Transcript

Transgenic Mice Expressing YellowFluorescent Protein Under Control of theHuman Tyrosine Hydroxylase Promoter

Eun Yang Choi,1 Jae Won Yang,1 Myung Sun Park,1 Woong Sun,2

Hyun Kim,2 Seung U. Kim,2,3 and Myung Ae Lee1*1Brain Disease Research Center, and Institute for Medical Sciences, Ajou University School of Medicine,Suwon, Korea2Department of Anatomy, Korea University College of Medicine, Seoul, Korea3Medical Research Institute, Chung-Ang University School of Medicine, Seoul, Korea4Division of Neurology, Department of Medicine, University of British Columbia, Vancouver,British Columbia, Canada

Pathogenesis of Parkinson’s disease and related cate-cholaminergic neurological disorders is closely associ-ated with changes in the levels of tyrosine hydroxylase(TH). Therefore, investigation of the regulation of the THgene system should assist in understanding the patho-mechanisms involved in these neurological disorders. Toidentify regulatory domains that direct human TH expres-sion in the central nervous system (CNS), we generatedtwo transgenic mouse lines in which enhanced yellowfluorescent protein (EYFP) is expressed under the controlof either 3.2-kb (hTHP-EYFP construct) human TH pro-moter or 3.2-kb promoter with 2-kb 30-flanking regions(hTHP-ex3-EYFP construct) of the TH gene. In the adulttransgenic mouse brain, the hTHP-EYFP constructdirects neuron-specific EYFP expression in various CNSareas, such as olfactory bulb, striatum, interpeduncularnucleus, cerebral cortex, hippocampus, and particularlydentate gyrus. Although these EYFP-positive cells wereidentified as mature neurons, few EYFP-positive cellswere TH-positive neurons. On the other hand, we coulddetect the EYFP mRNA expression in a subset of neu-rons in the olfactory bulb, midbrain, and cerebellum, inwhich expression of endogenous TH is enriched, withhTHP-ex3-EYFP transgenic mice. These results indicatethat the 3.2-kb sequence upstream of the TH gene is notsufficient for proper expression and that the 2-kbsequence from the translation start site to exon 3 is nec-essary for expression of EYFP in a subset of catechola-minergic neurons. VVC 2012 Wiley Periodicals, Inc.

Key words: tyrosine hydroxylase; promoter; EYFP;catecholaminergic neuron; transgenic mice

Tyrosine hydroxylase (TH) catalyzes the rate-limit-ing step of hydroxylating tyrosine to dihydroxyphenyla-lanine (DOPA) in the synthesis of catecholamine neuro-transmitters (Nagatsu et al., 1964). In the central nervoussystem (CNS), TH is expressed in dopaminergic(DAergic) neurons of the substantia nigra (SN), ventraltegmentum, hypothalamus, and olfactory bulb; in norad-

renergic neurons of the locus ceruleus and lateral teg-mental system; and in adrenergic neurons of the brain-stem (Zigmond et al., 1989). The mechanisms of THgene expression have been intensively studied, becausecatecholamines play fundamental and important role inneurophysiology and pathogenesis of neurodegenrativediseases, including Parkinson’s disease (PD). AberrantTH gene expression is also associated with psychiatricdisorders, such as schizophrenia, bipolar disorder, andside effects caused by alcoholism. More importantly,degeneration and cell death of TH-positive DAergicneurons in the SN are the major cause of PD. A recentstudy has reported that TH alterations and SN neuropa-thology arte also implicated in Huntington’s disease(Yohrling et al., 2003).

