From the Division of Human Genetics, Children's Hospital Research
Foundation, Children's Hospital Medical Center,
Cincinnati, Ohio 45229-3039
Prosaposin is the precursor of four low molecular
weight sphingolipid-activating proteins (SAPs) or saposins. These four
proteins function as intracellular activators of several lysosomal
enzymes involved in the degradation of glycosphingolipids, and
prosaposin itself has neurite outgrowth effects. Expression of
prosaposin is regulated in a temporal and spatial manner with
expression in specific brain neurons and visceral cell types. Here a
major regulatory fragment was characterized within 310 bp 5' to the transcription start site. Using electrophoretic mobility shift assay
(EMSA) and DNA footprinting, members of the Sp family (Sp1, Sp3, and
Sp4), the orphan nuclear receptor (ROR
), and an unknown transcription factor (U; TGGGGGAG) were shown to bind to this region.
To evaluate the role of such transcription factor binding sites for
this locus, a series of mutant constructs was generated within this
region, and their function was evaluated in cultured NS20Y
neuroblastoma cells. A 3' Sp1 site, a 5' Sp1/U cluster and the ROR
binding sites were functional. The data are consistent with a model in
which the factors that bind to the Sp1/U cluster and RORE site interact
negatively to diminish promoter activity to a background level that is
determined primarily by the 3' Sp1 site. These interactions depend on
the tissue-specific repertoire of transcription factors leading to
differential expression of this locus.
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INTRODUCTION |
Prosaposin is a multifunctional protein with specific intra- and
extracellular functions. It is the precursor of four 80-amino acid
glycoprotein activators (1-4). In humans, rats, and mice the
prosaposin mRNA encodes in tandem the highly similar saposins A, B,
C, and D that participate in the sequential degradation of
glycosphingolipids to sphingosine and fatty acids (2-5). In the rat,
prosaposin is designated SGP-1, sulfated glycoprotein 1, since it is a
major sulfated glycoprotein in Sertoli cells (5). In addition to
prosaposin's essential role in glycosphingolipid degradation, it has
ex vivo neurite outgrowth effects. When placed in the media
surrounding neuroblastoma cells, prosaposin facilitates neurite
outgrowth (6-8), and prosaposin facilitates in vivo
regeneration of the sciatic nerve following injury (9). Prosaposin also functions in glycosphingolipid transfer between artificial membranes (10). Targeted disruption of the murine prosaposin gene resulted in a
complex phenotype including severe central nervous system disease and
widespread storage of multiple sphingolipids (11).
The human gene for prosaposin has been partially characterized to
contain 13 (or 14) exons and 12 (or 13) introns (12, 13). An
alternatively spliced 9-bp1
"exon" is present in the saposin B region of the prosaposin gene (12). The 13 (or 14) exons are spread over ~30 kb of human chromosome 10. Interestingly, the prosaposin gene, a presumed "housekeeping" gene, displays temporally and spatially regulated expression (14). Despite the ubiquitous role of lysosomal hydrolases in all tissue types, the level of prosaposin expression is highly dependent on the
cell type and maturation. The highest levels of expression are in
specific neurons of the adult cerebrum, the Purkinje cell layer of
the cerebellum, and neurons of the lateral regions of the spinal cord
(14). Components of the hind brain also show higher levels of mRNA
expression early in embryogenesis (15).
Previously, we characterized the 5' region of the murine prosaposin
gene including the first exon that contains the translation initiation
site (16). The first intron of the mouse prosaposin locus is unusually
large, almost 15 kb, and constitutes nearly 60% of the gene. Like many
other housekeeping genes, the promoter region of the murine prosaposin
gene is "TATA-less" and GC rich. The prosaposin gene has a major
and a minor transcription start site. Transfection of deletion
constructs containing reporter genes into NS20Y, NIH-3T3, or SF-7
(Sertoli) cells showed positive and negative regulatory elements within
2,400 bp 5' to the transcription start sites (16).
In this paper, we report the characterization of a major regulatory
fragment located within 310 bp 5' to the murine prosaposin gene
transcription start site. DNA footprinting, electrophoretic mobility
shift assays (EMSA), and site-directed mutagenesis were used to analyze
in vitro the interaction of transcription factors that bind
to this fragment. Sp members 1, 3, and 4; ROR
(the orphan nuclear
receptor); and an unknown transcription factor (U) were identified to
be involved in the regulation of the murine prosaposin gene. A complex
interaction of multiple transcription factors is proposed to modulate
gene expression from one essential Sp1 binding site within 16 bp 5' to
the transcription start site.
