The 3′ Flanking Region of the Human Tyrosine Hydroxylase Gene Directs Reporter Gene Expression in Peripheral Neuroendocrine Tissues

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  • Journal of NeurochemistryRaven Press, Ltd . . New York((, 1995 International Society for Neurochernistry

    The 3' Flanking Region of the Human Tyrosine HydroxylaseGene Directs Reporter Gene Expression in Peripheral

    Neuroendocrine Tissues

    Shou C . Wong, Mark A. Moffat, George T. Coker, *John P. Merlie, and Karen L. O'Malley

    Departments of Anatomy and Neurobiology and *Pharmacology, Washington University Medical School,St. Louis, Missouri, U.S.A .

    Abstract : Cell type-specific expression of the catechola-mine synthetic enzyme, tyrosine hydroxylase (TH), ap-pears to be mediated in part by cis-acting elements lo-cated at the 3' end of the human gene . Further delinea-tion of this region indicated sequences corresponding toa CACGTG motif significantly stimulated transcription ofa heterologous promoter in various cell types . Mutationof this site led to a complete loss of activity. DNase foot-printing, gel retardation, and UV cross-linking experi-ments indicated that a 74-kDa cellular factor(s) boundspecifically to the CACGTG motif in the pheochromocy-toma cell line PC12 . The size of this protein and its patternof expression are compatible with those of the CACGTGbinding protein TFE3 . Transgenic animals were createdusing a 261-bp human TH 3' fragment encompassing theCACGTG motif in front of a thymidine kinase promoter/chloramphenicol acetyltransferase reporter gene . In threelines of mice this fragment was sufficient to direct a pat-tern of mRNA expression in peripheral neuroendocrinetissues that mimicked TH mRNA distribution . However,these sequences were not sufficient for CNS-specificpatterns of expression . Thus, multiple cell type-specificenhancers may regulate TH gene expression in theCNS and periphery . Key Words : Cell type specificity-Helix-loop-helix leucine zipper-Tyrosine hydroxy-lase-Transgenic mice-Gene regulation-3' enhancer .J . Neurochem. 65, 23-31 (1995) .

    Tyrosine hydroxylase (TH) is the rate-limiting en-zyme in the biosynthesis of catecholamines . The ex-pression of the TH gene is restricted to dopaminergic,noradrenergic, and adrenergic cells in the CNS as wellas in sympathetic ganglia and adrenal medullary cellsin the periphery . Such a restricted tissue distributionsuggests the possible involvement of cis-acting se-quences whose association with positive or negativeregulatory factors results in the tissue-specific expres-sion of the TH gene . Little is known about the molecu-lar mechanisms underlying either the CNS or periph-eral expression of this gene . However, several recentstudies have begun to focus on identifying tissue-spe-


    cific elements and transcriptional factors responsiblefor the observed distribution . For example, we andothers (Cambi et al ., 1989 ; Gandelman et al ., 1990 ;Yoon and Chikaraishi, 1992 ; Wong et al ., 1994) havefound elements within the 5' promoter region of therat TH gene that appear to confer tissue-specific ex-pression in transient transfection paradigms .

    In contrast, the human TH (hTH) 5' region failedto exhibit a cell type-specific response (Gandelman etal ., 1990) . Because other cell type-specific enhancershave been localized downstream from their transcrip-tional start sites, e.g ., immunoglobulins (Dariavach etal ., 1991 ; Pongubala and Atchison, 1991), the globingene (Purucker et al ., 1990), histone genes (Zhao etal ., 1990), the myosin light chain gene (Rosenthal etal ., 1990), and the hypoxia gene (Beck et al ., 1991),we tested fragments spanning the human TH loci fortheir efficacy in conferring "tissue specificity" ona heterologous promoter . Previously, we reported thata 759-bp region immediately 3' of exon 13 is cap-able of activating a heterologous promoter in an orien-tation-independent and neuroendocrine-specific man-ner (Gandelman et al ., 1990) . Further truncation ofthese sequences suggested an E box motif (CACGTG)---230 by downstream of exon 13 was important forthe in vitro response (Gandelman et al ., 1990) . Subse-quently, Kaneda et al . (1991 ) reported that constructscontaining 2,500 by of human TH 5' sequences as

    Received October 17, 1994; revised manuscript received Decem-ber 23, 1994 ; accepted January 12, 1995 .

