(Received for publication, March 3, 1995; and in revised form, May 1, 1995)
From the
The gene for P-selectin glycoprotein ligand (PSGL-1) has been
cloned from a human placenta genomic DNA library. A single intron of
approximately 9 kilobases was found in the 5`-untranslated region and
the complete coding region resides in exon 2. The genomic clone differs
from the cDNA clone isolated from HL-60 cells in that it encodes an
extra copy of the decameric repeat located in the extracellular domain
of PSGL-1. Further analysis indicated that the PSGL-1 genes of HL-60
and U-937 cells contain 15 repeats, whereas the PSGL-1 genes of
polymorphonuclear leukocytes, monocytes, and several other cell lines
contain 16 repeats. Transfection experiments did not indicate a
functional difference between these two variants of PSGL-1. The two
previously observed PSGL-1 mRNA species of 2.5 and 4 kilobases most
likely arise from differential utilization of polyadenylation signal
sequences. The organization of the PSGL-1 gene closely resembles those
of CD43 and human platelet glycoprotein GPIb, both of which have
an intron in the 5`-noncoding region, a long second exon containing the
complete coding region, and TATA-less promoters. The gene for human
PSGL-1, which has been designated SELPLG by the Human Gene
Nomenclature Committee, was mapped to chromosome 12q24 using Southern
blot analysis of DNA from a set of human-mouse cell hybrids, and
fluorescent in situ hybridization on metaphase chromosome
spreads.
P-selectin glycoprotein ligand (PSGL-1) To date little
is known about the organization and attributes of the human PSGL-1 gene (SELPLG). In a previous study, two PSGL-1 mRNA transcripts (a
predominant species of approximately 2.5 kb and a minor 4-kb species)
were identified in PMNs and HL-60 cells(1) . Furthermore,
Southern blot analysis indicated that the PSGL-1 gene is present as a
single copy in the human genome, suggesting that the two mRNAs are
alternative transcripts derived from the same gene, resulting from
either alternative splicing events or differential utilization of
promoter and/or polyadenylation signal sequences(1) . The
presence of multiple mRNA transcripts for PSGL-1 raises questions about
the organization of the PSGL-1 gene, in particular with respect to the
number and placement of introns. Although the coding regions of the
majority of eukaryotic genes are interrupted by introns, a number of
integral membrane proteins lack introns in their coding region. In this
regard, the glycoproteins genes can be divided into two classes. The
first class represents genes whose coding region is contained on one
long exon. Examples of this class are human and mouse CD43 and human
GPIb Another
potentially interesting question concerns the chromosomal localization
of the PSGL-1 gene. The genes for P-selectin, E-selectin, and
L-selectin as well as the L-selectin ligand CD34 all map to chromosome
1(13, 14) . The clustering of the selectins and their
counter-receptor(s) to the same chromosome has obvious evolutionary
implications and therefore it is of interest to ascertain whether
PSGL-1 also maps to chromosome 1. Here we report the isolation, genomic
organization, and chromosomal mapping of the human PSGL-1 gene.
In situ fluorescent hybridization on
metaphase chromosome spreads was done according to the method of
Trask(22) . The PSGL-1 gene fragment XP2.4 was labeled with
digoxygenin-11-dUTP (Boehringer Mannheim) by random
priming(23) . Digital images were obtained using a Nikon
epifluorescence microscope coupled to a cooled CCD camera
(Photometrics). The images were merged and enhanced using the IP Lab
Spectrum image analysis software supported on an Apple Macintosh IIci.
Figure 1:
Organization of the human PSGL-1 gene.
Exon 1 and 2 are represented by boxes, the shaded area represents the coding sequence starting at the ATG codon in exon
2, and the open areas represent the 5`- and 3`-untranslated
regions. The intron and flanking sequences are shown by thick
horizontal lines. Two overlapping genomic clones
Figure 2:
Nucleotide sequence of the human PSGL-1
gene. Position +1 corresponds to the first nucleotide of the
PSGL-1 cDNA (Sako). The sequences shown include 440 nt of 5`-flanking
sequence, exon 1 (nt 1-54), part of the 9-kb intron (lowercase letters), exon 2 (nt 55-2075, including the
complete coding region), and additional 3`-flanking sequences. The
amino acid sequence is shown in the one-letter code. The potential
binding sites for Ets (dashed line) and Sp1 (
Correct splicing of the intron and linking of exons 1 and 2, as
observed in the PSGL-1 cDNA clone derived from an HL-60 library, also
occurs in RNA from different sources. RT-PCR on freshly isolated human
PMN, monocyte, and HL-60 RNA with oligonucleotides spanning the intron
yielded the expected PCR product in all three cases (data not shown).
