(Received for publication, May 10, 1995; and in revised form, July 19, 1995)
From the
Senile plaques are primarily comprised of deposits of the
-amyloid protein derived from larger amyloid precursor proteins
(APPs). APP is a member of a gene family, including amyloid
precursor-like proteins APLP1 and APLP2.
Using interspecific mouse backcross mapping, we localized the mouse APLP2 gene to the proximal region of mouse chromosome 9, syntenic with a region of human 11q.
We cloned an 1.2-kilobase mouse genomic fragment
containing the APLP2 gene promoter. The APLP2 promoter lacks a typical TATA box, is GC-rich, and contains
several sequences for transcription factor binding. S1 nuclease
protection analysis revealed the presence of multiple transcription
start sites. The lack of a TATA box, the presence of a high GC content,
and multiple transcription start sites place the APLP2 promoter in the class of promoters of ``housekeeping
genes.''
Regulatory regions within the promoter were assayed by
transfection of mouse N2a and Ltk cells with
constructs containing progressive 5`-deletions of the APLP2 promoter fused to the bacterial chloramphenicol acetyl transferase
(CAT) reporter gene. A minimal region that includes sequences 99 bp
upstream of the predominant transcription start site of the APLP2 promoter was sufficient to direct high levels of CAT expression.
Senile plaques and neurofibrillary tangles constitute two of the
neuropathological hallmarks of Alzheimer's disease. The
predominant constituent of senile plaques is the 4-kDa -amyloid
peptide, derived from larger amyloid precursor proteins
(APPs)(
)(1, 2) . APP is a member of a
larger gene family including amyloid precursor-like proteins APLP1 and
APLP2(3, 4, 5, 6, 7, 8) .
Notably, APLP2 shares considerable sequence homology with APP with the
exception of the
-amyloid
domain(5, 7, 8) . In earlier studies, we
demonstrated that APLP2 matures through the same unusual
secretory/cleavage pathway as APP. Furthermore, APLP2 pre-mRNAs are alternatively spliced to generate at least four
alternatively spliced transcripts(9, 10) . Using in situ hybridization and reverse transcriptase-polymerase
chain reaction (RT-PCR) approaches, we and others have demonstrated
that in most adult tissues, APLP2 and APP mRNAs were
expressed at similar, if not identical, levels. There are several
exceptions; notably, in liver APP mRNA is essentially
undetectable, but APLP2 mRNA is fairly
abundant(5, 7, 9, 11) . In recent
studies, we have also demonstrated that specific alternatively spliced APLP2 mRNAs are differentially expressed in the olfactory
epithelium(12) . Moreover, APLP2 is highly enriched in
olfactory sensory axons and axon terminals in glomeruli. On the other
hand, APP is expressed, albeit at lower levels, in olfactory
sensory neurons and to a lesser extent in sensory axons. This suggests
that APLP2 and APP are regulated differentially in
selected neuronal populations.
In order to assess whether the
differential levels of APLP2 and APP expression may
be a reflection of differences in sequence elements contained within
respective promoters, we cloned and characterized an 1.2-kb
fragment of the mouse APLP2 gene promoter. The mouse APP promoter has been characterized previously(13) . We show
that the mouse APLP2 gene promoter contains several features
characteristic of promoters of ``housekeeping genes''; these
include the lack of a typical TATA box, the presence of a high GC
content, and multiple transcription start sites. These latter features
of the APLP2 promoter are similar to features described for
mouse, rat, and human APP promoter
regions(13, 14, 15, 16, 17) .
We assessed whether the APLP2 promoter contained positive or
negative regulatory elements by transfecting mouse neuroblastoma (N2a)
cells and mouse fibroblast (Ltk
) cells with
constructs containing progressive 5`-truncated promoter fragments of
the APLP2 gene fused with the reporter gene chloramphenicol
acetyl transferase (CAT). We demonstrate that CAT expression remains
fairly constant across different deletion constructs in both N2a and
Ltk
cells and that a fragment representing just 99 bp
upstream of the predominant transcription start site is sufficient to
direct high levels of transgene expression in both cell lines.
Interestingly, 5`-deletion studies of the human, mouse, and rat
promoters also revealed that
100 bp of the respective promoters
can drive high levels of expression of reporter
genes(13, 15, 18) .
A description of the probes and restriction fragment length polymorphisms for the loci linked to APLP2 including low density lipoprotein receptor (Ldlr), preproenkephalin (Penk), and E26 avian leukemia oncogene (Ets1) has been reported previously (21) . Recombination distances were calculated as described (22) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
One
positive phage contained a 14-kb SalI-SalI fragment,
which included 2.8 kb of sequence upstream of the translation start
codon. A 2.84-kb SalI-HindIII restriction fragment
from this phage was subsequently subcloned into Bluescript
KS (Stratagene) to generate plasmid pAPLP2P and
partially sequenced with Sequenase (U.S. Biochemical Corp.). Sequences
were analyzed for putative transcription factor binding sites using a
MacVector version 4.1 software package.
