Tau promoter activity in neuronally differentiated P19 cells

Brain Research 874 (2000) 1–9 www.elsevier.com / locate / bres

Research report

Tau promoter activity in neuronally differentiated P19 cells Alice Heicklen-Klein 1 , Stella Aronov 2 , Irith Ginzburg* Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel, 76100 Accepted 23 May 2000

Abstract Tau proteins are encoded by a single gene which is regulated by a unique promoter. The proximal 196 base pairs of the tau 59 flanking region confers tau protein with neuronal specific expression and nerve growth factor inducibility. We tested tau promoter activity in neuronally differentiated embryonal carcinoma cells, the P19 mouse blastoderm cell line. In these experiments, we examined the temporal expression pattern of the tau promoter and compared it to other viral and cellular promoters. Tau promoter activity increases significantly with differentiation, specifically during neurite initiation. In addition, tau promoter activity in neuronally differentiated P19 cells was significantly greater than all five of the other neuronal or non neuronal promoters tested. All other promoters displayed low levels of promoter activity throughout retinoic acid induced neuronal differentiation of P19 cells. Taken together, our results suggest that the tau promoter is a good choice for ectopic expression of exogenous genes in P19 cells, which serves as a differentiating neuronal model system.  2000 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Gene structure and function: general Keywords: Tau; Promoter activity; P19 cells; Neuronal differentiation

1. Introduction Tau, a microtubule associated protein, is encoded by a single gene and is expressed primarily in neurons [1,5,10,24,28,29,45,52]. The tau mRNA transcript undergoes complex and regulated alternative splicing, giving rise to six isoforms [12,21,60]. The tau proteins range in size from 48 to 67 KDa [9]. Tau, which is localized to the cell body and axon, functions in assembly and stabilization of microtubules [6,11,25,33,48]. The 59 flanking region of the tau gene was isolated in our lab from rat brain [52]. The proximal 196 base pairs of the tau promoter confers neuronal specific expression of tau in the rat adrenal pheochromocytoma cell line, PC12 [23,26]. Furthermore, a burst in tau promoter expression is observed upon nerve growth factor (NGF) induction of PC12 cells into sympathetic-like neurons [26]. This burst *Corresponding author. Tel.: 1972-8-934-2799; fax: 1972-8-9344131. E-mail address: [email protected] (I. Ginzburg). 1 Contributed equally to this work. 2 Contributed equally to this work.

in tau transcription corresponds with the transcription dependent stage of neurite initiation in PC12 cells [8]. P19 embryonal carcinoma cells originated from seven day mouse blastoderm and are multipotent cells that can differentiate into all three germ layers [38,39]. Treatment with retinoic acid (RA) and aggregation of cells leads to differentiation into neurons, glia and fibroblasts [30]. Neuronal cells are then selected for by treatment with mitotic inhibitors which eliminates the glia and fibroblast dividing cells. P19 cells were chosen as a model system to study tau promoter expression since, these cells are among the few neuronal cell lines that can be differentiated in culture into polarized cells with defined axons and dendrites [17] and they express high molecular weight microtubule associated proteins in a pattern corresponding to the developmental pattern in situ [59]. Moreover, P19 cells possess a primarily cholinergic phenotype [30,47]. The cholinergic nervous system is the primary site of insoluble tau protein accumulation, a hallmark of Alzheimer’s disease (AD) [4,21]. In order to study gene expression in model systems, transfections are employed and activity levels monitored. The introduction of DNA constructs encoding for ectopic

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02539-7

2

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9

proteins into differentiated non mitotic neuronal cells is technically difficult, and results varying depending on transfection method, cell type and promoter selection. Viral promoters are frequently used to transfect DNA into cell lines. Unfortunately, these viral promoters are inhibited to low or non detectable levels of expression following RA induced neuronal differentiation in P19 cells and may even interfere with diffferentiation [7,22,42,63,65]. In addition to viral promoters, a number of other promoters, neuronal and non neuronal, have been utilized to express exogenous proteins in RA treated and untreated P19 cells [34,40,43,51,57,62]. The relative efficiency of many of these promoters is unknown. We stably transfected P19 cells with the tau promoter upstream of the luciferase reporter gene to examine tau promoter efficiency and expression pattern in these cells. We compared the temporal expression pattern of the tau promoter in P19 cells during neuronal differentiation with our previous results in PC12 cells [56]. In both cell lines, there is a basal level of tau promoter activity that increases upon neurite initiation, accentuating tau’s role in assembling microtubules in neurites. In addition, we compared tau promoter efficiency with other neuronal and non neuronal promoters in P19 stable cell lines in order to find a promoter that will efficiently express ectopic proteins in neuronal cells. We were also interested in the temporal expression patterns of the various promoters so that ectopic proteins could be expressed at desired developmental stages. Promoters of various genes were stably transfected into P19 cells and their activity levels measured. The promoters tested were: neuronal promoters, tau and GAP-43 / B50 (two promoter types); cellular ubiquitous promoters, MHCI and p53; and a viral promoter, CMV, frequently used to express exogenous proteins. The use of a strong exogenous viral promoter in cell lines may disrupt the balance of the cell, producing artificial results and even lead to cell death [2]. Two overlapping GAP-43 promoters were used as neuronal promoters because GAP-43 protein, like tau protein, is a known marker of axons, expressed during axonal growth and regeneration [17]. The increase in GAP-43 protein expression is temporally similar to that observed for tau except, that GAP-43 protein levels rise at the onset of neuronal differentiation and then are attenuated in mature neurons whereas tau protein levels continue to increase with neurite elongation [44,52]. Our results show that the cellular promoters, MHCI and p53, and the viral CMV promoter had constant low levels of promoter activity in the multipotent stage and throughout RA induced P19 differentiation. Furthermore, the GAP-43 promoters had high levels of promoter activity prior to differentiation, which was substantially attenuated with differentiation. In contrast, the tau promoter expression level rose significantly with RA induced differentiation, peaking between four to 6 days when neurites

