- Email: [email protected]
Expression of amygdaloid precursor protein in a neuronal cell line: functional activity of proximal regulatory elements
Molecular Brain Research, 19 (1993) 246-250 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
246
BRESM 80184
Expression of amyloid precursor protein in a neuronal cell line: functional activity of proximal regulatory elements M a r t i n B o u r b o n n i ~ r e a a n d J o s e p h i n e N a l b a n t o g l u a,b a Department of Neurology and Neurosurgery, McGill Unicersity, Montreal, Que. (Canada) and b McGill Center for Studies in Aging, Montreal, Que. (Canada) (Accepted 20 April 1993)
Key words: Amyloid precursor protein; Gene regulation; DNA-binding protein
To study cell type-specific regulation of the gene for amyloid precursor protein (APP), we have analysed the human APP promoter for transcriptional activity and interaction with nuclear proteins in the neuronal cell line NG108-15. Sequences from -203 to + 104 were sufficient for promoter function and regulatory elements within this region (-128 to -63) were identified by DNase l protection experiments. These results suggest that essentially the same proximal elements are recognized by nuclear factors from both neuronal and nonneuronal cells.
The amyloid precursor protein (APP) represents a family of proteins of 695 to 770 amino acids which are generated through alternative splicing of a single-copy gene located on chromosome 217'12'13'2°'2t'e4'2s. Proteolytic cleavage of one or more of these isoforms results in the production of a 39 to 42 amino acid peptide labelled /3-amyloid/A4 protein which accumulates in insoluble deposits in the brain of individuals suffering from Alzheimer's disease, Down syndrome (trisomy 21), and, to a lesser extent, in normal agings,ls. The APP transcripts have been detected in a variety of tissues and organisms6,7,21.24,25. Both APP695 and APP751 are relatively abundant in human and rodent brain Is. In mouse, the expression of the APP gene is first detected during development of the nervous system and persists postnatally 4.26. A 3.8 kilobase (kb) upstream fragment of the human APP gene has been sequenced by Salbaum et al. 22 and shown to be typical of the promoters of housekeeping genes with no CAAT or TATA boxes but with several GC boxes and multiple transcriptional start sites. When the human and mouse genes are compared, the high level of homology observed at the protein level also extends into the 5' regulatory sequences 9. The APP gene is regulated by IL-1s, N G F 16 and heat shock I
indicating that constitutive expression of the gene can be modulated by growth factors and external stimuli. Since overexpression of the APP gene, through its duplication, may be involved in the neuropathology observed in Down syndrome, we have been studying the regulation of APP gene expression. We are particularly interested in the cell type-specific regulation of APP, identification of functionally important cis.acting sequences, and characterization of the trans-acting protein factors which bind them. In this report, we show that sequences previously reported to be active in HeLa cells are also active in a neuronal cell line and that the same sequences are recognized by nuclear factors in this cell line. To test for the presence of transcriptional activity, we cloned 5' genomic DNA fragments of various lengths upstream of the bacterial chloramphenicol acetyl transferase gene (CAT). These constructs were transfected into the NG108-15 cell line, a hybrid neuroblastoma × glioma cell line which exhibits a cholinergic neuronal phenotype and can be induced to extend neurites when exposed to dibutyryl cAMP 17. This cell line expresses APP constitutively (data not shown). The smallest construct, in which CAT expression was driven by 203 base pairs (bp) of APP upstream sequence
Correspondence: J. Nalbantoglu, Montreal Neurological Institute, 7301 University Street, Montreal, Que., H3A 2B4 Canada. Fax: (1) (514) 398-7371.
247
(shown previously to contain the minimal promoter required for expression in HeLa 19 and PC12 cells 14) (Fig. 1A), had transcriptional activity in both NGI08-15 cells and NG108-15 cells treated with dibutyryl cAMP (Fig. 1B). Thus these sequences are also sufficient for basal promoter function in the NG108-15 cell line and moreover, the transcriptional activity is not altered upon differentiation of the cells with dibutyryl cAMP (Fig. 1B).