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EXPERIMENTAL PROCEDURES |
Materials--
The following materials were from commercial
sources: WizardTM PCR Preps DNA kit, pGL2B Luciferase
Reporter Vectors, the luciferase assay system, and the
-galactosidase enzyme assay system (Promega, Madison, WI);
restriction enzymes and Taq DNA polymerase (New England
Biolabs, Beverly, MA); SequenaseTM version 2.0 DNA
sequencing kit (U.S. Biochemical Corp.); oligonucleotide synthesis,
poly(dI-dC), poly(dA-dT), NAP-10, and NAP-5 columns (Amersham Pharmacia
Biotech); anti-Sp1, -Sp2, -Sp3, and -Sp4 antibodies (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA); QuickChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA); GeneClean Kit (BIO 101, Inc., Vista, CA); QIAGEN Plasmid Midi Kit (QIAGEN, Chatsworth, CA);
Microcon microconcentrator 3 (Amicon, Inc, Beverly, MA); Lipofectamine,
Opti-MEM serum-free medium, DMEM (Life Technologies, Inc.); Monolight
2010 luminometer (Analytical Luminescence Laboratory; San Diego, CA);
isotope, [
-32P]ATP (NEN Life Science Products). Cell
line NS20Y was from Dr. Marshall Nirenberg (National Institutes of
Health).
Double-stranded Oligonucleotides--
Single-stranded
oligonucleotides were synthesized on an Amersham Pharmacia Biotech DNA
synthesizer. After purification with NAP-10 columns, the complementary
oligonucleotides were heated at 95 °C for 5 min in the annealing
buffer (20 mM Tris, pH 7.4, 2 mM
MgCl2, and 50 mM NaCl) and then cooled to room
temperature. The annealed oligonucleotides were purified by
eletrophoresis in polyacrylamide gels.
Nuclear Extract Preparation--
Nuclear extracts were prepared
using a miniextract procedure (17) with minor modifications. Nuclear
extracts were desalted and concentrated using Microcon
microconcentrator 3 (Amicon, Inc). Protein concentrations were
estimated by the Bradford method (18).
EMSA--
The double-stranded oligonucleotides were labeled with
[
-32P]ATP and T4 polynucleotide kinase. The probes
were purified on NAP-5 columns. The labeled double-stranded
oligonucleotides were incubated with 3-6 µg of nuclear extract at
room temperature for 20 min in 20 µl containing 5% glycerol, 1 mM Mg Cl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 50 mM NaCl, 10 mM
Tris-HCl (pH 7.5), and 0.5 mM fresh phenylmethylsulfonyl
fluoride. Poly(dI-dC) or poly(dA-dT) was used as heterologous
competitor in the reaction (800 ng/reaction). For antibody supershift
assays, the extract was incubated overnight at 4 °C with the
specific anti-Sp antibodies. Extract-antibody mixtures were then
incubated with the probe. Bound and free probes were resolved by
nondenaturing electrophoresis in 6% polyacrylamide gels.
DNase I Footprinting--
Polymerase chain reaction was used to
generate 5'-end-labeled footprinting probes of the prosaposin promoter
region (408 bp). A prosaposin deletion construct containing +98 bp to
310 bp was used as a template, and vector-specific (GLP1 and GLP2)
primers were from Promega. The DNase I protection assays were performed with sense and antisense strands as described (19). The DNA binding
reactions (50 µl) were done at 4 °C in 10 mM Tris, pH 7.5, 5 mM MgCl2, 50 µM EDTA, 75 mM KCl, 12% glycerol, 0.5 mM dithiothreitol, and 0.2 mM fresh phenylmethylsulfonyl fluoride. Bovine
serum albumin (20 µg) without nuclear extract was used as a control.
Nuclear extract (30 µg) or controls were first incubated with 1 µg
of poly(dI-dC) for 15 min, and then 20,000 cpm of probe was added and
incubated for 45 min. These reaction mixtures were subjected to DNase I
digestion for 1 min at room temperature. The controls had 0.1 unit of
DNase I/reaction while the nuclear extracts contained 0.4, 0.8, 1.2, or
1.6 units of DNase I/reaction. The DNA fragments were resolved by
electrophoresis in 6% polyacrylamide, 7 M urea gels. The
probe, cleaved at guanine and adenine with formic acid and piperidine
(20), was used as a size marker.
Site-directed Mutagenesis--
Deletion constructs (16) were
used as templates to create mutant constructs using the QuickChange
site-directed mutagenesis kit (Stratagene). Briefly, site-directed
mutations were generated by polymerase chain reaction using sense and
antisense mutagenic oligonucleotide primers and Pfu
DNA polymerase. The polymerase chain reaction product generated
in this system was a nicked circular strand that was then digested with
DpnI. The methylated, wild-type parental DNA template was
digested, and the circular, nicked double-stranded DNA was transformed
into XL2-Blue ultracompetent cells. Plasmid DNA from positive clones
was digested with XhoI and HindIII and then
recloned into the pGL2-basic vector. Each mutant construct was verified
by DNA sequencing. The mutagenic oligonucleotide primers are listed in
Table I. The primer pairs and templates used for each mutation construct are in Table
II.