initiation is observed (P,0.01 on day 5). The temporal expression pattern of the tau promoter in P19 cells was similar to that observed previously in PC12 regardless of the inducer utilized to induce neuronal differentiation: RA or NGF, respectively [56]. Our results suggest that the tau promoter is amenable for ectopic expression of exogenous proteins in RA induced P19 cells especially during neurite initiation.

2. Materials and methods

2.1. Constructs Previous results demonstrated that the proximal portion of the tau promoter (2196 to 166) conferred preferred neuronal expression and NGF responsiveness [26]. This tau promoter fragment was cloned upstream of the luciferase reporter gene in the p20 vector. The MHCI promoter was taken from Ehrlich et al. [15]. The p53 promoter was a gift from Dr. Moshe Oren, Weizmann Institute of Science, Israel. The GAP-43 promoters were kindly provided by Dr. Loes H. Schrama, University of Utrecht, Netherlands [14]. We tested the entire 1000 base pairs upstream of the GAP-43 translation start site (P1 and P2) and P2 separately, since P2 promoter activity increases with RA administration. All promoter constructs were linked upstream of the luciferase reporter gene. The puromycin gene downstream of the pgk promoter, used for selection in stable transfections, was kindly provided by Dr. Peter W. Laird, The Netherlands Cancer Institute. Tau cDNA [31,53] was cloned in frame downstream of green fluorescent protein (GFP) in the pEGFP vector of Clonetech.

2.2. Cell cultures and transfections P19 cells were grown as previously described [16] in alpha medium, 10% fetal calf serum, 100units / ml penicillin, 100 mg / ml Streptomycin, 2 mM L-glutamine, and 5% CO 2 . Cells were diluted approximately every other day following treatment with 0.18units / ml trypsin and 0.024% EDTA. Cells were plated at a density of 1310 5 / 60 mm petri dish 1 day prior to transfection. Cotransfections were performed with a total of 5 mg DNA consisting of puromycin downstream of the pgk promoter and a second vector with the desired promoter upstream of the luciferase gene at a 1:3 molar ratio, respectively. Transfections were performed using 20 ml lipofectin per plate as specified by the manufacturer (Gibco BRL). Transfections for each construct were repeated with different DNA preparations to account for variance between transfections and DNA preparations. Stable lines were selected for by adding 1 mg / ml puromycin to the medium for a minimum of 3

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9

weeks. After selection with puromycin, multiple plates with abundant foci were obtained and used for neuronal differentiation and luciferase experiments. In non differentiated stable lines, luciferase activity tests demonstrated relatively constant values for each promoter.

2.3. RA induced differentiation Following selection, stable lines of P19 cells were differentiated as specified by Jones–Villeneuve et al. [30] with some modifications. Briefly, cells were aggregated on 100 mm bacteriological plates for 4 days in the presence of 1 mM all trans retinoic acid (Sigma). The cells were trypsinized and plated on culture-grade plates coated with poly-L-lysine 25 mg / ml (Sigma) at a density of 1310 4 – 10 6 without Retinoic acid. Cytosine b-D-Arabinofuranoside (Sigma), a mitotic inhibitor, was added on day five for 48 h and the culture was sustained for 10 days to 2 weeks. At this stage, the culture consisted of approximately 90% neuronal cells.

2.4. Confocal microscopy analysis of P19 cells Retinoic acid differentiated P19 cells either untransfected or stably transfected with a GFP-tau cDNA construct were grown for 10 days on coverslips covered with poly-Llysine 25 mg / ml (Sigma). The cells were fixed with 4% paraformaldehyde in 4% sucrose for 20 min at room temperature. They were then permeabilized by incubation for 3 min in 0.5% Triton X-100, washed three times with PBS, and blocked overnight at 48C with 1 or 10% BSA. For immunochemical analysis P19 cells were incubated with anti-tubulin monoclonal antibodies, tubulin 2.1, (1:100) (Sigma) or with polyclonal anti-MAP-2 a / b (1:100) (kindly provided by Craig C. Garner, University of Alabama, USA) for 16 h at 48C. They were then washed three times, each for 15 min with PBS and incubated for 2 h at room temperature with CY3 labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA). The coverslips were then mounted with Mowiol and visualized with the MRC-1024 confocal laser scanning imaging system (Bio-Rad, Richmond CA) at 403 objective, using GFP optimized filters or red filters for tau or tubulin and MAP-2, respectively. The images were analyzed using the MRC-1024 confocal imaging software system.

