- Email: [email protected]
Conserved elements in the 5′ regulatory region of the amyloid precursor protein gene in primates
Neuroscience Letters 226 (1997) 203–206
Conserved elements in the 5′ regulatory region of the amyloid precursor protein gene in primates Rosa Adroer a, Cristina Lo´pez-Acedo a, Rafael Oliva a , b ,* a
Human Genome Laboratory, Molecular Genetics Research Group, Faculty of Medicine, University of Barcelona, C/ Casanova 143, 08036, Barcelona, Spain b Servei de Gene`tica. Hospital Clı´nic i Provincial de Barcelona, Barcelona, Spain Received 13 January 1997; revised version received 9 April 1997; accepted 9 April 1997
Abstract Oligonucleotides corresponding to conserved sites between the human and mouse amyloid precursor protein (APP) genes have been used to polymerase chain reaction (PCR) amplify and sequence the promoter region of the APP gene from chimpanzee, gorilla, orang-utan, papio and African green monkey. Several novel conserved potentially-regulatory sequences of the APP gene have been detected after alignment of the APP promoter sequences: an apolipoprotein E-B1 (APOE-B1) element at position −450, also present in the APOE gene, two activator protein-2 (AP-2) sites at positions −450 and −301 and an intermediate early-1 gene (IE1) site at position −280. These elements are conserved in all mammalian APP promoter sequences studied. Additionally a previously detected heat shock element (HSE) at position −317, and an activator protein-1 (AP-1) site at position −350 are also conserved. Knowledge of the essential regulatory elements at the APP gene constitute the basis for understanding its transcriptional control and subsequent model studies. 1997 Elsevier Science Ireland Ltd. Keywords: Amyloid precursor protein promoter; Amyloid precursor protein expression; Regulation; Apolipoprotein E-B1; Activator protein-1; Activator protein-2; Heat shock element
The amyloid precursor protein (APP) gene is located on chromosome 21 [9,12,31], consists of 18 exons and spans more than 170 kb [35]. Alternative splicing of the gene generates different mRNA: APP695, APP714, APP751 and APP770 [13]. A fragment of the APP, the bA4 peptide, constitutes the majority of the deposits of the senile plaques, one of the principal hallmarks in Alzheimer’s disease (AD) [7,33]. Several APP gene mutations have been described causing AD in some cases of early onset familial AD [3,8, 10,18,19]. However understanding the physiological expression and processing of the APP gene is also relevant to the majority of AD cases as bA4 deposition is one of the initial neuropathological changes in AD [27]. The human APP gene promoter has been sequenced and multiple potential binding sites associated with potentiallyregulatory elements have been described [25]. It has been observed that the APP gene lacks canonical TATA and * Corresponding author. Tel.: +34 3 4021877; fax: +34 3 4035260; e-mail: [email protected]
CAAT boxes and has a high GC content (72%) upstream from the multiple transcription start points. This configuration is typical of promoters of the so called ‘housekeeping’ genes [25], which are genes known to be expressed in all tissues of an organism [20]. The APP gene from rat and mouse have also been sequenced [4,11]. Upstream from the start codon, homology between rat and mouse promoters is 89%, and between rat and human is 82% [4]. However rat and mouse do not spontaneously develop dementia and age related amyloid deposition. A better model for AD related APP deposition has been shown in non-human primates where neuronal APP is considered to be one source of Ab deposits in the brain of aged individuals [15,22]. Thus we decided to sequence the APP gene promoter from five different nonhuman primates in order to identify possible conserved regulatory elements which could constitute the basis for subsequent model studies. Genomic DNA was isolated from different species: chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orang-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0283-8
204
R. Adroer et al. / Neuroscience Letters 226 (1997) 203–206