Figure 3:
Primer extension analysis of the
transcription start site(s) of the PSGL-1 gene. The 5`-end-labeled
primer complementary to nt 27-56 in exon 1 was hybridized to PMN
total RNA (lane 1) and HL-60 poly(A+) RNA (lane
2) and extended with reverse transcriptase as described under
``Experimental Procedures.'' The extended products were
analyzed on a 12% polyacrylamide sequencing gel with
Polyoma/DdeI fragments as size markers. Major bands are
indicated by arrows.
Additional mapping
of the minor 4-kb mRNA species was performed by Northern blot analysis
of HL-60 poly(A
Figure 4:
Northern blot analysis of PSGL-1 mRNAs.
Northern blots containing 3 µg of HL-60 poly(A
Figure 5:
PCR analysis of the repeat region in
PSGL-1. The repeat region of PSGL-1 was amplified by RT-PCR of mRNA
templates or PCR of cloned and genomic DNA templates as described under
``Experimental Procedures.'' The templates analyzed include:
cDNA clone pPL85 (lane 1), genomic clone
There does not appear to be a direct functional role
for the extra decameric repeat. The extra repeat was introduced into
the PSGL-1 cDNA expression vector pPL85, generating pPL85-R16. COS
cells cotransfected with vectors encoding the
Fluorescent in situ hybridization on metaphase chromosome
spreads was done with a 2.4-kb PSGL-1 genomic DNA probe (XP2.4 in Fig. 1) and the results are shown in Fig. 6. The double
fluorescent signals on both chromosomes were found only at 12q24 in 17
out of 20 metaphase spreads (85%) examined and on no other chromosome.
Three out of 20 spreads showed double fluorescent signals on only one
chromosome. These results localize SELPLG to 12q24.
Figure 6:
Mapping of the human PSGL-1 gene by in
situ hybridization. Metaphase chromosome spreads were hybridized in situ with the genomic PSGL-1 probe XP2.4. A partial spread
is illustrated. Double fluorescent signals are indicated by arrows.
A single 23-kb human PSGL-1 gene was isolated on two
overlapping clones derived from a human placenta genomic library. No
clones were isolated from any additional genomic loci, consistent with
the existence of a single human PSGL-1 gene and in agreement with
previously described Southern hybridization analysis of human genomic
DNA(1) . The human PSGL-1 gene contains a single intron of
approximately 9 kb located 5 bp upstream of the ATG initiation codon,
and the complete coding region is contained within exon 2. In contrast,
many members of the mucin gene family, such as glycophorin, episialin,
or MUC-1, and the L-selectin ligands CD34 and GlyCAM-1, are encoded by
multiple exons(9, 11, 12, 25) . Although
unusual, the lack of introns in the coding region has been reported for
other integral membrane proteins, such as the human and mouse
sialomucin CD43 and the human blood platelet membrane glycoprotein
GPIb Analysis of possible transcription
initiation sites also indicated features shared between the PSGL-1 gene
and the genes for CD43 and GPIb The two
previously identified PSGL-1 mRNA transcripts of 2.5 and 4 kb appear to
arise from the single gene by differential utilization of
polyadenylation sequences. Analysis of the 3` end of PSGL-1 mRNAs
indicated that the major mRNA species utilizes the polyadenylation
signal at position 2075. This finding (together with the 5` start
mapping data) would predict a mRNA species of approximately 2.1 kb, not
including the poly(A) tail. This is in agreement with the major PSGL-1
mRNA on Northern blots which was sized at approximately 2.5
kb(1) . The minor 4-kb PSGL-1 mRNA hybridized to sequences
located 3` of the polyadenylation signal at position 2075, suggesting
that the size difference of the two mRNA species may primarily arise
from utilization of different polyadenylation sequences. A similar
situation has been reported for the 1.9- and 4.3-kb mRNA species
observed for CD43(35) . The PSGL-1 gene in HL-60 and U-937
cells encodes 15 repeats of a 10-amino acid consensus
sequence(1) . This is in contrast to our human placenta genomic
PSGL-1 clone, as well as the PSGL-1 in all other cells and cell lines
tested, including the recently reported leukocyte PSGL-1
cDNA(5) , which contain 16 repeats. However, the number of
consensus repeats does not appear to correlate with PSGL-1 function.