Additional promoter deletions were prepared by PCR using the following sense primers linked to a HindIII site, GCCAAGCTTCACGGTCTACCCGCGAAG, GCCAAGCTTAGCCTCGGGTCCAGAG, GCCAAGCTTGAGTCGGTGTATCCGTGCT, and GCCAAGCTTGTTATGCCGGCTCGTATTG, respectively, with antisense primer BamHI+62 in the presence of pAPLP2P. The resulting 334-bp HindIII-BamHI (-272 to +62), 302-bp HindIII-BamHI (-240 to +62), 222-bp HindIII-BamHI (-160 to +62), and 161-bp HindIII-BamHI (-99 to +62) fragments were ligated to HindIII-BamHI-digested pBLCAT3 to generate plasmids pAPLP2PCAT-272, pAPLP2PCAT-240, pAPLP2PCAT-160, and pAPLP2PCAT-99, respectively. pRSVCAT(28, 29) , including the Rous sarcoma virus long terminal repeat as a promoter, was used as a positive control, and pBLCAT3 containing no insert was used as a negative control.
To assay
for CAT activity, 20 µg of cell lysate was incubated in the
presence of 1.1 mM acetyl CoA, 100 nCi of
[C]chloramphenicol (60 mCi/mmol) in 0.22 M Tris/HCl, pH 7.7, at 37 °C for 45 min. Acetylated and
nonacetylated forms of chloramphenicol were extracted with 0.5 ml of
ethyl acetate and separated by ascending silica gel thin-layer
chromatography in chloroform:methanol (95:5) at room temperature.
Thin-layer chromatography sheets were then air-dried, and acetylated
and nonacetylated forms of chloramphenicol were quantified using a
PhosphorImager. The percentages of monoacetylated forms of
chloramphenicol were plotted for each construct and normalized to the
CAT activity of pRSVCAT. Each construct was tested in three separate
transfections, and standard error of the mean was determined.
For
transfections of mouse fibroblast Ltk cells, cells
were plated at a density of 0.2
10
cells/well in a
6-well dish. Cells were transiently transfected with 4.26 µg of
pBLCAT3 or equivalent molar amounts of CAT plasmids containing various
5`-deletions of the APLP2 promoter adjusted to 7 µg with
empty vector DNA. 20 µg of cell lysate was used for CAT assays.
Recent studies have indicated that APP is a member of a larger gene family that includes APLP1 and APLP2. The physiological function(s) and regulation of the APP-related proteins is not well understood. In this study, we mapped the genomic location of APLP2 and have analyzed the APLP2 promoter for the presence of potential regulatory sequences that may be involved in transcriptional activity of the APLP2 gene.
Figure 1:
APLP2 maps in the proximal
region of mouse chromosome 9. APLP2 was placed on mouse
chromosome 9 by interspecific backcross analysis. The segregation
patterns of APLP2 and flanking genes in 152 backcross animals
that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 152 animals
were typed. Each column represents the chromosome identified
in the backcross progeny that was inherited from the (C57BL/6J M. spretus)F
parent. The black boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of the M. spretus allele. The
number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 9 linkage
map showing the location of APLP2 in relation to linked genes
is shown at the bottom of the figure. Recombination
distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes
are shown to the right. References for the human map positions
of loci cited in this study can be obtained from the Genome Data Base,
a computerized database of human linkage information maintained by the
William H. Welch Medical Library of the Johns Hopkins University
(Baltimore, MD).
We have compared our interspecific map of chromosome 9 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (provided from the Mouse Genome data base, a computerized data base maintained at The Jackson Laboratory, Bar Harbor, ME). APLP2 mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown).
The proximal region of mouse chromosome 9 shares regions of homology with human chromosomes 19p, 8q, and 11q (summarized in Fig. 1). The recent assignment of APLP2 to 11q23-q25 (31) confirms and extends the synteny between mouse chromosome 9 and human 11q.
Figure 2:
Mapping of the 5` termini of APLP2 mRNA. A, diagram of S1 nuclease protection assay. A
533-bp KpnI-HindIII restriction fragment from the
mouse APLP2 promoter was subcloned into Bluescript
KS. The locations of restriction sites are with
respect to the translation start site (ATG). S1 nuclease probe was
prepared by
P end labeling at HindIII after
linearizing the clone with HindIII. B, S1 nuclease
protection assay of poly(A)
RNA from CHO cells (lane 1), of total RNA from mouse thymus, heart, brain, liver,
kidney, lung, testes, and spleen (lanes 2-9,
respectively). tRNA served as negative control (lane 10).
Marker (M) DNA fragments are in
bp.
