Molecular Brain Research, 22 (1994) 121-131 121 © 1994 Elsevier Science B.V. All rights reserved 0169-328X/94/$07.00 BRESM 70716 Protein-DNA interactions in the promoter region of the amyloid precursor protein (APP) gene in human neocortex W.J. Lukiw a,,, E.I. Rogaev a,b, L. Wong a, G. Vaula a,c, D.R.C. McLachlan a, P. St George Hyslop a a Division of Neurology, Departments of Physiology and Medicine, Tanz Neuroscience Building, Center for Research In Neurodegeneratice Disease, 6 Queen's Park Circle West, University of Toronto, Toronto, Canada M5S IA8, b National Research Center of Mental Health, Laboratory of Molecular Brain Genetics, Academy of Medical Sciences, Zagorodnoe sh. 2/2, Moscow, Russia 113152, c Department of Neurology, Instituto di Clinica delle Malatie del Sistema Nervoso, Universita di Torino, via Cherasco 15, Torino, Italy 10126 (Accepted 17 August 1993) Key words: Alzheimer's disease; Amyloid; AP1; Brain gene regulation; Promotor structure; SP1; TFIID We have investigated protein-DNA interactions in the proximal promotor of the human amyloid precursor protein (APP) gene in temporal lobe neocortical nuclei isolated from control and Alzheimer disease (AD) affected brains. We report that the human APP 5' promotor sequence from -203 to+55 bp, which has been previously reported to contain essential regulatory elements for APP gene transcription, lies in a deoxyribonuclease I, micrococcal nuclease- and restriction endonuclease-sensitive, G+C-rich nucleosome-free gap flanked both 5' and 3' by typical nucleosome structures. As analyzed by electrophoretic mobility shift assay, this extended internucleosomal linker DNA is heavily occupied by nuclear protein factors, and interacts differentially with nuclear protein extracts obtained from HeLa and human brain neocortical nuclei. This suggests that the chromatin conformation of the APP gene promoter may vary in different cell types, and may correlate with differences in APP gene expression. Human recombinant transcription factors AP1, SP1 and TFIID (but not AP2 or brain histones H1, H2B and H4) interact with the - 203 to + 55 bp of the human APP promotor sequence. Only minor differences were observed in the chromatin structure of the immediate APP promotor between non-AD and AD affected neocortical nuclei, suggesting either that post-transcriptional processes, or that regulatory elements lying elsewhere in the APP gene may be important in the aberrant accumulation of the APP gene product. INTRODUCTION The 5' promoter region of the amyloid protein precursor (APP) gene has been characterized at the nucleotide sequence level for both the murine 1~ and the human ts,3s APP genes. In addition, the human APP promotor has been partially investigated functionally using reporter gene assays in transfected cells t4,3t'32. The murine and human APP promotor sequences have an unusually high C + G content. For instance in the human promotor, sequences between -203 and + 55 bp, which contain essential regulatory elements for APP gene transcription, are 79.5% C + G. Further- more, in the human promoter there are 30 CpG dinu- cleotides between -203 and + 1 bp, indicating the presence of a major CpG island in the promotor of this 'housekeeping' gene 6'45. This functional domain, which lacks TATA or CAAT elements, contains four C + G boxes 12-15 bp in length putatively recognized by the transcriptional activator SP1, and an imperfect AP1 consensus sequence recognized by the c-JUN-c-JUN homodimer and the c-JUN-c-FOS heterodimer 16'3s. S1 nuclease mapping has shown that the major transcrip- tion start site (+ 1) for human APP is flanked by multiple minor transcription start sites between -130 and + 36 bp 14. Reporter assays using different frag- ments of the human APP promoter transfected into HeLa cells reveal that DNA sequences from -97 to -87 bp and -65 to -47 bp confer transcriptional activity 3~ while domains upstream from -150 bp are associated with a negative regulatory function for APP gene expression 15. Little, however, is known about the * Corresponding author. Room 118, Tanz Neuroscience Building, Center for Research in Neurodegenerative Disease, University of Toronto, Toronto, M5S IA8 Canada. Fax: (1) (416) 978-1878. SSDI 0169-328X(93)E0156-T