein; CREM, cAMP-responsive element modulator; CMV, cytomegalovirus; HMG, high mobility group; HNF, hepatocyte nuclear factor; LUC, luciferase; Sox, SRY-type HMG box protein; SRY, Sex-determining Y chromosome protein; TSBR, testis-specific binding region; kb, kilobase pair; bp, base pair; PCR, polymerase chain reaction; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride.
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