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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-1378fje. |
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,
,2
* Medical University of South Carolina, Charleston, South Carolina, USA; and
Departments of Psychiatry and
Medical and Molecular Genetics, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA
2 Correspondence: 791 N. Union Drive, Indianapolis, IN 46206, USA. Email: dlahiri{at}iupui.edu
SPECIFIC AIMS
The amyloid plaque invariably deposited in Alzheimer’s disease (AD) is composed of an 
42-residue peptide (amyloid ß peptide, Aß) obtained by cleavage of the larger Aß precursor protein (APP) by proteases, referred to as ß and 
secretase. The aim of this study is to understand the genomic organization of ß secretase, ß site APP-cleaving enzyme (BACE) gene, and the position of exons and introns. Another aim is to study transcriptional regulation of the BACE gene, including structure of the regulatory region. We determined whether its structure resembles that of a housekeeping gene, and/or can be regulated by different cell-type specific factors, metallic ions and stress-related agents. We report cloning and detailed analysis of 3765 bp of the promoter region of BACE and 364 bp of the 5' untranslated region of BACE mRNA (5'-UTR). GenBank accession number of sequence is AY542689.
PRINCIPAL FINDINGS
1. Molecular cloning of the BACE1 5'-flanking region
The BACE1-containing BAC clone RP11-677N11 was identified by BLAST searching of the RP1146 human genomic library, and BACE1 promoter was PCR amplified with either oligomers BACE1F and BACE1R or BACE2F and BACE2R. Two clones of different sizes were further analyzed. The larger clone, p4.1BACEP-SEAP contained a 4.1 kb fragment of the BACE1 5' flanking region. The smaller clone, p2.4BACEP-SEAP contained a 2.4 kb fragment of the BACE1 5' flanking region that was coterminous at the 3' end with the 4.1 kb fragment (Fig. 1
).
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2. Genomic organization of BACE: structure of intron-exon regions
Reported BACE cDNA clones were either 2.5 kb or 5.9 kb long. BLAST alignment of BACE cDNA revealed that these clones varied in length of their 3'-UTR. Two BACE mRNAs are synthesized as 9 exons and 8 introns from an 30.6 kb region of chromosome 11q23.2-11q23.3. Additional introns were not observed in this region of BACE. A third mRNA has been seen in Northern blots of BACE. Searching the EST database suggested that longer cDNAs exist that terminate closer to the 3'-end of the genomic sequence, although boundaries have not been defined. Presence of multiple mRNAs that terminate at different sites is likely to be due to variable polyadenylation of mRNAs.
In addition to BACE1 proteins of 501 amino acid residues, three alternatively spliced forms of BACE1 of 432, 457, and 476 residues have also been described in the literature. These forms arise by alternative and adventitious splicing of exons 3 and 4 and deletion of 25, 44, or 69 residues between the two conserved active site domains of BACE1. These alternatively-spliced BACE1 forms have been found to be inactive against APP substrate.
Boundaries and sizes of the 9 coding exons are highly conserved, as would be expected, between mice and humans. In contrast, there is greater variation in the size of introns, as routinely observed for several other genes. Brief analysis of the first intron for possible transcription factor binding sites with TESS and MatInspector revealed thousands of possible sites, including nearly 1200 putative sites shared with the BACE promoter region (BACEP). The intron has at least five predicted ORFs: two in forward and three in reverse orientation. One of these putative ORFs has a predicted 72% homology with a 375 residue segment of human neuronal thread protein.
3. Transcription start site (TSS)
The BACE open reading frame is synthesized in mRNAs that fall into two sizes: 2.2–2.5 kb and 5.9 kb. Among multiple cDNAs cloned and sequence reported in the literature, three presented the sequence starting from its 5'-UTR. These clones begin 449, 452, or 457 bp from the translation start site. BLAST search of the EST library available at NCBI revealed no longer clones. This suggests that TSS is 457 bp from the translation start site as used in Table 1
and Fig. 1
. An alternate TSS at 691 bp from translation start has been reported while our report was in preparation.
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4. Chromosomal localization and genomic alignment
MegaBLAST database search of GenBank with BACEP sequence as a probe demonstrated matches with previously published human BACE mRNA and with human chromosome 11q clones CMB9-94B1 and CTD-2336M4. Genomic MegaBLAST against the human genome placed the sequence at complement of positions 20734562 to 20730273 on chromosome 11 and between chromosome bands 11q23.3 and 11q23.2. A 163 bp region from the GenBank human BACE1 genomic sequence (NCBI build 34) was not found in our BACEP sequence. Eleven substitutions and two single-base insertions also appear in our sequence in comparison to genomic sequence. None of these single-base differences correspond to previously characterized SNPs. Alignment with the prototype GenBank mRNA sequence revealed that the BACEP sequence ends 81 bp upstream of the human BACE1 gene "ATG" translation start.