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Table II
Templates and primer pairs for polymerase chain reaction mutagenesis of
transcription factor binding sites
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Transient Transfection and Reporter Gene Assays--
Functional
assays of reporter constructs were performed using transient
transfection of NS20Y cells (16). Confluent NS20Y cells were seeded at
density of 105 cells/well into 12-well plates and incubated
for 18 h at 37 °C. For each construct, 0.75 µg of test DNA
and 0.25 µg of
-galactosidase DNA were diluted into 50 µl of
OPTI-MEM serum-free medium. This solution then was mixed with 4 µl of
Lipofectamine in 50 µl of DMEM. The mixture was incubated at room
temperature for 30 min to allow DNA-liposome complexes to form. The
complexes were then mixed with 0.4 ml of DMEM and overlaid onto the
DMEM-rinsed cells. After incubation for 5 h at 37 °C, the
transfection mixtures were removed, and 1 ml of complete DMEM (with
10% fetal serum and 1% penicillin/streptomycin) was added to the
cells. At 65 h after transfection, the cells were washed twice
with phosphate-buffered saline by centrifugation (1000 × g, 5 min) and then incubated in the 200 µl of reporter
lysis buffer (25 mM Tris-phosphate, pH 7.5, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol,
1% Triton X-100) at room temperature for 15 min with shaking. The cell
lysate was collected, transferred into microcentrifuge tubes,
vigorously agitated (15 s), and centrifuged (12,000 × g, 2 min). The supernatants were transferred to fresh tubes
for luciferase and
-galactosidase activity assays. For luciferase
assays, lysate (20 µl) was mixed with luciferase assay reagent (100 µl). Light emission was quantified at room temperature. For the
-galactosidase assay, lysate (10 µl) was diluted into lysis buffer
(150 µl) and mixed with 150 µl of 2 × assay solution (120 mM Na2HPO4, 80 mM
NaH2PO4, 2 mM MgCl2,
100 mM
-mercaptoethanol, and 1.33 mg/ml
O-nitrophenyl-
-O-galactopyranoside). The
mixture was incubated (37 °C, 20 min) and stopped with 1 M Na2CO3 (0.5 ml). The absorbance
was determined at 420 nm. Luciferase activity was normalized to
-galactosidase activity for each lysate collected from the
individual transfection experiments. The results represent the means of
three independent experiments conducted in triplicate.
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RESULTS |
DNA Footprint Analysis--
A 408-bp segment (
310 to +98) from
the 5' end of the murine prosaposin gene contained promoter activity in
a number of different cell types (16). This promoter region is shown in
Fig. 1 (GenBankTM accession
number AF037437). Sequence analysis of the 310 bp 5' to the
transcription start site revealed four potential Sp1 binding sites,
three of which are overlapping in a 5' cluster, and GATA, AP-1, and
ROR
binding sites.

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Fig. 1.
Partial DNA sequence of Exon 1 (in
boldface type) and 5'-flanking regions of the mouse
prosaposin gene. The numbering of nucleotides in the text is
referenced to the major transcription start site (large
arrow). The smaller arrow is a minor transcription
start site (16). The translation initiation codon is boxed.
Putative transcription factor binding sites, analyzed here, are
underlined and labeled with appropriate consensus binding
sequences.
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DNA footprint analysis of this region (Fig.
2) showed three DNase I protected regions
when using nuclear extracts from NS20Y cells and the sense probe. The
control reactions contained bovine serum albumin. The most 3'-protected
region (
32 to
12) is GC-rich and contains a Sp1 binding site. In
particular, the AP-1 site did not footprint. A strongly protected
region was at
184 to
164. This region has high homology to the
ROR
binding site (also termed ROR response element (RORE)) that is
composed of a half-site PuGGTCA preceded by a 6-bp AT-rich region (21,
22). A weakly protected region was at
267 to
294 and contained
three overlapping Sp1 binding sites. Using the antisense probe, similar
results were obtained, but weaker protection was observed at the RORE and the 3' Sp1 binding sites (data not shown).

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Fig. 2.
DNase I footprint analysis of the prosaposin
promoter region shown in Fig. 1. The in vitro DNase I
digestion pattern of the sense strand of the promoter region was in the
presence of NS20Y nuclear extract or of bovine serum albumin
(BSA). The triangle indicates an increasing
amount of DNase I in the digestion. The G+A lane is the
specific Gilbert sequencing reaction products for G and A. The
boxes on the right indicate the regions protected
from DNase I digestion by NS20Y cell nuclear extract. The filled
rectangles are strongly protected regions. The hatched
box was a region that was less strongly protected. Analyses were
in 238 to +1 (A) and 213 to 300 (B).