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Fig. 1. Characterization of the proximal promoter region of the human APP gene. A: schematic representation of the BamHl/ Xmalll fragment ( - 2 0 3 to + 104) used in the transfection experiments. The open arrows indicate the Mspl sites used to generate fragments for electrophoretic mobility shift analysis. The stippled boxes represent the 6 copies of the 9 bp GC-rich element, the solid box marks the putative AP-1 binding site. X: Xmalll; B: BamHl. The arrow marks the m~or transcriptional initiation site. Based on Saibaum et al. 22. B: functional activity of the proximal upstream region of the human APP gene in NG108-15 cells. The CAT construct depicted in A) was transfected into NG108-15 cells (lane 2) and NG108-15 cells treated with dibutyryi cAMP (lane 4). The corresponding mock transfections were carried out in parallel (lanes 1 and 3). The transfections using lipofectin (Gibco/BRL) were carried out according to the manufacturer's instructions and the CAT assays were performed as described 23 except that the reaction was allowed to proceed for 16 h in presence of 8 mM acetyI-CoA. NGI08-15 cells were cultured for 3 days in the presence of 1 mM dibutyryl cAMP prior to transfection and maintained in its presence during the transfection. Transfection efficiency was standardized through co-transfection with a RSV/3gal plasmid.
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Fig. 2. Electrophoretic mobility shift analysis of the 48 bp Mspl fragment ( - 1 0 0 to -52) in the presence of nucli~ar factors from NGI08-15 cells. A: both NGI08-15 cells (lane 1) and NGI08-15 cells treated with dibutyryl cAMP (lane 2) contain nuclear binding activity. B: competition of mobility shift (lane 2) by pre-incubation of nuclear extracts with an excess of 2.5-fold (lane 4), 10-fold (lane 5) and 50-fold (lane 6) of unlabelled 48 bp fragment. Note that no competition is observed in the presence of a 70-fold excess of unlabelled Spl double-stranded oligonucleotide (lane 3). Free probe (in the absence of nuclear extracts) is shown in lane 1. The arrowhead represents free probe whereas the arrow marks bound complex. in this experiment, Mspl restriction fragments were end-labelled with [a-32p]dCTP and [a-32p]dGTP using Klenow polymerase and were purified on a polyacrylamide gel (10%). The binding reaction took place at room temperature in a volume of 15/tl (4% glycerol, 1.25 mM MgCI 2, 0.5 mM DTT, 50 mM NaCI and 10 mM Tris-HCI pH 7.5) in presence of I/~g of poly dl-dC and 6 ~g of crude nuclear extracts 2. The probe (10,000 cpm) was added 10 min after extract and incubated another 20 min. When cold specific competitor ~,as present it was added 10 min before the labelled fragment. The Spl oligonucleotide (Promega) had the following sequence: 5'.ATI'CGATCGGGGCGGGGCGAG-3'. The fragments were resolved on a 5% polyacrylamide gel containing 2.5% glycerol. Electrophoresis was performed at 10 mA for 3 h in 0.5 x TBE with circulating cold water.