HL-60 and U-937 cells bind P-selectin as do freshly isolated PMNs,
monocytes, and THP-1 cells, all of which contain 16 copies of the
repeat. In contrast, the lymphocytic cell lines Ramos, CEM, MOLT-4, and
SB, which also express PSGL-1 with 16 repeats, do not bind
P-selectin. Mapping of the PSGL-1 gene to chromosome 12 prompted a search of the
genome data base for colocalized disease markers or genes. Although the
genes for interferon- In summary, the
organization of the PSGL-1 gene is strikingly similar to those of CD43
and platelet glycoprotein GPIb
The table is compiled from
Southern analysis of 29 mouse-human hybrids as described under
``Experimental Procedures.'' Scoring was determined by the
presence (+) or absence (-) of a human PSGL-1 band on the
blots compared with the presence or absence of human chromosomes in
each hybrid. A 0% discordance indicates matched segregation of the
probe with a human chromosome.
(
)<
/a>is the high affinity counter-receptor for P-selectin
on myeloid cells and stimulated T
lymphocytes
(
)(1
, which also share several other common features such as a
promoter lacking the typical TATA box(6, 7, 8) .
The second class represents genes whose coding region consists of
multiple exons, and the promoters of these genes generally (albeit with
a few exceptions) resemble the typical eukaryotic promoter containing a
TATA consensus sequence. Examples of this class include glycophorin,
CD4, and the L-selectin ligands GlyCAM-1 and
CD34(9, 10, 11, 12) .
Cells and Cell Lines
The tumor cell lines HL-60,
U-937, THP-1, Ramos, CEM, MOLT-4, and SB were obtained from the
American Type Culture Collection and maintained in RPMI 1640 containing
10% fetal calf serum (Sigma). Human polymorphonuclear leukocytes (PMNs)
were isolated from whole blood by centrifugation over a Mono-Poly
Resolving Medium Ficoll-Hypaque density gradient (ICN Biomedicals,
Costa Mesa, CA). Peripheral blood mononuclear cells were isolated from
heparinized blood of normal donors by centrifugation over a
Ficoll/Hypaque density gradient (Sigma), and monocytes were
subsequently isolated by adherence to plastic.Isolation and Sequencing of Genomic Clones Encoding Human
PSGL-1
A human placenta genomic library in the Fix II
vector (Stratagene, La Jolla, CA) was plated at
20,000
plaques/plate, and nitrocellulose replicas of the plates were
hybridized according to standard procedures(15) . The PSGL-1
cDNA probe comprising nucleotides (nt) 60-389, was amplified by
polymerase chain reaction (PCR) and labeled to a specific activity of 5
10
cpm/µg with [
P]dCTP
(Amersham Corp.) using the random-primed DNA labeling kit (Boehringer
Mannheim). Filters were hybridized for 16 h at 65 °C with the
labeled probe in 6
SSC, 10
Denhardt's solution,
0.1% SDS, and 50 µg/ml denatured, sheared salmon sperm DNA. The
filters were washed three times for 30 min in 0.3
SSC, 0.1% SDS
at 65 °C and exposed to film. Duplicate positive plaques were
rescreened until purified plaques were obtained, and the
DNAs
were prepared using a Qiagen lambda kit (Qiagen, Chatworth, CA). Ten
independent
clones were obtained and rescreened with PSGL-1
sequence-specific oligonucleotide probes, which were end-labeled using
[
-
P]ATP and T4 polynucleotide kinase.
Clones
2 and
4 were characterized by restriction mapping, and
suitable restriction fragments were subcloned into the pBluescript
vector (Stratagene). Subclones containing PSGL-1 gene exons and
flanking regions were sequenced using the Applied Biosystems Taq DyeDeoxy Terminator Kit and ABI 373 autosequencer(16) . DNA
sequences were analyzed using the Sequencher software package (Gene
Codes, Ann Arbor, MI).
