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HEMATOPOIESIS
Localization of distal regulatory domains in the megakaryocyte-specific platelet
basic protein/platelet factor 4 gene locus
Chunyan Zhang, Michael A. Thornton, M. Anna Kowalska, Bruce S. Sachis, Michael Feldman,
Mortimer Poncz, Steven E. McKenzie, and Michael P. Reilly
The genes for the related human (h) chemokines, PBP (platelet basic protein) and
PF4 (platelet factor 4), are within 5.3 kilobases (kb) of each other and form a
megakaryocyte-specific gene locus. The
hypothesis was considered that the PBP
and PF4 genes share a common distal
regulatory region(s) that leads to their
high-level megakaryocyte-specific expression in vivo. This study examined
PBP and PF4 expression in transgenic
mice using 4 distinct human PBP/PF4
gene locus constructs. These studies
showed that within the region studied
there was sufficient information to regu-
late tissue-specific expression of both
hPBP and hPF4. Indeed this region contained sufficient DNA information to lead
to expression levels of PBP and PF4
comparable to the homologous mouse
genes in a position-independent, copy
number–dependent fashion. These studies also indicated that the DNA domains
that led to this expression were distinct
for the 2 genes; hPBP expression is regulated by a region that is 1.5 to 4.4 kb
upstream of that gene. Expression of
hPF4 is regulated by a region that is
either intergenic between the 2 genes or
immediately downstream of the hPF4
gene. Comparison of the available human
and mouse sequences shows conserved
flanking region domains containing potential megakaryocyte-related transcriptional factor DNA-binding sites. Further
analysis of these regulatory regions may
identify enhancer domains involved in
megakaryopoiesis that may be useful in
the selective expression of other genes in
megakaryocytes and platelets as a strategy for regulating hemostasis, thrombosis, and inflammation. (Blood. 2001;98:
610-617)
© 2001 by The American Society of Hematology
Introduction
Platelet basic protein (PBP) and platelet factor 4 (PF4) are
related, platelet-specific chemokines that are expressed at high
levels during megakaryopoiesis and stored in platelet ␣-granules.1 They represent 3% to 5% of total protein releasate from
platelets on a molar basis. PBP is N-terminally cleaved to
␤-thromboglobulin (␤-TG) and then to neutrophil-activating
peptide-2 (NAP-2).2-4 This final product binds and activates the
chemokine receptor CXCR2 on neutrophils.5 What role this
chemokine has in thrombosis and inflammation is unclear.
Unlike PBP and other chemokines, PF4 appears to bind mostly
to large, negatively charged molecules such as heparin, and
though many biologic functions have been attributed to PF4, its
true role in vivo is unknown.6-9
Although the biologic roles of these chemokines need further
investigation, their genes offer an opportunity to understand
megakaryocyte-specific expression. Like many other genes
encoding chemokines, both the PBP and PF4 genes are encoded
by 3 exons, and are preceded by a TATA box in the immediate
5⬘-flanking region.10,11 Transient transcriptional studies with the
immediate 5⬘-flanking region defined a PU.1-binding promoter
region upstream of the PBP gene,12 similar to a thrombopoietininduced enhancer domain upstream of the platelet-specific
[alpha]IIb gene.13 Several silencers and promoter domains have
been similarly defined in the immediate 5⬘-flanking region of the
PF4 gene, including one GATA-1 binding site.14,15 Reporter
gene constructs with 245 bp of the immediate 5⬘-flanking region
of the hPF4 gene showed that this region contained sufficient
information to drive tissue-specific expression of a LacZ
reporter construct16 and that 1.1 kb of rat PF4 promoter can
drive vigorous tissue-specific expression.17 However, neither of
these constructs had sufficient information to drive positionindependent, copy number–dependent expression.
The genes for PBP and PF4 are closely linked on chromosome 4 with the PBP gene upstream and both transcripts in the
same 5⬘ to 3⬘ orientation.18-20 Because the 2 genes are both
expressed at high levels in developing megakaryocytes, we
asked whether the 2 genes are regulated by a common distal
regulatory region, similar to those located flanking the ␣- and
␤-globin gene loci that are involved in erythroid-specific
expression.21 To address these issues, we made a series of
transgenic mice with various lengths of the hPBP or the hPF4
genes (or both). Our studies localized distinct regions involved
in achieving high level, copy number–dependent expression, at
least one for each gene. These potential regulatory regions
contain highly conserved domains between human and mouse,
which were found to harbor consensus DNA binding sequences
of known megakaryocyte-specific transcription factors.
From the The Children’s Hospital of Philadelphia, Department of Pediatrics and
Pathology and Laboratory Medicine at the University of Pennsylvania, and the
Cardeza Foundation for Hematologic Research, Departments of Medicine and
Pediatrics, Jefferson Medical College, Philadelphia, Pennsylvania.
Reprints: Chunyan Zhang, The Children’s Hospital of Philadelphia, Abramson
Research Center, Rm 314, 34th St and Civic Center Blvd, Philadelphia, PA
19104; e-mail: [email protected].
Submitted October 24, 2000; accepted March 15, 2001.
Supported in part by grants from the American Society of Hematology (to C.Z.), the
National Institutes of Health HL40387 (to M.P.), HL40387 (to S.E.M.), and HL61865
(to S.E.M. and M.P.R.), and the Nemours Foundation (to S.E.M. and M.P.R.).