To identify possible sites for DNA binding proteins within this region, we performed an electrophoretic mobility shift analysis on subfragments of the 203 bp clone. The highest affinity binding was observed with a 48 bp Mspl fragment ( - 1 0 0 to -52) in the presence of nuclear extracts prepared either from NG108-15 cells or NG108-15 cells treated with dibutyryl cAMP (Fig. 2A). The extent of binding increased proportionally with the amount of nuclear extract (6-18 ~g) used in the incubation when the bound complex was quantitated by phosphorimage analysis on a Molecular Dynamics phosphorimager (data not shown). The specificity of this binding was verified by a competition assay
248 in which the nuclear extracts were incubated with an excess of unlabelled DNA prior to the addition of the labelled 48 bp fragment. A 10-fold excess of the unlabelled 48 bp fragment effectively diminished the observed shift(s) (Fig. 2B, lane 5). However, no competition occurred in the presence of a 70-fold excess of an Spl-srecific double-stranded oligonucleotide (Fig. 2B, lane 3.', indicating that the observed shift was not due to recognition of the putative Spl site present in this fragment. We also observed a weak mobility shift of the adjacent 25 bp Mspl fragment ( - 1 2 3 to - 9 8 ) which contains two GC boxes (data not shown). The specific sequences bound by the nuclear proteins were identified by DNase I footprinting experiments on a BamHl/Xmalll fragment, spanning - 2 0 3 to + 104, and end-labelled on the non-coding strand. As shown in Fig. 3, the strongest DNase I footprinting activity detected in NG108-15 cells covers the - 128 to - 6 3 region of the promoter. In HeLa cells, sequences from - 9 4 to - 3 5 act as a transcriptional activa .r with three domains which interact with DNA-binding proteins as determined by DNase I footprinting analysis (A: - 6 3 to -47; B: - 8 2 to -75; and C: - 9 7 to - 8 7 ) 19. In NG108-15 nuclear extracts, the footprinted region encompasses both the B element consisting of a pyrimidine tract, the C element characterized by a GC-palindrome as well as the GC-rich distal sequences ( - 1 3 3 to - 122) described by Pollwein et al. tg. The nuclear factors which bind these elements have not been identified. Since some common regions were footprinted by both NG108.15 and HeLa nuclear extracts, we verified that factors of similar size classes were bound to the 48 bp fragment ( - 100 to - 5 2 ) containing the core of the protected sequence. The labelled DNA-protein complex was UV cross-linked and electrophoresed on SDS-PAGE. Labelled polypeptides in the 30 kilodalton (kDa) range were detected in both HeLa and NG108-15 nuclear extracts (Fig. 4). Although the binding affinity of the NG108-15 nuclear extract seems to be lower, these proteins have considerable footprinting activity as seen in Fig. 3. Similarly to what has been reported in HeLa cells t9, we do not observe any protection from DNase I digestion of the region around - 4 5 , the putative AP-1 binding site. However, the presence of the AP-1 binding activity may be dependent on external stimuli and the subsequent upregulation of c-fos. The footprinting experiments also revealed specific binding to the 2 GC boxes situated at -123 to -104. Although GC boxes have been reported to be Spl binding sites ~°, the mobility shifts observed with the 48 bp and the 25 bp fragments could not be competed out
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.12s Fig. 3. DNAse I footprinting analysis in the absence (lane 1) and presence (lane 2) of nuclear extract from NGI08-15 cells. Footprinting of the end-labelled Xmalll/BamHl fragment was performed under the same buffer conditions as those of the electrophoretic mobility shift assay. The binding took place in a ritualvolume of 50/zl with 20,000 cpm of probe (10 s cpm/ng) in presence of 5 #g of poly dl-dC and 60 #g of crude nuclear extract. After 20 rain of binding, 50 #! of solution A (5 mM CaCi 2, 10 mM MgCI2) was added, DNAse I' digestion was performed at room temperature for 60 s and stopped with 100 #! of solution B (20 mM EDTA, 1% SDS, 100 ~g/ml tRNA). The products were phenol-chloroform extracted, ethanol precipitated and then resolved on a 6% denaturing poly. acrylamide gel.