RNA Isolation and Northern Blot Analysis
Total RNA
was purified using the guanidine isothiocyanate/cesium chloride cushion
method(15) . Poly(A) mRNA was purified using
the PolyATtract® mRNA isolation system II (Promega, Madison, WI).
RNA was fractionated by 1.0% formaldehyde-agarose gel electrophoresis
and transferred to nitrocellulose via capillary blotting. The PSGL-1
cDNA or gene fragments to be used as probes were amplified by PCR and
labeled to a specific activity of at least 1
10
cpm/µg with [
P]dATP and
[
P]dCTP and hybridized to filters as described
above.
Rapid Amplification of cDNA Ends (RACE)
Rapid
amplification of cDNA 5` and 3` ends was performed as described by
Innis et al.(17) . Briefly, three primers were used for
the 3`-RACE experiment. The dT-adapter, a 35-mer
containing an oligo(dT) stretch (17 residues) linked to a unique 18-nt
adapter, was used to prime reverse transcription of total RNA isolated
from PMNs, using the AMV Reverse Transcriptase System (Life
Technologies, Inc.). The 18-nt adapter primer and a PSGL-1-specific
primer, a 19-mer corresponding to nt 1564-1582, were subsequently
used to amplify the cDNA by PCR. PCR products were analyzed on a 1%
agarose gel. For 5`-RACE experiments, HL-60 poly(A
)
RNA and PMN total RNA were reverse-transcribed using a PSGL-1-specific
18-mer (antisense to nt 294-311 in exon 2). The first-strand
reaction products were extended with dATP and terminal transferase and
subsequently amplified by PCR using a mixture of the
dT
-adapter and the 18-nt adapter as the 5` primers and a
PSGL-1-specific 21-mer (antisense to nt 243-263) as the 3`
primer. The PCR products were analyzed on a 1.5% agarose gel and
identified by Southern hybridization with a PSGL-1 oligonucleotide
probe corresponding to nt 1-14 in exon 1.
Primer Extension Mapping
Primer extension was done
according to described methods(15) . In brief, a 30-mer
5`-ACCACCGTGCTCAGCAGAGCATGGGACAGC-3` (antisense to nt 27-56 in
exon 1) was end-labeled to a specific activity of 8 10
cpm/pmol as described above. Approximat
ely 5 ng of labeled primer
was hybridized to 3 µg of poly(A
) RNA (HL-60) or 3
µg total RNA (PMNs) at 48 °C overnight. The extension reaction
was done using the AMV Reverse Transcriptase System (Life Technologies,
Inc.) with 20 units of AMV reverse transcriptase (Life Sciences, St.
Petersburg, FL) for 2 h at 48 °C. The extension products were sized
by electrophoresis on a denaturing 12% polyacrylamide gel using
Polyoma/DdeI fragments as size markers.
PCR Analysis of the Repeat Region
The number of
repeats in PSGL-1 from different cell types was determined by PCR using
primers that flank the repeat region. The 5` primer corresponds to nt
381-400, and the 3` primer is antisense to nt 880-903 of
the PSGL-1 gene, resulting in PCR products of 522 bp and 492 bp,
representing 16 and 15 copies of the repeat, respectively. mRNA
templates were analyzed by reverse transcription PCR (RT-PCR), using
the reverse transcriptase reaction described above to generate a cDNA
template for PCR amplification. The PCR reactions contained 1 ng
of cDNA or genomic DNA template and were performed according to
standard procedures (17) using Taq DNA polymerase
(Promega). The PCR products were sized by electrophoresis on a 2.5%
NuSieve (FMC Bioproducts, Rockland, ME), 0.5% agarose gel.
Chromosomal Mapping of the PSGL-1 Gene
(SELPLG)
Mapping of the human PSGL-1 gene was achieved by
probing Southern blots of DNAs from 29 mouse-human somatic cell hybrids
that were made from 17 unrelated human cell lines and 4 mouse cell
lines(18, 19, 20) . The hybrids were
characterized by karyotypic analysis and by mapped enzyme markers (18,
20). Chromosome translations with no intact chromosome present were not
tabulated for the percent discordance. Cell hybrid DNA was digested
with XbaI and subjected to Southern analysis using the P-labeled PSGL-1 cDNA probe comprising nt 60-389
(21). Control DNA from human and mouse gave a single human XbaI hybridizing fragment of 8 kb, with no cross-hybridization
with mouse DNA.