2.5. Luciferase assay The luciferase activity assay was performed as previously described by Heicklen-Klein et al. [26]. In brief, stably transfected cell lines at different stages of differentiation were washed three times with phosphate-buffered saline (PBS) and lysed in 100 ml of 100 mM KPO 4 buffer (pH 7.8), containing 1 mM DTT and 0.5% (v / v) Triton X-100. The luciferase assay buffer contained 100 mM Tris Acetate (pH 7.8), 10 mM MgOAc, 100 mM EDTA, 2 mM ATP

3

(pH 7.0) and 74 mM luciferin. Activity tests were performed in a Biocounter M2500 luminometer (Lumac). Luminescence was read for 20 s immediately after luciferin assay buffer was injected. Luciferase values were standarized for protein concentrations. Differentiation of cell lines and activity assays were performed a minimum of three times for each construct tested. Differences between constructs and days of differentiation were tested using the Student T-tests and one-way analysis of variance (ANOVA).

2.6. RT-PCR RNA was extracted from nondifferentiated P19 cells and P19 cells differentiated with RA for three to ten days employing the RNA Pure kit (Promega). The extracted RNA was reverse transcribed with 200 units M-MLV reverse transcriptase (Promega), random hexamers 0.125 ng / ml, 2 ml reverse transcriptase 10X buffer, 20 units RNasin (Promega) and 0.125 mM dNTPs in a final volume of 20 ml and extended for an hour at 378C. PCR was performed with 5 ml of the reverse transcription reaction, 2.5 units Taq polymerase (Promega), 5 ml Taq polymerase 10X buffer, 0.05 mM dNTPs, 8.3 mM MgCl 2 and 20 mM of each of the specific primers in a final volume of 50 ml. PCR amplification was performed by denaturing at 948C for 4 min, and then 29 cycles as follows: denaturing at 948C for 1 min, annealing at 568C for 1 min and then extension at 728C for 2 min. A final cycle at 728C for 5 min concluded the PCR. The samples were then separated on a 1% (wt / vol) agarose gel and viewed under UV light. Intensities of tau transcripts were normalized to a GAPDH internal control. The primers used for the RT-PCR of P19 cells throughout differentiation were as follows: for tau, 59ATGGCTGAACCCCGCCAG-39 (1–18) and 59CGGGTGGGCGGCGTTGGTAGG-39 (442–463) [53]and for GAPDH, 59 -GCCATCAACGACCCCTTCAT-39 (118–137) and 59-TTCACACCCATCACAAACAT-39 (412–431) [61] .

2.7. Immunoblot analysis of P19 protein extracts Proteins were extracted from P19 pelleted cells in 1 volume of lysis buffer (50 mM Tris pH 8.5, 1% triton X-100, 5 mM EDTA, 0.15 M NaCl, 50 mg / ml PMSF) and cleared of cell debris by centrifugation for 10 min at 16,0003g at 48C. Protein samples (25 mg) were resolved by SDS-gel electrophoresis, transferred to nitrocellulose filters, and reacted with either tau-1 monoclonal antibody (1:10,000) [5]or anti-MAP-2 monoclonal antibody, AP-20, (1: 200) (Sigma) at 48C for 16 h. They were then visualized by peroxidase-conjugated goat anti-mouse secondary antibodies obtained from Jackson ImmunoResearch (West Grove, PA) at room temperature for 1 h and developed using ECL chemiluminescence procedure.

4

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9

3. Results

3.1. P19 cell differentiation and polarity

gates of cell bodies with protruding neurites along with numerous individual cell with axons and dendrites. When individual cells are examined by specific axonal and dendritic markers, the segregation of tau and MAP-2 is observed. Tau protein is localized to the soma and the axon whereas MAP-2 protein is localized to the soma and dendrites (Fig. 1B–E). MAP segregation to various compartments of the developing neuron is one of the first hallmarks of neuronal polarity.