610
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
Materials and methods
DNA clones studied
The HindIII fragment used to make the Short-PBP transgenic animals was
isolated from a ␭ phage EMBL3A clone that had been previously
characterized by us.10 The Long-PBP and ␭-PBP/PF4 were also isolated
from this same clone as depicted in Figure 1. PF4-Only was isolated as an
EcoRI fragment from a P1 genomic clone (Genome Systems, St Louis, MO).
The mouse (m) PF4 gene was isolated from mouse 129 SV ␭ phage
genomic library (Stratagene, La Jolla, CA) using a short region of mPF4
flanking sequence generously provided by Katya Ravid (Boston University,
Boston, MA) by standard phage library screening techniques as previously
described by us.10,20 Universal T7 primer as well as sequence-specific
primer was used to complete the sequence. Additional 5⬘ and 3⬘ sequences
were obtained from direct sequencing of an mPBP/PF4⫹ BAC clone
isolated by Genome Systems. Dot-matrix homology plot analysis was done
by comparing our sequences of the hPBP/PF4 gene locus and supplemental
sequences contained within the GenBank public database at www.ncbi.nih.gov, with our complete sequences of the mPBP/PF4 gene locus. Comparative analysis was performed using the DNAsis-Mac version 2.0 (Hitachi
Software Engineering, San Bruno, CA) on an Apple Macintosh G4
(Cupertino, CA). Length of comparison was set to 20 bp with a 14-bp
match. Analysis of the conserved domains for sequences for consensus
GATA, NF-E2, and Ets binding sites was done using Transcription Element
Search Software (TESS) developed by the University of Pennsylvania
Computational Biology Informatics Laboratory (Philadelphia, PA).
Establishment of transgenic mice
REGULATED EXPRESSION OF PBP AND PF4 GENES
611
inserts using a Qiaex gel extraction kit (Qiagen, Valencia, CA). The DNAs
were then precipitated with ethanol and resuspended in Elutip buffer and
passed through Elutip columns (Schleicher & Schuell, Keene, NH).
Transgenic mice were generated by pronuclear injections following standard methods at the University of Pennsylvania Transgenic Mice Core
Facility. Genomic DNA was isolated from mouse tails using QIAamp DNA
Mini Kit (Qiagen). Positive animals were detected by polymerase chain
reaction (PCR) analysis of tail DNA using primer pairs for hPF4 or hPBP or
both described below and carried out under the following conditions for 30
cycles with Taq polymerase: denaturing 94°C for 25 seconds, annealing
53°C for 40 seconds, and extension at 72°C for 50 seconds. Amplified
products were size fractionated on an agarose gel, ethidium stained, and
visualized with UV light.
Positive founder lines were also analyzed by Southern blots as
previously described by us.20 Genomic DNA was digested with EcoRI and
separated on a 0.8% (wt/vol) agarose gel along with 5 and 10 pg unlabeled
probe DNA as controls. Two probes were radiolabeled using a Random
Primers Labeling Kit (Boehringer Mannheim, Indianapolis, IN). One was a
1.8-kb hPBP genomic fragment to detect the transgene and the other 1.3 kb
fragment was an m␣IIb fragment used as a positive mouse genomic control.
The filter was washed in 2 ⫻ sodium chloride sodium citrate (SSC)/0.1%
(wt/vol) (sodium dodecyl sulfate [SDS]) at room temperature, 0.2 ⫻
SSC/0.1% (wt/vol) SDS at 65°C, and finally 0.1 ⫻ SSC/0.1% (wt/vol) SDS
at 65°C, and then exposed on a Phosphorimaging screen (Molecular
Dynamics, Sunnyvale, CA). The intensity of bands on film was analyzed by
Imagequant PhosphorImager software (Molecular Dynamics). The copy
number was determined by comparing the intensity of transgene hPBP band
to the genomic control m␣IIb band. All studies were approved by the
Animal Care and Use Committee of the Children’s Hospital of Philadelphia, the University of Pennsylvania, and Thomas Jefferson University.
Cesium chloride gradient purified plasmid and ␭ clone DNAs were cut with
appropriate restriction enzymes and run on TAE agarose gel to recover
Reverse transcription–polymerase chain reaction analysis
Figure 1. Characterization of the human and mouse PBP/PF4 gene locus and
transgenic constructs. (A) A dot-matrix comparison of the 2 gene loci calculated at
20 bp with 14-bp match. The exons/introns are shown in gray and the arrows
indicating the 5⬘33⬘ transcription orientation. (B) A stick figure of the human
PBP/PF4 gene locus in humans is shown at the top with a partial restriction map. The
constructs used in these studies including the nomenclature are shown. Their copy
number per haploid genome is also indicated. H indicates HindIII; S, Sal I; E, EcoRI.
The arrow indicates the direction of transcription.
Tissues examined for RNA expression included the brain, lung, thymus,
heart, liver, spleen, small intestine, adrenal, kidney, testes, skeletal muscle,
marrow, and purified platelets. Other than the platelets, tissues were
obtained after sacrificing the animals by cervical dislocation. The organs
were repeatedly rinsed with normal saline to remove residual blood. Tissues
were homogenized in RNA Stat-60 (Tel-Test, Friendswood, TX) using a
pellet pestle (Kontes Glass, Vineland, NJ). RNA samples were then
extracted with chloroform and precipitated in isopropanol. The final RNA
pellet was dissolved in water.
