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Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins

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Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins
  Testis Expression of Hormone-sensitive Lipase Is Conferred by aSpecific Promoter That Contains Four Regions Binding TesticularNuclear Proteins* (Received for publication, December 9, 1998, and in revised form, January 19, 1999) Re´gis Blaise‡, Jacques Grober‡§, Philippe Rouet‡, Genevie`ve Tavernier‡ ¶ , Dominique Daegelen ¶ ,and Dominique Langin‡   From  ‡  INSERM Unit 317, Institut Louis Bugnard, Universite´ Paul Sabatier, Hoˆpital Rangueil, F-31403 Toulouse Cedex 4and  ¶  INSERM Unit 129, Institut Cochin de Ge´ne´tique Mole´culaire, Faculte´ de Me´decine, 24 Rue du Faubourg Saint Jacques, F-75014 Paris Cedex, France The testicular isoform of hormone-sensitive lipase(HSL tes ) is encoded by a testis-specific exon and 9 exonscommon to the testis and adipocyte isoforms. In mouse,HSL tes  mRNA appeared during spermiogenesis in roundspermatids. Two constructs containing 1.4 and 0.5 kilo-base pairs (kb) of the human HSL tes  gene 5  -flanking region cloned upstream of the chloramphenicol acetyl-transferase gene were microinjected into mouse oo-cytes. Analyses of enzyme activity in male and femaletransgenic mice showed that 0.5 kb of the HSL tes  pro-moter was sufficient to direct expression only in testis.Cell transfection experiments showed that CREM   , atestis-specific transcriptional activator, does not trans-activate the HSL tes  promoter. Using gel retardation as-says, four testis-specific binding regions (TSBR) wereidentified using testis and liver nuclear extracts. Thetestis-specific protein binding on TSBR4 was selectivelycompeted by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed inpost-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. More-over, binding of the high mobility group domain of Sox5induced a bend within TSBR4. Together, our resultsshowed that 0.5 kb of the human HSL tes  promoter bindSox proteins and contain cis-acting elements essentialfor the testis specificity of HSL. Hormone-sensitive lipase (HSL) 1 is a triacylglycerol lipaseand a cholesterol esterase expressed at high levels in adipo-cytes, testes, and adrenals (1–3). In adipocytes, HSL catalyzesthe rate-limiting step in the hydrolysis of triglycerides intofatty acids and glycerol (4). HSL activation is mediated throughphosphorylation by the cAMP-dependent protein kinase (5). Inrat testis, HSL mRNA and protein are expressed in the semi-niferous tubuli and not in interstitial cells with a stage-depend-ent pattern corresponding to the appearance of haploid germcells (3, 6). Several isoforms of HSL produced by a single genehave been characterized (2, 7, 8). Human adipose tissue ex-presses 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 se-quences revealed that human adipocyte and testis HSL(HSL tes ) are 775 and 1076 amino acids long, respectively.HSL tes  differs from the adipocyte form by a unique NH 2 -termi-nal region. Elucidation of the HSL gene organization showedthat nine coding exons are common to both forms. The addi-tional sequence in HSL tes  is encoded by a 1.2-kb-long testis-specific exon (3, 7). When a gene is expressed in somatic tissuesand in germ cells, tissue-specific expression often results fromalternate promoter use (10, 11). The promoter of the adipocyteform of HSL (9) is located 13 kb downstream of the HSL tes 5  -flanking region suggesting that the expression of the differ-ent forms of HSL is controlled by several tissue-specificpromoters.During spermatogenesis, specialized transcriptional mecha-nisms ensure stage-specific gene expression in the germ cells.The factors controlling gene expression in post-meiotic germcells are beginning to be elucidated. Several germ cell-specificputative transcription factors have been cloned, but targetgenes have been identified only for a few of them. CREM    is aproduct of the CREM gene that acts as a transcriptional acti- vator responsive to the cAMP signaling pathway (12). Severaltarget genes for CREM   -mediated activation