P19 cell were induced for 4 days with RA, non neuronal cells eliminated and allowed to differentiate into neuronal cells. The resulting culture consisted of 90% neuronally differentiated P19 cells with defined axons and dendrites. Fig. 1A illustrates the morphology of differentiated P19 cells as visualized by immunochemical staining with antitubulin antibodies. The RA treated P19 cell culture at the final stages of neuronal differentiation consists of aggre-

3.2. Promoter activity throughout P19 cell neuronal differentiation

Fig. 1. Morphology and MAP segregation in neuronally differentiated P19 cells. (A) Tubulin staining (red) in P19 cells differentiated for 9 days. The open arrow head denotes an aggregate and the solid arrow head denote a single cell with cell body and neurites. Bar Scale is 50 mm. (B–E) P19 cells were stably transfected with GFP-tau protein, neuronally differentiated for 9 days, and MAP segregation analyzed by confocal microscopy. Bar Scale is 5 mm. (B) GFP-tau protein is visible (green) in the soma and axon of a single cell. (C) MAP-2 visualized (red) in the soma and dendrite of the same cell. (D) Computer merge of GFP-tau (green) and MAP-2 (red) proteins: GFP-tau and MAP-2 colocalize (orange) in the soma however, GFP-tau and MAP-2 segregate to the axon and the dendrite, respectively. (E) Phase of the same P19 cell. In B–E open arrows denote axons and solid arrows denote dendrites.

Tau promoter activity during the course of P19 neuronal development was compared to the luciferase activity obtained for five other neuronal and non neuronal promoters stably transfected into P19 cells. The cellular promoters, MHCI and p53, and the viral CMV promoter had constant low levels of promoter activity in the multipotent nondifferentiated stage and throughout RA induced P19 differentiation. The p53 promoter retained an even lower level of promoter activity than either the MHCI or CMV promoters in treated and untreated P19 cells. (Table 1 and Fig. 2A). The promoter of the GAP-43 gene has been characterized and was shown to include two tandem promoters, P1 and P2 [14]. When the first 1000 base pairs of the 59 flanking region, containing both P1 and P2, were previously tested by Fukuchi et al. [19] the activity level in neuronally differentiated and multipotent P19 cells was relatively stable. However, the distal P1 promoter was inhibited by RA in P19 whereas the proximal P2 promoter was stimulated by RA and indeed the majority of GAP-43 mRNA isolated from a late stage of development, 8 days, of rat brain was found to be transcribed from P2 [14]. The GAP-43 promoters were found here to have expression levels comparable or higher than those of tau in the nondifferentiated stage. Following RA induced differentiation, both of the GAP-43 promoters expression levels were significantly attenuated (P,0.01) and were significantly less than tau’s expression level (P,0.01) (Table 1 and Fig. 2B). Tau promoter activity rose significantly with RA induced differentiation, peaking between four to six days when the cells are plated and neurites initiation is observed (P,0.01 on day 5) (Table 1 and Fig. 2). The temporal pattern of expression observed for the tau promoter corresponds with that observed in PC12 cells [52]. Tau promoter expression at 5 days of neuronal differentiation is significantly higher (P,0.01) than any of the other tested promoters throughout neuronal differentiation (Table 1 and Fig. 2). Of the promoters assayed here, tau is the most compatible promoter for the ectopic expression of exogenous proteins in RA induced P19 cells during neurite initiation, which marks an early stage of neuronal differentiation.

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9

5

Table 1 Luciferase promoter activity in P19 cells during the course of retinoic acid induced neuronal differentiation a

a

Values are arbitrary units / mg6standard deviation. Days denotes days of differentiation.

3.3. Endogenous tau mRNA and protein levels Endogenous tau mRNA levels throughout differentiation were analyzed by the RT-PCR method. Low levels of tau mRNA can be observed in non differentiated P19 cells but attain substantial levels at 4 days of RA treatment just before neurites initiate. These findings are indicative of a putative role of tau protein in microtubule assembly during axon outgrowth. Tau mRNA levels plateau between 6 and 12 days, decreasing between 12 and 14 days (Fig. 3). To determine the expression pattern and appearance of endogenous tau and MAP-2 proteins as neuronal differentiation progresses, immunoblot analysis was performed. Tau protein is first detected between four to six days of RA differentiation in P19 cells, as was seen by Falconer et al. [16]. Tau protein progressively increases as differentiation progresses. MAP-2 protein was detected in differentiated P19 cells following a similar time course as tau protein (Fig. 4) [59].

4. Discussion Our results show that P19 cells can serve as a model system to study neuronal differentiation of cells with defined axons and dendrites. Finley et al. [17], also demonstrated that RA treated P19 cells are polar cells with axons and dendrites using the segregation of GAP-43 and microtubule associated protein-2 (MAP-2) protein, respectively. The findings here reinforce our previous results where induction of tau expression in PC12 cells resulted in neurite extension [25]. Furthermore, we demonstrate that the treatment of P19 cells with RA, which induces neuronal differentiation of P19 cells, increases tau promoter activity (Figs. 2 and 3). It has been shown previously in P19 cells that RA increases

