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Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2002-05-1307.
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Blood, 15 November 2002, Vol. 100, No. 10, pp. 3588-3596
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Identification of distal regulatory regions in the human
 IIb gene locus necessary for consistent, high-level
megakaryocyte expression
Michael A. Thornton,
Chunyan Zhang,
Maria A. Kowalska, and
Mortimer Poncz
From the Department of Pediatrics, University of
Pennsylvania School of Medicine, Philadelphia.
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Abstract |
The  IIb/ 3-integrin receptor is present at high levels only in
megakaryocytes and platelets. Its presence on platelets is critical for
hemostasis. The tissue-specific nature of this receptor's expression
is secondary to the restricted expression of IIb, and studies of the
IIb proximal promoter have served as a model of a
megakaryocyte-specific promoter. We have examined the IIb gene locus for distal regulatory elements. Sequence comparison between
the human (h) and murine (m) IIb loci revealed high levels of
conservation at intergenic regions both 5' and 3' to the IIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity
mapping defined tissue-specific hypersensitive (HS) sites that
coincide, in part, with these conserved regions. Transgenic mice
containing various lengths of the hIIb gene locus, which included or
excluded the various conserved/HS regions, demonstrated that the
proximal promoter was sufficient for tissue specificity, but that a
region 2.5 to 7.1 kb upstream of the hIIb gene was necessary
for consistent expression. Another region 2.2 to 7.4 kb downstream of
the gene enhanced expression 1000-fold and led to levels of hIIb
mRNA that were about 30% of the native mIIb mRNA level. These
constructs also resulted in detectable hIIb/m3 on the platelet
surface. This work not only confirms the importance of the proximal
promoter of the IIb gene for tissue specificity, but also
characterizes the distal organization of the IIb gene locus and
provides an initial localization of 2 important regulatory regions
needed for the expression of the IIb gene at high levels
during megakaryopoiesis.
(Blood. 2002;100:3588-3596)
© 2002 by The American Society of Hematology.
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Introduction |
It is known that IIb/3 is a prototypic member
of the integrin superfamily of cell surface receptors that include the
receptors for such ligands as von Willebrand factor, fibrinogen,
vitronectin, fibronectin, and collagen.1 IIb/3 is
found at high levels only on the surface of megakaryocytes and
platelets.2 The approximately 80 000 copies of
IIb/3 found on the platelet surface make it the most abundant
receptor present, representing half of the total molar number of
receptors.3 Normal activity of this receptor is critical
for platelet function during thrombus development.4
The tissue specificity of the IIb/3 receptor is determined by
IIb. Whereas 3 is expressed as part of the v/3 vitronectin receptor on many tissues, IIb expression is limited to hematopoietic tissue and achieves high levels of expression only in developing megakaryocytes.5,6 In both the human and mouse genomes,
there exists a single IIb gene (ITGA2B), which
contains 30 exons and spans a distance of about 18 kb along chromosomes
17 and 11, respectively.7,8 In vitro studies in
megakaryocytic cell lines have defined important tissue-specific regulatory domains in the immediate 5'-flanking region,
including proximal ( 55 bp upstream from the transcriptional start
site) and distal (480 bp) GATA-1 DNA-binding sites, each associated
with an adjacent Ets-binding site,9,10 as well as a
potential silencer region located between the 2 GATA/Ets motifs.11
These in vitro studies, which determined that about 600 bp of 5'
flanking of the IIb gene proximal promoter region is
sufficient to support tissue-specific expression, have been consistent
with in vivo transgenic murine studies. In those reports, as little as
787 bp of the 5'-flanking region of the IIb gene was used to
drive thymidine kinase (TK) toxigene expression in the megakaryocytes of transgenic mice.12 Using ganciclovir as the toxic
agent, these studies demonstrated TK expression predominantly in the bone marrow. In these studies, the relative level of expression of the
reporter gene was not determined, as even low levels of TK expression
would make cells susceptible to ganciclovir. Hence, these in vivo
studies, although indicating domains important for tissue and
hematopoietic lineage-restricted expression, did not define the
specific extent of regulatory domains required to constitute a
complete, functional IIb gene locus, capable of withstanding position effects and hence generating high mRNA and protein levels.