utan (Pongo pygmaeus), African green monkey (Cercopithecus patas) and papio (Papio doguera). Polymerase chain reaction (PCR) primers were designed from conserved regions derived from the comparison of human [25], mouse [11] and rat [4] genes. The primer sequences were: PAPP1: 5′-GCTTTTGACGTTGGGGGTTA-3′ (position −538 bp) and PAPP2: 5′-AACAGTGGGAGGGAGAGTCT-3′ (position −245 bp). The amplification reaction contained 100 ng of DNA, 5 pmol of each primer, dNTPs at 200 mM, 1.5 mM MgCl2, 10 mM Tris–HCl pH 8.3, 50 mM KCl and 1 unit Taq polymerase in a final volume of 25 ml. Amplification conditions of the APP gene promoters was successful using touchdown PCR [5]. Initial denaturation 5 min at 94°C, 38 cycles: 1 min at 94°C, 1 min annealing at Tm (from 68°C to 50°C, 18 cycles, and 20 additional cycles at 50°C) and 1 min at 72°C followed by a final elongation of 10 min at 72°C. A product of the expected size was obtained for the homologous regions of chimpanzee, gorilla, orang-utan, papio and African green monkey. Sequencing of these amplification products [23] revealed their corresponding promoter sequences (Fig. 1). Subsequently all sequences were aligned (DNASTAR computer software package) to the human and mouse sequences in order to detect conserved potentially-regulatory regions. Additionally all sequences were compared to a regulatory sequence database (Signal Scan program) [21]. Interestingly we have identified a conserved sequence at position −450 to −442 common to all primates including human (Fig. 1) with a very high homology to the apolipoprotein E-B1 (APOE-B1) element (11 nucleotides match). This APOE-B1 element is known to be protected in footprinting experiments in the human apolipoprotein E gene (abbreviated APOE to refer to the gene and ApoE to refer to the protein) where it behaves as an enhancer [29]. It has been demonstrated that the presence of the allele 4 (e4) of the APOE gene is a risk factor for the AD [1,2,26,30,32]. ApoE is a protein that is also present in senile plaques and in congophilic angiopathy in AD patients. It is known that the ApoE and b-amyloid are closely associated ‘in vitro’ [30].Our results suggests that the APOE and the APP genes could share through this site a common transcription factor perhaps explaining in part the coexistence of both gene products. A sub-sequence of this APOE-B1 element present in the APP gene also has homology to the AP-2 element and to the adenine phosphoribosyltransferase (aprt) gene element (octanucleotide GCCCCACC) where it is necessary for transcription [20]. This potential AP-2 site had already been described in the mouse [11]. Downstream the APOE-B1 site, we have identified two different new potential binding sites for activator protein-2 (AP-2) located on positions −301 and −450 (Fig. 1). There are no strictly conserved sequence motifs related to AP-2 binding sites. The consensus sequence is highly variable: CCCMNSSS. This suggests that AP-2 is not linked with a particular DNA binding protein and that a variety of com-
binatorial associations with other promoter binding factors is possible [17]. At position −350 (Fig. 1) there is a potential regulatory binding site for activator protein-1 (AP-1) highly conserved in all primates. The AP-1 binding site was initially described interacting with enhancer elements in wild type human metallothionein IIA (hMTIIA), simian virus 40 (SV40) and human collagenase genes [14]. The AP-1 sequence is recognised by the Fos family, a protein complex encoded by c-fos that acts as an inducible regulator of gene expression [24]. 3′ to the AP-1 site, a heat shock element (HSE) is present at position −317. A trans-acting factor called heat shock activator protein has been described which is induced only by heat shock stimulation. Binding competition studies indicate that the same factor is able to bind to HSE’s of all heat shock genes [34]. The HSE and the AP-1 sites are highly conserved in all primates suggesting that they could be functional in gene regulation in those species. At position −280 we found a conserved 6 bp motif
Fig. 1. Alignments of APP promoter sequences from all species analysed. ce, African green monkey; ch, chimpanzee; go, gorilla; or, orang-utan; pa, papio; hu, human [25]; mo, mouse [11]. APRT, AP-2, APOE, AP-1, HSE and IE1 potential binding sites are boxed. CpG dinucleotides are underlined.