have been identi-fied in haploid germ cells, most notably the gene encoding protamine 1, a nucleoprotein that replaces histones and pro-motes nuclear condensation (13, 14). Moreover, targeted dis-ruption of the CREM gene results in a complete block of germcell differentiation at the first steps of spermiogenesis (15, 16).Thus, CREM    may govern a coordinated regulation of geneexpression in post-meiotic germ cells.In this study, we investigated the molecular mechanismsthat control the testis-specific expression of HSL tes . During spermatogenesis, HSL tes  mRNA was expressed in haploid germcells concomitantly with protamine 1 mRNA. We show that 0.5kb of the HSL tes  promoter was sufficient to drive testis-specificexpression in transgenic mice. In cell transfection experiments,transactivation of the HSL tes  promoter was independent of thecAMP signaling pathway. Four regions bound nuclear proteins * The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“ advertisement ” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact. The nucleotide sequence(s) reported in this paper has been submittedto the GenBank TM /EBI Data Bank with accession number(s) AJ132272. § Present address: Laboratoire de Nutrition, Ecole Nationale Su-pe´rieure de Biologie Applique´e a` la Nutrition et a` l’Alimentation, Dijon,France.   To whom correspondence should be addressed: INSERM U317, In-stitut Louis Bugnard, Baˆtiment L3, CHU Rangueil, F-31403 ToulouseCedex 4, France. Tel.: (33) 5 62172950; Fax: (33) 5 61331721; 1 The abbreviations used are: HSL, hormone-sensitive lipase; HSL tes ,testicular hormone-sensitive lipase; CAT, chloramphenicol acetyltrans-ferase; C/EBP, CCAAT-enhancer binding protein; CRE, cAMP-respon-sive 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 chromosomeprotein; TSBR, testis-specific binding region; kb, kilobase pair; bp, basepair; PCR, polymerase chain reaction; DTT, dithiothreitol; PMSF,phenylmethylsulfonyl fluoride. T HE  J OURNAL OF  B IOLOGICAL  C HEMISTRY   Vol. 274, No. 14, Issue of April 2, pp. 9327–9334, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.  Printed in U.S.A. This paper is available on line at  9327   b  y g u e  s  t   on N o v e m b  e r 1 2  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   present in testis and not in liver. One of the region bound Soxproteins expressed in post-meiotic germ cells raising the pos-sibility that Sox proteins are involved in the transactivation of the HSL tes  promoter. EXPERIMENTAL PROCEDURES  Northern Blot Analyses—  A mouse HSL DNA probe (477 bp) wasgenerated by PCR on mouse genomic DNA with primers located in thefirst adipocyte coding exon 5  -ATG GAT TTA CGC ACG ATG ACA CAG-3   and 5  -TAG CGT GAC ATA CTC TTG CAG GAA-3  . Proen-kephalin (227 bp) and protamine 1 (207 bp) cDNA probes were gener-ated by reverse transcription-PCR on mouse testis total RNA using 5  -GAC AGC AGC AAA CAG GAT GA-3   and 5  -TTC AGC AGA TCGGAG GAG TT-3  , and 5  -AGC AAA AGC AGG AGC AGA TG-3   and5  -AGA TGT GGC GAG ATG CTC TT-3   primers, respectively. PCRreactions 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 sequencesto published sequences was checked by automatic DNA sequencing (Applied Biosystems).Total testis RNA was prepared from prepuberal and sexually maturemice by a single-step guanidinium thiocyanate phenol/chloroform ex-traction (18). RNA samples (25   g) were separated on a 1% agarose, 2.2 M  formaldehyde gel, transferred, and UV cross-linked to a nylon mem-brane (Nytran, Schleicher & Schuell). Equal loading of the differentlanes was checked by ethidium bromide staining of the gel and byhybridization with a rat   -actin probe. Membranes were pre-hybridizedfor 1 h in hybridization buffer (500 m M  Na 2 HPO 4 , 1 m M  EDTA, 7% SDS,1% bovine serum albumin) and then hybridized overnight in 10 ml of the same buffer containing 1.5 10 6 cpm/ml HSL and proenkephalincDNA probes and 10 6 cpm/ml protamine cDNA probe. After hybridiza-tion, membranes were washed twice with 0.3  M  NaCl, 30 m M  tri-sodiumcitrate, 0.1% SDS 20 min at room temperature and once with 30 m M NaCl, 3 m M  tri-sodium citrate, 0.1% SDS for 30 min at 65 °C. Mem-branes were subjected to digital imaging (Molecular Dynamics).  