with an Spl-specific oligonucleotide (Fig. 2B and data not shown). However, Spl may also function by interacting directly with other nuclear factors, without binding to its recognition sequence 3. GC boxes can also be recognized by the ETF transcription factor 11. This protein is directly implicated in the regulation of promoters without TATA boxes, through an affinity for se-
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Fig. 4. Cross-linking of the 48 bp Mspl fragment to proteins of NG108-15 and HeLa nuclear extracts. Polypeptides of approximately 30 kDa were detected in lane 2 (NGI08-15) and lane 3 (HeLa) (arrow). Molecular weight markers (14C-labelled) are shown in lane 1 and free probe in lane 4. Nuclear extracts (18/~g) were incubated as described in Fig. 2 with end-labelled 48 bp Mspl fragment in the presence of 3 /~g of poly dl-dC in a total volume of 45 /tl. The reaction mixture waa then exposed to uv light (254 nm) for 60 min. The samplt~ was resuspended in SDS sample buffer and electrophoresed on a 10% polyacrylamide gel (SDS-PAGE). The gel was dried and analysed on a Molecular Dynamics phosphorimager.
quences containing a core of 5'-GGGG-3' or 5'-CCCC3' nucleotides lj. It remains to be seen which factors bind the GC boxes found in the APP promoter. In summary, these results suggest that essentially the same proximal c/s-elements are recognized by nuclear factors from both neuronal and nonneuronal cells. More distal sequences may be involved in the tissuespecific modulation of APP expression. We would like to acknowledge Danielle Desmarais and Guy Charron for technical advice. This work was funded by the Medical Research Council of Canada (MA-10407). M.B. is supported by a studentship from the Alzheimer Society of Canada. J.N. is a Research Scholar of the Fonds de ia recherche en sant~ du Quebec. 1 Abe, K., St. George-Hyslop, P.H., Tanzi, R.E. and Kogure, K.,
Induction of amyloid precursor mRNA after heat shock in cultured human lympboblastoid cells, Neurosci. Lett., 125 (1991) 169-171. 2 Dignam, J.D., Lebovitz, R.M. and Roeder, R.G., Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucl. Acids ICes., 11 (1983) 1475-1489. 3 Elder, G.A., Liang, Z., Li, C. and Lazzarini, R.A, Targeting of Spl to a non-Spl site in the human neurofilament (H) promoter via an intermediary DNA-binding protein, Nucl. Acids Res., 20 (1992) 6281-6285.
4 Fisher, S., Gearhart, G.D. and Oster-Granite, Expression of the amyloid precursor protein gene in mouse oocytes and embryos, Proc. Natl. Acad. Sci. USA, 88 (1991) 1779-1782. 5 Glenner, G.G. and Wong, C.W., Initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. ICes. Commun., 120 (1984) 885-890. 6 Goedert, M, Neuronal localization of amyloid/3 protein precursor mRNA in normal brain and Alzheimer's disease, EMBO J., 6 (1987) 3627-3632. 7 Goldgaber, M., Lerman, M.I., McBride, O.W., Saffiotti, U. and Gajdusek, D.C., Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease, Science, 235 (1987) 877-880. 8 Goldgaber, D., Harris, H.W., Hia, T., Maciag, T., Donneily, R.J., Jacobsen, J.S., Vitek, M.P. and Gajdusek, D.C., Interleukin 1 regulates the synthesis of amyloid beta-protein precursor mRNA in human endothelial cells, Proc. Natl. Acad. Sci. USA, 87 (1990) 7606-7610. 9 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 amylcid precursor-encoding gene in mouse, Gene, 112 (1992) 189-195. 10 Kadonaga, J.T., Courey, AJ., Ladika, J. and Tjian, R., Distinct regions of Spl modulate DNA binding and transcriptional activation, Science, 242 (1988) 1566-1570. 11 Kageyama, R., Merlino, G.T. and Pastan, I., Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box, J. Biol. Chem., 264 (1989) 15508-15514. 12 Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and MiillerHill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736. 