R. Adroer et al. / Neuroscience Letters 226 (1997) 203–206
(CTTTCC; Fig. 1) that is present in the immediate early 1 (IE1) gene from human cytomegalovirus. This motif is also present in mouse cytomegalovirus, SV40, lymphotropic papovavirus and immunoglobuline k gene enhancers as well as human immunodeficiency virus long terminal repeat [6]. The sequenced region of the promoter also contains 16 CpG dinucleotides (−478, −456, −434, −422, −415, −405, −380, −371, −360, −354, −337, −331, −321, −315, −295 and −262). Six of them (−422, −415, −360, −331, −315, −295) are highly conserved in all of the species, and six are conserved in all primates (−478, −456, −434, −405, −371, −262). Some of them have been previously reported to be unmethylated [16], suggesting that cytosine methylation does not seem to control protein/DNA interaction at the APP promoter in the brain of healthy individuals. Older non-human primates develop senile plaques and cerebral vessels with amyloid, so they could be good candidates to be animal models for studying AD [28]. Here, we have reported the sequences of the promoter region of five primates and identified, previously unreported, conserved potential regulatory DNA binding sites: the APOE-B1 site at position −450, the AP-2 sites at positions −301, −374, and the IE1 site at position −280. In the light of the complex pattern of expression of the APP gene in most tissues, it is not surprising that the APP gene contains multiple regulatory elements. The possibility to further clarify their respective role through ‘in vitro’ or ‘in vivo’ experiments is now open. Supported by grants from the Fondo de Investigaciones Sanitarias (FIS96/0658) to R.O. and from the European Union Biomed-2 project BMH4-CT96-0554 to the European Chromosome 21 consortium. [1] Adroer, R., Santacruz, P., Blesa, R., Lo´pez-Pousa, S., Ascaso, C. and Oliva, R., Apolipoprotein E4 allele frequency in Spanish Alzheimer and control cases, Neurosci. Lett., 189 (1995) 182–186. [2] Blesa, R., Adroer, R., Santacruz, P., Ascaso, C., Tolosa, E. and Oliva, R., High apolipoprotein E e4 allele frequency in age-related memory decline, Ann. Neurol., 39 (1996) 548–551. [3] Chartier-Harlin, M.-C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, P., Hardy, J. and Mullan, M., Early-onset Alzheimer’s disease caused by mutations at codon 717 of the b-amyloid precursor protein gene, Nature, 353 (1991) 844–846. [4] Chernak, J.M., Structural features of the 5′ upstream regulatory region of the gene encoding rat amyloid precursor protein, Gene, 133 (1993) 255–260. [5] Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K. and Mattick, J.S., ‘Touchdown’ PCR to circumvent spurious priming during gene amplification, Nucleic Acids Res., 19 (1991) 4008. [6] Ghazal, P., Lubon, H., Fleckenstein, B. and Hennighausen, L., Binding of transcription factors and creation of a large nucleoprotein complex on the human cytomegalovirus enhancer, Proc. Natl. Acad. Sci. USA, 84 (1987) 3658–3662. [7] Glenner, G.G., Alzheimers disease, Encycl. Human Biol., 1 (1991) 209–216. [8] Goate, A., Chartier-Harlin, M.-C., Mullan, M., Brown, J. and Crawford, F. et al., Segregation of a missense mutation in the amy-
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
205
loid precursor protein gene with familial Alzheimer’s disease, Nature, 349 (1991) 704–706. Goldgaber, D., Lerman, M.I., McBride, O.W., Saffiotti, U. and Gajdusek, D.C., Characterisation and chromosomal localisation of a cDNA encoding brain amyloid of Alzheimer’s disease, Science, 235 (1987) 877–880. Hendriks, L., Van Duijn, C.M., Cras, P., Cruts, M., Van Hul, W., Van Harskamp, F., Warren, A., McInnis, M.G., Antonarakis, S.E., Martin, J.-J., Hofman, A. and Van Broeckhoven, C., Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the bamyloid precursor protein gene, Nature Genet., 1 (1992) 218–221. Izumi, R., Yamada, T., Yoshikai, S., Sasaki, H., Hattori, M. and Sakaki, Y., Positive and negative regulatory elements for the expression of the Alzheimer’s disease amyloid precursor-encoding gene in mouse, Gene, 112 (1992) 189–195. Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and Mu¨llerHill, B., The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733–736. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H., Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitory activity, Nature, 331 (1988) 530–532. Lee, W., Mitchell, P. and Tjian, R., Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements, Cell, 49 (1987) 741–752. Martin, L.J., Sisodia, S.S., Koo, E.H., Cork, L.C., Dellovade, T.L., Weidemann, A., Beyreuther, K., Masters, C. and Price, D.L., Amyloid precursor protein in aged nonhuman primates, Proc. Natl. Acad. Sci. USA, 88 (1991) 1461–1465. Milici, A., Salbaum, J.M. and Beyreuther, K., Study of the Alzheimer’s A4 precursor gene promoter region by genomic sequencing using Taq polymerase, Biochem. Biophys. Res. Commun., 69 (1990) 46–50. Mitchell, P.J., Wang, C. and Tjian, R., Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen, Cell, 50 (1987) 847–861. Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L., Winblad, B. and Lannfelt, L., A pathogenic mutation for probable Alzheimer’s disease in the APP gene at the N-terminus of b-amyloid, Nature Genet., 1 (1992) 345–347. Murrell, J., Farlow, M., Ghetti, B. and Benson, M.D., A mutation in the amyloid precursor protein associated with hereditary Alzheimer’s disease, Science, 254 (1991) 97–99. Park, J. and Taylor, M.W., Analysis of signals controlling expression of the Chinese hamster ovary aprt gene, Mol. Cell. Biol., 8 (1988) 2536–2544. Prestridge, D.S., SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements, Comput. Appl. Biosci., 7 (1991) 203–206. Price, D.L., Walker, L.C., Martin, L.J. and Sisodia, S.S., Amyloidosis in ageing and Alzheimer’s disease, Am. J. Pathol., 141 (1992) 767–772. Queralt, R. and Oliva, R., Protamine 1 gene sequence from the primate Saguinus imperator isolated with PCR using consensus oligonucleotides, Nucleic Acids Res., 19 (1991) 5786. Rauscher, F.J., Sambucetti, L.C., Curran, T., Distel, R.J. and Spiegelman, B.M., Common DNA binding site for Fos protein complexes and transcription factor AP-1, Cell, 52 (1988) 471–480. Salbaum, J.M., Weidemann, A., Lemaire, H.-G., Masters, C.L. and Beyreuther, K., The promoter of Alzheimer’s disease amyloid A4 precursor gene, EMBO J., 7 (1988) 2807–2813. Saunders, A.M., Strittmatter, W.J. and Schmechel, D., St. GeorgeHyslop, P.H., Pericak-Vance, M.A., Joo, S.H., Rosi, S.H., Gusella, B.L., Crapper-McLachlan, D.R., Alberts, M.J., Hulette, C., Crain, B., Goldgaber, D. and Roses, A.D., Association of apolipoprotein E allele e4 with late-onset familial and sporadic Alzheimer’s disease, Neurology, 43 (1993) 1467–1472.
206
R. Adroer et al. / Neuroscience Letters 226 (1997) 203–206
[27] Selkoe, D.J., Amyloid b-protein precursor and the pathogenesis of Alzheimer’s disease, Cell, 58 (1989) 611–612. [28] Selkoe, D.J., Bell, D.S., Podlisny, M.B., Price, D.L. and Cork, L.C., Conservation of brain amyloid proteins in aged mammals and humans with Alzheimer’s disease, Science, 235 (1987) 873–876. [29] Smith, J.D., Melia´n, A., Leff, T. and Breslow, J.L., Expression of the human apolipoprotein E gene is regulated by multiple positive and negative elements, J. Biol. Chem., 263 (1988) 8300–8308. [30] Strittmatter, W.J., Saunders, A.M., Schmechel, D., Pericak-Vance, M., Enghild, J., Salvesen, G.S. and Roses, A.D., Apolipoprotein E: high-avidity binding to b-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease, Proc. Natl. Acad. Sci. USA, 90 (1993) 1977–1981. [31] Tanzi, R.E., Gusella, J.F., Watkins, P.C. and Bruns, G.A.P., St. George-Hyslop, P., Van Keuren, M.L., Patterson, D., Pagan, S., Kurnit, D.M. and Neve, R.L., Amyloid b protein gene: cDNA,
[32]
[33]
[34]
[35]
mRNA distribution, and genetic linkage near the Alzheimer locus, Science, 235 (1987) 880–884. Ueki, A., Kawano, M., Namba, Y., Kawakami, M. and Ikeda, K., A high frequency of apolipoprotein E4 isoprotein in Japanese patients with late-onset nonfamilial Alzheimer’s disease, Neurosci. Lett., 163 (1993) 166–168. Van Broeckhoven, C.L., Molecular genetics of Alzheimer disease: identification of genes and gene mutations, Eur. Neurol, 35 (1995) 8–19. Wu, C., Wilson, S., Walker, B., Dawid, I., Paisley, T., Zimarino, V. and Ueda, H., Purification and properties of Drosophila heat shock activator protein, Science, 238 (1987) 1247–1253. Yoshikai, S., Sasaki, H., Doh-ura, K., Furuya, H. and Sakaki, Y., Genomic organisation of the human amiloyd beta-protein precursor gene, Gene, 87 (1990) 257–263.