Plasmid Constructs—  A 1.6-kb  Hin dIII/   Bgl II human DNA genomicrestriction fragment was isolated from a cosmid clone containing theentire human HSL gene (3). The fragment was subcloned into the  Hin dIII and  Bam HI sites of pBluescript and sequenced by automaticDNA sequencing (Applied Biosystems). It contained 1.4 kb of the 5  -flanking region upstream of the testis-specific exon. The construct wasdigested with  Hin dIII and  Xba I, and the 1.6-kb fragment was ligatedupstream of the chloramphenicol acetyltransferase (CAT) gene into thepromoterless pCAT-basic vector (Promega) (p1.4HSLtesCAT). The1.6-kb  Hin dIII/   Bgl II DNA genomic restriction fragment was also clonedupstream 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  Sma I to producep0.5HSLtesLUC. The  Sma I site used to generate p0.5HSLtesLUC andthe  Ava I site used to generate the microinjected fragment 0.5HSLtes-CAT (see below) are overlapping. The complete 1-kb CREM    cDNA (12)was subcloned into the expression vector pSVSport (Life Technologies,Inc.). Transgenic Mice— The two transgenes were prepared by digesting p1.4HSLtesCAT with  Hin dIII and  Bam HI or with  Ava I and  Bam HI togive, respectively, 1.4HSLtesCAT and 0.5HSLtesCAT. These two frag-ments were isolated on agarose gel by electroelution and purified using an elutip-d column (Schleicher & Schuell). Transgenic mice were pro-duced by microinjection of the transgenes into the pronuclei of fertilizedB6D2/F1 mouse eggs (19). Microinjected embryos were transferred topseudo-pregnant B6-CBA/F1 female mice and carried to term. Screen-ing of the positive transgenic animals was performed with DNA pre-pared from tail samples using Southern blot or PCR using as senseprimer an oligonucleotide located in the human HSL tes  5  -flanking sequence and as antisense primer an oligonucleotide located in the CATgene. Subsequent generation of heterozygous mice were produced bymating transgenic mice with wild type B6-CBA/F1 mice. The transmis-sion of the transgene was  50% in the progeny of all founders indicat-ing Mendelian transmission. Protein extracts for CAT assays wereprepared from hemizygous transgenic mice. Briefly, tissues were rap-idly frozen in liquid nitrogen and homogenized in 0.5 ml of 250 m M  Tris,pH 7.6, 5 m M  EDTA, and 1 m M  DTT. Homogenates were heated 7.5 minat 65 °C and centrifuged at 4 °C for 15 min at 13,000 rpm. Supernatantswere kept for CAT and protein analyses (17, 20).  RNase H Mapping— Ninety   g of RNA from transgenic sexuallymature mouse testis or 1   g of human testis poly(A)  RNA (CLON-TECH) were lyophilized and resuspended in 10   l of RNase H buffer (20m M  Tris, pH 7.5, 10 m M  MgCl 2 , 100 m M  KCl, 0.1 m M  DTT, 5% sucrose)containing 10 pmol of the human-specific single strand antisense oligo-nucleotide 5  -GTA GAG TAA CTA AGG AGT TG-3   (nt 197 to 179downstream of the transcriptional start site). After 10 min at 70 °C,hybridization was performed for 30 min at 37 °C. Then, 40   l of RNaseH buffer containing 7 units of RNase H (Amersham Pharmacia Biotech)were added, and digestion was carried out for 45 min (21). The digestionproducts were separated on a polyacrylamide-urea gel after ethanolprecipitation. The gel was washed twice in 7% formaldehyde, 9 m M  Trisborate, 0.2 m M  EDTA, and RNA was passively transferred onto a nylonmembrane. Hybridization was performed as described above using a 32 P-labeled probe corresponding to the 197 bp located downstream of the transcription start site. Cell Transfection Experiments— JEG3 cells grown in 28-cm 2 plateswere transfected using Fugene-6 (Boehringer Mannheim) with 700 ng of p0.5HSLtesLUC or pCRE-LUC (Stratagene), 700 ng of CREM   -pSVSport or pSVSport, 700 ng of pFC-PKA, an expression vector en-coding 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 encod-ing   Renila  luciferase was used to normalize transfection efficiency.Cells were treated 44 h post-transfection with 1 m M  dibutyryl cAMP(Sigma) when specified. Cells were harvested 48 h post-transfection forFirefly and  Renilla  luciferase activity determinations according to themanufacturer’s instructions (Promega).  