Isolation and Genomic Structure of the Human PSGL-1
Gene
The PSGL-1 gene was isolated by hybridization of a human
placenta genomic library using a P-labeled cDNA probe
comprising nucleotides 60-389 of PSGL-1(1) . Ten
independent genomic clones were isolated after screening approximately
10
phages. Hybridization of these 10 clones with a
P-labeled 14-mer derived from the 5` end of the PSGL-1
cDNA revealed that only two clones were positive, suggesting that a
large intron interrupted the PSGL-1 gene separating the sequences
defined by the two probes. Two overlapping clones, denoted
2 and
4, were found to contain inserts of approximately 13 and 16 kb,
respectively, and were selected for further fine mapping of the gene.
The
2 clone, which hybridized only to the PSGL-1 probe used in the
primary screen, and the
4 clone, which hybridized with both
probes, were digested with HindIII, PstI, or XbaI and subcloned into the pBluescriptII vector. By this
procedure several subclones, designated pPst4.5A, pPst4.5B, pHind5.0,
pHind5.2, and pXba8, were generated (Fig. 1). Restriction enzyme
mapping and hybridization of the generated fragments with
oligonucleotides derived from the 5` terminus (nt 1-14) and 3`
terminus (nt 1636-1649) of the PSGL-1 cDNA clone further
established the genomic map for human PSGL-1 as presented in Fig. 1. Indeed, the PSGL-1 gene contains an approximately 9-kb
intron inserted in the 5`-noncoding region between nt 54 and nt 55,
based on the numbering system of the original PSGL-1 cDNA
clone(1) . The coding region which starts at nt 60 is fully
encoded by exon 2 and does not contain any introns (Fig. 2).
4 and
2
are shown with restriction maps for HindIII (H) and XbaI (X), respectively. The subclones pHind5.2,
pHind5.0, pPst4.5A (containing sequences derived from the
4 clone
and the
phage arm), pPst4.5B, and pXba8 were generated. The
2.4-kb fragment XP2.4, which contains exon 2, is
indicated.
) are indicated. The extra repeat encoding 10 amino acids
is boxed. The major polyadenylation signal is underlined and the Alu sequence is indicated between
brackets.
Preliminary Analysis of the Transcription Initiation
Region of the Human PSGL-1 Gene
The putative transcription
initiation region of the PSGL-1 gene was defined by 5`-RACE and by
primer extension on RNA isolated from human PMNs and HL-60 cells.
5`-RACE was performed with a primer complementary to nt 243-263
in exon 2 as described under ``Experimental Procedures.''
Hybridization of the PCR products with an oligonucleotide probe derived
from exon 1 identified a broad band of 280-340 bp from PMN as
well as HL-60 RNA templates (data not shown). Considering that the
requisite dT adapter is 35 nt long, this tentatively
places the transcription initiation region between -42 and
+18. Primer extension analysis of human PMN and HL-60 RNA was
performed using a 30-mer complementary to nt 27-56 in exon 1.
Several primer extension products were observed including two
predominant bands between 55 and 70 nt in length, suggesting initiation
from multiple sites in the region from -14 to +1 (Fig. 3). The region immediately upstream from these
transcriptional start sites contains no TATA or CAAT box, but is
GC-rich and contains putative binding sites for the regulatory elements
Sp1 and Ets (Fig. 2).
Analysis of the 3` Termini of Human PSGL-1
mRNAs
Two PSGL-1 transcripts, a predominant mRNA of
approximately 2.5 kb and a minor 4-kb mRNA, have previously been
identified in PMNs and HL-60 cells(1) . The 3`-noncoding region
of the PSGL-1 gene contains a polyadenylation signal at position 2075
as indicated in Fig. 2. Mapping of the PSGL-1 mRNA species from
PMNs by 3`-RACE with an oligonucleotide corresponding to nt
1564-1582 identified a predominant PCR product of approximately
520 bp, indicating that the major PSGL-1 mRNA species utilizes this
polyadenylation signal sequence (data not shown).) RNA with two different PSGL-1 probes. Fig. 4shows that the PSGL-1 cDNA probe, comprising nt
60-389, hybridized with the 2.5-kb mRNA and also (albeit very
weakly in this exposure) with the 4-kb mRNA (lane A). The
genomic DNA probe, comprising nt 2080-2459 located 3` of the
polyadenylation signal, only hybridized to the 4-kb mRNA (lane
B), suggesting that the 4-kb mRNA utilizes a downstream, yet
unidentified, polyadenylation signal sequence. The region of the PSGL-1
gene that is unique for the 4-kb transcript contains an Alu sequence (24) as shown in Fig. 2.
