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
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 .
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.
MATERIALS AND METHODS
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' : 3