Conserved elements in the 5′ regulatory region of the amyloid precursor protein gene in primates Rosa Adroer a, Cristina Lo´pez-Acedo a, Rafael Oliva a , b ,* a
Human Genome Laboratory, Molecular Genetics Research Group, Faculty of Medicine, University of Barcelona, C/ Casanova 143, 08036, Barcelona, Spain b Servei de Gene`tica. Hospital Clı´nic i Provincial de Barcelona, Barcelona, Spain Received 13 January 1997; revised version received 9 April 1997; accepted 9 April 1997
Abstract Oligonucleotides corresponding to conserved sites between the human and mouse amyloid precursor protein (APP) genes have been used to polymerase chain reaction (PCR) amplify and sequence the promoter region of the APP gene from chimpanzee, gorilla, orang-utan, papio and African green monkey. Several novel conserved potentially-regulatory sequences of the APP gene have been detected after alignment of the APP promoter sequences: an apolipoprotein E-B1 (APOE-B1) element at position −450, also present in the APOE gene, two activator protein-2 (AP-2) sites at positions −450 and −301 and an intermediate early-1 gene (IE1) site at position −280. These elements are conserved in all mammalian APP promoter sequences studied. Additionally a previously detected heat shock element (HSE) at position −317, and an activator protein-1 (AP-1) site at position −350 are also conserved. Knowledge of the essential regulatory elements at the APP gene constitute the basis for understanding its transcriptional control and subsequent model studies. 1997 Elsevier Science Ireland Ltd. Keywords: Amyloid precursor protein promoter; Amyloid precursor protein expression; Regulation; Apolipoprotein E-B1; Activator protein-1; Activator protein-2; Heat shock element
The amyloid precursor protein (APP) gene is located on chromosome 21 [9,12,31], consists of 18 exons and spans more than 170 kb [35]. Alternative splicing of the gene generates different mRNA: APP695, APP714, APP751 and APP770 [13]. A fragment of the APP, the bA4 peptide, constitutes the majority of the deposits of the senile plaques, one of the principal hallmarks in Alzheimer’s disease (AD) [7,33]. Several APP gene mutations have been described causing AD in some cases of early onset familial AD [3,8, 10,18,19]. However understanding the physiological expression and processing of the APP gene is also relevant to the majority of AD cases as bA4 deposition is one of the initial neuropathological changes in AD [27]. The human APP gene promoter has been sequenced and multiple potential binding sites associated with potentiallyregulatory elements have been described [25]. It has been observed that the APP gene lacks canonical TATA and * Corresponding author. Tel.: +34 3 4021877; fax: +34 3 4035260; e-mail: [email protected]
CAAT boxes and has a high GC content (72%) upstream from the multiple transcription start points. This configuration is typical of promoters of the so called ‘housekeeping’ genes [25], which are genes known to be expressed in all tissues of an organism [20]. The APP gene from rat and mouse have also been sequenced [4,11]. Upstream from the start codon, homology between rat and mouse promoters is 89%, and between rat and human is 82% [4]. However rat and mouse do not spontaneously develop dementia and age related amyloid deposition. A better model for AD related APP deposition has been shown in non-human primates where neuronal APP is considered to be one source of Ab deposits in the brain of aged individuals [15,22]. Thus we decided to sequence the APP gene promoter from five different nonhuman primates in order to identify possible conserved regulatory elements which could constitute the basis for subsequent model studies. Genomic DNA was isolated from different species: chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orang-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0283-8
204
R. Adroer et al. / Neuroscience Letters 226 (1997) 203–206