)
RNA/lane were hybridized under high stringency conditions with a
P-labeled PSGL-1 cDNA probe comprising nt 60-389 (A) or a PSGL-1 genomic DNA probe comprising nt
2081-2460 (B). The major 2.5-kb and minor 4-kb PSGL-1
mRNAs are indicated.
An Extra Repeat Unit is Encoded in the PSGL-1 Gene of
Many Cell Types
The PSGL-1 cDNA clone (pPL85) derived from an
HL-60 cDNA library encodes 15 consecutive repeats of a 10-amino acid
consensus sequence(1) . Our genomic clone derived from human
placenta, however, contains an extra sequence of 30 nucleotides (boxed in Fig. 2) located in the second repeat unit and
thus encodes 16 such repeats. RT-PCR analysis with primers spanning the
repeat region allowed for rapid analysis of the number of repeats in
PSGL-1 mRNA from different cell types. Similarly, the number of repeats
in PSGL-1 genomic DNA from different sources was determined by PCR
analysis. Fig. 5shows that freshly isolated HL-60 RNA contains
15 repeats (lane 3) similar to the original PSGL-1 cDNA clone
pPL85 (lane 1), eliminating the possibility that the cDNA
clone had lost one repeat due to a cloning artifact. Identical results
were obtained for RNA isolated from the monocytic cell line U-937 (lane 7). In contrast, 16 repeats were found in the genomic
clone 4 (lane 2) and in mRNA isolated from human PMNs (lane 5) and monocytes (lane 6) and several human
cell lines, such as THP-1 (lane 8) and Ramos, CEM, MOLT-4, and
SB (not shown). To investigate whether splicing was responsible for
this phenomenon, genomic DNA isolated from HL-60 cells was analyzed by
PCR. Fig. 5(lane 4) shows that the HL-60 PSGL-1 gene
encodes just 15 repeats. Similarly, the PSGL-1 gene of U-937 cells only
encodes 15 repeats (data not shown). Sequence analysis of the repeat
region in the PSGL-1 gene from U-937 cells confirmed that the U-937 and
HL-60 PSGL-1 genes are identical and are therefore lacking the same
repeat unit.
4 (lane
2), HL-60 RNA (lane 3), HL-60 genomic DNA (lane
4), human PMN RNA (lane 5), human monocyte RNA (lane
6), U-937 RNA (lane 7), and THP-1 RNA (lane 8).
The PCR products representing 16 or 15 copies of the repeat sequence
were separated by agarose gel
electrophoresis.
(1,3/1,4)-fucosyltransferase gene, and pPL85 or pPL85-R16 bound
equally well to P-selectin in vitro (data not shown).
Chromosomal Localization of the Human PSGL-1 Gene
(SELPLG)
Human chromosomal localization of the PSGL-1 gene was
performed in a two-step procedure employing analysis of a human-mouse
somatic cell hybrid panel and chromosomal fluorescent in situ hybridization. Southern analysis of XbaI-digested DNA
from 29 human-mouse hybrids with the human PSGL-1 cDNA probe comprising
nt 60-389 detected a single 8-kb fragment in human DNA. This
human PSGL-1 probe did not cross-hybridize with mouse DNA. The results
in Table Ishow that only chromosome 12 gave 0% discordance,
indicating that SELPLG localizes to chromosome 12.
(6, 7, 8) . These genes, like the
PSGL-1 gene, lack introns in the coding region yet also have an intron
in the 5`-untranslated region.