In this regard, we wished to pursue the molecular basis of
IIb gene expression in vivo, by focusing not only on the
proximal promoter region, but also examining more distal intergenic
regions in the IIb gene locus. The difficulty in examining these
intergenic regions lies in their broad size. One technique for
localizing regulatory domains within a large expanse of DNA sequence is
phylogenetic footprinting. In this approach, nucleotide sequences of 2 or more species are compared. Functionally important intergenic
regulatory regions are less likely to diverge than the remaining
intergenic sequence and hence are highlighted by their own
conservation.13,14 A second approach for defining
functional cis-regulatory elements takes advantage of the
fact that regulatory regions are often embedded in less compacted
chromatin as reflected in their greater susceptibility to digestion by
general endonucleases such as deoxyribonuclease (DNase)
I.15,16 For example, in the upstream region of the -globin gene locus, there are multiple DNase I hypersensitive (HS)
sites that comprise a region essential for high-level expression of the
various -globin genes.17-19 A third approach involves
transgenic animals containing various lengths of a gene and its
flanking regions. Such studies can define important distal elements
that regulate tissue specificity, copy number dependency, and levels of
expression relative to the native gene.18,20-22 These
intergenic regions are often not only functionally important, but also
conserved between species. For example, transgenic studies of the
platelet basic protein (PBP)/platelet factor 4 (PF4) double gene locus, identified a region between 2.5 and 4.5 kb upstream of the human PBP
gene that appears to be an important enhancer of PBP expression and
that is also highly conserved between the human and mouse PBP/PF4 gene
loci.22
In the studies described below, we use phylogenetic footprinting to
compare the human and mouse flanking sequences and define a series of
intergenic conserved regions. Several of these regions also coincide
with tissue-specific, DNase I HS sites. Using these potential
functional domains as a guide, transgenic mice were made with segments
of the human (h) IIb gene locus, encompassing increasing 5' and 3'
lengths. These studies collectively define a limited domain surrounding
the hIIb gene that is required for achieving consistent high
levels of hIIb message levels and detectable surface expression of
the hIIb receptor on murine platelets. Thus, these studies represent
the first localization of important distal regulatory elements flanking
the megakaryocyte-specific IIb gene.
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Materials and methods |
Phylogenetic footprinting and sequence analysis
Sequence determination and analysis of the human and murine
IIb gene locus were performed as previously
described.8,23 Generated sequences were submitted to the
GenBank public database at www.ncbi.nih.gov, and include the following
accession numbers: AF170316, AF169829, AF160252, and AF489555. Using these original sequence files together with both complete and incomplete BAC genomic sequence files available at GenBank (accession numbers: AC007722, AC003043, AC025326, a murine chromosome 11 BAC
clone-RP11-621L13) and Celera Databases (scaffold number: GA_x5J8B7W82RK: 6500001.6874571; a partially complete mIIb locus scaffold), we compiled composite sequences of both the human and murine
sequences, encompassing for each a 200-kb sequence domain. These files
were then used for a homology comparison of orthologous regions using
the nucleotide sequence comparison program, Visual Tools for Alignment
(VISTA/AVID [www-gds.lbl.gov/vista]). Length of comparison was set to
100 bp with a minimum of 50% match. Specific regions within the 200-kb
region were compared using the Basic Local Alignment Search Tool
(BLAST).24 Specific subregions were also compared to the
public expressed sequence tag (EST) database at www.ncbi.nih.gov
using BLAST. A 10-kb subregion extending from +7.8 kb within the IIb
3'-intergenic domain was also analyzed using GeneMachine gene
prediction software available at
http://genome.nhgri.nih.gov/genemachine. This program allows
users to query multiple exon and gene prediction programs in an
automated fashion and incorporates BLAST analysis as well in assessing
final predictions. Additionally, the Transcript Assembly Program (TAP)
available at http://sapiens.wustl.edu/~zkan/TAP/ was used to
delineate gene structures using genomically aligned EST sequences.