We previously reported that a 3.2-kb sequence ofthe human TH gene promoter contains functional pro-moter and cis elements and effectively regulates celltype-specific expression (T.E. Kim et al., 2003). Toidentify regulatory domains that direct human THexpression in the CNS, we generated transgenic mice,hTHP-EYFP, in which enhanced yellow fluorescentprotein (EYFP) is expressed under control of the 3.2-kblength of human TH promoters. Earlier studies of THpromoter in transgenic mice have shown that a sequence

Contract grant sponsor: BK21 Program of the Ministry of Education and

Human Resource Development; Contract grant sponsor: KOSEF/BDRC

Ajou University from the Korean Ministry of Science and Technology;

Contract grant sponsor: Neurobiology Research Program grant from the

Korean Ministry of Science and Technology; Contract grant sponsor: Stem

Cell Research Center of the 21st Century Frontier Research Program

(SC3090) from the KoreanMinistry of Science and Technology.

*Correspondence to: Myung Ae Lee, PhD, Brain Disease Research

Center, and Institute for Medical Sciences, Ajou University School of

Medicine, Suwon, Korea 442-749. E-mail: [email protected]

Received 22 March 2012; Accepted 15 April 2012

Published online in Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/jnr.23085

Journal of Neuroscience Research 00:000–000 (2012)

' 2012 Wiley Periodicals, Inc.

of 5–11 kb is required for high-level expression of thereporter in catecholamine neurons (Sasaoka et al., 1992;Min et al., 1994; Liu et al., 1997; Trocme et al., 1998;Sawamoto et al., 2001; Matsushita et al., 2002; Kessleret al., 2003). Other studies have demonstrated that thehuman TH promoter of 2.5 kb, including the entireexon–intron structure with 0.5 kb of the 30-flankingregion, is sufficient for tissue-specific expression of TH intransgenic mice (Kaneda et al., 1991). Therefore, toinvestigate further the role of hTH gene in tissue-specificexpression, we generated an additional transgenic mouseline, hTHP-ex3-EYFP, which contains the sameupstream region as hTHP-EYFP, and 2-kb sequencefrom the translation start site to exon 3. Our currentobservations suggest that the 3.2-kb sequence upstream ofthe TH gene is not sufficient for proper expression andsuggest the importance of the 2-kb sequence from thetranslation start site to exon 3 for the expression of down-stream genes in a subset of catecholaminergic neurons.

MATERIALS AND METHODS

Construction of Transgenes With EYFP Reporter

Human TH promoter fragment of 3.2 kb produced bySalI-KpnI restriction enzyme digestion of THP4434-pGEM3zf1 was inserted upstream of the EYFP gene inpEYFP plasmid. For normal transcription of the EYFP gene,it was directly connected with the EYFP gene using Quik-Change II site-directed mutagenesis kits (Stratagene, La Jolla,CA). A 0.7-kb fragment of the SV40 poly-A gene frompcDNA3.1/His/lacZ was inserted into the ApoI site down-stream of EYFP to stabilize mRNAs, resulting in the hTHP-EYFP construct. To generate hTHP-ex3-EYFP construct, weconnected a 3.2-kb human TH upstream genomic fragmentand a 2-kb sequence from the translation start site nucleotide76 of exon 3 to the EYFP coding sequence.

Generation and Genotyping of hTH-EYFP TransgenicMice

Transgenic mice were generated by pronuclear microin-jection of fertilized (C57BL/6J 3 DBA/2J) F2 mouse oocytes.To identify founder mice, the genotypes of all offspring wereanalyzed by polymerase chain reaction (PCR). GenomicDNA was prepared from tail biopsies. The thermocycle pro-file for PCR amplification was 1 min at 948C, 1 min at 608C,and 2 min at 728C for 22 cycles to distinguish homozygousmice from heterozygous mice. The primers for PCR analysiswere sense primer for human TH gene, 50-TTTAG-GAAAGGTCCCAGGGG-30; antisense primer for transgene,50-TTGGAGAGACCT TTGCAG TT-30, to yield a 640-bpproduct. The PCR products were separated on a 1.5% agarosegel, stained with ethidium bromide, and quantitatively ana-lyzed in Image Gauge 4.0 (Fuji Film, Tokyo, Japan).