. Primer extension and 5`-RACE
experiments were employed to identify the transcription initiation
sites of the PSGL-1 gene in PMNs and HL-60 cells. These preliminary
analyses indicated that transcription may initiate from multiple sites
just upstream of exon 1. Our data does not exclude the possibility that
transcription of the PSGL-1 gene originates from other promoters as
well, since those 5`-RACE PCR products would fail to hybridize with the
exon 1 probe. A complete understanding of possible other promoter usage
will require sequence analysis and mapping of cloned RACE PCR products.
The PSGL-1 promoter region tentatively identified in this study lacks
consensus sequences such as TATA and CAAT, which are general components
of eucaryotic promoters. Several features of the PSGL-1 promoter, e.g. absence of TATA and CAAT boxes, multiple transcription
initiation sites, high G + C content (55%), and a potential Sp1
binding site, resemble those of ``housekeeping''
genes(26) . However, several tissue-specific promoters that lack
TATA and CAAT boxes, but are nonetheless regulated, have been described
recently, including the promoters for human GPIb
and
CD43(8, 27, 28) . Other examples of TATA-less
promoters include those for the integrins CD11a and CD11b and for
CD4(29, 30, 31) . Potential binding sites for
transcriptional activators, such as Sp1, PU.1, Ets, and AP-2, have been
identified in these promoter regions and are thought to direct
tissue-specific gene expression(30, 31) . The PSGL-1
promoter has potential binding sites for Sp1 and Ets and therefore may
very well fall in this category of promoters(32, 33) .
Indeed PSGL-1 expression appears to be regulated in a cell
type-specific manner, since PSGL-1 transcripts have been found in
myeloid and lymphocytic cells
but not in the hepatoblastoma
cell line HepG2 (1) or the human lung fibroblast line
MRC-5.
(
)This is consistent with obser
vations by
others that cell surface PSGL-1 can be detected on leukocytes, but not
on fibroblasts and keratinocytes(5, 34) .
Moreover, COS cells cotransfected with
(1,3/1,4)-fucosyltransferase and recombinant PSGL-1 with either 15
or 16 copies of the repeat exhibited indistinguishable binding to
P-selectin. The PSGL-1 gene in the promyelocytic leukemia cell line
HL-60 and the histiocytic lymphoma cell line U-937 are lacking the same
copy of the 10-amino acid repeat in both alleles, yet these cell lines
are unrelated. It is not clear how this loss of a repeat has occurred
in the PSGL-1 genes in both cell lines, although this may have been
caused by some form of polymorphic variation. Other examples of
polymorphic and allelic variations in the repeat regions of mucin-type
glycoproteins, such as the carcinoma-associated mucin episialin and
human intestinal mucin, have been reported(36, 37) .
, protein phosphatase 1, and aldehyde
dehydrogenase 2 also map to 12q24, the search did not reveal any
readily apparent phenotypes that could be attributed to abnormal PSGL-1
expression(21, 38, 39) . The chromosomal loci
for several other mucin-type glycoproteins and cell adhesion molecules
have been mapped and appear to be clustered. In particular, the genes
that encode the selectin family of leukocyte adhesion molecules are
clustered on chromosome 1(13) . The gene for P-selectin (SELP) maps to chromosome 1q21-24, as do genes for
L-selectin (SELL), E-selectin (SELE), and
episialin(13, 40) . In addition, CD34, a ligand for
L-selectin, also maps to chromosome 1q(14) . The PSGL-1 gene,
however, does not map to this cluster of selectin and selectin ligand
genes, and neither is the PSGL-1 gene linked to the organizationally
related genes for CD43 and GPIb
. The gene for human CD43 is
located on chromosome 16p11.2, close to the genes encoding the
subunits of the leukocyte integrins LFA-1, Mac-1, and p150,95, and
human GPIb
maps to chromosome
17p12-ter(29, 41, 42) .
. Each of these genes contains a
single intron in the 5`-untranslated region and a long exon containing
the complete coding region. The promoter regions lack the typical TATA
and CAAT elements, and the genes are flanked by Alu sequences.
Although PSGL-1, CD43, and GPIb
are all sialic acid-carrying O-glycosylated proteins, their amino acid sequences are
unrelated and their genes do not appear to be linked in the genome.
Table: Segregation of PSGL-1 with human chromosomes in
XbaI-digested human-mouse cell hybrid DNA
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U25955 [GenBank]and U25956[GenBank].