Analysis of aligned conserved sequence regions for consensus
transcription factor-binding sites, was done using both the rVISTA
program (www.gds.lbl.gov/vista) and the Transcription Element Search
Software (TESS) developed by the University of Pennsylvania (PENN)
Computational Biology Informatics Laboratory (http://www.cbil.upenn.edu/).
DNase I HS studies
Human erythroleukemia (HEL) and Children's Hospital Research
Foundation (CHRF) 288-11 cells were the 2 megakaryocytic
IIb-expressing cell lines25,26 used in these DNase I HS
site studies. The fibroblast line HeLa27 and the gastric
carcinoma cell line SNU-128 were studied as
non-IIb-expressing lines. HEL, HeLa, and SNU-1 cells were obtained
from American Type Culture Collection (Rockville, MD). CHRF cells were
obtained from Dr Michael Liebman (University of Cincinnati, OH). HEL,
SNU-1, and HeLa cells were cultured in RPMI 1640, 10% fetal bovine
serum (FBS), 1% penicillin/streptomycin, and L-glutamine
(Invitrogen, Carlsbad, CA). CHRF cells were cultured as above
but with 20% FBS. All cell lines were split 24 hours before each DNase
I HS experiment. Preparation of cell nuclei for DNase I treatment was
as previously described.29 Nuclei from
1 × 108 cells were centrifuged for 10 minutes at
500g and were suspended in 4.5 mL DNase I buffer (10 mM Tris
[tris(hydroxymethyl)aminomethane], pH 7.5, 10 mM NaCl, 5 mM
MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM sodium
butyrate, and 1 mM CaCl2). Aliquots of 500 µL nuclei were
added to 500 µL DNase I buffer containing DNase I enzyme (Invitrogen)
varying from 0 to 3.4 µg/mL. The tubes were gently mixed, then placed
at 37°C for 5 minutes, and then replaced back on ice. Genomic DNA was
recovered and digested further with either EcoRI or
BamHI restriction enzymes for Southern blot analysis as
previously described.29,30 The probes used for detection were produced via polymerase chain reaction (PCR) amplification. Primers used to generate these probes were the following
sense/antisense pairs: IIb exon 1 (P1)7:
5'-CCTGTGGAGGAATCTGAA-3'/5'-TCCTGCTCTCTCCCAATAC-3'; IIb
exon 4 (P2)7: 5'-CAATCGGGGGCAGGGACAC-3'/5'-CAAGCCGTCGCGAGTGGG-3'; and IIb exon 30 (P3)7:
5'-GAGTACAGTGGGCTTCATGTTCT-3'/5'-CCCTGGCAGTGACTCTCTCGTTCA-3'. A
previously described genomic  clone containing the hIIb locus served as template for the reactions.23
Transgenic constructs
The SalI fragment used to make the
2.5hIIb transgenic animal lines was isolated from
a Dash (Stratagene, La Jolla, CA) bacteriophage clone
subcloned from P1(clone no. 1147), an hIIb-containing P1 clone previously described.23 This construct extends from
the Dash vector polylinker SalI site through 2.5 kb of
the 5'-flanking region, 17.35 kb of coding exon/intron sequence, and
2.2 kb of the 3'-flanking sequence up to the 3'-polylinker
SalI site and has a total length of 22 052 bp. The
EcoRV/AflII restriction fragment used to make the
7.1hIIb transgenic animal lines was isolated from
an hIIb pWE15 cosmid clone. This fragment extends from a vector
pWE15 polylinker EcoRV site through 7.1 kb of the
5'-flanking region to 2.1 kb downstream of the hIIb gene
stop codon, ending at an endogenous AflII site with a total
length of 26 559 bp. The final transgenic construct
3'+hIIb is an EcoRV/PvuI
restriction fragment, derived from the same cosmid clone described
above. It is identical with the 7.1hIIb
construct, except that the 3'-flanking region is 5.2 kb longer,
extending to a polylinker PvuI site in pWE15 with a total
length of 31 759 bp.