Immunohistochemistry

The animals were perfused with 4% paraformaldehyde(PFA) in 0.1 M phosphate buffer, and brains were removedand fixed in the same fixative at 48C for 16 hr. After washingin phosphate-buffered saline (PBS) for 20 min, the brains were

equilibrated in 30% sucrose in PBS and frozen in dry ice. Thefrozen brains were cut into 30-lm sections with a CM 3000cryostat (Leica Microsystems, Milan, Italy), and the sectionswere permeabilized and blocked with PBS containing 3% goatserum and 0.2% Triton X-100 for 2 hr, then stained with pri-mary antibodies at 48C overnight. After three washes withPBS, the sections were incubated for 1 hr at room temperaturewith Texas red- or Cy3-conjugated secondary antibodies(1:200–500; Vector, Burlingame, CA) in PBS containing 3%bovine serum albumin. The sections were then mounted on aglass slide with PermaFluor (Thermo Shandon, Pittsburgh,PA). Fluorescence images were obtained under a confocal laserscanning microscope (Olympus, Tokyo, Japan). Primary anti-bodies used were TH (1:500, sheep; Pel-Freeze, Rogers, AR),NeuN (1: 400, mouse mAb; Chemicon, Temecula, CA), dou-blecortin (DCX; 1:500, rabbit; Chemicon), calretinin (CR;1:500, rabbit; Chemicon), and calbindin (CB; 1:500, mousemAb; Chemicon). For immunodetection of EYFP, we usedanti-GFP antibody (1:100, rabbit; BD Bioscience, San Jose,CA) and biotinylated anti-rabbit antibody (1:200; Vector), anABC kit, and a diaminobenzidine (DAB) staining kit (Vector).

RT-PCR

Total RNA was extracted from various tissues of mousebrain using RNeasy mini kit (Qiagen, Valencia, CA). Twomicrograms of RNA was reverse transcribed with SuperscriptII Reverse Transcriptase (Invitrogen, Carlsbad, CA) in thepresence of random primers, according to the manufacturer’sinstructions. The resulting cDNA was amplified by PCRusing primers specific for human TH and EYFP. Primerswere TH forward 50-CTGAGCCATGCCCACCCCC-GACGCCACC AC-30 and two EYFP reverse 50-TGAA-GAAGATGGTGC GCTCCTGGAC-30 and 50-GGTTCAC-CAGGGTGTCGC CC-30. Amplification was performed in aPTC-200 thermal cycler (MJ Research, Toronto, Ontario,Canada), and conditions were 30 cycles of 1 min at 948C, 1min at 658C, and 2 min at 728C. Amplified products wereseparated on a 2% agarose gel containing ethidium bromide.

Southern Blot Analysis

PCRs were performed using cDNAs from brain tissuesof hTHP-ex3-EYFP transgenic mice. PCR products wereresolved on a 1.2% agarose gel and transferred to a positivelycharged nylon membrane (Roche Diagnostics, Indianapolis,IN). The blot was hybridized overnight at 428C in hybridiza-tion buffer with a human TH-gene-derived 400-bp XbaIprobe that had been labeled with 32P-dCTP (3,000 Ci/mmol)by DNA polymerase extension of random hexamers (Boeh-ringer Mannheim, Indianapolis, IN).

In Situ Hybridization

Mouse TH cDNA (nucleotides 180–1382 of GenBankaccession No. BC053706) was obtained from the KoreanGenBank and EYFP cDNA from pEYFP plasmid (Promega,Madison, WI). These cDNAs were cloned into the pGEMT-easy plasmid (Promega) and used for riboprobe synthesis.Sections from wild-type and transgenic brains (coronal andsagittal, 12 lm thick) were collected in serial order using a

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cryostat (Leica), fixed in 4% paraformaldehyde (PFA), andhybridized with radiolabeled riboprobes. Hybridization andwashing conditions were described previously (Kim et al.,1994). For autoradiography, the slides were exposed at 2208Cto b-Max film (Amersham, Arlington Heights, IL) for 4 days.For darkfield and brightfield microscopy, the slides were dippedin NTB2 nuclear track emulsion (Eastman Kodak, Rochester,NY) and incubated at 48C for 1 week; sections were thendeveloped, lightly counterstained with cresyl violet (FisherScientific, Seoul, Korea), and overlaid with coverslips.