Preparation of Liver and Testis Nuclear Extracts— Total nuclei ex-tracts were performed as described by Howard  et al.  (22) with modifi-cations. Four adult rat testis and 10–15 mg of adult rat liver (perfusedwith 0.9% NaCl to wash out blood) were washed in ice-cold salinecontaining 0.1 m M  PMSF, decapsulated, and minced with scissors in 40ml of homogenization buffer (10 m M  HEPES, pH 8, 1 m M  EDTA, 25 m M KCl, 0.5 m M  spermidine, 0.15 m M  spermine, 10% glycerol, 0.5 m M  DTT,0.5 m M  PMSF, 0.1 m M  benzamidine, 1   g/ml leupeptin, 1   g/ml pep-statin, 2   g/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 thencompleted to 80 (testis) and 100 ml (liver) with homogenization buffer,and 28-ml aliquots were layered over 10-ml cushions of the same bufferin SW28 tubes. The tubes were centrifuged at 27,000 rpm for 1 h at4 °C. The supernatants were carefully removed, and the tube wallswere washed with water and dried. Nuclei pellets were resuspended in3 (testis) or 5 ml (liver) of buffer A (20 m M  HEPES, pH 8, 100 m M  EDTA,8.8 m M  MgCl 2 , 25% glycerol, 0.5 m M  spermidine, 0.15 m M  spermine,0.14  M  NaCl) using an all-glass Dounce homogenizer (pestle B). Analiquot was diluted 100 times in 0.5% SDS, and the absorbance at 260nm was measured. The nuclear suspension was diluted at 40  A 260  unitsper ml. An equal volume of buffer B (20 m M  HEPES, pH 8, 100 m M EDTA, 8.8 m M  MgCl 2 , 25% glycerol, 0.5 m M  spermidine, 0.15 m M  sperm-ine, 0.7  M  NaCl) was added dropwise, and the extract was gently shakenfor 45 min. The viscous lysate was then centrifuged at 35,000 rpm for1.5 h at 4 °C to pellet the chromatin. Solid (NH 4 ) 2 SO 4  was progressivelyadded (0.4 g/ml) to the supernatant and dissolved by gentle mixing. After incubation 45 min on ice, the precipitated proteins were centri-fuged in an SW60 rotor at 37,000 rpm for 30 min at 4 °C. The pelletswere resuspended in 200   l of dialysis buffer (20 m M  HEPES, pH 8, 1.2m M  EDTA, 60 m M  KCl, 25% glycerol, 1 m M  DTT, 0.5 m M  PMSF) for 40  A 260  units of nuclear lysate (see above). The protein extract was dia-lyzed twice for 2 h against 200 ml of the dialysis buffer without DTT andPMSF. The precipitate, formed during dialysis, was discarded by a10-min centrifugation at 10,000 rpm at 4 °C. The protein extract wasfrozen in small aliquots in liquid nitrogen and stored at  80 °C. Proteinconcentrations ranged between 5 and 10 mg/ml. Gel Retardation Assays— Single strand oligonucleotides (35 bp) cov-ering 0.5 kb of the testis HSL promoter were gel-purified. Other oligo-nucleotides 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 TGCGCC ACG ATG-3   (26). 100 ng of single strand oligonucleotides were5  -end-labeled using T4 polynucleotide kinase (Eurogentec) and[   - 32 P]ATP (  4000 Ci/mmol). After heat inactivation of the kinase, thelabeled oligonucleotides were annealed to 300 ng of the complementarystrand oligonucleotides. Labeled double strand oligonucleotides werepurified with the QIAquick nucleotide removal kit (Qiagen).  32 P-La-beled DNA (1 ng at approximately 100,000 cpm/ng) was incubated onice for 30 min with testis (10   g) or liver (8   g) nuclear extracts in atotal reaction buffer volume of 25   l containing 10 m M  HEPES, pH 7.9,60 m M  KCl, 0.1 m M  EDTA, 1 m M  DTT, 4 m M  spermidine, 5 m M  MgCl 2 , Testis-specific Expression of Hormone-sensitive Lipase 9328   b  y g u e  s  t   on N o v e m b  e r 1 2  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   12% glycerol, and 1   g of poly(dI-dC) (Amersham Pharmacia Biotech) or0.5   g of poly(dI-dC) and 0.5   g of poly(dG-dC) (Amersham PharmaciaBiotech). DNA-protein complexes were resolved on 6% nondenaturing polyacrylamide gels at 10 V/cm for 3 h in a 23 m M  Tris borate, pH 8, 0.5m M  EDTA migration buffer. Polyacrylamide gels were dried under vacuum and subjected to digital imaging (Molecular Dynamics). Gelretardation assays were also performed with Sox proteins. A truncatedform of the trout orthologue of Sox6 deleted of the leucine zipper regionwas produced in a reticulocyte lysate-coupled transcription-translationsystem (Promega) using the pCMV/Sox-LZ(D105–356) vector and theempty pRc/CMV vector (27). Six   l of the reaction mixture were used ingel retardation assays. A peptide containing the high mobility group(HMG) box of mouse Sox5 (28) was produced in  Escherichia coli  as aglutathione  S -transferase fusion protein and purified using glutathi-one-Sepharose beads and bovine thrombin. The purity of the peptidewas checked on SDS-polyacrylamide gel electrophoresis. Fifty ng of purified protein and 0.5 ng of   32 P-labeled double strand oligonucleotidewere used in gel retardation assays. Circular Permutation Assay—  Annealed synthetic oligonucleotidescontaining the TSBR4 region (Fig. 4) were cloned into the  Xba I site of the circular permutation vector pBend2 (29). Circularly permuted DNA fragments were made by cleavage with the restriction enzymes indi-cated in Fig. 8  A , dephosphorylated using calf intestine phosphatase(Eurogentec), and gel-purified. The DNA fragments were 5  -end-labeledusing T4 polynucleotide kinase and [   - 32 P]ATP, and gel-purified. Bind-ing reactions were performed for 20 min at room temperature with 10ng of Sox5 peptide, 50 000 cpm of DNA in a reaction buffer containing 10 m M  HEPES, pH 7.9, 60 m M  KCl, 1 m M  EDTA, 1 m M  DTT, and 12%glycerol. The reactions were electrophoresed on 8% nondenaturing poly-acrylamide gels at 10 V/cm for 4–5 h. Bend parameters were calculatedaccording to Thompson and Landy (30). RESULTS  HSL tes  Expression in Germ Cells— The developmental ex-pression of HSL tes  mRNA was examined by Northern blotanalysis of testis total RNA from mice at different ages. Inrodents, the time at which a transcript appears during the firstwave of spermatogenesis in prepuberal animal can be used toidentify the spermatogenic cell type in which transcriptioninitiates (31). The levels of proenkephalin and protamine 1mRNA were therefore determined. In rodents, somatic andspermatogenic cells expressed a 1.4- and a 1.7-kb proenkepha-lin mRNA, respectively. The testis-specific proenkephalinmRNA is expressed at high levels in late pachytene spermato-cytes, and protamine 1 mRNA expression appears in roundspermatids (32–34). The proenkephalin germ cell form wasdetected from day 21 on (Fig. 1). HSL tes  and protamine 1mRNAs appeared on day 24. Densitometric analyses of thebands showed that the kinetics of HSL tes  and protamine mRNA expression were very similar (data not shown), suggesting anexpression of both genes in haploid round spermatids.  Analysis of Tissue-specific Expression in Transgenic Mice— To investigate whether the 5  -flanking region of thehuman HSL tes  specific exon contained cis-acting sequences in- volved in tissue-specific expression, we generated transgenicmice with 1.4HSLtesCAT and 0.5HSLtesCAT constructs.Three (A, B, and C) and two (D and E) lines were generatedfrom the large and small constructs, respectively. High levels of CAT activity were detected in testis and epididymis from sex-ually mature mice (between 60 and 90 days old) for the twotransgenes (Table I). The activity in epididymis was ascribed tosperm since, when mature sperm is washed from the epididy-mis, CAT activity was between 200 and 800 cpm/min/mg pro-tein in the collected fluid. HSL enzymatic activity and proteinwas also detected in the collected fraction indicating the ex-pression of HSL in sperm after spermiation (data not shown).No apparent variation in CAT activity was observed in theoffspring of the founders and subsequent generations (data notshown). These data provided evidence that 0.5 kb of the 5  -flanking region are sufficient to drive expression of the CATgene in testis. Next, we sought to determine if the 5  -flanking regions conferred tissue-specific expression. In males, CAT ac-tivity levels were very low in all non-gonadal tissues. In fe-males, the low level of CAT activity seen in all tissues wascomparable to the level detected in tissues of non-transgenicmale and female mice (data not shown). Therefore, the se-quences present in the first 0.5 kb of the human HSL tes  pro-moter are critical for specific expression in testis. CAT activitywas also determined in