tau protein levels [16]. Moreover, RA regulates a variety of promoters in P19 cells at least partially by the activation of AP-2 [13,46,49,64]. The presence of nine AP-2 binding motifs on the tau promoter fragment tested in this study suggests that the increase in tau protein expression following RA treatment results at least in part from increased transcription activation by AP-2. Both neuronal and non neuronal promoters have been utilized in P19 cells. Fukucki et al. [19] compared the activity of nine different promoters transiently transfected into P19 cells treated and untreated with RA. Only one of these promoters, a chimera between the cytomegalovirus (CMV) enhancer and the b-actin promoter, had a reasonable level of expression. The chimera of a viral enhancer and a cellular promoter is inaccessible and of questionable biological significance. Of the eight low level expressing promoters six were viral, which as stated previously, are problematic in P19 neuronally differentiated cells. Of the two remaining low level expressing promoters one was the nerve growth factor receptor which is neuronal specific but endogenously expressed in P19 cells only after RA treatment and predominantly in glia cells [55]. The second is the amyloid precursor protein (APP) promoter which is expressed in neurons and has an appreciable biological significance in AD. Tau protein and b-amyloid are the principle components of the neurological lesions of AD [4,21]. It has been documented that the transcriptional regulation of tau and b-amyloid are both disrupted in AD [3,27,32]. The b-amyloid promoter was found by Fukuchi et al. [19] to be basely expressed prior to induction in P19 cells but was induced eight fold upon exposure to RA. The tau and APP promoters both are induced by RA in P19 cells, are GC rich, lack a TATA box motif and possess multiple transcription initiation sites [52,54]. These similarities may imply a common pathway of transcriptional activation which malfunctions in AD.

6

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9

Fig. 3. RT-PCR analysis of endogenous tau mRNA levels throughout the course of neuronal differentiation in P19 cells. In the control (ctrl), PCR was performed without the reverse transcriptase step to ensure that the sample was not contaminated with DNA. GAPDH was used as an internal control to normalize the results.

with RA induction of P19 cells. A previous work reported that GAP-43 promoter activity increased for the P2 promoter, decreased for the P1 promoter and the sum of these two promoter (P11P2) activities remained constant during neuronal differentiation of P19 cells [14]. One feasible explanation for this discrepancy is the difference between activities tested following transient transfections, performed by Eggen et al. [14] and activities tested in stable cell lines, performed here. Transient transfection in neuronally differentiating P19 cells were found in our lab to be technically difficult, yielding results with large variances (data not shown). Differences in promoter activities were also documented between transient and stable transfections for the retinoblastoma promoter in P19 cells and the actin promoter in Ltk 2 fibroblast cell line and in BC3H-1 mouse myogenic cell line [18,56] Furthermore, McBurney et al. demonstrated that intragenic regions of the pgk-1 locus had

Fig. 2. Summary of the luciferase promoter activity observed during retinoic acid induced P19 neuronal differentiation. (A) Luciferase promoter activity of the non neuronal promoters; CMV, MHCI, p53; compared to tau promoter activity. (B) Luciferase promoter activity of the neuronal promoters: Tau, 1000 base pairs of the GAP-43 59 flanking region (P11P2), and the 39 portion of the GAP-43 promoter (P2). Days of differentiation are as in Table 1. Note, that the tau promoter activity at 5 days of differentiation is significantly greater (P,0.01) than the tau promoter activity in nondifferentiated P19 cells and is significantly greater than the activity observed for all other promoters during the course of neuronal differentiation of P19 cells (P,0.01). ND5 nondifferentiated. RA5retinoic acid.

In this work, we tested the promoter expression levels of both neuronal and non neuronal promoters in stably transfected P19 cell lines during neuronal differentiation. The use of stable cell lines circumvents the problems of low transfection efficiencies in neuronally differentiated cell lines as well as the fluctuations observed in transient transfection assays. Our data show that GAP-43 promoter activity decreases

Fig. 4. Immunoblot analysis of endogenous microtubule associated protein levels during the course of neuronal differentiation in P19 cells. (A) Tau protein levels visualized with the tau-1 antibody. (B) MAP-2 protein levels visualized with the AP-20 (MAP-2) antibody.

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9

conflicting effects on promoter activity in transient and stably transfected P19 cells [37]. The difference in transfected DNA copy number, nucleosome structure and the site of incorporation into the chromosome differentially effect the level of transcription from stably and transiently transfection DNA. However, the molecular mechanism that accounts for the contradicting results frequently obtained with transient and stable transfection remains to be explored. All three neuronal promoters were active in non differentiated P19 cells. Interestingly, a number of other neuronal promoters were also reported to be active in non differentiated P19 cells [35,41,50,51,58] suggesting a unique mechanism in P19 non differentiated cells that allows their aberrant expression. However, tau protein was not expressed in P19 cells until 4–6 days of RA treatment suggesting that the tau mRNA observed here by RT-PCR in non differentiated cells is not translated. Previously, tau mRNA has been shown not to be translated in newborn rodent brains [20,36] suggesting a putative mechanism to explain the lag between tau mRNA transcription and tau protein translation in neuronally differentiating P19. The CMV promoter was used as a reference point for relative promoter activity levels since it was used here and by Fukuchi et al. [19] who examined the relative levels of activity for a number of promoter in P19 cells. CMV was found by Fukuchi et al. [19] to have the second highest expression level while in this report, tau promoter activity was significantly greater than that obtained for CMV both in nondifferentiated and RA differentiated P19 cells. Therefore it can be suggested that the tau promoter would be an efficient promoter to ectopically express various proteins in neuronally differentiated P19 cells particularly during neurite initiation. The study of tau promoter regulation and aberrations in its function are of particular importance in view of its critical role in axon formation and several dementias, such as Alzheimer’s disease.