Blood was drawn from mice by cardiac puncture and spun at 200g for
10 minutes to obtain platelet-rich plasma (PRP). The platelets were
obtained by centrifugation at 800g for 10 minutes. Platelet RNA were
isolated using RNA STAT-60 as described above. Reverse transcription–
polymerase chain reaction (RT-PCR) was done using the Superscript II
Reverse Transcriptase Kit (Life Technologies, Gaithersburg, MD) following the procedures outlined by the manufacturer (which includes no
RT-enzyme control samples). RT-PCR used the following sets of sense/
antisense primers:
hPBP: 5⬘-ATGAGCCTCAGACTTGATAC-3⬘/5⬘-ATCAGCAGATTCATGACCTG-3⬘10
mPBP: 5⬘-GCCTGCCCACTTCATAACCTC-3⬘/5⬘-GGGTCCAGGCACGTTTTTTG-3⬘
hPF4: 5⬘-ATCGCACTGAGCACTGAGATC-3⬘/CTATATAGCAAATGCACACACG-3⬘11
mPF4: 5⬘-GTCCAGTGGCACCCTCTTGA-3⬘/5⬘-AATTGACATTTAGGCAGCTGA-3⬘
m␣IIb: 5⬘-GGCTGGAGCACACCTATGAGCT-3⬘/5⬘-CTCAACCTTGGGAGATGGGCTG-3⬘22
mHPRT: 5⬘-CACAGGACTAGAACACCTGCH-3⬘/5⬘-GCTGGTGAAAAGGACCTCT-3⬘23
The use of RT-PCR to obtain quantitative data regarding the expression
of the transgene relative to an endogenous gene requires comparison of
products at points when both are in the linear range of amplification. For
quantitative studies, the antisense primers were 5⬘-labeled with fluorescent
dye Cy-5 (Integrated DNA Technologies, Coralville, IA). Preliminary
experiments showed that the human and mouse PCR products were in the
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
ZHANG et al
linear range of amplification between cycles 14 to 20. Therefore, PCR
reaction mixtures were typically divided into several 15-␮L aliquots before
initiation of the reaction. PCR was performed at 94°C for 2 minutes,
followed by cycles at 94°C for 25 seconds, 50°C for 40 seconds, and 72°C
for 40 seconds in a PTC-100 Programmable Thermal Controller (MJ
Research, Watertown, MA). At selected cycles aliquots from hPF4 and
mPF4 or hPBP and mPBP reactions were removed from the thermocycler
and placed on ice. The Cy-5–labeled products were then run on a 10% TBE
Ready Gel (Bio-Rad Laboratories, Hercules, CA) and analyzed with a
Storm imaging system and Imagequant PhosphorImager software (Molecular Dynamics). The log of the signal intensity of each band was plotted
against the cycle number to confirm that the amplification was linear. The
signal intensity of the human product was normalized with that of the
mouse product to compare the transgene expression levels among the
different founder lines. Inclusion of RNase free DNase I (1 U/10 ␮L
reaction, Life Technologies) or DNase free RNase A (1 U/10 ␮L reaction,
Sigma, St Louis, MO) were done in the RT step as controls to verify the
RNA nature of the amplified material. Total platelet-derived RNA (1 ␮g)
was added to a PCR tube to which 1 ␮L 10 ⫻ reaction buffer (200 mM
Tris-HCl, pH 8.4, 20 mM MgCl2; 500 mM KCl) and 1 ␮L RNase A or
DNase I enzyme was added (total volume ⫽ 10 ␮L). Each tube was
incubated at 37°C for 30 minutes. The reaction was stopped by adding 1 ␮L
25 mM EDTA (pH 8.0) and heating at 70°C for 15 minutes. Oligo dT primer
was then added and the RT reaction was followed as described above.
To control for differences in efficiency between the primer pairs, the
mPBP and mPF4 and the hPBP and hPF4 complementary DNAs (cDNAs)
were all cloned into a single pBluescript SK⫹ vector (Stratagene).
Following the same procedure as described above, the semiquantitative
PCR was done using this multigene construct as template. The differences
of intensity of the amplified cDNA run on the gel, hPBP versus mPBP and
hPF4 versus mPF4 at the same PCR cycles in the range of exponential
growth of PCR reaction, were used to determine the differences of the primer
pairs amplification efficiency. This information was then used to normalize the
expression values determined in the quantitative RT-PCR studies.