testis of 25- and 60-day-old mice fromlines A and D. Four animals were analyzed per line at bothages. In young mice, the levels of CAT activity were 21    3cpm/min/mg protein for line A and 18  1 cpm/min/mg proteinfor line D. In older mice, CAT activity levels were 818  36 and862    43 cpm/min/mg protein, respectively. The marked in-crease in CAT activity showed that the transgenes were ex-pressed in post-meiotic germ cells.In order to check if the transcriptional start site of thechimeric genes expressed in transgenic mice and of the endog-enous human HSL tes  gene were identical, we performed RNaseH mapping analyses with human-specific oligonucleotides onRNAs from human and transgenic mouse testis (Fig. 2). In bothtissues, a band of    175 nucleotides was detected. The datashow that the human HSL tes  promoter in transgenic mice usedthe same initiation site as the endogenous human promoter.Moreover, the length of the 5  -noncoding region deduced fromRNase H mapping corresponded to the size (277 nucleotides)found using 5  -rapid amplification of cDNA ends PCR (3).  HSL tes  Promoter Activation Is cAMP- and CREM    -independ- ent— It has been shown that CREM    binds to cAMP-responsiveelements (CREs) and stimulates transcription of several germcell-specific genes (12, 13). CREM    functions as a transcrip-tional activator after phosphorylation by cAMP-dependent pro-tein kinase. Computer-based (35) and visual analyses did notreveal apparent consensus sequences for CREs in the HSL tes promoter. Since functional CRE-like sites can substantiallydiverge from the palindromic sequence TGACGTCA (36), wewished to determine whether CREM    and cAMP had an effecton HSL tes  transcriptional activity. Because of the lack of hap-loid germ cell lines, cotransfection experiments were performedinJEG3,ahumanchoriocarcinomacelllinebearinganefficientcAMP-dependent transduction pathway (13). To ensure that F IG . 1.  HSL tes  mRNA expression during development in mice.  RNA blots were prepared with 25   g 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 cellproenkephalin;  sProenk , somatic proenkephalin. Testis-specific Expression of Hormone-sensitive Lipase  9329   b  y g u e  s  t   on N o v e m b  e r 1 2  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   our transfection system was valid to study cAMP-dependenttransactivation, we used a control CRE-LUC vector containing four copies of CREs upstream of a minimal promoter. Thisreporter construct was strongly cAMP-inducible whether thecellsweretreatedwithdibutyrylcAMP,astableandpermeableanalogue of cAMP, or cotransfected with an expression vectorfor the catalytic subunit of the cAMP-dependent protein ki-nase. This result was predictable because JEG3 cells containendogenous CREB. CREM    coexpression resulted in a furtherincrease of cAMP-induced luciferase activity. The results ob-tained with 0.5HSLtesLUC were strikingly different. No sig-nificant increase in luciferase activity was observed showing that the HSL tes  promoter does not represent a cellular target of CREM/CREB transregulatory function. Testis Nuclear Protein-binding Sites within the Human HSL tes  Promoter— Transgenic analyses demonstrated that the0.5-kb region located upstream of the transcriptional start sitewas sufficient to confer testis-specific expression. To assessdirectly whether sequences within the human HSL tes  promoterbound nuclear proteins present in testis, a series of   in vitro DNA binding studies was performed. The strategy used con-sisted in designing 20 overlapping double strand oligonucleo-tides spanning the entire region (Fig. 4). Each of the 20 oligo-nucleotides was used to map interaction sites for factorspresent in nuclear extracts prepared either from rat testis orfrom rat liver, an organ that does not express HSL. Four probesbound 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 testistranscription factors. TSBR4 contained a sequence AACAAAG(Fig. 4) that has been shown to bind members of the SRY/Soxprotein family (37). The testis-specific binding on TSBR4 (Fig.6) was competed by mSRY/Sox oligonucleotide but not by signaltransducer and activator of transcription