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EMSA--
To confirm the DNA footprinting results, EMSA was
performed with several double-stranded oligonucleotides (Table
III) that covered the protected regions.
When EMSA was conducted with a probe (oligonucleotide 1, O1) covering
bases
35 to
3, two major DNA-protein complexes were observed with
NS20Y cell nuclear extracts (Fig. 3,
A and B (lanes 1 and 2)).
Formation of DNA-protein complexes was competed by an excess of
unlabeled O1, but not by MO1, an oligonucleotide containing a mutated
Sp1 binding site (Table III; Fig. 3B, lanes 3 and
4). The addition of excess cold Sp1 consensus oligonucleotide competed with labeled O1 for binding to the protein(s) (Fig. 3B, lane 5). When purified Sp1 protein was
incubated with the same probe, a single DNA-protein complex was
detected (Fig. 3B, lane 6). O3 was synthesized to
cover three overlapping Sp1 binding sites from
298 to
263. Three
DNA-protein complexes were resolved when this probe was incubated with
NS20Y cell nuclear extracts (Fig. 3C, lanes 1 and
2). The formation of DNA-protein complexes was competed off
by the addition of excess cold O3 (Fig. 3C, lane
3). However, excess Sp1 consensus oligonucleotides competed off
only the slowest migrating DNA-protein complex. This indicated that
other transcription factors also bind to this region and that their
binding is independent of Sp1 binding (Fig. 3C, lane 4). The strong footprint from
184 to
164 covered a consensus ROR
binding site (also termed RORE). However, there was a GATA binding site just 5 bp 3' to RORE. To determine which binding site was
functional, we conducted EMSA with oligonucleotides 2 (O2) and 4 (O4)
that cover the GATA binding site and RORE, respectively (Table III;
Fig. 4A). No DNA-protein
complexes were observed when O2 was incubated with NS20Y cell nuclear
extract (Fig. 4B). Several DNA-protein complexes were
detected with O4 and NS20Y nuclear extracts (Fig. 4C,
lanes 1 and 2). The formation of these
DNA-protein complexes was prevented by the addition of excess cold O4,
but not MO4 (Fig. 4C, lanes 3 and 4).
MO4 contains a mutated ROR
binding site (Table III). These results
indicate that RORE is functional but that the GATA binding site is
not.

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Fig. 3.
EMSA with O1 ( 35 to 3 bp) and O3 ( 298
to 263 bp). Labeled oligonucleotides were incubated with NS20Y
cell nuclear extract in the presence or absence of a 100-fold excess of
unlabeled oligonucleotides. Poly(dI-dC) (800 ng) was added to each
reaction. A, schematic diagram of the prosaposin sequences
in Fig. 1 with three 5'-overlapping Sp1 and a U site in a cluster. The
arrow represents the major transcription start site. The
locations of O1 and O3 within this region are indicated by the
underline. B, EMSA with O1. The legend
above each lane indicates the contents of each
reaction in addition to radiolabeled O1. The molar excess (100×) over
the amount of radiolabeled O1 is indicated for cold O1, MO1 (a mutated
O1 with destroyed Sp1 binding site), or Sp1 (a consensus Sp1). The
arrows indicate the major shifted complexes. Lane
1 contains only radiolabeled O1. C, EMSA with O3. The
legend above indicates the contents of the reactions in each
lane in addition to radiolabeled O3. Lane 1 contains probe
only. Lanes 2, 3, and 4 contain NS20Y
nuclear extract. The arrows indicate DNA-protein complexes.
F is the unbound probe.
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Fig. 4.
EMSA with O2 ( 168 to 133 bp) and O4
( 186 to 159 bp). Double-stranded labeled oligonucleotides were
incubated with NS20Y nuclear extract in the presence or absence of
100-fold molar excess of unlabeled O2 or O4. Poly(dI-dC) (800 ng) was
in each reaction. A, a schematic diagram of the nucleotide
sequences in Fig. 1 with the locations of O2 and O4
underlined. B, EMSA with O2. Lane 1 contains probe only, and lane 2 contains the probe and NS20Y
nuclear extract. C, EMSA with O4. Each lane contains
radiolabeled probe, NS20Y nuclear extract, and the indicated additions.
Lane 1, probe only; lane 2, no addition;
lane 3, 100-fold excess unlabeled O4; lane 4,
100-fold excess unlabeled MO4. The MO4 has the RORE consensus site
obliterated. The arrows indicate DNA-protein complexes.
F, unbound probe.