RESULTS

Generation of Human TH-EYFP Transgenic Mice

To determine whether the TH upstream sequencedirects its expression in a cell-specific and developmen-tally regulated manner, we fused the 50-flanking regionwith or without the first three exons of the human THgene to the EYFP reporter gene (hTHP-ex3-EYFP andhTHP-EYFP constructs, respectively; Fig. 1A). For thehTHP-EYFP construct, the fusion gene consists of a3.2-kb hTH upstream genomic fragment (starting at thehTH translation initiation site), the EYFP gene, and apolyadenylation site derived from the SV40 gene (Fig.

1A). For the hTHP-ex3-EYFP construct, we used a3.2-kb hTH upstream genomic fragment and a 2-kbsequence from the translation start site to exon 3(Fig. 1A).

We have generated transgenic mice, using thesetwo constructs, with the aim of identifying a region (orregions) that transactivates TH transcription specificallyin the brain. PCR analyses of tail DNAs identified fiveand three independent transgenic lines that passed theirtransgenes onto their offspring for the hTHP-EYFP andthe hTHP-ex3-EYFP constructs, respectively (Fig. 1B).Three of five hTHP-EYFP lines (named hTHP-EYFP7,-9, and -131) and three of hTHP-ex3-EYFP linesshowed mRNA expression in the brain (data notshown). To assess the tissue specificity of EYFP expres-sion, we tested the EYFP mRNA expression by RT-PCR (Fig. 1C). EYFP mRNA was expressed strongly inthe hippocampus, cerebral cortex, and olfactory bulb(OB) and only weakly in the midbrain of hTHP-EYFPtransgenic mice. By contrast, EYFP mRNA was ratherubiquitously observed in most brain regions of hTHP-ex3-EYFP mice. These brain-region-dependent expres-sion patterns are distinct from the distribution of endog-enous TH mRNA. When tissue specificity of EYFP

Fig. 1. Human TH-EYFP transgenic mice. A: Schematic of thehuman TH-EYFP and TH-ex3-EYFP transgene constructs. The 3.2-kb SalI-Eco52I fragment from the 50-flanking region of human THgene was fused with EYFP cDNA (yellow box) and a polyadenyl-ation sequence to generate the TH-EYFP construct. The TH-ex3-EYFP construct was generated to analyze the function of genomicsequences other than the 50-flanking region on TH gene expressionin vivo and includes the region from 23174 to exon 3 of the THgene, the EYFP reporter gene, and polyadenylation signals. B: PCR-

based genotype screening for the generation of transgenic mice. The640-bp band corresponds to the human TH gene. DW, distilledwater; WT, wild type; PC, positive control. C: RT-PCR analysis ofhuman TH promoter-driven mRNA expression from various mousebrain regions of transgenic mice. HC, hippocampus; Ctx, cerebralcortex; MB, midbrain; OB, olfactory bulb; St, striatum; VTA, ventraltegmental area; CB, cerebellum cortex; LC, locus ceruleus; M, DNAsize marker. [Color figure can be viewed in the online issue, whichis available at wileyonlinelibrary.com.]

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expression was assessed via fluorescence microscopy,strong EYFP fluorescence was found in several brainregions of hTHP-EYFP mice.

Characterization of hTHP-EYFP Transgenic Mice

Next we addressed the cellular-level expression ofEYFP signals in brain tissue sections of hTHP-EYFPtransgenic mice by epifluorescence or laser scanningconfocal microscopy (Fig. 2). Because the hTHP-EYFP7line showed the strongest EYFP fluorescence signals, allexperiments were performed with this transgenic mouseline. Strong EYFP staining was seen in the anterior ol-factory nucleus, olfactory granule cell layers, CA1–3region, and dentate gyrus (DG) of the hippocampus, cer-ebellum, and cerebral cortex, whereas no staining wasfound in the globus pallidus or substantia nigra (SN; Fig.2). On the other hand, the slices prepared from the non-transgenic mice showed no signals (data not shown).Expression of EYFP in the adult brain is summarized inTable I.