13 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. 14 Lahiri, D.K. and Robakis, N.K., The promoter activity of the gene encoding Alzheimer/3-amyloid precursor protein (APP) is regulated by two blocks of upstream sequences, lffol. Brain Res., 9 (1991) 253-257. 15 Masters, C.L., Simms, G., Weinmann, N.A., Multhaup, G., McDonald, B.L. and Beyreuther, K., Amyloid plaque core protein in Alzheimer's disease and Down's syndrome, Proc. Nad. Acad. Sci. USA, 82 (1985) 4245-4249. 16 Mobley, W.C., Neve, R.L., Prusiner, S.B. and McKinley, M.P., Nerve growth factor increases mRNA levels for the prion protein and the beta.amyloid protein precursor in developing hamster brain, Proc Natl. Acad. Sci. USA, 85 (1988) 9811-9815. 17 Nelson, P., Christian, C. and Nirenberg, M., Synapse formation between clonal neuroblastoma x glioma cells and striated muscle cells, Proc. Natl. Acad. Sci. USA, 73 (1976) 123-127. 18 Neve, R.L., Finch, E.A. and Dawes, L.R., Expression of the Alzheimer amyloid precursor gene transcripts in the human brain, Neuron, 1 (1988) 669-677. 19 Pollwein, P., Masters, C.L. and Beyreuther, K., The expression of the amyloid precursor protein (APP) is regulated by two GC-elements in the promoter, Nucl. Acids Res., 20 (1992) 63-68. 20 Ponte, P., Gonzales-DeWhitt, P., Schilling. J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, L, Fuller, F. and CordeU, B., A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors, Nature, 331 (1988) 525-527. 21 Robakis, N.K., Ramakrishna, N., Wolfe, G. and Wisniewski, H.M., Molecular cloning and characterization of a eDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides, Proc. Natl. Acad. Sci. USA, 84 (1987) 4190-4194. 22 Salbaum, J.M., Weidemann, A., Le,maire, H.-G., Masters, C.L. and Beyreuther, K., The promoter of Alzheimer's disease amyioid A4 precursor gene, EMBO J., 7 (1988) 2807-2813. 23 Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning. A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989.
250 24 Tanzi, R.E., Gusella, J.F., Watkins, P.C., Bruns, G.A.P., St. George-H~lop, P., van Keuren, M.L., Patterson, D., Pagan, S., Kurnit, D.M. and Neve, R.L., Amyloid/3 protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus, Science, 235 (1987) 880-883. 25 Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L, Gusella, J.F. and Neve, R.L., Protease inhibitor domain
encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease, Nature, 331 (1988) 528-530. 26 Wirak, D.O., Bayney, R., Kundel, C.A., Lee, A., Scangos, G.A., Trapp, B.D. and Unterbeck, A.J., Regulatory region of human amyioid precursor protein (APP) gene promotes neuron-specific gene expression in the CNS of transgenic mice, EMBO J., 10 (1991) 289-296.
246
BRESM 80184
Expression of amyloid precursor protein in a neuronal cell line: functional activity of proximal regulatory elements M a r t i n B o u r b o n n i ~ r e a a n d J o s e p h i n e N a l b a n t o g l u a,b a Department of Neurology and Neurosurgery, McGill Unicersity, Montreal, Que. (Canada) and b McGill Center for Studies in Aging, Montreal, Que. (Canada) (Accepted 20 April 1993)
Key words: Amyloid precursor protein; Gene regulation; DNA-binding protein
To study cell type-specific regulation of the gene for amyloid precursor protein (APP), we have analysed the human APP promoter for transcriptional activity and interaction with nuclear proteins in the neuronal cell line NG108-15. Sequences from -203 to + 104 were sufficient for promoter function and regulatory elements within this region (-128 to -63) were identified by DNase l protection experiments. These results suggest that essentially the same proximal elements are recognized by nuclear factors from both neuronal and nonneuronal cells.