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The Sp1 multigene family includes Sp1 and three other genes encoding
the Sp2, Sp3, and Sp4 proteins. The consensus binding sequences of
these proteins are very similar (23, 24). No cross-reaction was
detected among the antibodies for the individual Sp proteins, and none
of them bound to O1 or O3 to form DNA-protein complexes (data not
shown). Polyclonal antibodies against Sp1, Sp2, Sp3, or Sp4 were used
for EMSA. Preincubation of anti-Sp1 or anti-Sp3 antibodies with NS20Y
cell nuclear extracts produced a DNA-protein supershift using O1 and O3
(Fig. 5). A similar shift was obtained
with Sp4 antibody using O1, but not with O3 (Fig. 5A,
lane 6). For O1, the Sp1 and Sp3 antibodies appeared to
supershift different bands and, by inference, interacted with different
proteins or complexes. The Sp1 antibody supershifted a slower migrating band (lane 3), whereas the Sp3 antibody supershifted a
faster migrating band (lane 5).

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Fig. 5.
Gel supershift assays using anti-Sp
antibodies. A, interaction of O1-protein complex with
anti-Sp antibodies. Radiolabeled O1 was used as the probe. All lanes
contain the radiolabeled probe. Lane 1, the radiolabeled O1
probe only; lanes 2-6, the probe and NS20Y nuclear extract
plus the indicated additions; lane 2, no addition;
lane 3, anti-Sp1 antibody; lane 4, anti-Sp2
antibody; lane 5, anti-Sp3 antibody; lane 6,
anti-Sp4 antibody. B, interaction of O3-protein complexes
with anti-Sp antibodies. Lane 1 contained only the
radiolabeled O3. Lanes 2-6 contained the probe, NS20Y
nuclear extract, and the indicated additions. Lane 2, no
addition; lane 3, anti-Sp1 antibody; lane 4,
anti-Sp2 antibody; lane 5, anti-Sp3 antibody; lane
6, anti-Sp4 antibody. The amount of antibody in each reaction is
0.5 µg. The whole reactions were incubated overnight at 4 °C. The
complexes were resolved in 6% polyacrylamide gels. The
arrows indicate the supershifts. The antibody added in each
lane is indicated at the top of the gels.
F, unbound probe.
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Definition of an Unknown Transcription Factor Binding Site--
In
the EMSA experiment using O3 as probe (Fig. 3C, lane
4), the addition of cold Sp1 consensus oligonucleotides eliminated the top band, but not the middle and bottom bands. This result shows
that a transcription factor(s), in addition to Sp1, binds to this
region and that this binding is independent of that for Sp1. In an
attempt to define this transcription factor binding site, a series of
mutant O3s were made to densely cover this region (Fig.
6A) and were used as probes
during EMSA. NS20Y nuclear extracts were used. Three similar bands were
obtained with the oligonucleotides 3, 3A-3D, and 3F. Oligonucleotide
3E produced only two supershifted bands, and one major band was
missing, i.e. a transcription factor binds to the mutated
region that was not covered in the oligonucleotide 3E region (Fig.
6B, lane 7). Competition assays using cold
oligonucleotides 3A-3F against their radioactive derivatives showed
that the bindings of each of the mutant O3s were specific (data not
shown). A search of the transcription factor binding site data bases
for the 3E region (TGGGGGAG) showed high homology to the myeloid zinc
finger (MZF) protein consensus binding sequences (25). Cold MZF
consensus oligonucleotides were synthesized and did not compete with
labeled O3 for the formation DNA-protein complexes (data not shown).
Thus, MZF is unlikely to bind to the region covered by O3. We defined this region as an unknown, or U, region (
270 to
277).

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Fig. 6.
Definition of the U binding site using EMSA.
A, the set of normal and mutant oligonucleotides used.
B, EMSA with each mutant O3 as probe. The conditions were as
in Fig. 3. Lane 1 contains radiolabeled O3 only. Lanes
2-8 contain nuclear extract and the radiolabeled O3 variants O3
and O3A-O3F in the respectively labeled lanes. Binding of each mutant
O3 was shown to be specific by competition assays (data not shown). The
arrows indicate DNA-protein complexes. F, unbound
probe.