    Address correspondence and reprint requests to Dr. K . L .O'Malley at Box 8108, Department of Anatomy and Neurobiology,Washington University Medical School, 660 South Euclid Avenue,St . Louis, MO 63110, U.S.A .The present address of Dr. S . C . Wong is Cytotherapeutics, Inc .,

    4 Richmond Square, Providence, RI 02906, U.S .A .The present address of Dr . G . T . Coker is Gliatech . Inc ., 23420

    Commerce Park Road, Cleveland, OH 44122, U.S .A .Abbreviations used: CAT, chloramphenicol acetyltransferase ;

    hINS, human insulin promoter region ; HLH, helix-loop-helix ;PCR, polymerise chain reaction ; TH, tyrosine hydroxylase ; TK,thymidine kinase .

  • 24

    well as the primary transcript and 3'-flanking (500-bp) sequences were sufficient for tissue-specific ex-pression in transgenic animals . In contrast, when 5 .0,2.5, and 0.2 kb of the 5'-flanking promoter region ofthe human TH gene were tested in transgenic mice, nocell type-specific gene expression was observed (Sa-saoka et al ., 1992) . These results suggested that se-quences downstream of the human TH promoter in-cluding 3'-untranslated sequences are important inachieving high-level, cell type-specific gene expressionin vivo . Because the full-length tissue-specific con-struct described by Kaneda et al . (1991 ) encompassedthe 3' domain we had previously defined, the in vivoresults are consistent with our transient assays . Con-versely, it is conceivable that the tissue culture para-digm used to define human TH cell type-specific ele-ments does not accurately reflect endogenous behavior,or that a combination of both 5' and 3' elements willbe important for accurate transcriptional regulation . Totest these hypotheses, we have further characterizedthe 3' elements in the human TH gene by both func-tional assays and in vivo paradigms. In this report wepresent evidence that the 3' enhancer region of thehuman TH gene contains sufficient regulatory informa-tion to direct an appropriate pattern of expressionin peripheral neuroendocrine cell types but not inthe CNS.


    Plasmid constructsThe vector pTK81-Luc vector obtained from Dr . S . K.

    Nordeen (University of Colorado) contains the herpes sim-plex virus type I thymidine kinase (TK) promoter in frontof the firefly luciferase reporter gene . The human 3' cle-ment-TK-luciferase clone J was constructed as previouslydescribed (Gandelman et al ., 1990) . Mutations within the Ebox were generated by the recombinant polymerasc chainreaction (PCR) method (Higuchi, 1990) using internal oli-gonucleotides 634/635. The human insulin promoter region(hINS) encompassing nucleotides -365 to --36 (Bell et al .,1982) was also cloned by PCR using the oligonucleotides393/395. All PCR constructs were sequenced to verily theaccuracy of the amplification process. To create the ,I-TK-chloramphenicol acetyltransferase (CAT) plasmid, the anal-ogous luciferase construct was digested to completion byBantHI and HindIll, filled in, size-selected in low-tneltinoagarose, and ligated into ptkCAT-342 . Because the TK pro-moter in ptk-CAT-342 contains the promoter sequence -- 108to +50, we truncated it to -81 to be consistent with theminimal promoter used in the previous studies (Gandelmanet al ., 1990) . The final plasmid was named ptk-CAT-.I-380 .The human TH 3' flanking sequence was previously Submit-ted to GcnBank under accession no . M93281 .

    Cell culturesThe rat pheochromocytoma cell line PC 12 and the human

    hepatotna cell line HepG2 were cultured as described (Gan-delman et al ., 1990) . 8TC-1 cells were derived froth theislets of transgenic animals expressing SV40 large T antigenunder the control of the insulin gene pronwter (Efrat et al .,1988) . These cells express large amounts of insulin and

    J- Neuroc'dAem., Vol. 65, No . /, /995

    S. C. WONG 117 AL .

    were obtained from Dr . R . Stein (Vanderhilt University ) ./3TC-I cells were 11rown in Dulhecco', Modified Ea~cle'smedium supplemented with 2.5Sc fetal call scrum and 10'/,horse scrum_