Acknowledgements This work was supported by grants from the Minerva Foundation, Germany; Nella and Leon Benoziyo Center for Neuroscience, Weizmann Institute of Science; and the Henry S. and Anne S. Reich Research Fund for Mental Health, Weizmann Institute of Science.

[4] [5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18] [19]

[20]

References [21] [1] A. Andreadis, W.M. Brown, K.S. Kosik, Structure and novel exons of the human tau gene, Biochemistry 31 (1992) 10626–10633. [2] D. Antic, N. Lu, J.D. Keene, Elav tumor antigen, Hel-N1 increases translation of neurofilament M mRNA and induces formation of Neurites in human teratocarcinoma cells, Genes Dev. 13 (1999) 449–461. [3] A.J.L. Barton, P.J. Harrison, A. Najlerahim, J. Heffernan, B.

[22]

7

McDonald, J.R. Robinson, D.C. Davies, W.J. Harrison, P. Mitra, J.A. Hardy, R.C.A. Pearson, Increased tau messenger RNA in Alzheimer’s disease hippocampus, Am. J. Path. 137 (1990) 497–502. K. Beyreuther, C.L. Masters, Tangle disentanglement, Nature 383 (1996) 476–551. L.I. Binder, A. Frankfurter, L.I. Rebhun, The distribution of tau polypeptides in the mammalian central nervous system, J. Cell Biol. 101 (1985) 1371–1378. L.I. Binder, A. Frankfurter, L.I. Rebhun, Differential localization of MAP-2 and tau in mammalian neurons in situ, Ann. N.Y. Acad. Sci. 466 (1986) 145–166. C.A. Boulter, E.F. Wagner, The effects of v-src expression on the differentiation of embryonal carcinoma cells, Oncogene 2 (1988) 207–214. D.E. Burstein, L.A. Greene, Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor, Proc. Natl. Acad. Sci. U.S.A. 75 (1978) 6059–6063. D. Couchie, A. Faivre-Bauman, J. Puymirat, J. Guilleminot, A. Tixier-Vidal, J. Nunez, Expression of microtubule-associated proteins during the early stages of neurite extension by brain neurons cultured in a defined medium, J. Neurochem. 47 (1986) 1255–1261. D. Drubin, S. Kobayashi, M. Kirschner, Association of tau proteins with microtubules in living cells, Ann. N.Y. Acad. Sci. 466 (1986) 257–286. D.G. Drubin, S.C. Feinstein, E.M. Shooter, M.W. Krischner, Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors, J. Cell Biol. 101 (1985) 1799–1807. D.G. Drubin, S. Kobayashi, S. Kellogg, M.W. Kirschner, Regulation of microtubule protein levels during cellular morphogenesis in nerve growth factor-treated PC12 cells, J. Cell Biol. 106 (1988) 1583– 1591. J.R.B. Dyck, L. Fliegel, Specific activation of the NA / H exchanger gene during neuronal differentiation of embryonal carcinoma cells, J. Biol. Chem. 270 (1995) 10240–10427. B.J.L. Eggen, H.B. Nielander, M.G.A. Rensen-de Leeuw, P. Schotman, W.H. Gispen, L.H. Schrama, Identification of two promoter regions in the rat B50 / GAP43 gene, Mol. Brain Res. 23 (1994) 221–234. R. Ehrlich, J.E. Maguire, D.S. Singer, Identification of negative and positive regulatory elements associated with a class I major histocompatility complex gene, Mol. Cell Biol. 8 (1988) 695–703. M.M. Falconer, C.J. Echeverri, D.L. Brown, Differential sorting of beta tubulin isotypes into colchicine-stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells, Cell Mot. Cytoskel. 21 (1992) 313–325. M.F.A. Finley, N. Kulkarni, J.E. Huettner, Synapse formation and establishment of neuronal polarity by P19 embryonic carcinoma cells and embryonic stem cells, J. Neurosci. 16 (1996) 1056–1065. N. Fregien, N. Davidson, Activating elements in the promoter region of the chicken beta actin gene, Gene 48 (1986) 1–11. K.-I. Fukuchi, M.G. Hearn, S.S. Deeb, A.C. Smith, N. Dang, J.I. Miyazaki, M. Bothwell, G.M. Martin, Activity assays of nine heterogenous promoters in neural and other cultured cells, In Vitro Cell Dev. Biol. 30A (1994) 300–305. I. Ginzburg, T. Scherson, D. Giveon, L. Behar, U.Z. Littauer, Modulation of mRNA for microtubule-associated proteins during brain development, Proc. Natl. Acad. Sci. 79 (1982) 4892–4896. M. Goedert, C.M. Wischik, R.A. Crowther, J.E. Walker, A. Klug, Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 4051–4055. C.M. Gorman, P. Rigby, D.R. Lane, Negative regulation of viral enhancers in undifferentiated embryonic stem cells, Cell 42 (1985) 519–526.