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Mutational Analysis--
The function and potential interaction of
the transcription factors that bind to the identified sites across this
region were evaluated by mutagenesis and transfection/expression
analyses (Fig. 7). For these studies,
luciferase was used as the reporter gene, and NS20Y cells were
transfected. Deletion constructs were made to contain
43,
114,
234, and
310 bp 5' and +98 bp 3' to the major transcription start
site (+1). The constructs are arranged in groups to display the effects
of the identified transcription factor binding sites up to the 5' Sp1/U
cluster. The results with the first eight constructs show that
destruction of the most 3' Sp1 site caused a
80% reduction in
activity. Lengthening of the construct from
43 to
234 bp resulted
in small (~15%) increases in absolute promoter activity until the
RORE site was included (~45% increase). Destruction of the RORE
alone reduced activity to nearly those observed with m114, whereas loss
of the Sp1 site alone or together with RORE mutagenesis reduced
activity to that observed with the m43m. These findings indicate that
within the context of the m234 construct the 3' Sp1 is nearly essential
for activity, and RORE adds an additional ~30% of activity. The
sequences between the 5' end of the Sp1 site and
114 have minor, but
consistent, positive effects, although this region did not footprint;
nor are these known transcription factor binding sites in this region. Also, construct m310mC, which contains an additional 5' 76 bp and a
destroyed 3' Sp1 site, gave promoter activity that was ~80% reduced
relative to the wild-type sequence (m310) and about 45% of that for
the shortest construct, m43. These results indicate the major
importance of the most 3' Sp1 (designated D) site to the promoter of
prosaposin.

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Fig. 7.
Mutagenesis of major transcriptionally active
binding sites of the prosaposin promoter. Constructs refer to
Table II where the designations and exact mutagenesis sequences are
indicated and refer to the primers in Table I. The symbols
refer to the designated transcription factor binding sites identified
as participating in transcriptional modulation by DNA footprinting
and/or EMSA (Figs. 2-6). The most 5' region represents three
overlapping Sp1 consensus sequences, the most 3' of which overlaps the
U (unknown) region. Together they constitute the Sp1/U cluster (Fig.
1). The Sp1 sites within m310 from 5' to 3' are labeled as
A, B, C, and D. The
open and closed symbols designate the wild-type
and mutagenized sequences. The mutations obliterate the indicated
binding sites. The horizontal lines represent sequences with
identified (i.e. GATA or AP-1) or no identifiable
transcription factor binding sites that are minor or nonfunctional by
DNA footprinting, EMSA, or expression analysis. The mutagenized
constructs are arranged in groups to highlight the effects of
obliteration of the Sp1D site alone or in the presence or absence of
wild-type or mutant RORE (first eight constructs) or the
effects of systematic destruction of one or more of the 5' Sp1/cluster
sites. This was with the RORE and Sp1D intact (seven
constructs). The next five constructs underwent similar
analyses as the previous seven but with the RORE site destroyed. The
last eight constructs underwent similar analyses with Sp1D
destroyed but with the RORE site intact (5 constructs) or destroyed (3 constructs). For each group, a promoter activity value of 100% was
assigned for the wild-type sequence, and the absolute value is provided
for the wild-type constructs for reference. All absolute values of
luciferase activity were normalized to -galactosidase activity in
lysates from individual co-transfection experiments. GL-2B and GL-2C
are promoterless and SV40 promoter/enhancer-driven plasmid constructs,
respectively, as negative and positive controls for the
experiments.
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The next series of constructs evaluated the effects of the 5' Sp1/U
cluster in the context of intact 3' Sp1 and RORE. For clarity, the
overlapping 5' Sp1 sites have been designed from 5' to 3' A, B, and C. Destruction of the Sp1 and U sites individually or in several
combinations led to increased activity over the wild-type sequence
(m310). The largest increases were found when site Sp1B and -C were
destroyed alone (m310mA) or together with U (m310mD). Obliteration of
Sp1A produced a small increase (m310mS), as did combinations of the
Sp1A, -B, and -C mutagenesis with (m310mG) or without (m310mI)
mutagenesis of U.
Similar analyses were conducted when the RORE site was mutated (m310mK
backbone). Compared with wild-type sequence, the mutation of RORE
enhanced activity, but the presence of additional mutations in the 5'
Sp1/U cluster modulated the observed level and pattern of increased
activity. Within the context of a mutant RORE, individual mutation of
Sp1C with or without Sp1B or U mutagenesis (m310mO or P) in the cluster
produced small decreases (20-50%) in activity. This contrasts with
90-160% increases in the context of a wild-type RORE. Destruction of
the Sp1A, -B, and -C sites (m310mQ) produced a 90% increase in
activity that was similar to that observed in the presence of a
wild-type RORE site. The addition of a mutated U site (m310mR) produced
the same level of activity as in a construct with an intact RORE
(m310mI).
The final series of mutant constructs were made on the m310 backbone
Sp1D destroyed. Compared with m310, this led to a substantial decrease
in overall promoter activity (20-94%). Mutagenesis of all Sp1 sites
and of the U and RORE sites (m310mM) gave absolute basal promoter
activities that were very similar to those in m43m, m114m, and m234m3.