    Transient transfectionAll cell lines were translected u,in`a the calcium phosphate

    method (Chen tuid ()kayama, 1987) . PC 12 ( I x 10 ( ') andTC-I ( 2 x 10' ) cells were tran,fected with 3 pgo of plasmidDNA, whereas HepG2 cell, ( 3 x 10' ) were transfected with2 Fig of pl,ismid DNA. All cells were incubated with thecalcium phosphate DNA precipitate lit 5 h . The PC'I 2 cell,were subsequently exposed to 20(7r glycerol for I min .HepG2 cells to 10'4 glycerol for 3 inin, and fITC-I cells to20 1/c glycerol Ior 2 min . At 4S h later, the cells were har-vested, and cell Ipsates were used for the luciferase assay Ll ."described by De Wet et al . ( 1987 ) . Lucilerase activitieswere normali/ed using RSV-/)gal a,, an internal control a,previously described (Gandelman et al ., 1990 : Won, et al .,1994) . Protein content was ntca .stn- ed by the assay of Brad-ford ( 1976) wing bovine serum alhumin as the standard .

    OligonucleotidesOligcmucleotidcs were ,yn(hcsi/ed by the Protein Chemis-

    try Core Facility at Washington University Medical School .For gel retardation assays, complenrenlary oligonucleotideswere synthesi/ed and then anucnlcd to frnm double-strandedoligonucleotides . Oli`aonuclccriides inclu(IC(I the 1ollowittg :338 (E box: 30-mcr) . 5'-AACi(i"IGGCiiAGC'CAC'GTCi-ACAGTGGGAGG,G-3' : 1104 (E box ; 20-mcr) . 5'-GGG-GAGC'CACGTCiAC'AG"fGG-3' ; 363 (mutan( E box ; 20-mcr), S'-CiCiGGACiC'AC'CiAG'fAC'A(iT(iG-3' : 360 (ca-nonical myc/USF/TFL CAC( TG motif (Blackwell et al . .1993)1, 5'-A l'AGGTCiTAGGC'('ACCi fGACCGGGTC~T-TC'C-3' ; 344 1 c,uuniic,tl MyoD C'ACC'T('j motif (Weintraut)et al ., 1991)1, 5'-GA'TC`C'CC'CCAACACCTGC'TG('CT-GA-3' : 342 (random) . 5'-AGGAAGCCGTGC'TA000Ci-TAGCC'TCTG f(1A-3' ; 441 IhTH (this study) I, 5'-CCCAAGC'I"fCTGAG(7"I"l'CCC'C`"F"hGG'fTC'GC'-3' ; 443 I h - I'H(this study)( . 5'-CC'C'GGA"f('C'CiC'f1GCTCC'AGAGGC'f-CGGGCA-3' : 393 I hINS (Bell et al . . 1982)1 . 5'-CCCA-AGCTTACAC;CAGC'GC'AAACiACK'CCC'G-3' : 395 I hINS(Bell et al ., 1982 )I, 5'-CC'C"AAiC"l'TCTC'AGyG000'A't-CTCCCCTAC'-3' :

    634 111TH

    (tlli, s(udy) I .

    5'-AAGCi-1'GGGAGCTG('CICAACAG"IC'CiGAGGCi-3' ; 291 (CAI'(Alton and Vapuck, 1979)1, 5'-AGCTAAGGAAGC'hA-AAATGG-3' ;


    I CA]




    1979)1,5'-AC'(~ I"I"f('ACi"pT"hGC "! C' .4 hGG-3' ;


    I myc (Kerk-hoff et al ., 1991 ) I, 5'-CiACiCiAC',~l'C' -l'CiGAACiAAl1T"I-(.'-GAGC'"fG-3' ; 741 I inyc ( KerkholI et al . . 1991 ) I . 5'-('-CGC'"f('C:ACA'I-AC'AGTCCh(iGA"fCiA

    805 1 TI h(Carr and Sharp. 1990) I . 5'-(1GAGATTCjA'I'C ;ATCi fCA'I'-TGA-3' ; 806 I TFE (Canr and Sharp. 1990)1 . 5'-GATG(i f-CC'C'C'I"l'G'TTCCAG-3' : 807 I l!SF ( Gregor et al . . 1990) 1 .5'-CTCAGCATAATGAAGF(i(i-3' : and 808 11 !SF (Grc-gor et al ., 1990)1, 5'-AGA'TC'"1 . lC . C . ACCTG'CTGT-3'.