Generation and initial analysis of transgenic animals
Transgenic mice were generated by pronuclear injections
following standard methods at the PENN Transgenic Mice Core Facility. Positive founder animals were detected by polymerase chain reaction (PCR) analysis of tail genomic DNA using multiple human-specific primer
pairs for hIIb.7 Murine PF4 genomic
primers22 were used as a control. These sense/antisense
primer pairs were as follows: hIIb-exon 14:
5'-AGGCCTCTGTCCAGCTAC-3'/5'-GCCATTCCAGCCTCCGTG-3'; and
mPF4:
5'-GTCCAGTGGCACCCTCTTGA-3'/5'-AATTGACATTTAGGCAGC-3'.
Positive founder lines with intact hIIb genes by Southern and PCR
analysis were further characterized for copy number by Southern blot.
Tail genomic DNA was digested with EcoRI and size separated
on a 0.8% (wt/vol) agarose gel. The Southern blot and copy number
determination procedures were as described before.22,23
Tissue and platelet RT-PCR analysis
RNA isolation and reverse transcriptase (RT)-PCR procedures for
all mRNA expression analyses, as well as a list of the 11 tissues
examined, have been described before in our previous transgenic studies.22 Briefly, using about 0.1 µg total platelet
RNA, or 1 µg RNA from other tissues, RT-PCR was done using the
SuperScript II Reverse Transcriptase Kit (Invitrogen) with
the following sets of sense/antisense primer pairs:
hIIb7: 5'-AGGCCTCTGTCCAGCTAC-3'/5'-G CCATTCCAGCCTCCGTG-3';
mIIb8:
5'-TCAAGACTCCCTGAATCCAACAC-3'/5'-GGGCTCCTCCAGTCTCTTCT-3'; mPBP22:
5'-GCCTGCCCACTTCATAACCTC-3'/ 5'-GGGTCCAGGCACGTTTTT-3'; mPF422:
5'-GTCCAGTGGCACCCTCTTGA-3'/5'-AATTGACATTTAGGCAGCTGA-3'; mHPRT31:
5'-CACAGGACTAGAACACCTGC-3'/5'-GCTGGTGAAAAGGACCTCT-3'; mKIAA05538:
5'-GACCCAAAGGTGCTTGTAAT-3'/5'-GAAAACTACCTCCAGGATGG-3'.
Control experiments included studies where no RT was added and others
treated with RNase A (Sigma, St Louis, MO) prior to the RT
step, were done to ensure that PCR bands seen in the experiments were
not generated via pre-RT sources of DNA.22 Also, control experiments to control for genomic DNA contamination were done where
the RNA was treated with DNase I (Invitrogen), as previously described.22 Quantitation results from DNase I-treated
versus DNase I-untreated samples did not vary significantly; hence,
all RT-PCR data presented below used untreated RNA samples.
To quantitate the level of hIIb transgene mRNA expression in
platelets relative to endogenous mIIb, we used methods previously described.18,20,22 Briefly, PCRs from first-strand cDNA of platelet RNA were set up using the primers described above and the
cycle number at which both genes were in their linear range of
amplification. Antisense primers were 5' labeled with a fluorescent dye
"Cy-5" (Oligos Etc, Wilsonville, OR). Preliminary
experiments were done to determine the detectable linear range of
amplification for the native mIIb gene and the hIIb
transgene for each founder line. Thereafter, a 100-µL PCR mixture was
divided into six 15-µL aliquots, one for each cycle beginning 3 cycles below midpoint of the detectable linear range and extending to 2 cycles above it, prior to initiating the PCR. PCR was performed at
94°C for 2 minutes, followed by cycles at 94°C for 25 seconds,
62°C for 36 seconds, 72°C for 55 seconds in a PTC-100 Programmable
Thermal Controller (MJ Research, Waltham, MA). For each
consecutive single cycle change, one of the PCR tubes for hIIb and
mIIb reactions was removed from the thermocycler and placed on ice.