Although we failed to observe EYFP-positive cellsin the SN, a subset of EYFP-positive cells in other brain

regions did exhibit TH immunoreactivity (IR; Fig. 3A–E). For instance, small numbers of EYFP-positive cellsin the ventral tegmental area (VTA), OB glomerulus,and raphe nucleus exhibited TH-IR, although mostEYFP1 neurons in these regions were also TH negative.Although most of the EYFP-positive cells did notexpress TH, they appear to maintain their neuronalidentity. Immnostaining of the neuron-specific nuclearprotein NeuN (Weyer and Schilling, 2003) demon-strated that most EYFP-positive cells in hTHP-EYFP7transgenic mice were NeuN positive (Fig. 4A–E):

Fig. 2. Regional patterns of EYFP expression in the brains of adultTH-EYFP transgenic mice. Microphotographs of immunohisto-chemically stained brain sections with anti-EGFP antibody areshown. EYFP fluorescence images in boxed regions are shown athigher magnification in insets. A: Anterior olfactory nucleus. B:Indusium griseum. C: Motor cortex. D: Dentate gyrus. E: CA1. F:Cerebellum. A–F: 312.5; insets, 3400. [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]

TABLE 1. Summary of Expression Pattern of Ttransgene in

hTHP-EYFP Mice

Wild-type mice

CNS regions

Tranasgenic

mice (EGFP) EYFP TH

Traditional catecholaminegic sites

Hypothalamus (A11–14)

Arcuate nucleus 1 11Periventricular nucleus 1 11Paraventricular nucleus 1 2 11Zona inceta 1 1111Olfactory bulb 1 1111Substantia nigra (A9) 11 1111VTA (A10) 1 1111Dorsal raphe nucleus 1 11Locus cereleus (A6) 1 1111A5 1 111A2 1 111Area postrema 1 11Adrenal gland 1 1

1 1111

Nontraditional catecholaminegic sites

Anterior olfactory nucleus 1111Infralimbic cortex 1111Insular cortex 111Somatic motor cortex 111Somatic sensory cortex 111Piriform cortex 1111Paraventricular thalamic nu 1N. accumbens 11Septum 1111Striatum 1Indusium griseum 111Amygdala 11Bed nucleus of stria terminalis 1Endopiriform nucleus 111Hippocampus 1111Interpeduncular nucleus 11Parabrachial nucleus 1Medial parabrachial nucleus 1Vestibular nucleus 1Superioir cerebellar peduncle 1Cerebella cortex 11

*The frequency was classified into four groups depending on the per-

centage of EYFP-positive cells: 1111, 75–100%; 111, 50–75%;

11, 25–50%; 1, 0–25%.

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Fig. 3. Confocal microscopic images of EYFP fluorescent cells stained with anti-TH antibody. Micefrom the TH-EYFP7 line were used for fluorescence imaging. Coronal sections of the brain werestained with anti-TH antibody and Cy3-conjugated secondary antibody. EYFP fluorescence (A1–E1,green), TH staining signal (A2–E2, red), and their merged image (A3–E3, yellow) in the various brainregions are indicated. The merged images show that cells with EYFP fluorescence are localized adjacentto cells with TH immunoreactivity, but there is limited colocalization. A limited number of EYFP fluo-rescent cells (D1, green) contain TH (D2, red), as shown by the merged image (D3, yellow). [Colorfigure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Fig. 4. EYFP-positive cells have neuronal cell properties. All EYFP-positive cells were co-localizedwith NeuN-positive clells, a neuron-specific nuclear protein. Expression of NeuN and EYFP are shownin motor cortex layer 2 (A1–A3), motor cortex layer 5 (B1–B3), CA layer (C1–C3), anterior olfactorynucleus (D1–D3), and glomerular layer of olfactory nucleus (E1–E3). Left panel, EYFP; middle panel,NeuN; right panel, merged images. [Color figure can be viewed in the online issue, which is availableat wileyonlinelibrary.com.]