5. Structural characterization of the 5'-flanking region
A total of 4129 bp of BACE 5' flanking promoter containing DNA was sequenced, including the majority of 5'-UTR. Analysis reveals an overall GC content of 47%. A 50 bp running average of GC content varies between 14% and 90% along the length of the sequence, reaching maximum levels at the 3' end of the sequence. 5' end of the sequence has one tandem repeat of two 159 bp segments with 84% mutual homology; the sequence also has an inverted repeat of two 274 bp segments with 85% mutual homology, separated by 1.8 kb of sequence. The upstream portion of this inverted repeat also takes part in a triple direct repeat, which has 74% homology over ClustalX aligned length. Each element of this triple repeat is also part of an inverted repeat with a 271 bp segment near the 5' end of the BACEP sequence. Homology of this quadruple repeat is 67% (Fig. 2
B).
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6. Transcription factor binding sites
While TATA-like sequences appear in the far-upstream region of the sequence, canonical "CAAT" and "TATA" boxes are not found within 1.5 kb of TSS. Sequence analysis of the promoter region and 5'-UTR reveals presence of multiple potential transcription factor binding sites. In 5'-UTR (+1 to +364), one ACE-like sequence, three AP2 sequences, one AP4 sequence, one ApoAI RP-1 sequence, one bHLH site, four GC boxes, two IL-6 responsive element sequences, one ONSF sequence, three GATA-1 sites, and eight SP1 binding sites were found (Table 1)
. The immediate promoter region and 5' flanking region (–3765 to –1) has, among other sites, 1 ACE-like sequence, 1 each of AP1, -2, -3, and -4 sequences, 1 ApoAI RP sequence, 2 bHLH sites, one C/EBP site, 1 CREB site, >30 possible GATA binding sites, 1 GC box, 2 glucocorticoid receptor sites, 1 HIF site, 3 HSF sequences, 13 octamer binding sites, 4 potential NF-
B sites, 2 progesterone response element sites, 2 ERE sites, 4 sites involved in retinoic acid recognition, 8 Smad3 binding sites, 1 USF site, and 11 putative Zeste binding sequences (Table 1)
. Consult full text for complete table.
7. Comparison of DNA sequence among species
Comparing the human sequence with corresponding genomic DNA from rat and mouse sequences showed varying amounts of interspecific similarity, approaching or exceeding 90% within ±100 bp of TSS, but rapidly falling to below 50% for most of the sequence before –150 bp, although there is a brief segment of >80% homology between –2000 bp to –1850 bp. This suggests that the region from –150 bp and downstream may play an important role in BACE gene expression and that it could be regarded as a common proximal region for all mammalian BACE genes.
The 163 bp gap found when aligning BACEP sequence with human genomic sequence did not appear when aligning BACEP with mouse and rat genomic sequences. Several putative transcription factors found by TESS search of human BACEP sequence also turned up in mouse and rat sequences, notably, AP2, CLS, CREB, GATA-1, MZF1, NF-B, SP1, and USF.
CONCLUSIONS AND SIGNIFICANCE
Aß accumulates to very high levels in AD brain and apparently plays an important role in its pathogenesis. BACE is an important target for drug development and for understanding metabolism of APP and brain-specific deposition of Aß peptide.
It is unlikely that BACE evolved primarily to make the potentially pathogenic Aß peptide; it may have some other physiological function(s). This possibility is now strengthened by our results showing that the structure of BACE promoter resembles with that of a "housekeeping gene" as it lacks characteristics of type II promoter, such as "CAAT" and "TATA" boxes, within 1.5 kb of TSS. Of particular interest is that a MZF1 putative zinc finger protein binding site that spans human TSS is preserved in rat and mouse sequences. Others have reported that mutating the MZF1 site does not effect basal expression in the rat gene. Nevertheless, it may participate in inflammatory responses mediated by cytokine binding to sites we have mentioned herein.
BACE promoter displays inducible characteristics in that it contains potential binding sites for several important transcription factors, such as AP1, AP2, CRE, ERE, GRE, and NF-B. Four of five NF-B sites predicted in BACEP appear in some segment of the triple direct repeat, suggesting a possible regulatory role for these repeats.
Taken together, the BACE gene contains basal regulatory elements, inducible features, and sites for regulated activity by various transcription factors. This study enables us to identify important regions for functional analysis of binding domains and neuron-specific expression. These studies will allow us to further examine the possible role of changes in the BACE promoter and AD pathogenesis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1378fje; 
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