All of the samples were then run on a 12-well 10% acrylamide Ready Gel
(Bio-Rad Laboratories, Hercules, CA), and bands were detected
with a STORM imaging system red light laser (Molecular Dynamics,
Sunnyvale, CA). The detected bands were then analyzed using ImageQuant
PhosphorImager software (Molecular Dynamics).22 The log of
the signal intensity of each band was calculated. The log value
difference for each band between 2 consecutive cycles was calculated,
with the expected log difference being 0.3 units (log102).
Those values, which fell into a range of 0.2- to 0.4-log difference
units/cycle, were considered within the linear range of amplification.
The raw signal intensity of those human PCR bands within the linear
range was normalized to that of the mouse product, also within the
linear range at the same cycle. The signals were further normalized for
differences in the ability of the primer/PCR conditions to amplify
hIIb and mIIb from equal molar amounts of the
appropriate cDNA control template as described before.22
These normalized values were used to compare transgene expression level
among the different founder lines.
Protein detection
Platelet-rich plasma (PRP) from human and mice blood was
isolated as described.22 For these studies, prostaglandin
E1 (Sigma) was added to a final concentration of 1 µM
prior to spinning down the platelets at 800g for 10 minutes
at room temperature. The pellets were washed twice in platelet buffer
(PB; 134 mM NaCl, 3 mM KCl, 0.3 mM NaH2PO4, 2 mM MgCl2, 5 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 5 mM glucose,
0.1% NaHCO3, pH 6.5) plus 1 mM EGTA
(ethyleneglycoltetraacetic acid) and resuspended in PB without
EGTA. The platelets were lysed by freezing and thawing 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). Twenty micrograms of the total platelet
protein was electrophoresed on a 4% to 12% gradient sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (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 hIIb protein was detected
using MAB1990 (Chemicon, Temecula, CA), a mouse anti-hIIb
monoclonal antibody. Western blot signals were visualized by
phosphoimaging of the enhanced chemiluminescence (Perkin-Elmer,
Wellesley, MA) signal on the STORM and quantitated using
Imagequant PhosphorImager software. For flow cytometric studies,
1 × 107 wild-type and transgenic mice platelets and
human platelets were prepared as previously described,32
using the monoclonal MAB1990 antibody32 as the primary
antibody and a fluorescein isothiocyanate (FITC)-labeled, goat
antimouse IgG (Sigma) as the secondary antibody.
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Results |
Phylogenic footprinting of the IIb gene locus
To search for upstream and downstream distal, intergenic
regulatory elements in the IIb gene locus, we used a cross-species sequence comparison to define evolutionarily constrained regions that
may reflect important functional domains. We previously reported on the
sequencing of an approximate 30-kb region surrounding the human and
murine IIb gene loci7,8,23 (GenBank accession numbers:
AF170316, AF169829, AF160252, and M33320). In the present study, we
have extended our sequencing of the murine locus (accession number:
AF489555) and used our collective sequences together with partially
complete public and private database sequences (accession numbers:
human IIb locus, AC007722 and AC003043; murine IIb locus,
AC025326; and Celera-scaffold number, GA_x5J8B 7W82RK:6500001.6874571)
to compile an ordered 200-kb stretch of both the human and murine
sequences surrounding their IIb gene loci. We then used the
VISTA/AVID global alignment program to examine this extended region for
conserved domains. Figure 1 shows a
subregion of this original 200-kb analysis centered on a 62-kb region
surrounding the IIb gene. (The complete 200-kb comparison is
included in the supplemental Figure 1 online.) The data clearly show
that conserved homologous regions exist not only within the coding
regions of the multiple genes found within the compared domains, but
also intergenically in the noncoding domains upstream and downstream of
these genes.
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| Figure 1.