Protein detection
Human and mouse platelets were isolated by differential centrifugation of
whole blood. Collection of blood from healthy human volunteers was
approved by the Institutional Human Review Board at the Children’s
Hospital of Philadelphia. Samples from both humans and mice were
collected in acid-citrate-dextrose (ACD) and centrifuged at 200g for 10
minutes at room temperature as previously described.24 PRP was then
collected and prostaglandin E1 (Sigma) was added to a final concentration
of 1 ␮M. Platelets were then obtained by centrifugation at 800g for 10
minutes at room temperature. The pellets were washed twice in 134 mM
NaCl, 3 mM KCl, 0.3 mM NaH2PO4, 2 mM MgCl2, 5 mM HEPES, 5 mM
glucose, 0.1% NaHCO3, and 1 mM EGTA, pH 6.5, and resuspended in the
same buffer except without EGTA. The platelets were then lysed by
freezing and thawing the sample twice. The protein concentration of each
lysate was determined by the Pierce BCA Protein Assay kit according to the
manufacturer’s instructions (Pierce, Rockford, IL). Approximately 20 ␮g
total platelet protein was electrophoresed on a 12% SDS-polyacrylamide
gel (SDS-PAGE) and stained with Coomassie blue. A duplicate SDS-PAGE
gel was transferred to a polyvinylidene difluoride (PVDF) Immobilon-P
Transfer Membrane (Millipore, Bedford, MA). The hPF4 protein was
detected using RTO, a mouse anti-hPF4 monoclonal antibody generously
provided by Gow Arepally (University of New Mexico, Albuquerque,
NM),25 and the hPBP protein was detected using a polyclonal rabbit
anti–hNAP-2 antibody (PEPRO Tech, Rocky Hills, NJ) that does not
cross-react with mouse PBP. Western blot signals were visualized by
enhanced chemiluminescence (NEN Life Science Products, Boston, MA) and
measured using Imagequant PhosphorImager Software (Molecular Dynamics).
Immunohistochemical staining for hPF4
Tissues from hPF4 transgenic and littermate control mice were immunostained for hPF4 expression using the anti-hPF4 monoclonal antibody RTO.
Briefly, formalin-fixed, paraffin-embedded 5-␮m sections were deparaf-
finized in xylene and rehydrated. Endogenous peroxidase activity was
quenched with 0.9% peroxide in methanol and unreactive sites blocked
with 10% goat serum in 1 ⫻ Automation buffer (Biomeda, Foster City, CA)
for 20 minutes at 37°C. Slides were then incubated overnight at 4°C with
RTO (1 ␮g/mL), washed in 1 ⫻ Automation buffer, and incubated with
biotinylated goat-antimouse antibody (Jackson Laboratories, West Grove,
PA) diluted 1:200 at 37°C. Slides were washed and incubated with
streptavidin–horseradish peroxidase (HRP; Research Genetics, Huntsville,
AL) for 30 minutes at 37°C, then were washed and stable DAB chromogen
(Research Genetics) was applied for 5 minutes at 20°C. Slides were
counterstained with dilute hematoxylin.
Results
Characterization of the PBP/PF4 gene locus
The fact that hPBP and hPF4 form a gene locus has been previously
described,20 suggesting that these 2 platelet-specific genes may be
coordinately regulated during megakaryopoiesis. We have now
completed the characterization of the human PBP/PF4 locus and
have cloned and characterized the mouse equivalent of the PBP and
PF4 genes (GenBank access numbers AF349465 and AF349466).
We found that the human genes are 5.3 kb apart. The murine
equivalents are also closely linked with both genes oriented in the
same 5⬘33⬘ orientation, but with only 3.2 kb in the mice intergenic
region. A dot-matrix comparison of the available human and
determined mouse sequences shows that homology exists not only
within the genes and the immediate 5⬘- and 3⬘-flanking regions, but
also within several more distal regions as well (Figure 1A). The
most 5⬘ of these conserved regions is actually another CXC
chemokine, ENA-78 in human26 and its apparent mouse homologue, LIX.27 This gene is also oriented in the same 5⬘33⬘
orientation as the PBP and PF4 genes. There are 8.9 kb between
ENA-78 and hPBP. No other gene was defined up to 40 kb 3⬘ to the
PF4 gene by our analysis. Although the human genome had been
shown to contain a nonfunctional duplication of the PBP/PF4 gene
complex, ␺PBP/PF4alt,10,20 we could not find such a duplication of the
mouse genes by analysis of genomic Southern blots (data not shown).
To study the genetic regulation of this region, 4 human
transgenic constructs from the hPBP/PF4 gene locus were studied
(Figure 1B). Short-PBP was a 3.2-kb HindIII fragment that
extended 1.4 kb upstream of the hPBP gene’s transcriptional start
site and 0.8 kb downstream of its poly A signal site, containing only
the immediately 5⬘- and 3⬘-conserved sequence around PBP.
Long-PBP contains an additional 3.0 kb of upstream region, which
includes the conserved region upstream of the PBP gene (Figure
1A). The ␭-PBP/PF4 construct includes all of Long-PBP, plus the
entire intergenic region, the hPF4 gene and 3.0 kb downstream,
including all of the conserved regions flanking both the PBP and
PF4 genes (Figure 1A). PF4-Only is an EcoRI fragment that begins
in the second exon of hPBP, and contains the entire intergenic
region, the hPF4 gene and 3.8 kb downstream. This construct is
similar to the ␭-PBP/PF4 construct, but lacks most of the PBP gene
and the conserved region upstream of the PBP gene.
Characterization of transgenic mice: genome copy number and
tissue-specific expression
The number of founder animals obtained for each construct is also
shown in Figure 1B. Except for the ␭-PBP/PF4 construct, at least 4
founders were obtained for each construct. Copy numbers per
haploid genome are also indicated in Figure 1B, and except for the
␭-PBP/PF4 construct, cover a range of copy numbers.