utan (Pongo pygmaeus), African green monkey (Cercopithecus patas) and papio (Papio doguera). Polymerase chain reaction (PCR) primers were designed from conserved regions derived from the comparison of human [25], mouse [11] and rat [4] genes. The primer sequences were: PAPP1: 5′-GCTTTTGACGTTGGGGGTTA-3′ (position −538 bp) and PAPP2: 5′-AACAGTGGGAGGGAGAGTCT-3′ (position −245 bp). The amplification reaction contained 100 ng of DNA, 5 pmol of each primer, dNTPs at 200 mM, 1.5 mM MgCl2, 10 mM Tris–HCl pH 8.3, 50 mM KCl and 1 unit Taq polymerase in a final volume of 25 ml. Amplification conditions of the APP gene promoters was successful using touchdown PCR [5]. Initial denaturation 5 min at 94°C, 38 cycles: 1 min at 94°C, 1 min annealing at Tm (from 68°C to 50°C, 18 cycles, and 20 additional cycles at 50°C) and 1 min at 72°C followed by a final elongation of 10 min at 72°C. A product of the expected size was obtained for the homologous regions of chimpanzee, gorilla, orang-utan, papio and African green monkey. Sequencing of these amplification products [23] revealed their corresponding promoter sequences (Fig. 1). Subsequently all sequences were aligned (DNASTAR computer software package) to the human and mouse sequences in order to detect conserved potentially-regulatory regions. Additionally all sequences were compared to a regulatory sequence database (Signal Scan program) [21]. Interestingly we have identified a conserved sequence at position −450 to −442 common to all primates including human (Fig. 1) with a very high homology to the apolipoprotein E-B1 (APOE-B1) element (11 nucleotides match). This APOE-B1 element is known to be protected in footprinting experiments in the human apolipoprotein E gene (abbreviated APOE to refer to the gene and ApoE to refer to the protein) where it behaves as an enhancer [29]. It has been demonstrated that the presence of the allele 4 (e4) of the APOE gene is a risk factor for the AD [1,2,26,30,32]. ApoE is a protein that is also present in senile plaques and in congophilic angiopathy in AD patients. It is known that the ApoE and b-amyloid are closely associated ‘in vitro’ [30].Our results suggests that the APOE and the APP genes could share through this site a common transcription factor perhaps explaining in part the coexistence of both gene products. A sub-sequence of this APOE-B1 element present in the APP gene also has homology to the AP-2 element and to the adenine phosphoribosyltransferase (aprt) gene element (octanucleotide GCCCCACC) where it is necessary for transcription [20]. This potential AP-2 site had already been described in the mouse [11]. Downstream the APOE-B1 site, we have identified two different new potential binding sites for activator protein-2 (AP-2) located on positions −301 and −450 (Fig. 1). There are no strictly conserved sequence motifs related to AP-2 binding sites. The consensus sequence is highly variable: CCCMNSSS. This suggests that AP-2 is not linked with a particular DNA binding protein and that a variety of com-
binatorial associations with other promoter binding factors is possible [17]. At position −350 (Fig. 1) there is a potential regulatory binding site for activator protein-1 (AP-1) highly conserved in all primates. The AP-1 binding site was initially described interacting with enhancer elements in wild type human metallothionein IIA (hMTIIA), simian virus 40 (SV40) and human collagenase genes [14]. The AP-1 sequence is recognised by the Fos family, a protein complex encoded by c-fos that acts as an inducible regulator of gene expression [24]. 3′ to the AP-1 site, a heat shock element (HSE) is present at position −317. A trans-acting factor called heat shock activator protein has been described which is induced only by heat shock stimulation. Binding competition studies indicate that the same factor is able to bind to HSE’s of all heat shock genes [34]. The HSE and the AP-1 sites are highly conserved in all primates suggesting that they could be functional in gene regulation in those species. At position −280 we found a conserved 6 bp motif
Fig. 1. Alignments of APP promoter sequences from all species analysed. ce, African green monkey; ch, chimpanzee; go, gorilla; or, orang-utan; pa, papio; hu, human [25]; mo, mouse [11]. APRT, AP-2, APOE, AP-1, HSE and IE1 potential binding sites are boxed. CpG dinucleotides are underlined.