    DNA binding assayElectrophorelic mobility shill assays and UV cross-linking

    were performed exactly a, dcscrihcd ( Won`_ et al ., 1994) .I)Nasel footprint analysis ~,,-as performed according to (hel'romega Core I "oothrinting Protocol (no. 137) using I0 Imolof probe and 50 /tg of (otal protein . The 299-bp probe wasgenerated by PCR using oligonucleotides 4-13 and 441 . Theresulting PCR product vas purilied front a 5'/r polyacryl-

  • amide gel, and the purified DNA fragment was subsequentlyradiolabeled using T4 polynucleotide kinasc ( Maniatis et al .,1982) . The sense strand probe was generated by digestingthe labeled DNA fragment with Hindlll .

    mRNA analysis by reverse transcription-MRTotal RNA was isolated from PC12 and HepG2 cell lines

    (Chomczynski and Sacchi, 1987), reverse-transcribed usingohgonucleotides 741, 806, and 808 for c-Myc, TFE3, andUSF, respectively (Krug and Berger, 1987), and MINIM'amplified according to the manufacturer's protocol ( l'crkinHmer Cetus, Alameda, CA, U.S .A .) . TFE oliponucleotideswere chosen such that TFE3 and TFEB would be ccruTnpli-fied . Amplification temperatures for the oligonucleotide set739/741 (c-Myc) were denaturation at 94C for I min, an-nealing at 63C Ior I min, and extension at 72C for I min.Amplification temperatures for oligonucleotide sets 805/806('TFE3) and 807/808 (USF) were 94 (1 min), 46 ( I min),and 72C ( I min) .To increase the sensitivity of the reverse transcription-

    PCR experiments, the 5' oligonucleotides were radiolabeledand included in the reaction mixture at a Specific activity of5 X 10' cpm/pmol . Typically, radioactive PCR productswere elect rophoresed on 5% polyacrylamidc gels andscanned with the Phosphorlmager SF ( Molecular Dynamics,Sunnyvale, CA, U.S .A .) . To confirm the identity of ampli-fied products, PCR Fragments were excised from the gel,extracted as described by Maniatis et al . ( 1982), and cyclesequenced using the AmpliTaq Cycle Sequencing Kit (Per-kin Elrner Cetus) .

    Production of transgenic miceThe 1,900-bp DNA fragment containing the J-TK-CAT

    sequence was generated by K/ml and Noel digestion andsize-selected by agarose electrophoresis (Maniatis et al .,1982) . DNA was purified using the EIuQuik kit accordingto the manufacturer's instruction (Schleicher and Schnell ) .The DNA was dissolved in in buffer 110 mM Tris-HC1 (pH 7.5), 10 mM NaCI . and 0.1 mM EDTA I at aconcentration o1 I N.g/ml and dialyzed against the samehul'ler . The DNA was microinjected into a single premuclcusof 12 h (C57BL/6J X CBA/J Fl hybrid) mouse crnhryosby standard methods (Hogan et al ., 1986) . Identification ofthe integration of the ,1=TK-CAT transgene in the mousegenorne was done by PCR using unpurified DNA extractsfrom tail digests according to the method of Hanley andMerlie ( 1991 ) .


    Our previous studies indicated tissue-specific ele-ments involved in hTH gene expression were down-stream of the last exon (Fig . I A) (Gandelman et al .,1990) . In particular, aCANNTG motif or E box withinthe maximal response region of fragment J (Gandel-man et al ., 1990) appeared to be important for elicitinghigh-level activity over basal level controls . Such se-quences constitute binding sites for a family of tran-scription factors containing helix-loop-helix (FILM)structural domains, such as Myc ( Kerkhoff et al .,1991 ), MyoD (Weintraub et al ., 1991 ), USF (Gregoret al ., 1990), and AP-4 (Hu et al ., 1990) . However,because the 3' end of the human TH gene is only 2.7kb 5' of the insulin gene (O'Malley and Rotwein,


    1988) (Fib . IA), the question arose as to whetherthe 3' elements previously identified might in fact beinvolved in the regulation of the human insulin gene .We tested this possibility by cloning the insulin pro-moter and determining whether the downstream THfragments enhanced the expression of an insulin pro-Inoter/luciferase construct in the mouse pancreatic 0-cell line /3TC-1 .

    Several Studies have indicated that cell type-specificregulation of the insulin gene is also controlled by CAN-NTG-binding transcription factors clustered...


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