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EYFP-expressing neurons represented 17.4% of neuronsin motor cortex layer 2, 42.3% in motor cortex layer 5,17.4% in CA1 of the hippocampus, 81.0% in the ante-rior olfactory nucleus, and 15.0% in the glomerular layerof OB. These results indicate that the 3.2-kb human THpromoter could drive EYFP expression in neuronal cellsof the adult CNS, although it is not sufficient for prop-erly driving reporter gene expression in TH-positiveneurons in the adult brain.

Because new neurons are continuously added inthe adult DG, next we asked whether EYFP expressionis related to the differentiation stage of the neurons inthe DG (Fig. 5). To address this issue, we performedimmunohistochemistry using doublecortin (DCX, amarker for newly born neurons), calretinin (CR, amarker for immature postmigratory neurons) and calbin-din (CB, a marker for mature neurons) in the adult DG(Muddanna, 2004; Sun et al., 2004). Whereas mostEYFP-positive cells were devoid of DCX- or CR-IR,they were doubly positive for CB (Fig. 5C1–C3), indi-cating that the 3.2-kb human TH promoter drives thedownstream gene expression in mature neuronal popula-tions.

Expression of EYFP mRNAs in hTHP-ex3-EYFPTransgenic Mice

In all three lines of hTHP-ex3-EYFP transgenicmice, we failed to identify any EYFP fluorescence (data

not shown), although EYFP mRNAs were detected byRT-PCR (Fig. 1C). As in rat and mouse, the humanTH locus spans 8 kb and contains 13 primary exons. Incontrast to rodent TH genes, the human gene undergoesalternative splicing, creating at least four different tran-scripts (Grima et al., 1987; Dumas et al., 1996; Fig. 6A).To determine whether human TH splicing variants werepresent in the transgenic mouse, RT-PCR and Southernblot analyses were performed (Fig. 6B–D). We used oli-gonucleotides complementary to the human TH andEYFP sequences as RT-PCR primers (Fig. 5B) andhuman TH cDNA as a hybridization probe (Fig. 6D).Unspliced mRNAs and single splicing variant of sizesimilar to that of human TH splicing variants type 1 ortype 2 form were strongly expressed in all transgeniclines, although there was no EYFP fluorescence (Fig.6D). These results indicate that EYFP proteins could notbe expressed because splicing of TH-ex3-EYFP mRNAswas not properly processed in the mouse or because ofincorrect reading frame.

To provide further information on the expressionof EYFP mRNA in the brain of hTHP-ex3-EYFPtransgenic mice, the distribution of EYFP mRNA wasexamined by using in situ hybridization histochemistry(Fig. 7B–D). Interestingly, in two different TH-ex3-EYFP transgenic lines (125 and 131 lines), EYFPmRNA expressions were detected in ventral tegmentumand glomerular layer of the OB, in which DAergic neu-

Fig. 5. EYFP-positive cells express calbindin but not doublecortin orcalretinin. A1–A3: There was no colocalization between doublecor-tin (DCX) (red) and EYFP (green). B1–B3: In addition, EYFP-posi-tive cells did not express calretinin (red), although the EYFP-express-

ing cell bodies were closely positioned with DCX- or calretinin-la-beled ones. C1–C3: EYFP-positive cells co-localized with cellsexpressing calbindin (red). [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

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rons are concentrated, although EYFP mRNA expres-sion was seen only marginally in the SN and locus ceru-leus. In addition, ectopic expression of EYFP mRNAwas observed in two TH-ex3-EYFP lines to differentextents. For instance, EYFP mRNA appeared to bepresent in the granule cell layer of the OB, cerebral cor-tex, hippocampus, and cerebellum, in which TH

mRNA is not strongly expressed. Therefore, it appearsthat regulatory elements conferring brain-region-depend-ent expression are present within the 2 kb of THgenomic sequences from translation start site to thirdexon, whereas sequences required for the suppression ofectopic expression do not appear to exist within ourtransgenic construct.