R. Adroer et al. / Neuroscience Letters 226 (1997) 203–206
(CTTTCC; Fig. 1) that is present in the immediate early 1 (IE1) gene from human cytomegalovirus. This motif is also present in mouse cytomegalovirus, SV40, lymphotropic papovavirus and immunoglobuline k gene enhancers as well as human immunodeficiency virus long terminal repeat [6]. The sequenced region of the promoter also contains 16 CpG dinucleotides (−478, −456, −434, −422, −415, −405, −380, −371, −360, −354, −337, −331, −321, −315, −295 and −262). Six of them (−422, −415, −360, −331, −315, −295) are highly conserved in all of the species, and six are conserved in all primates (−478, −456, −434, −405, −371, −262). Some of them have been previously reported to be unmethylated [16], suggesting that cytosine methylation does not seem to control protein/DNA interaction at the APP promoter in the brain of healthy individuals. Older non-human primates develop senile plaques and cerebral vessels with amyloid, so they could be good candidates to be animal models for studying AD [28]. Here, we have reported the sequences of the promoter region of five primates and identified, previously unreported, conserved potential regulatory DNA binding sites: the APOE-B1 site at position −450, the AP-2 sites at positions −301, −374, and the IE1 site at position −280. In the light of the complex pattern of expression of the APP gene in most tissues, it is not surprising that the APP gene contains multiple regulatory elements. The possibility to further clarify their respective role through ‘in vitro’ or ‘in vivo’ experiments is now open. Supported by grants from the Fondo de Investigaciones Sanitarias (FIS96/0658) to R.O. and from the European Union Biomed-2 project BMH4-CT96-0554 to the European Chromosome 21 consortium. [1] Adroer, R., Santacruz, P., Blesa, R., Lo´pez-Pousa, S., Ascaso, C. and Oliva, R., Apolipoprotein E4 allele frequency in Spanish Alzheimer and control cases, Neurosci. Lett., 189 (1995) 182–186. [2] Blesa, R., Adroer, R., Santacruz, P., Ascaso, C., Tolosa, E. and Oliva, R., High apolipoprotein E e4 allele frequency in age-related memory decline, Ann. Neurol., 39 (1996) 548–551. [3] Chartier-Harlin, M.-C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, P., Hardy, J. and Mullan, M., Early-onset Alzheimer’s disease caused by mutations at codon 717 of the b-amyloid precursor protein gene, Nature, 353 (1991) 844–846. [4] Chernak, J.M., Structural features of the 5′ upstream regulatory region of the gene encoding rat amyloid precursor protein, Gene, 133 (1993) 255–260. [5] Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K. and Mattick, J.S., ‘Touchdown’ PCR to circumvent spurious priming during gene amplification, Nucleic Acids Res., 19 (1991) 4008. [6] Ghazal, P., Lubon, H., Fleckenstein, B. and Hennighausen, L., Binding of transcription factors and creation of a large nucleoprotein complex on the human cytomegalovirus enhancer, Proc. Natl. Acad. Sci. USA, 84 (1987) 3658–3662. [7] Glenner, G.G., Alzheimers disease, Encycl. Human Biol., 1 (1991) 209–216. [8] Goate, A., Chartier-Harlin, M.-C., Mullan, M., Brown, J. and Crawford, F. et al., Segregation of a missense mutation in the amy-
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
205
loid precursor protein gene with familial Alzheimer’s disease, Nature, 349 (1991) 704–706. Goldgaber, D., Lerman, M.I., McBride, O.W., Saffiotti, U. and Gajdusek, D.C., Characterisation and chromosomal localisation of a cDNA encoding brain amyloid of Alzheimer’s disease, Science, 235 (1987) 877–880. Hendriks, L., Van Duijn, C.M., Cras, P., Cruts, M., Van Hul, W., Van Harskamp, F., Warren, A., McInnis, M.G., Antonarakis, S.E., Martin, J.-J., Hofman, A. and Van Broeckhoven, C., Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the bamyloid precursor protein gene, Nature Genet., 1 (1992) 218–221. Izumi, R., Yamada, T., Yoshikai, S., Sasaki, H., Hattori, M. and Sakaki, Y., Positive and negative regulatory elements for the expression of the Alzheimer’s disease amyloid precursor-encoding gene in mouse, Gene, 112 (1992) 189–195. Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and Mu¨llerHill, B., The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733–736. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H., Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitory activity, Nature, 331 (1988) 530–532. Lee, W., Mitchell, P. and Tjian, R., Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements, Cell, 49 (1987) 741–752. Martin, L.J., Sisodia, S.S., Koo, E.H., Cork, L.C., Dellovade, T.L., Weidemann, A., Beyreuther, K., Masters, C. and Price, D.L., Amyloid precursor protein in aged nonhuman primates, Proc. Natl. Acad. Sci. USA, 88 (1991) 1461–1465. Milici, A., Salbaum, J.M. and Beyreuther, K., Study of the Alzheimer’s A4 precursor gene promoter region by genomic sequencing using Taq polymerase, Biochem. Biophys. Res. Commun., 69 (1990) 46–50. Mitchell, P.J., Wang, C. and Tjian, R., Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen, Cell, 50 (1987) 847–861. Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L., Winblad, B. and Lannfelt, L., A pathogenic mutation for probable Alzheimer’s disease in the APP gene at the N-terminus of b-amyloid, Nature Genet., 1 (1992) 345–347. Murrell, J., Farlow, M., Ghetti, B. and Benson, M.D., A mutation in the amyloid precursor protein associated with hereditary Alzheimer’s disease, Science, 254 (1991) 97–99. Park, J. and Taylor, M.W., Analysis of signals controlling expression of the Chinese hamster ovary aprt gene, Mol. Cell. Biol., 8 (1988) 2536–2544. Prestridge, D.S., SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements, Comput. Appl. Biosci., 7 (1991) 203–206. Price, D.L., Walker, L.C., Martin, L.J. and Sisodia, S.S., Amyloidosis in ageing and Alzheimer’s disease, Am. J. Pathol., 141 (1992) 767–772. Queralt, R. and Oliva, R., Protamine 1 gene sequence from the primate Saguinus imperator isolated with PCR using consensus oligonucleotides, Nucleic Acids Res., 19 (1991) 5786. Rauscher, F.J., Sambucetti, L.C., Curran, T., Distel, R.J. and Spiegelman, B.M., Common DNA binding site for Fos protein complexes and transcription factor AP-1, Cell, 52 (1988) 471–480. Salbaum, J.M., Weidemann, A., Lemaire, H.-G., Masters, C.L. and Beyreuther, K., The promoter of Alzheimer’s disease amyloid A4 precursor gene, EMBO J., 7 (1988) 2807–2813. Saunders, A.M., Strittmatter, W.J. and Schmechel, D., St. GeorgeHyslop, P.H., Pericak-Vance, M.A., Joo, S.H., Rosi, S.H., Gusella, B.L., Crapper-McLachlan, D.R., Alberts, M.J., Hulette, C., Crain, B., Goldgaber, D. and Roses, A.D., Association of apolipoprotein E allele e4 with late-onset familial and sporadic Alzheimer’s disease, Neurology, 43 (1993) 1467–1472.
206
R. Adroer et al. / Neuroscience Letters 226 (1997) 203–206
[27] Selkoe, D.J., Amyloid b-protein precursor and the pathogenesis of Alzheimer’s disease, Cell, 58 (1989) 611–612. [28] Selkoe, D.J., Bell, D.S., Podlisny, M.B., Price, D.L. and Cork, L.C., Conservation of brain amyloid proteins in aged mammals and humans with Alzheimer’s disease, Science, 235 (1987) 873–876. [29] Smith, J.D., Melia´n, A., Leff, T. and Breslow, J.L., Expression of the human apolipoprotein E gene is regulated by multiple positive and negative elements, J. Biol. Chem., 263 (1988) 8300–8308. [30] Strittmatter, W.J., Saunders, A.M., Schmechel, D., Pericak-Vance, M., Enghild, J., Salvesen, G.S. and Roses, A.D., Apolipoprotein E: high-avidity binding to b-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease, Proc. Natl. Acad. Sci. USA, 90 (1993) 1977–1981. [31] Tanzi, R.E., Gusella, J.F., Watkins, P.C. and Bruns, G.A.P., St. George-Hyslop, P., Van Keuren, M.L., Patterson, D., Pagan, S., Kurnit, D.M. and Neve, R.L., Amyloid b protein gene: cDNA,
[32]
[33]
[34]
[35]
mRNA distribution, and genetic linkage near the Alzheimer locus, Science, 235 (1987) 880–884. Ueki, A., Kawano, M., Namba, Y., Kawakami, M. and Ikeda, K., A high frequency of apolipoprotein E4 isoprotein in Japanese patients with late-onset nonfamilial Alzheimer’s disease, Neurosci. Lett., 163 (1993) 166–168. Van Broeckhoven, C.L., Molecular genetics of Alzheimer disease: identification of genes and gene mutations, Eur. Neurol, 35 (1995) 8–19. Wu, C., Wilson, S., Walker, B., Dawid, I., Paisley, T., Zimarino, V. and Ueda, H., Purification and properties of Drosophila heat shock activator protein, Science, 238 (1987) 1247–1253. Yoshikai, S., Sasaki, H., Doh-ura, K., Furuya, H. and Sakaki, Y., Genomic organisation of the human amiloyd beta-protein precursor gene, Gene, 87 (1990) 257–263.