The amyloid precursor protein (APP) represents a family of proteins of 695 to 770 amino acids which are generated through alternative splicing of a single-copy gene located on chromosome 217'12'13'2°'2t'e4'2s. Proteolytic cleavage of one or more of these isoforms results in the production of a 39 to 42 amino acid peptide labelled /3-amyloid/A4 protein which accumulates in insoluble deposits in the brain of individuals suffering from Alzheimer's disease, Down syndrome (trisomy 21), and, to a lesser extent, in normal agings,ls. The APP transcripts have been detected in a variety of tissues and organisms6,7,21.24,25. Both APP695 and APP751 are relatively abundant in human and rodent brain Is. In mouse, the expression of the APP gene is first detected during development of the nervous system and persists postnatally 4.26. A 3.8 kilobase (kb) upstream fragment of the human APP gene has been sequenced by Salbaum et al. 22 and shown to be typical of the promoters of housekeeping genes with no CAAT or TATA boxes but with several GC boxes and multiple transcriptional start sites. When the human and mouse genes are compared, the high level of homology observed at the protein level also extends into the 5' regulatory sequences 9. The APP gene is regulated by IL-1s, N G F 16 and heat shock I
indicating that constitutive expression of the gene can be modulated by growth factors and external stimuli. Since overexpression of the APP gene, through its duplication, may be involved in the neuropathology observed in Down syndrome, we have been studying the regulation of APP gene expression. We are particularly interested in the cell type-specific regulation of APP, identification of functionally important cis.acting sequences, and characterization of the trans-acting protein factors which bind them. In this report, we show that sequences previously reported to be active in HeLa cells are also active in a neuronal cell line and that the same sequences are recognized by nuclear factors in this cell line. To test for the presence of transcriptional activity, we cloned 5' genomic DNA fragments of various lengths upstream of the bacterial chloramphenicol acetyl transferase gene (CAT). These constructs were transfected into the NG108-15 cell line, a hybrid neuroblastoma × glioma cell line which exhibits a cholinergic neuronal phenotype and can be induced to extend neurites when exposed to dibutyryl cAMP 17. This cell line expresses APP constitutively (data not shown). The smallest construct, in which CAT expression was driven by 203 base pairs (bp) of APP upstream sequence
Correspondence: J. Nalbantoglu, Montreal Neurological Institute, 7301 University Street, Montreal, Que., H3A 2B4 Canada. Fax: (1) (514) 398-7371.
247
(shown previously to contain the minimal promoter required for expression in HeLa 19 and PC12 cells 14) (Fig. 1A), had transcriptional activity in both NGI08-15 cells and NG108-15 cells treated with dibutyryl cAMP (Fig. 1B). Thus these sequences are also sufficient for basal promoter function in the NG108-15 cell line and moreover, the transcriptional activity is not altered upon differentiation of the cells with dibutyryl cAMP (Fig. 1B).
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Fig. 1. Characterization of the proximal promoter region of the human APP gene. A: schematic representation of the BamHl/ Xmalll fragment ( - 2 0 3 to + 104) used in the transfection experiments. The open arrows indicate the Mspl sites used to generate fragments for electrophoretic mobility shift analysis. The stippled boxes represent the 6 copies of the 9 bp GC-rich element, the solid box marks the putative AP-1 binding site. X: Xmalll; B: BamHl. The arrow marks the m~or transcriptional initiation site. Based on Saibaum et al. 22. B: functional activity of the proximal upstream region of the human APP gene in NG108-15 cells. The CAT construct depicted in A) was transfected into NG108-15 cells (lane 2) and NG108-15 cells treated with dibutyryi cAMP (lane 4). The corresponding mock transfections were carried out in parallel (lanes 1 and 3). The transfections using lipofectin (Gibco/BRL) were carried out according to the manufacturer's instructions and the CAT assays were performed as described 23 except that the reaction was allowed to proceed for 16 h in presence of 8 mM acetyI-CoA. NGI08-15 cells were cultured for 3 days in the presence of 1 mM dibutyryl cAMP prior to transfection and maintained in its presence during the transfection. Transfection efficiency was standardized through co-transfection with a RSV/3gal plasmid.