VISTA/AVID global alignment of
the IIb gene locus.
The homology between the human and mouse IIb gene locus is shown for
homology 50% or more using the VISTA program. The x-axis defines 62 kb
of human sequence, whereas the vertical axis indicates the percentage
homology between this and the orthologous murine sequence. The 75%
homology level, a point halfway between the minimal and maximal
homology shown, is denoted. The IIb gene is located near the
center of the figure in a 5'  3' orientation. Annotations for gene
nomenclature are indicated and also have their 5' 3'
transcriptional direction and exon structure shown. Numbering in
kilobase pairs is shown beginning at the transcriptional start site of
the IIb gene (position zero, "0"). The sequences upstream of the
IIb genes' start site are indicated as negative numbers and
those downstream as positive numbers. Sequences that are 70% or more
identical over a continuous 100-bp stretch have shaded peaks. Stretches
of homology extending less than 100 bp are shown but are not shaded.
Homology within coding regions is shown in black, within the 5'- or
3'-untranslated regions is striped, and within the intergenic and
intronic, noncoding region is shown in gray. The 2 vertical arrows,
"1" and "2," highlight homology regions of potential
physiologic importance discussed in this paper. Predicted exons for the
newly defined hCP44813 gene are shown above the gray-shaded
homology matches, formerly thought to be areas of noncoding sequence.
Gray-filled exons within hCP44813 denote predicted alternatively
spliced exon locations. GRN indicates Granulin gene.
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In the 5'-intergenic region between the IIb and the
KIAA0553 genes, there are 2 conserved domains. The first is
a double peak between positions O and 2 kb (Figure 1), and this
double peak is consistent with the previously reported homology between the rat and human IIb immediate promoter regions.33
Further upstream is a small conserved domain, positioned at about 4 kb (Figure 1, vertical arrow "1"). Analysis of this 5'-intergenic human sequence for Alu consensuslike sequences34
demonstrated that there were several Alu repeat elements within this
5'-intergenic region, but these do not overlap with the conserved
domains described above (data not shown).
A homology comparison of the 3'-intergenic region between the
IIb gene and the previously reported downstream gene,
Granulin,8,23 defined several noncoding
conserved loci (Figure 1). The region most proximal to the
IIb gene is positioned 18.0 to 18.6 kb downstream of the
hIIb start site (Figure 1) and is about 65% conserved. Further
downstream from this homologous region is a conserved domain extending
from 21.5 to 23.3 kb downstream of the hIIb start site or 4.3 to 6.1 kb downstream of the IIb stop codon (Figure 1, vertical arrow
"2"). This domain is conserved up to 85% between human and murine
sequences. Downstream of this conserved region is a highly conserved
domain between +26 to +36 kb. Analysis using the GeneMachine program, a
gene prediction program,35 suggested that this homologous
region defines another gene. BLAST comparison of this about 10-kb
region against the public EST database yielded multiple matches for
mRNAs expressed in diverse tissues, but predominantly brain and heart
tissue in the human, rat, and mouse databases. BLAST comparison of the
highest scoring ESTs from the public database against the Celera human
protein database yielded a 99% match with hCP44813, a Celera-defined
protein of unknown function that corresponds to a 1500-bp Celera human
mRNA transcript hCT21474. Using the TAP36 to analyze this
domain, we showed that the Celera human transcript subdivides with
perfect identity into 9 separate domains in the human IIb sequence,
each flanked by canonical splice junctions (data not shown) and each conserved in the murine sequence. Hence, we conclude that a previously unrecognized gene coding for the hCP44813 protein lies 8.5 kb downstream of the IIb gene in the same 5' 3'
orientation as the IIb gene.