Figure 3: DNA sequence of the 5`-regulatory region of the mouse APLP2 gene. Base numbers are relative to the predominant transcription start site, 89 bp upstream of the translation start site. Consensus sequences for putative transcription factors are overlined, including the GC element (GCE) and GC factors (GCF). Exon 1 (bold) is followed by 120 bp of intron 1.
The DNA sequence upstream of the predominant transcription start site contains a CAAT box (-135 in antisense orientation) but lacks a typical TATA box (Fig. 3). The promoter has a high GC content, specifically between positions -1 and -300 (68%) and -500 and -700 (69%). Multiple consensus sequences for transcription factor binding sites are present in the entire region, including one AP-1, two AP-2s, five GC boxes, one GC element, two GC factors, and seven SP-1 sites. Similar putative transcription factor binding sites are found in the APP promoter, however, at different locations with respect to the transcription start site(13, 14, 16, 17) . Furthermore, the APP promoter contains sites for transcription factors not present in the APLP2 promoter, including a potential heat shock element and an overlapping AP-1/AP-4 site(14, 16, 18) , suggesting that the transcriptional regulation of APLP2 and APP genes may be dissimilar. The presence of multiple transcription start sites, the absence of a typical TATA box, the high GC content, and the presence of GC-rich boxes places the APLP2 promoter in the class of promoters of housekeeping genes; these include the human, rat, and mouse APP genes (13, 14, 16, 17) , the adenosine deaminase gene(32) , the dihydrofolate reductase gene(33) , and the hamster prion gene(34) .
Recently, the upstream AP-1 site (position -350 with respect to the predominant transcription start site) in the APP promoter has been implicated in protein kinase C mediated up-regulation of APP gene expression(35) . The AP-1 binding activity is thought to be composed of Jun-Jun homodimers. Interleukin-1, nerve growth factor, and retinoic acid, agents known to increase APP gene expression, have been shown to induce c-jun and c-fos expression and cause transcriptional activation of target genes through AP-1 sites(36, 37, 38, 39) . Furthermore, interleukin-1 effects are thought to involve protein kinase C activation(40) . It remains to be determined if APLP2 gene expression is also regulated by interleukin-1, nerve growth factor, and retinoic acid, particularly in view of the presence of a potential AP-1 site located at position -982.
Figure 4:
CAT assay
of APLP2 promoter in mouse N2a and Ltk cells. A, EtBr-stained agarose gel of XhoI-digested RT-PCR products derived from mouse N2a (lane
1) and mouse Ltk
cells (lane 2).
Plasmids containing mouse APP and mouse APLP2 cDNA
were used as positive controls (lanes 3 and 4,
respectively). Marker (M) DNA fragments are in bp. B,
diagram of 5`-truncated APLP2 promoter constructs used for
transfection into mouse N2a and Ltk
cells (constructs
1-6). Sites of termination of these constructs are indicated with
respect to the predominant transcription start site. C, CAT
assays of APLP2 promoter constructs 1-6 in N2a cells.
CAT activity was determined as the percentage of monoacetylated forms
of chloramphenicol. CAT activity of the promoter construct containing
the long terminal repeat of the Rous sarcoma virus (RSV) was
assigned the activity of 100%, and the activities of the constructs
carrying 5`-deletions of the APLP2 promoter were expressed as
values relative to the Rous sarcoma virus construct. The vector
pBLCAT3, containing CAT in the absence of any promoter fragment, was
used as a negative control (BL). Each construct was tested in
three separate sets of transfections, and the standard error of the
mean is indicated by the error bars. D, CAT assays of APLP2 promoter constructs 1-6 in Ltk
cells; otherwise as in panel C above.
Progressive 5`-deletions from position
-971 to position -99, with respect to the predominant
transcription start site, had no significant effect on promoter
activity in either of the two cell lines tested. These findings suggest
that in N2a and Ltk cells, 99 bp of the APLP2 promoter are sufficient for directing high levels of promoter
activity. Similarly, studies that analyzed progressive 5`-deletions of
the APP promoter from human, mouse, and rat have shown that
reporter gene expression levels remained fairly constant up to
approximately 100 bp upstream of the predominant transcription start
site(13, 15, 18) .
In summary, we have
localized APLP2 to the proximal region of mouse chromosome 9,
characterized 1.2 kb of the APLP2 promoter, and shown it
to contain features characteristic of promoters in the class of
housekeeping genes. We further showed that 99 bp upstream of the
predominant transcription start site are sufficient to direct high
levels of promoter activity.
Given the similarities in overall structure of the APLP2 and APP promoters and the minimal sequence requirements for transcription initiation, it is highly likely that additional sequence elements distal to the regions analyzed here are responsible for differential expression of APLP2/APP in specific neuronal populations or systemic organs (i.e. liver). Further studies will be directed toward using transgenic strategies with larger genomic fragments to clarify these issues with the eventual goal of identifying transcription factors responsible for mediating basal level of APLP2 gene expression.
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