Fig. 6. Splicing of human TH gene in hTHP-ex3-EYFP mice. A:Schematic illustration of the genomic structures of human TH and ofthe typical alternatively spliced variants. In humans, the RNA from asingle TH gene is alternatively spliced to generate four different iso-forms (types 1–4). Human TH type 1 is most similar to the rat THgene. B: PCR primer sets. Locations and directions of primers areshown above the genomic structures. C: Expected PCR product

sizes for the four splicing variants of the hTHP-ex3-EYFP gene witheach primer set shown in B. D: Southern blot assays in hTH-ex3-EYFP transgenic mice. RT-PCR products were generated fromtransgenic mice (left panel) and analyzed by Southern blot analysiswith 32P-labeled N-terminal probe of human TH cDNA (rightpanel). [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

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DISCUSSION

The present study provides insights into importantaspects of how specificity of expression is conferred inthe human TH promoter in vivo. Human TH promoterof 3.2 kb was previously isolated from a genomic DNAlibrary and sequenced (T.E. Kim et al., 2003). Compara-tive analysis of the sequences of TH promoters inhuman, mouse, and rat led to the identification of onlytwo small evolutionarily conserved regions, which havesimilar positions in the promoters of the three species(T.E. Kim et al., 2003). Similar observations were madein previous studies analyzing and comparing cell-type-specific expression of various DNA elements of thehuman and rat TH promoters (Gandelman et al., 1990;Kessler et al., 2003).

Human TH sequences contain several scaffold/ma-trix attachment regions (S/MAR; Lenatorski and Goc,2002), which may form a subcellular structure involvedin chromatin organization and result in a particular tis-sue-specific gene regulation (van Wijnen et al., 1993).The human TH sequence contains S/MARs at positions2186/216, 1645/1755, and 1835/1945 (the secondand third of which are in the first intron of the humanTH gene; Lenartorski and Goc, 2002). Interestingly, theS/MAR at 1645/1755 also encodes for a microsatelliterepeat (TCAT) termed HUMTH01, which acts as a

silencer for human TH gene expression by interactingspecifically with the transcription factors ZNF191,HBP1, and probably AP-1 (Albanese et al., 2001; Mel-oni et al., 2002). ZNF191 is a zinc finger protein,whereas HBP1 is an HMG box transcription factor. Pre-vious studies have reported the intronic colocalization ofS/MARs with enhancer or silencer regulatory elementsin many systems other than the human TH promoter(Cockerill and Garrard, 1986; Boulikas, 1995; Forresteret al., 1999). The two S/MARs at 1645/1755 and1835/1945 were present in the TH intronic transgenicmice, because the human TH gene was fused to theEYFP cDNA. This seems to indicate that, in hTHP-ex3-EYFP-transgenic mice, the S/MARs at 1645/1755 and 1835/1945 are sufficient to repress ectopicgene expression and confer partial specific gene expres-sion at least in the ventral tegmentum. It is likely thatthe two S/MARs encoded in the first intron of thehuman TH gene could improve the degree of specificityof the human TH promoter in vivo.

A previous study has revealed that the five evolu-tionarily conserved regions of the human TH promotermodulate its transcriptional activity in a tissue-specificmanner only in human neuronal progenitor cells and didnot confer any specificity in mouse cells intrinsicallyexpressing TH or after induction with differentiation

Fig. 7. Expression of transgenic EYFP mRNA in hTHP-ex3-EYFPtransgenic mice. Coronal sections of brains of wild-type (A,B) andhTHP-ex3-EYFP transgenic (C,D) mice were radioactively labeledby in situ hybridization and examined via autoradiography. A: Distri-bution patterns of endogenous mouse TH mRNA (dark grains) as

revealed by a TH antisense probe. B: Hybridization of coronal sec-tions of wild-type brain with a EYFP antisense probe does not pro-duce a signal. C,D: Distribution patterns of transgenic EYFP mRNAas revealed by an EYFP antisense probe in hTHP-ex3-EYFP125 and-131 lines, respectively.