DNase I HS mapping of the 5'- and 3'-intergenic regions of IIb
As an additional method for defining distal cis
regions that could potentially be involved in controlling the
platelet-specific expression of the IIb gene, we also
carried out DNase I sensitivity mapping on chromatin from active
(megakaryocytic) hIIb-expressing tissue versus those from inactive
(nonmegakaryocytic) nonexpressing tissue. Nuclei were isolated and
subjected to limited DNase I digestion followed by EcoRI
restriction and analysis by Southern blot. A series of bands were seen
on genomic Southern blots with a probe P1, covering IIb exon 1 (Figure 2A,C). The 12.3-kb, full-length EcoRI band disappears at increasing DNase I concentrations
in both the megakaryocytic and nonmegakaryocytic cell lines (Figure 2A). A band representing a DNase I HS at position 6.4 kb upstream of
the IIb gene (HS IV), that was common to both cell lines, was
identified, and increased in signal strength with higher concentrations of DNase I and then was digested away at the highest concentrations. The band for HS III, located directly below HS IV in the HeLa sample,
is absent at the lowest DNase I concentration then subsequently appears
and persists up to the highest DNase I concentration. Note also that HS
III observed in the HeLa cell blot and not in the megakaryocytic HEL
cell blot was actually also observed in HEL cell studies in other
experiments (data not shown). An additional series of bands, ranging
from about 3.1 to about 4.0 kb in length (HS I-II), appeared in the HEL
cell study in a DNase I-dependent fashion, but were absent in the HeLa
study (Figure 2A,C). Here, we describe HS I as a composite of 2 closely
localized bands. The lowest molecular weight band is 3.1 kb and appears
only transiently in HEL (second lane from left), but not at the lowest
DNase I concentration, indicating its dependence on DNase I. The second band, which is slightly longer and more intense (~3.7 kb), appears at
the same point as the 3.1-kb band and is eventually digested away at
the higher DNase I concentrations. Consistent results were observed
using an additional restriction enzyme, BamHI, with HEL,
CHRF (megakaryocytic), and HeLa cell lines and using a probe P2,
covering exon 4 (data not shown, but see Figure 2C). The
BamHI studies also showed an additional tissue-specific HS
site in the immediate promoter region (HS "P" in Figure 2C). It is
of interest that the tissue-specific HS domains I/II and "P"
overlap with the conserved 5'-intergenic regions at about 3.7 and 0.5 kb, respectively, shown in Figure 1. Figure 2, panels B and C, show
DNase I HS mapping of the 3'-intergenic domain of the IIb
gene. HEL nuclei and nuclei from SNU-1, a human gastric,
non-IIb-expressing cell line, were treated as above. SNU-1 cells
were used over HeLa cells because they were suspension rather than
adherent cells. Analysis by Southern blot studies using probe P3 based
on hIIb exon 30 (Figure 2B) revealed in the SNU-1 cells the expected
12.5 kb. This band decreased in intensity at increasing DNase I
concentrations, but no additional bands were seen (Figure 2B-C). In HEL
cells, the 12.5-kb EcoRI fragment also was visualized, but
there were, in addition, a series of HS segments appearing at the
mid-concentration range of DNase I, which correspond to regions of
increased DNase I susceptibility centered around 18.5 kb (HS V), 20.3 kb (HS VI), and 21.6 kb (HS VII; Figure 2B-C). Because these HS regions
were absent in the SNU-1 cells, these HS domains appear to be tissue
specific. Although HS region V is very faintly represented, we believe
it is an HS domain. First, it appears in both lanes 3 and 4 (from left
side of the blot). Second, in other experiments using a lower
concentration range of DNase I it also appeared with even greater
presence (data not shown). This suggests that it may be a short-lived
series of fragments present only within a narrow time period. It
appears that HS domains V to VII do not exist as clear distinct bands, but rather as a broader series of DNase I/EcoRI bordered
fragments within each HS domain. In contrast to restriction sites that
have a single cleavage point within a 4- to 8-nucleotide recognition sequence, HS sites can vary in size from 200 bp to more than 800 bp,37 and are nonspecifically cleaved. Hence, it is likely
that HS V-VII in Figure 2B represent larger extended HS domains. These domains are not present at the lowest DNase I concentration, then faintly appear within the second lane from the left of the HEL blot
reaching maximum intensity in the third lane. Note also that none of
the broad bands are present at the highest DNase I level. These types
of broad, tissue-specific HS domains have been reported to be present
in enhancer-containing regions of other genes such as the MyoD
muscle-specific enhancer,38 which is localized about 20 kb
upstream of the gene. In this enhancer the broad HS domains corresponded to a series of tandem transcription factor-binding sites.