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Fig. 2. Electrophoretic mobility shift analysis of the 48 bp Mspl fragment ( - 1 0 0 to -52) in the presence of nucli~ar factors from NGI08-15 cells. A: both NGI08-15 cells (lane 1) and NGI08-15 cells treated with dibutyryl cAMP (lane 2) contain nuclear binding activity. B: competition of mobility shift (lane 2) by pre-incubation of nuclear extracts with an excess of 2.5-fold (lane 4), 10-fold (lane 5) and 50-fold (lane 6) of unlabelled 48 bp fragment. Note that no competition is observed in the presence of a 70-fold excess of unlabelled Spl double-stranded oligonucleotide (lane 3). Free probe (in the absence of nuclear extracts) is shown in lane 1. The arrowhead represents free probe whereas the arrow marks bound complex. in this experiment, Mspl restriction fragments were end-labelled with [a-32p]dCTP and [a-32p]dGTP using Klenow polymerase and were purified on a polyacrylamide gel (10%). The binding reaction took place at room temperature in a volume of 15/tl (4% glycerol, 1.25 mM MgCI 2, 0.5 mM DTT, 50 mM NaCI and 10 mM Tris-HCI pH 7.5) in presence of I/~g of poly dl-dC and 6 ~g of crude nuclear extracts 2. The probe (10,000 cpm) was added 10 min after extract and incubated another 20 min. When cold specific competitor ~,as present it was added 10 min before the labelled fragment. The Spl oligonucleotide (Promega) had the following sequence: 5'.ATI'CGATCGGGGCGGGGCGAG-3'. The fragments were resolved on a 5% polyacrylamide gel containing 2.5% glycerol. Electrophoresis was performed at 10 mA for 3 h in 0.5 x TBE with circulating cold water.
To identify possible sites for DNA binding proteins within this region, we performed an electrophoretic mobility shift analysis on subfragments of the 203 bp clone. The highest affinity binding was observed with a 48 bp Mspl fragment ( - 1 0 0 to -52) in the presence of nuclear extracts prepared either from NG108-15 cells or NG108-15 cells treated with dibutyryl cAMP (Fig. 2A). The extent of binding increased proportionally with the amount of nuclear extract (6-18 ~g) used in the incubation when the bound complex was quantitated by phosphorimage analysis on a Molecular Dynamics phosphorimager (data not shown). The specificity of this binding was verified by a competition assay
248 in which the nuclear extracts were incubated with an excess of unlabelled DNA prior to the addition of the labelled 48 bp fragment. A 10-fold excess of the unlabelled 48 bp fragment effectively diminished the observed shift(s) (Fig. 2B, lane 5). However, no competition occurred in the presence of a 70-fold excess of an Spl-srecific double-stranded oligonucleotide (Fig. 2B, lane 3.', indicating that the observed shift was not due to recognition of the putative Spl site present in this fragment. We also observed a weak mobility shift of the adjacent 25 bp Mspl fragment ( - 1 2 3 to - 9 8 ) which contains two GC boxes (data not shown). The specific sequences bound by the nuclear proteins were identified by DNase I footprinting experiments on a BamHl/Xmalll fragment, spanning - 2 0 3 to + 104, and end-labelled on the non-coding strand. As shown in Fig. 3, the strongest DNase I footprinting activity detected in NG108-15 cells covers the - 128 to - 6 3 region of the promoter. In HeLa cells, sequences from - 9 4 to - 3 5 act as a transcriptional activa .r with three domains which interact with DNA-binding proteins as determined by DNase I footprinting analysis (A: - 6 3 to -47; B: - 8 2 to -75; and C: - 9 7 to - 8 7 ) 19. In NG108-15 nuclear extracts, the footprinted region encompasses both the B element consisting of a pyrimidine tract, the C element characterized by a GC-palindrome as well as the GC-rich distal sequences ( - 1 3 3 to - 122) described by Pollwein et al. tg. The nuclear factors which bind these elements have not been identified. Since some common regions were footprinted by both