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
The RT-PCR analysis of brain, lung, thymus, heart, liver,
spleen, small intestine, adrenal, kidney, testes, skeletal muscle,
marrow, and purified platelets was done using species-specific
primers for hPBP, hPF4, mPBP, mPF4, m␣IIb, and mHPRT.
Expression of the hPBP or hPF4 transgenes and the murine
megakaryocyte markers mPBP, mPF4, and m␣IIb was limited to
platelets, and the 2 hematopoietic tissues, spleen and bone marrow,
with no detectable ectopic expression in any of the other tissues
examined (data not shown). The ubiquitous mHPRT was readily
detectable in these tissues as a positive housekeeping gene control
(data not shown). An ethidium bromide gel of the RT-PCR data
from platelet RNA, for hPBP and mPBP message, is shown in
Figure 2A and for hPF4 and mPF4 in Figure 3A from transgenic
and wild-type mice. In Figures 2A and 3A, the amplifications were
done for 30 cycles and are only roughly quantitative. The observed
genomic bands seen in several lanes in Figure 2A were only
variably present and were not seen at the more limited cycle
numbers used for quantitative analysis in Figure 2B. DNase
pretreatment prior to RT-PCR also eliminated the genomic bands
(data not shown).
Interestingly, none of the Short-PBP constructs expressed hPBP
message well (Figure 2A and solid squares in Figure 2B). Among
the 4 founders, only the 8 and 17 copy lines had any detectable
hPBP, and this amounted to less than 1% of concurrently expressed
Figure 2. RT-PCR analysis of hPBP versus mPBP. (A) Ethidium bromide analysis
of platelet RT-PCR products for mPBP (top) and for hPBP (bottom) for all of the
founder lines studied plus wild-type (WT) mouse platelets and the appropriate cDNA
control are included. The founder lines are numbered by transgenic copy. The PCR
amplification was carried out for 30 cycles. The anticipated cDNA band is indicated by
a large arrow. The genomic DNA band is indicated by a smaller arrow and is only
occasionally prominent when there is no or little RNA message. No cDNA bands were
seen when the RT step was skipped or when RNase A was included in the RT step
(data not shown). WT indicates wild-type litter mates; M, ␾X HaeIII marker. (B)
Relationship between the copy number for the PBP-expression transgenic lines
Short-PBP, Long-PBP, and ␭-PBP/PF4 and the level of hPBP versus mPBP message
are shown. The relative quantity of hPBP to mPBP message was determined using
Cy5-labeled primers in the amplification step and measuring final product quantitatively with a Storm imager. Analysis was done of the PCR products within the
logarithmically amplifying range of cycles (usually 14-20 cycles) corrected for the
relative efficiency of the 2 sets of primer pairs; f, Short-PBP ; F, Long-PBP ; 䉬,
␭-PBP/PF4. Data are shown for average ⫾ 1 SD. Experiments were repeated 3
separate times.
REGULATED EXPRESSION OF PBP AND PF4 GENES
613
Figure 3. RT-PCR analysis of hPF4 versus mPF4. (A) Ethidium bromide analysis
as in Figure 2A, but for PF4. (B) Relationship between the copy number of the
transgene and the platelet level of message of human versus mouse PF4 was
determined for the PF4-expression lines ␭-PBP/PF4 and PF4-Only. The determination of the relative quantity of hPF4 to mPF4 message was as described in Figure 2B.
䡺, PF4-Only ; 䉬, ␭-PBP/PF4. Data are shown for average ⫾ 1 SD. Experiments were
repeated 3 separate times.
mPBP when analyzed over the exponential range of amplification
and then corrected for efficiency of amplification of different
primer pairs (Figure 2B). Furthermore, 2 other founder lines of the
Short-PBP construct with similar or higher copy numbers (7 or 22
copies) did not have any detectable expression. Thus, although the
Short-PBP construct may drive tissue-specific expression, it does
so inefficiently and not in a copy number–dependent fashion.
To see if the inclusion of the conserved region further upstream
of the hPBP gene was involved in the regulated expression of PBP,
the Long-PBP constructs that contained this region were examined
(Figure 1A). All of the transgenic lines from this construct
expressed high levels of hPBP message (Figure 2A) that on
quantitation was approximately equal to endogenous mPBP expression on a per copy basis (Figure 2B). The 2 ␭-PBP/PF4 lines,
which have the same 3.0 kb of sequence upstream of hPBP, also
expressed high levels of hPBP message. Expression per gene copy
tends to plateau at high copy numbers in transgenic mice, but it
appears that the addition of more 3⬘ sequence did not further
increase PBP message levels detected. Together, these constructs
suggest that the distal 5⬘-flanking region (between 4.4 and 1.4 kb) is
necessary and perhaps sufficient to achieve high-level expression of
hPBP gene in a tissue-specific and copy number–dependent fashion.