and HNF3 oligonu-cleotides. The mSRY/Sox oligonucleotide contains an AACAATsequence with high affinity for mouse SRY, Sox5, and Sox6 (27,28, 38–40). An efficient competition was observed that wasmaximal with a 30-fold excess of mSRY/Sox oligonucleotide.Binding of the testis-specific nuclear proteins to TSBR4 wasincreased when poly(dG-dC) was added as nonspecific compet-itor (Fig. 6), a feature suggesting interaction of HMG domainproteins such as Sox proteins with A-T pairs in the minorgroove of the DNA helix (41, 42). A short form of Sox5 and Sox6is expressed in mice in post-meiotic germ cells (27, 38). Binding  F IG . 2.  Analysis of 5  -untranslated region of HSL tes  mRNA inhumanandtransgenicmicetestes. Total RNA (90   g) from sexuallymature transgenic mouse testis and 1   g of poly(A)  RNA from humantestis were digested by RNase H in the presence of an antisenseoligonucleotide complementary to the 5  -untranslated region of hu-man HSL tes  mRNA. The reaction mixture was denatured and electro-phoresed on a polyacrylamide-urea gel. The resulting blot was hy-bridized with a  32 P-labeled DNA probe located 5   of the antisenseoligonucleotide on human HSL tes  cDNA. RNA size markers were pro-duced by  in vitro  transcription and labeled by incorporating [ 32 P]UTPinto the reaction mixture.F IG . 3.  Effect of CREM   , cAMP-dependent protein kinase, andcAMPonp0.5HSLtesLUCvectorinJEG3cells. Thep0.5HSLtesLUC vector contains 0.5 kb of the HSL tes  promoter in the promoterlessluciferase vector (pGL3basic). The pCRE-LUC reporter construct con-tains four copies of cAMP-responsive elements upstream of a minimalpromoter linked to the luciferase gene. The reporter constructs werecotransfected with expression vectors encoding the catalytic subunit of the cAMP-dependent protein kinase (  PKA ) and CREM   . Cells weretreated for 4 h with 1 m M  dibutyryl cAMP ( db cAMP ), a stable andpermeable analogue of cAMP, as indicated. Results are expressed rel-ative to the activity of pGL3basic treated under the same conditions.Data represent means    S.D. of three experiments performed induplicate.T  ABLE  I Chloramphenicol acetyltransferase (CAT) activity of 1.4HSLtesCAT and of 0.5HSLtesCAT in transgenic mice 1.4HSLtesCAT and 0.5HSLtesCAT contain 1.4 and 0.5 kb of the testicular HSL 5  -flanking region upstream of the CAT gene, respectively. Datarepresent means of CAT activity determined on tissues from at least two mice and are expressed as cpm/min/mg protein. Skel., skeletal muscle; Adi., adipose tissue; Epi., epididymis. ND, not determined. Males Line Brain Heart Liver Kidney Gut Stomach Spleen Lung Skel. Adi. Testis Epi. 1.4HSLtesCAT A 7 11 1 2 2 5 1 10 1 21 750 450B 12 5 3 7 1 0 4 5 8 15 830 NDC 1 4 10 3 6 3 8 2 7 6 200 ND0.5HSLtesCAT D 2 4 0 2 1 5 6 4 0 15 800 550E 1 2 5 3 8 12 7 5 6 14 700 ND Females Line Brain Heart Liver Kidney Gut Stomach Spleen Lung Skel. Adi. Uterus Ovary 1.4HSLtesCAT A 8 5 6 12 2 4 2 3 4 7 21 5B 7 15 7 4 0 4 8 0 1 15 2 00.5HSLtesCAT D 9 11 0 1 0 6 7 20 6 26 9 0 Testis-specific Expression of Hormone-sensitive Lipase 9330   b  y g u e  s  t   on N o v e m b  e r 1 2  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   of recombinant Sox5 HMG box peptide and  in vitro  translatedSox6 protein on TSBR4 was studied using linker scan mu-tagenesis (Fig. 7). The HMG domain of Sox5 bound the mSRY/ Sox probe containing the AACAAT sequence and TSBR4 butnot a C/EBP recognition motif (Fig. 7  B ). Mutation of the AA-CAAAG sequence of TSBR4 strongly decreased the binding.Because Sox6 homodimers do not bind DNA (27), a truncatedform of Sox6 deleted of the leucine zipper region was producedby  in vitro  translation. As previously reported (27), incubationof the unprogrammed lysate (data not shown) and of the pro-grammed lysate of the empty vector pRc/CMV with labeleddouble strand oligonucleotides resulted in retardation of theprobe (Fig. 7 C ). The binding appeared to be due to endogenousDNA binding factor of the reticulocyte lysate. When the pCMV/ Sox-LZ(D105–356) vector was used, an additional binding com-plex was detected. This binding was abolished by mutation of the AACAAAG sequence. These