NG108.15 and HeLa nuclear extracts, we verified that factors of similar size classes were bound to the 48 bp fragment ( - 100 to - 5 2 ) containing the core of the protected sequence. The labelled DNA-protein complex was UV cross-linked and electrophoresed on SDS-PAGE. Labelled polypeptides in the 30 kilodalton (kDa) range were detected in both HeLa and NG108-15 nuclear extracts (Fig. 4). Although the binding affinity of the NG108-15 nuclear extract seems to be lower, these proteins have considerable footprinting activity as seen in Fig. 3. Similarly to what has been reported in HeLa cells t9, we do not observe any protection from DNase I digestion of the region around - 4 5 , the putative AP-1 binding site. However, the presence of the AP-1 binding activity may be dependent on external stimuli and the subsequent upregulation of c-fos. The footprinting experiments also revealed specific binding to the 2 GC boxes situated at -123 to -104. Although GC boxes have been reported to be Spl binding sites ~°, the mobility shifts observed with the 48 bp and the 25 bp fragments could not be competed out
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.12s Fig. 3. DNAse I footprinting analysis in the absence (lane 1) and presence (lane 2) of nuclear extract from NGI08-15 cells. Footprinting of the end-labelled Xmalll/BamHl fragment was performed under the same buffer conditions as those of the electrophoretic mobility shift assay. The binding took place in a ritualvolume of 50/zl with 20,000 cpm of probe (10 s cpm/ng) in presence of 5 #g of poly dl-dC and 60 #g of crude nuclear extract. After 20 rain of binding, 50 #! of solution A (5 mM CaCi 2, 10 mM MgCI2) was added, DNAse I' digestion was performed at room temperature for 60 s and stopped with 100 #! of solution B (20 mM EDTA, 1% SDS, 100 ~g/ml tRNA). The products were phenol-chloroform extracted, ethanol precipitated and then resolved on a 6% denaturing poly. acrylamide gel.
with an Spl-specific oligonucleotide (Fig. 2B and data not shown). However, Spl may also function by interacting directly with other nuclear factors, without binding to its recognition sequence 3. GC boxes can also be recognized by the ETF transcription factor 11. This protein is directly implicated in the regulation of promoters without TATA boxes, through an affinity for se-
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21.5-
Fig. 4. Cross-linking of the 48 bp Mspl fragment to proteins of NG108-15 and HeLa nuclear extracts. Polypeptides of approximately 30 kDa were detected in lane 2 (NGI08-15) and lane 3 (HeLa) (arrow). Molecular weight markers (14C-labelled) are shown in lane 1 and free probe in lane 4. Nuclear extracts (18/~g) were incubated as described in Fig. 2 with end-labelled 48 bp Mspl fragment in the presence of 3 /~g of poly dl-dC in a total volume of 45 /tl. The reaction mixture waa then exposed to uv light (254 nm) for 60 min. The samplt~ was resuspended in SDS sample buffer and electrophoresed on a 10% polyacrylamide gel (SDS-PAGE). The gel was dried and analysed on a Molecular Dynamics phosphorimager.
quences containing a core of 5'-GGGG-3' or 5'-CCCC3' nucleotides lj. It remains to be seen which factors bind the GC boxes found in the APP promoter. In summary, these results suggest that essentially the same proximal c/s-elements are recognized by nuclear factors from both neuronal and nonneuronal cells. More distal sequences may be involved in the tissuespecific modulation of APP expression. We would like to acknowledge Danielle Desmarais and Guy Charron for technical advice. This work was funded by the Medical Research Council of Canada (MA-10407). M.B. is supported by a studentship from the Alzheimer Society of Canada. J.N. is a Research Scholar of the Fonds de ia recherche en sant~ du Quebec. 1 Abe, K., St. George-Hyslop, P.H., Tanzi, R.E. and Kogure, K.,
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