The 4 PF4-Only founder lines expressed high levels of hPF4
message, comparable to endogenous mPF4 levels of message
(Figure 3A,B). The level of expression was position independent,
copy number dependent (Figure 3B) with the construct with the
highest copy number having the highest level of expression. The 2
␭-PBP/PF4 constructs also expressed hPF4 at a high level and
seemingly also copy number dependent (Figure 3A,B). Comparison of the 14 copy ␭-PBP/PF4 and the 10 copy PF4-Only
expression levels suggests that the ␭-PBP/PF4 construct drives
perhaps an additional 3-fold increase in expression.
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PBP and PF4 protein expression by the transgenic lines
Although considerable effort was made to quantitatively analyze
mRNA level in the above experiments, we also measured hPBP and
hPF4 protein levels as a secondary determination of expression
(Figures 4 and 5). Human platelet proteins from 4 volunteers were
used as a positive control. Levels of PBP and PF4 expression were
done on a per milligram basis rather than on a per platelet basis
because mouse platelets are much smaller than human platelets.28
Protein concentrations were determined by optical density measurements and confirmed by Coomassie blue stained SDS-PAGE gels
(data not shown).
Figure 4A is an immunoblot of platelet proteins from 4 human
volunteers and from the Long-PBP and ␭-PBP/PF4 lines. The
double bands seen with human platelets in Figure 4A likely
represents differential N-terminal cleavage products of hPBP
protein.29 The lower band has the mobility of recombinant NAP-2
(data not shown), and the upper band is likely to be ␤-TG. In
humans, it appears to be variably cleaved to ␤-TG and NAP-2, and
in mice, the predominant product from hPBP appears to be ␤-TG
and not NAP-2. These differences suggest that there is differential
cleavage of hPBP between human and mice platelets. Not shown is
that platelets from the Short-PBP constructs had no detectable
hPBP, consistent with their low levels of hPBP message. The level of
hPBP expression increased in the same order as the level of hPBP
message seen, although the degree of increase was blunted (Figure
4A,B). This may be due to a limitation in achievable protein expression,
or perhaps excess hPBP cannot be efficiently packaged into the platelet
␣-granules and is lost by the developing megakaryocytes.
Figure 5A is an immunoblot of platelet proteins from the same
human volunteers and from the PF4-Only and ␭-PBP/PF4 lines.
Both sets of transgenic lines have detectable hPF4, and the levels
increased in the same order as their hPF4 platelet RNA level
Figure 5. Immunodetection of hPF4 in the transgenic mice platelets. (A)
SDS-PAGE gels of equal quantities of platelet protein were immunoblotted with an
anti-hPF4 monoclonal antibody RTO that does not recognize mPF4 proteins. Platelet
proteins from the transgenic lines PF4-Only and ␭-PBP/PF4 were studied. Control
lanes include wild-type mice and 4 different normal human volunteers (a through d).
(B) The relative expression of hPF4 in the transgenic mice to the average value in the
4 human samples per milligram total platelet protein was determined and compared
to its copy number. The dotted line is that average human platelet hPF4 value.
䡺 PF4-Only ; 䉬, ␭-PBP/PF4. Data are shown for average ⫾ 1 SD.
(Figure 5B). Again, the degree of increase in protein was blunted
compared to the degree of increase in message level, perhaps for
the same reasons as mentioned above concerning hPBP.
It should be noted that levels of hPBP and hPF4 protein found in
a number of lines equal that or exceed that of the proteins found in
the tested human samples. For hPBP, the maximum level expressed
was 1.5 times the level seen in the human controls, and for hPF4,
the maximum level seen was 2.5 times the level seen in the 4
controls. None of the lines studied had decreased viability (being
transmitted at the expected mendelian frequency) or had abnormalities in their blood counts (data not shown).
Immunohistochemical staining for hPF4
Figure 4. Immunodetection of hPBP in the transgenic mice platelets. (A)
SDS-PAGE gels of equal quantities of platelet protein were immunoblotted with an
anti–NAP-2 polyclonal antibody that does not recognize mPBP proteins. Platelet
proteins from the transgenic lines Long-PBP and ␭-PBP/PF4 were studied. Control
lanes include wild-type mice and 4 different normal human volunteers (a through d).
(B) The relative expression of hPBP in the transgenic mice to the average value in the
4 human samples per milligram total platelet protein was determined and compared
to its copy number. The dotted line is the average human platelet hPBP value. F,
Long-PBP ; 䉬, ␭-PBP/PF4. Data are shown for average ⫾ 1 SD. Experiments were
repeated 3 separate times.
Tissues from PF4 transgenic and wild-type control mice were
immunostained for hPF4 expression using RTO. Only the bone
marrow and spleen showed positive immunostaining, consistent
with the RT-PCR studies mentioned above (data not shown). Figure
6 shows that in the spleen, which is a hematopoietic tissue in mice,
only mature megakaryocytes show significant staining in the
10-copy PF4-Only and in the 21-copy ␭-PBP/PF4 animals,
whereas megakaryocytes were unstained in the wild-type control.
Of interest is that the platelets in the ␭-PBP/PF4 spleen are so
intensely stained that their punctate staining allows one to readily
distinguish the red pulp, which contains circulating platelets, from
the white pulp.