Comparison of m310mC and m310mN shows a 3-fold increase in activity of
m310mN that has a mutagenized RORE. Thus, the m310mN reflects the basal
activity of the 5' Sp1/U cluster and that an intact RORE has a negative
influence. The U region (m310mL) supports promoter activity at a level
that is nearly equivalent to that observed with m114, i.e.
with only Sp1D present. Taken in totality, these results support a
promoter region within the first 310 bp 5' to the transcription start
site that has complex, and mostly negative, interactions between
elements. Each of these promoter elements alone has substantial ability
to promote prosaposin expression and modulates basal promoter activity
on the Sp1D background.
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DISCUSSION |
The multifunctional properties of prosaposin, its intracellular
and extracellular functions, and the temporal and spatial regulation of
its locus provided the rationale for the current studies to define the
elements that facilitate expression of the prosaposin gene. Of
lysosomal loci, prosaposin (14), lysosomal acid lipase (26), and acid
-glucosidase (27) have shown sufficient temporal and spatial
variation in transcript levels to permit differential detection by
in situ hybridization. Thus, elucidation of the control
elements for transcription of the prosaposin locus is essential to
understanding the relationship of this regulation, biological function,
and the modulation of its activities in various tissues. For the
current studies, we have focused on the 310-bp 5' to the
transcriptional start site, since initial analysis in transgenic mice
indicates that this is an important region for promoter activity in the
central nervous system. The present results indicate a potential for
complex regulation based upon the milieu of transcription factors
present in various tissues. The current studies define an essential Sp1
binding site (Sp1D) just 5' to the transcription start site and a 5'
functional Sp1/U cluster (Sp1A, -B, and -C, and an unknown factor (U)),
and RORE. All of these participate in the modulation of prosaposin
transcriptional activity. Furthermore, Sp1 and Sp3 interact at sites in
the Sp1/U cluster to modulate prosaposin transcriptional activity. Sp1, Sp3, and Sp4 bind to the Sp1D site. Other potential binding sites in
this region, i.e. AP-1 and GATA, were nonfunctional and
apparently play little role in the modulation of transcriptional
activity of this locus.
The most 3' Sp1 site, just 16 bp upstream from the major
transcriptional start site, is essential to promoter activity. From EMSA, the Sp1, Sp3, and Sp4 members of the Sp family of transcription factors bind to this region. Although Sp1 and Sp3 are ubiquitously expressed with some temporal and spatial regulation of expression (28),
Sp4 expression appears to be limited to brain and the reproductive
system (29). Thus, the binding of Sp1, Sp3, and Sp4 at the Sp1D site
depends on the transcription factor milieu that may determine the
tissue specific expression of prosaposin. The NS20Y line used here has
cholinergic properties and prosaposin exerts neurite outgrowth effects
in this particular NS20Y subculture (8). Also, a high level of
expression of the prosaposin gene was observed in NS20Y cells by
immunofluorescence analyses.2
Thus, we selected this cell type to assess the specific effects of our
constructs in nervous system tissues, since primary cultures of various
neurons are not clearly adaptable to transfection assays. It appears
that the NS20Y cells contain at least some complement of the endogenous
transcription factors present in specific neurons in the brain and
probably are not representative of all specific neuronal types
throughout the brain substance. Ongoing transgenic analyses in these
laboratories show that the Sp1D site is also essential to prosaposin
expression in transgenic animals (30). Based upon the ubiquitous
expression of Sp1 and Sp3, we would propose that the Sp1D site is
responsible for basal expression of prosaposin in a variety of tissues.
Thus, the basal expression provided by this Sp1 site could be modulated
by varying and competitive Sp3 or Sp4 levels. The Sp1D site would
provide for necessary lysosomal functions of the derived saposins on
glycosphingolipid hydrolases throughout the body. This Sp1 site would
require interaction with other promoter components for tissue-specific
modulation of transcription and activity observed throughout
development and in a variety of cellular types, particularly in the
brain and reproductive system.