8

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9

[23] L.A. Greene, A. Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. U.S.A. 73 (1976) 2424–2428. [24] Y. Gu, F. Oyama, Y. Ihara, Tau is widely expressed in rat tissue, J. Neurochem. 67 (1996) 1235–1244. [25] R. Hanemaaijer, I. Ginzburg, Involvement of mature tau isoforms in the stabilization of neurites in PC12 cells, J. Neurosci. Res. 30 (1991) 163–171. [26] A.J. Heicklen-Klein, I. Ginzburg, Tau promoter regions conferring neuronal precursor specificity and nerve growth factor responsiveness, J. Neurochem. (2000) in press. [27] G.A. Higgins, D.A. Lewis, S. Bahmanyar, D. Goldgaber, D.C. Gajdusek, W.G. Young, J.H. Morrison, M.C. Wilson, Differential regulation of amyloid-b-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease, Proc. Natl. Acad. Sci. 85 (1988) 1297–1301. [28] A. Himmler, Structure of the bovine tau gene: alternatively spliced transcripts generate a protein family, Mol. Cell. Biol. 9 (1989) 1389–1396. [29] C. Janke, M. Holzer, J. Klose, T. Arendt, Distribution of isoforms of the microtubule-associated protein tau in gray and white matter areas of human brain: a two-dimensional gel electrophoretic analysis, FEBS Lett. 379 (1996) 222–226. [30] E.M.V. Jones-Villeneuve, M.W. McBurney, K.A. Rodgers, V.I. Kalnins, Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glia cells, J. Cell Biol. 94 (1982) 253–262. [31] S. Kaech, B. Ludin, A. Matus, Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins, Neuron 17 (1996) 1189–1199. [32] S. Khatoon, I. Grundke-Iqbal, K. Iqbal, Brain levels of Microtubuleassociated protein tau are elevated in Alzheimer’s disease: a radioimmuno-slot-blot assay for nanograms of the protein, J. Neurochem. 59 (1992) 750–753. [33] N.W. Kowall, K.S. Kosik, Axonal disruption and aberrant localization of tau protein characterize the neurophil pathology of Alzheimer’s disease, Ann. Neurol. 22 (1987) 639–643. [34] F.A.E. Kruyt, G.E. Folkers, A.J.M. Walhout, B.-J.M. van der Leede, P.T. van der Saag, E1 functions as a coactivator of retinoic aciddependent retinoic acid receptor-beta2 promoter activation, Mol. Endocrinol. 7 (1993) 604–615. [35] S. Lu, H.H. Loh, L.-N. Wei, Studies of dual promoters of mouse kappa-opiod receptor gene, Mol. Pharmacol. 52 (1997) 415–420. [36] G. Mangin, D. Couchie, C.C.-B. Nunez, Timing of expression of tau and its encoding mRNAs in the developing neocortex and cerebellum of the mouse, J. Neurochem. 53 (1989) 45–50. [37] M.W. McBurney, S. Fournier, K. Jardine, L. Sutherland, Intragenic regions of the murine pgk-1 locus enhance integration of transfected DNAs into genomes of embryonal carcinoma cells, Somat. Cell Mol. Genet. 20 (1994) 515–528. [38] M.W. McBurney, E.M.V. Jones-Villeneuve, M.K.S. Edwards, P.J. Anderson, Conrol of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line, Nature 299 (1982) 165– 167. [39] M.W. McBurney, B.J. Rodgers, Isolation of male embryonic carcinoma cells and their chromosome replication patterns, Devel. Biol. 89 (1982) 503–508. [40] M.W. McBurney, L.C. Sutherland, C.N. Adra, B. Leclair, M.A. Rudnicki, K. Jardine, The mouse pgk-1 gene promoter contains an upstream activator sequence, Nucl. Acids Res. 19 (1991) 5755– 5761. [41] M.B. McCormick, R.M. Tamimi, L. Snider, A. Asakura, D. Bergstrom, S.J. Tapscott, neuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family, Mol. Cell Biol. 16 (1996) 5792–5800. [42] S. Minucci, D.J. Zand, A. Dey, M.S. Marks, T. Nagatsa, J.F. Grippo, K. Ozato, Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells, Mol. Cell Biol. 14 (1994) 360–372.