Discussion
Regulation of megakaryocyte-specific genes has been studied by
our laboratory and others to gain insights into the mechanisms
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
Figure 6. Immunohistochemical localization of PF4. Spleens were stained with
the monoclonal anti-PF4 RTO as the primary antibody. Each spleen was counterstained with hemotoxylin. Panel A represents immunohistochemical studies of the
spleen from a wild-type mouse, whereas panel B is from a 10 copy PF4-Only mouse,
and panel C is from a 21 copy ␭-PBP/PF4 mouse. The arrows point to some, but not
all, of the megakaryocytes in each field. Original magnification 20 ⫻.
underlying megakaryocyte differentiation. Given the paucity of
megakaryocytes in bone marrow these studies have previously
heavily required the use of cell lines with some, but not complete,
megakaryocytic features.30-32 Previous studies of megakaryocytespecific gene expression in transgenic mice have provided additional insights into this process. However, these studies used
reporter gene constructs, which in themselves often contain cryptic
regulatory components. Further, these studies have not dealt with
the important issues of expression level in megakaryocytes, and
copy number dependence and position independence.
Our studies have focused on the PBP/PF4 gene locus because
both genes are highly expressed, megakaryocyte-specific chemokines and are physically linked to one another on human chromosome 4.20 The studies presented show this tight linkage to be
conserved for the mPBP and mPF4 genes. Further, our studies are
the first to show close physical linkage to an additional related
CXC chemokine gene ENA-78/LIX, located 8.9 and 6.9 kb
upstream of the transcription start site of hPBP and mPBP genes,
respectively. ENA-78 is also expressed in, but not restricted to,
megakaryocytes. These data are consistent with our initial supposition that this region could potentially represent a common expression locus for several platelet-specific chemokines.
We also proposed that given their proximity, the PBP and PF4
genes might possess common distal regulatory elements. Phylogenetic footprinting data demonstrated in Figure 1A show that not
only are PF4, PBP, and ENA-78 highly conserved across mammalian species, but there are a number of regions outside of the coding
regions that are highly conserved. Our data show that the PBP and
PF4 genes each contain distinct regulatory regions in their flanking
regions that allow for their copy number–dependent and positionindependent expression. A region upstream to the PBP gene was
necessary for these features for PBP gene expression and may also
contribute to an approximate 3-fold enhanced expression of the
PF4 gene. Despite this apparent expression augmentation, copy
number–contingent high levels of PF4 expression can be achieved
independent of this element, being regulated by the flanking
regions surrounding the PF4 gene.
Further studies are needed to define the molecular basis by
which these 5⬘-flanking regions regulate the efficient expression of
these 2 genes. Such important regulatory elements are likely to be
conserved, and indeed, phylogenetic footprinting data analyses for
other genes have been useful in localizing important distal regulatory elements,33,34 and may apply to our data as well. For example,
our expression data show that the Short-PBP and Long-PBP
constructs differ only in that the Long-PBP includes the region 1.4
to 4.4 kb upstream of the hPBP gene. Thus, this region is necessary
for high-level tissue-specific and copy number–dependent expres-
REGULATED EXPRESSION OF PBP AND PF4 GENES
615
sion of the hPBP gene. Within this region is a highly conserved
approximate 700-bp domain, showing about 60% nucleotide
conservation (Figures 1A and 7A). We analyzed this region in the
mouse and human sequences for transcription factor consensus
binding sequences implicated in the control of megakaryocytic and
hematopoietic gene regulation, focusing on GATA-1, NF-E2, Ets,
PU.1, and AML-1 binding sites.35-47 Of note is a homologously
conserved NF-E2 site beginning 3696 (2151) bp upstream of the
PBP start site (with the mouse distance in parenthesis), and a
GATA-1 site 3140 (1644) bp upstream (Figure 7A). Whether either
site is biologically relevant will now need to be tested. Additionally, several regions contain other GATA-1 and Ets binding sites
that are present within about 60 bp of each other in the mouse and
human sequences but that may nevertheless be functionally significant. Studies of enhancers have classically demonstrated independence for physical attributes such as distance and orientation. It is
possible that there is no evolutionary constraint in this context to
keep these binding sites precisely aligned, but rather generally
present within the boundaries of the enhancer domain.
Expression of hPF4 from all animals, including the 4 PF4-Only
founder lines and the 2 ␭-PBP/PF4 lines, was tissue specific.
Previous transgenic studies with 245 bp and 1.1 kb of the
immediate 5⬘-flanking region of the mouse and rat PF4 promoters
driving Lac Z reporter genes showed excellent tissue specificity,
but the relative degree of expression and copy number dependency
and position independence were not well studied.16,17 The present
studies with PF4-Only show that the inclusion of the entire PBP
and PF4 intergenic region, the intact PF4 gene itself, and 3.8 kb of
3⬘-flanking sequence was sufficient to achieve megakaryocyterestricted high level, copy number–dependent and positionindependent expression, comparable to the endogenous mPF4
gene. Interestingly, these flanking regions contain several interspecies conserved domains, including an intergenic region beginning
4989 (2728) bp upstream of the hPF4 transcriptional start site
(Figure 7B), and a region beginning 1018 (942) bp downstream of
the hPF4 gene (Figure 7C). Analysis of these regions again show
about 60% nucleotide conservation in these regions. A search for
consensus DNA binding sites as described above found multiple
consensus sequences for GATA, NF-E2, Ets, PU.1, and AML-1
binding with one homologously conserved site, which was a GATA
consensus binding site 1716 (1705) bp downstream of the PF4
gene. The functional relevance of these and other sites not readily
apparent from the above analysis will require further investigation.