data show that testis Sox pro-teins produced  in vitro  and from nuclear extracts bind TSBR4. The HMG Box of Sox5 Bends a Testis-specific Binding Re- gion— Sox proteins as other proteins containing HMG domainsinduce a marked bend within the DNA (23, 37). Therefore, weinvestigated whether the HMG box of Sox5 was able to modifyTSBR4 DNA curvature using a circular permutation assay.TSBR4 DNA was cloned into the pBend2 vector (29). Digestionof the resulting plasmid with various restriction endonucleasesgave DNA probes of almost identical sizes and base composi-tion but with TSBR4 at variable distances from the end of theprobe (Fig. 8  A ). Fig. 8  B  shows the result of a gel retardationassay with recombinant Sox5 HMG box peptide and the differ-ent DNA probes. The retarded complexes migrated with amobility that was inversely correlated to the distance betweenthe binding site and the end of the probe, a relationship char-acteristic of proteins that bend their target DNA (43). The ratiobetween the fastest and the slowest migrating species was usedto estimate the extent of DNA distortion (Fig. 8 C ). The centerof the bending mapped to the AACAAAG motif of TSBR4.Using the empirical equation proposed by Thompson andLandy (30), the angle of DNA bending was estimated between65 and 70°. DISCUSSION In this paper, we show that the proximal 5  -flanking regionof the HSL gene functions as a testis-specific promoter andbinds testis nuclear proteins. HSL tes  mRNA appears in roundspermatids concomitantly to protamine 1 mRNA (Fig. 1). Thisresult is in agreement with  in situ  hybridization data obtainedin rat which showed that HSL tes  mRNA was detected in stages X–XIV of spermatogenesis (6). As shown for many genes ex-pressed during spermatogenesis, the HSL tes  protein accumula-tion is delayed to stages XIII-VIII corresponding to late sper-matids (3, 44). The similar stage-specific expression patternobserved for HSL tes  and protamine 1 mRNAs and other tran-scripts suggests the presence of common regulatory mecha-nisms. Since CREM    plays an important role during the firststeps of spermiogenesis as a transcriptional activator, wechecked whether this transcription factor transactivates theHSL tes  promoter. Cell transfection experiments (Fig. 3) similarto 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 HSL tes  promoter transactivation. An indi-rect role of CREM    that is essential for a complete differenti-ation of haploid germ cells (15, 16) cannot, however, be ruledout,  e.g.  through the control of expression of a transcriptionfactor activating the HSL tes  promoter.The lack of appropriate male haploid germ cell line led us touse transgenic mice to investigate the transcriptional regula-tion of HSL tes . We demonstrate here that 0.5 kb of the regionflanking the HSL tes -specific exon govern testis expression intransgenic mice (Table I). Analysis of a large number of tissuesin male and female transgenic mice showed the strict testisexpression of the transgene. The testis form of HSL that ischaracterized by larger mRNA and protein species than theother isoforms has only been detected in testis (1–3). Moreover,the 25-day-old transgenic mice showed very little CAT activitycompared with the 60-day-old animals. The data in transgenicmice are therefore in agreement with the pattern and timing of expression of HSL tes .In order to determine testis-specific DNA-protein interac-tions on the HSL tes  promoter 0.5-kb region, gel retardationassays were performed using overlapping double strand oligo-nucleotides (Fig. 4). Four regions were shown to bind testisnuclear proteins absent in liver nuclear extracts (Fig. 5). One of them, TSBR4, contained a DNA sequence motif AACAAAGrecognized by the HMG domain of SRY/Sox proteins (23, 39).Competition experiments revealed that TSBR4 bound a testis F IG . 4.  Sequence of 0.5 kb of the HSL tes  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 . Testis-specific Expression of Hormone-sensitive Lipase  9331   b  y g u e  s  t   on N o v e m b  e r 1 2  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om 
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