[43] D.J. Morassutti, W.A. Staines, D.S.K. Magnuson, K.C. Marshall, M.W. McBurney, Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum, Neuroscience 59 (1994) 753–763. [44] E. Nedivi, G.S. Basi, I.V. Akey, J.H.P. Skene, A Neural-Specific GAP-43 core promoter located between usual DNA elements that interact to regulate its activity, J. Neurosci. 12 (1992) 691–704. [45] R.L. Neve, P. Harris, K.S. Kosik, D.M. Kurnit, T.A. Donlon, Identification of cDNA clones for the human microtubule-associated protein tau and chromosomal localization of the genes for tau and microtubule associated protein 2, Mol. Brain Res. 1 (1986) 271– 280. [46] N. Ohkawa, Y. Konishi, M. Shimada, Y. Makino, S. Yoshikawa, K. Mikoshiba, T. Tamura, Activation of the mouse inositol 1,4,5triphosphate receptor type 1 promoter by AP-2, Gene. 229 (1999) 11–19. [47] D. Parnas, M. Linial, Cholinergic properties of neurons differentiated from an embryonal carcinoma cell-line (P19), Int. J. Devl. Neurosci. 13 (1995) 767–781. [48] I. Peng, L.I. Binder, M.M. Black, Biochemical and immunological analyses of cytoskeletal domains of neurons, J. Cell Biol. 102 (1986) 252–262. [49] J. Philipp, P.J. Mitchell, U. Malipiero, A. Fontana, Cell type-specific regulation of expression of transcription factor AP-2 in neuroectodermal cells, Dev. Biol. 165 (1994) 602–614. [50] C.S. Pinal, V. Cortessis, A.J. Tobin, Multiple elements regulate GAD65 transcription, Dev. Neurosci. 19 (1997) 465–475. [51] V.A. Pospelov, T.V. Pospelova, J.-P. Julien, AP-1 and Knox-24 transcription factors activate the neurofilament light gene promoter in P19 embryonal carcinoma cells, Cell Growth Differ. 5 (1994) 187–196. [52] E. Sadot, A. Heicklen-Klein, J. Barg, P. Lazarovici, I. Ginzburg, Identification of a tau promoter region mediating tissue-specificregulated expression in PC12 cells, J. Mol. Biol. 256 (1996) 805– 812. [53] E. Sadot, J. Marz, J. Barg, L. Behar, I. Ginzburg, Complete sequence of the 39UTR region of tau from rat central nervous system, J. Mol. Biol. 241 (1994) 325–331. [54] J.M. Salbaum, A. Weidemann, H.-G. Lemaire, C.L. Masters, K. Beyreuther, The promoter of Alzheimer’s disease amyloid A4 precursor gene, EMBO J. 7 (1988) 2807–2813. [55] A.M. Salvatore, M. Cozzolino, G. Nicola, S. Galanti, A. Levi, S. Alema, Neuronal differentiation of P19 embryonal cells exhibits cell-specific regulation of neurotrophin receptors, Neuroreport 6 (1995) 873–877. [56] R.S. Slack, P.A. Hamel, T.S. Bladon, R.M. Gill, M.W. McBurney, Regulated expression of the retinoblastoma gene in differentiating embryonal carcinoma cells, Oncogene 8 (1993) 1585–1591. [57] R. St-Arnaud, J.M. Moir, Wnt-1 inducing factor-1: a novel G / C box-binding transcription factor regulating the expression of Wnt-1 during neuroectodermal differentiation, Mol. Cell Biol. 13 (1993) 1590–1598. [58] S. Takashima, Y. Yoshida, T. Kanematsu, N. Kojima, S. Tsuji, Genomic structure and promoter activity of the mouse polysialic acid synthase (mST8Sia IV/ PST) gene, J. Biol. Chem. 273 (1998) 7675–7683. [59] Y. Tanaka, K. Kawahata, T. Nakata, N. Hirokawa, Chronological expression of microtubule-associated proteins (MAPs) in EC cell P19 after neuronal induction by retinoic acid, Brain Res. 596 (1992) 269–278. [60] K.K. Teng, I.S. Goergieff, J.M. Aletta, J. Nunez, M.L. Shelanski, L.A. Greene, Characterization of a PC12 cell subclone (PC12-C41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis, J. Cell Sci. 106 (1993) 611–626. [61] J.Y. Tso, X.H. Sun, K.S. Reece, R. Wu, Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase

A. Heicklen-Klein et al. / Brain Research 874 (2000) 1 – 9 cDNAs: genomic complexity, Nucl. Acids Res. 13 (1985) 2485– 2502. [62] T. Uetsuki, K. Tagagi, H. Sugiura, K. Yoshikawa, Structure and expression of the mouse necdin gene, J. Biol. Chem. 271 (1996) 918–924. [63] A. Wiederkehr, P. Caroni, Widely used enhancer of eukaryotic expression vectors is strongly and differentially regulated in fibroblast, myoblast, and teratocarcinoma cell lines, Exp. Cell Res. 219 (1995) 664–670.

9

[64] T. Williams, R. Tjian, Analysis of the DNA-binding and activation properties of the human transcription factor AP-2, Genes Dev. 5 (1991) 670–682. [65] J.-X. Wu, E.D. Adamson, Inhibition of differentiation in P19 embronal carcinoma cells by the expression of vectors encoding truncated or antisense EGF receptor, Dev. Biol. 159 (1993) 208– 222.