It is intriguing that the ␭-PBP/PF4 transgenics have a further
increase in hPF4 expression in transgenic animals compared to the
PF4-Only transgenics. This could be the result of their having
higher copy numbers although the highest PF4-Only had 10 copies
and the lowest ␭-PBP/PF4 had 14 copies, yet had 3 times the
message level. It is possible that the 5⬘-enhancer region 1.4 to 4.4
upstream of the hPBP gene also regulates hPF4 gene expression.
Additional ␭-PBP/PF4 lines with lower copy numbers and a
truncated version of ␭-PBP/PF4, lacking the region from 1.4 to 4.4
kb upstream of hPBP would be needed to see whether the PBP
upstream enhancer domain increases PF4 as well as PBP expression. Conversely, additional 3⬘ sequence of the PBP gene, including the intergenic region and the region downstream of the PF4
gene do not contribute to the level of PBP expression.
Our study clearly shows we have defined important domains for
high level, tissue-specific expression within the flanking regions of
the PBP and PF4 genes. With the identification of ENA-78 gene
immediately upstream of the PBP/PF4 gene locus, the question of
whether there is a larger megakaryocyte-specific gene locus
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616
ZHANG et al
BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
Figure 7. Comparison of the homologous domains in the flanking regions of the PBP/PF4 locus. A comparison of the conserved regions, other than within the coding
areas or the immediate 1.0 kb of 5⬘-flanking region, of the mouse and human PBP and PF4 genes are shown. A “兩” refers to an identical nucleotide. Consensus DNA binding
sites for a number of megakaryocyte-specific transcriptional factors are highlighted and the names of the transcriptional factors and their orientations are shown. Panel A
represents the conserved domain upstream of the PBP gene, and negative numbering begins at the PBP transcriptional start site.11 Panel B represents the conserved
intergenic region, and negative numbering begins at the PF4 transcriptional start site.10 Panel C represents the conserved region downstream of the PF4 gene, and numbering
begins at the PF4 transcriptional start site.
involved in the regulation of all 3 genes is raised. Given that all
known CXC chemokines localize to a single locus on human
chromosome 420,48,49 and that our studies show that ENA-78, PBP,
and PF4 are clustered and transcribed in the same 5⬘33⬘ direction,
this possibility becomes more likely. Further, although ENA-78
was first recognized in epithelial cells, it is highly expressed in
platelets,50,51 and nonquantitative RT-PCR suggests that its platelet
RNA message is as abundant as PBP.51 Because other CXC
chemokines are also expressed in platelets,52 we believe further
analysis of the CXC chemokine locus may elucidate other physical
and functional links for additional CXC chemokine genes to the
PBP/PF4 gene locus.
For over 40 kb downstream of the PF4 gene, we found no other
CXC chemokine genes. Whether this means that the ENA-78/PBP/
PF4 genes are at one end of the CXC chemokine locus is not clear
yet. Furthermore, we have yet to determine the precise relative
location of the inactive, duplicated ␺PBP/PF4alt human gene locus,
which is within the same human chromosome 4 locus.20 This
duplication, though within the CXC chemokine gene locus, is not
closely linked to the functional PBP/PF4 locus.
The protein levels of hPBP and hPF4 in the founder lines are
consistent with the semiquantitative RT-PCR data. The 2 ␭-PBP/
PF4 lines have the highest PBP and PF4 RNA and protein levels.
The expressions of hPBP and hPF4 in these and a number of other
lines are equivalent or higher than PBP and PF4 expressions in
human platelets both by immunoblot and tissue immunostaining.
Whether these animal lines might be useful to study the biologic
and pathobiologic functions of hPBP and hPF4 in mouse models is
being pursued. We have already used the highest expressing PF4-Only
construct to develop and characterize a mouse model for the pathologic
state heparin-induced thrombocytopenia. We found that an accurate
mouse model of the disease requires the expression of hPF4 in vivo.53
Therefore it is apparent that these constructs are useful not only to better
understand the regulation of PBP and PF4 gene expression, but also to
study the human proteins that are expressed.
Acknowledgments
We wish to thank Dr Gowthami Arepally at the University of New
Mexico, Albuquerque, NM, for providing the mouse anti-hPF4
monoclonal antibody RTO, and Dr Katya Ravid, Boston University, Boston, MA, for sharing unpublished sequences with us. We
would also like to thank Zheng Cui, Diana Cassel, and Saul Surrey
at the Thomas Jefferson University as well as Jean Richa at the
Transgenic Mouse Facility of the University of Pennsylvania for
their technical help.
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BLOOD, 1 AUGUST 2001 䡠 VOLUME 98, NUMBER 3
REGULATED EXPRESSION OF PBP AND PF4 GENES
617
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Blood. In press.
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2001 98: 610-617
doi:10.1182/blood.V98.3.610
Localization of distal regulatory domains in the megakaryocyte-specific
platelet basic protein/platelet factor 4 gene locus
Chunyan Zhang, Michael A. Thornton, M. Anna Kowalska, Bruce S. Sachis, Michael Feldman, Mortimer
Poncz, Steven E. McKenzie and Michael P. Reilly
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