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factors (Romano et al., 2005). In this context, the possi-bility that the TH gene regulation might be achievedthrough different mechanisms in different species isappealing. The low degree of homology seems to be akey factor, insofar as it reflects substantial diversegenomic organization between the human and therodent TH promoters. Interestingly, a recent study hasalso shown that S/MARs in the TH gene might beinvolved in differential TH gene regulation among vari-ous species (Lenartowski et al., 2003). A comparativeanalysis indicated that, in contrast to the human THgene, the association between the bovine TH gene andnuclear matrix was not tissue specific, although the posi-tion of the matrix-binding region is conserved in bothsystems (Lenartowski and Goc, 2002; Lenartowski et al.,2003). This finding indicates that TH gene regulationmight indeed be achieved by different mechanisms inthe human and bovine models.

In the present study, as previously reported (Berodet al., 1987), TH mRNAs in the brains of wild-typemice were detected specifically in SN, ventral tegmen-tum, and OB, which are the predominant DAergicnuclei in the midbrain. In situ hybridization in sectionsof transgenic brain clearly demonstrated high levels ofexpression of the human TH promoter-driven mRNAsin these nuclei of the VTA (Fig. 6C), which indicatesthat the transgene was expressed in a partially region-specific manner in the brains of transgenic mice. As withthe rodent gene, the human TH locus spans 8 kb andcontains 13 primary exons. In contrast to rodent TH,the human gene seems to undergo alternative splicing,creating at least four different transcripts, demonstratingthe heterogeneity in human mRNAs (Grima et al.,1987; Dumas et al., 1996). Although RT-PCR andSouthern blot analyses showed one significant splicingproduct that corresponded to the type 1 or type 2human TH mRNAs, we could not detect the EYFPprotein in the mouse brain. This result may indicate thatthe fundamental cellular machinery necessary for alterna-tive splicing could not recognize human TH-EYFPmRNAs in the mouse brain and so could not producemultiple forms of mRNAs.

Several groups have found reporter gene expressiondriven by various lengths of human TH promoters intransgenic mice lines. Kaneda et al. (1991) indicated thatthe reporter gene expression is totally colocalized withTH immunoreactivity in transgenic mice, with an 11-kbDNA fragment of the TH gene containing 2.5 kb of 50upstream region, the entire exon–intron structure, and0.5 kb of the 30 flanking region. Sasaoka et al. (1992)constructed three transgenic lines in which the 5-kb,2.5-kb, and 0.2-kb sequences upstream of the humanTH gene were fused to the bacterial chloramphenicolacetyltransferase (CAT) gene to generate TC50, TC25,and TC02, respectively. Although CAT expression inthe TC50 line colocalized with endogenous TH expres-sion, reporter gene expression in the TC25 line did not,although both lines showed high levels of ectopicexpression. Even in the TC02 line, there was no re-

porter gene expression in either the catecholaminergicor the noncatecholaminergic regions. Recently, Kessleret al. (2003) reported that, in transgenic mice in whichthe flanking 11 kb fused to the reporter GFP, robusttransgene expression was detected in most catecholami-nergic tissues with the notable exceptions of the adrenalmedulla and locus ceruleus. Conversely, ectopic expres-sion of GFP was detected in several regions within theCNS that have consistently been observed by theauthors using shorter transgenic constructs derived fromrat or human TH (Nagatsu et al., 1994; Liu et al., 1997;Trocme et al., 1998; Schimmel et al., 1999). Taken to-gether, these results show that the 3.2-kb sequenceupstream of TH is insufficient for proper expression andthat the sequence from the translation start site to exon3 can lead to TH expression in a subset of catecholami-nergic neurons in several brain regions, including ventraltegmentum, but lacks some regulatory elements thatattenuate ectopic expression.

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EYFP Expression Driven by Human TH Promoter 11

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