The Sp1A, -B, and -C; U; and RORE sites appear to have a primarily
negative regulatory effects on the basal activity of Sp1D in NS20Y
cells. This conclusion is based upon the findings that individual
mutations of these binding sites lead to an enhanced activity of the
reporter gene when the Sp1D site is functional. However, when the Sp1D
site is mutated, these upstream sites have some positive effects on the
reporter gene transcription and can provide a substantial level
expression. Mutation of all of the sites, the 5' Sp1/U cluster, RORE,
and the Sp1D, nearly obliterates activity. The negative modulatory
effects on the Sp1D site of these upstream sites are supported by the
relatively weak protection in DNA footprinting of the most Sp1A, -B,
and -C sites and the U site. Indeed, an explanation for this relatively
weak footprint is the presence of competing transcription factors Sp1
and Sp3, and potentially U, for this region leading to a less clear
footprint than might be anticipated. Strong and very distinct
footprints are obtained in the 5' Sp binding regions with purified Sp1
protein. The EMSA assays clearly show specific binding to sites in this Sp1/U cluster region and competition between Sp1 and Sp3. Although the
Sp1C and U sites overlap, EMSA analysis indicated they have separable
activities. The U site is not an MZF binding site as is evident from
competition assays with cold oligonucleotides. Thus, the transcription
factor binding to the U site remains unidentified. However, the
mutagenesis, supershift, and competition assays support interaction of
Sp1, Sp3, and U in a complex manner for the binding sites at this
upstream region. The mutagenesis of individual Sp1 and the U sites
within this cluster leads to increased promoter activity over the
wild-type sequence. The largest increases were observed with
mutagenesis of the Sp1B and -C sites in the cluster with or without U
mutations. Lower promoter activity was found with two of these cluster
Sp1 sites, with or without U being obliterated. These results indicate
a negative interaction between the factors binding to the Sp1B or -C
sites and the U site in the cluster. The increase in absolute activity
between m234 and m310 shows a positive overall effect of the presence
of this 5' cluster. The RORE has additive effects (~23%) on promoter
activity in the context of the Sp1D site. In constructs containing the
5' Sp1/U cluster, the effects are less than expected. For example,
m310mK increased promoter activity relative to wild type when RORE was mutated; i.e. this indicates a negative interaction between
factors binding at RORE and the 5' Sp1/U cluster. The greater activity with m310mQ than m310mG suggests that negative interaction is primarily
via the Sp1A and -B sites in the cluster, but U is not involved. Thus,
it is likely that the tissue milieu of transcription factors that
compete for these upstream sites could provide a variety of suppressive
interactions for the Sp1D site and modulate the background activity
provided by an unmodulated Sp1D site.
The ROR
nuclear receptor is expressed primarily in neurons of the
brain and provides a tempting target for the neuronal specific expression modulation of prosaposin. In particular, the recent identification of the ROR
gene deletion in the staggerer
mouse indicates the profound effects of the absence of this receptor (31). In the staggerer mouse, Purkinje cell development and migration as well as olivo-pontine neurons degenerate progressively through adulthood (32). Thus, some target sequences for ROR
have a
maintenance and, potentially, trophic effect upon these specific types
of neurons during brain development. The massive destruction of neurons
in the brains of the prosaposin knockout make it difficult to assess
trophic effects of prosaposin deficiency in that animal model, but the
staggerer mouse may provide an interesting model system for
the evaluation of prosaposin trophic effects on cerebellar development
and maintenance.
The mutagenesis and expression studies support a complex model of
prosaposin promoter function within the first 310 bp 5' to the
transcription start site. A model is proposed in Fig.
8 that indicates a basal functional
activity of the 3' Sp1 site and a combination of transcription factor
interactions at the more upstream sites that are primarily negative in
their modulatory functions. The negative effects are principally
mediated by a negative (competitive) interaction of factors binding to
the Sp1/U cluster, since enhanced promoter activity is observed when
any of the sites in this cluster are obliterated. RORE factors also negatively interact with this cluster, since enhanced activity of m310
is apparent when RORE is destroyed. For the Sp1/U cluster, we showed
competition of Sp1 and Sp3. We would propose that such competition
could be extended to Sp4 at the Sp1D site and that the occupancy of
various transcription sites, depending upon the chromatin and DNA
structure in the region, could interfere or interact with each other to
down- or up-regulate the basal Sp1D site function. These studies have
not defined factors that are clearly tissue-specific for the modulation
of prosaposin function in neurons, the reproductive tract, or specific
epithelial cells throughout the body. Our previous in vitro
studies indicate negative regulatory elements within the first 741 bp
5' to the transcription start site and the large first intron (~15
kb) provide additional targets for tissue specificity and/or
facilitator functions of prosaposin expression in more physiologic
systems. However, our preliminary transgenic data (30) show that the
first 310 bp 5' to the start site contains elements that promote
prosaposin expression in neurons of the central nervous system and that
the interaction of the factors described here must have a significant effect on the modulation of regional prosaposin expression in that
organ system.

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Fig. 8.
Proposed model of transcriptional regulation
of the murine prosaposin locus. The binding proteins are shown
below, the binding sites are shown on the
line, and the interactive effects of the binding proteins
are indicated above. The Sp1D site has a basal functional
activity and under normal circumstances is essential for promoter
activity. Sp1, Sp3, and Sp4 proteins can bind to this site
independently. The factors binding to the Sp1A, -B, and -C and U and
RORE interact negatively with each other. This may result from crowding
of the factors or other structural effects. Sp1 and Sp3 proteins can
bind to the 5' Sp1 cluster. Individually, the factors that bind to the
5' Sp1/U cluster and RORE can activate the transcription of the
prosaposin gene even in the absence of Sp1D site. The occupancies of
transcription factor binding sites vary among different tissues, which
make this region drive tissue-preferential expression.
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