Perhaps related to this, it appears that some of the tissue-specific HS
domains of our IIb 3' locus overlap with conserved intergenic domains in Figure 1. In particular, the HS region VII overlaps with the
conserved domain at +22 kb (Figure 1, vertical arrow "2").
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| Figure 2.
DNase 1 HS studies of the 5'- and 3-intergenic regions
. (A) Nuclei from megakaryocytic HEL and nonmegakaryocytic
HeLa cells were digested with DNase I nuclease at increasing
concentrations (wedges). The genomic DNA was then isolated, digested
with EcoRI restriction enzyme, and analyzed by Southern
blotting. The blots shown were hybridized to probe P1 covering hIIb
exon 1. The size marker is indicated as well as the detected HS sites,
which are denoted with Roman numerals. (B) Same as in panel A but for
SNU-1 and HEL cells, and the probe P3 covered hIIb exon 30. (C) The
hIIb gene locus is shown schematically at the top with its
transcriptional start site indicated as well as the orientation of the
genes shown. Restriction sites of importance are indicated with E
indicating EcoRI and B, BamHI. The full-length
fragment anticipated after EcoRI digestion for the studies
in panels A and B and a schema of the observed HS sites (vertical
arrows) within the cell lines tested are shown. P1 indicates probe for
EcoRI blot in panel A; P2, probe for BamHI blot
(not shown); and P3, probe for EcoRI blot in panel B. Similar results were seen on at least 2 independent studies for each
probe, cell line, and endonuclease restriction conditions.
|
|
Human IIb transgenic mice
The identification of at least 5 tissue-specific DNase I HS
domains located in the flanking intergenic regions of IIb, some of
which coincide with conserved domains, suggests the presence of
corresponding control elements that might be essential for establishing
appropriate transcriptional activation and critical for generating high
levels of hIIb on the surface of murine platelets. To test this, a
series of transgenic mice containing one of 3 different hIIb
constructs were made (Figure 3). These
include the shortest construct 2.5hIIb, which extends
from 2.5 kb upstream of the hIIb gene to 2.2 kb downstream of the
stop codon; 7.1hIIb, which extends from 7.1 kb upstream
of the hIIb gene to 2.1 kb downstream of the stop codon; and
the longest construct, 3'+hIIb, which is identical to the
7.1hIIb construct at the 5' end, but extends an
additional 5.2 kb downstream. Five founder lines for each
construct were evaluated. Copy number was determined and covered a wide
range from low to high copy number per haploid genome for each
construct (Figure 3). To begin our expression analysis, we investigated
our founder lines to determine if any or all of the transgenic lines
were expressing hIIb mRNA at some level. Because our genome copy
number range varied extensively, we anticipated similarly varied levels
of mRNA expression. Hence, we initially used nonquantitative PCR at a
high cycle number to detect the presence of any platelet hIIb mRNA.
All 15 founder lines had detectable hIIb mRNA in platelets by
nonquantitative RT-PCR analysis after 33 PCR cycles, well beyond the
linear/exponential range of amplification (Figure
4A). In the linear range, at 20 PCR
cycles, only the endogenous mIIb mRNA and the 3'+hIIb
mRNA samples produced visible bands on ethidium gel analysis (Figure 4A, top panel, and data not shown). Interestingly, the
3'+hIIb line produced visible bands even as low as 14 PCR
cycles, suggesting its expression level was significantly greater than
the 2.5 hIIb and 7.1 hIIb lines (data not
shown). None of these lines expressed hIIb mRNA in any
tissue other than platelets, bone marrow, and spleen, consistent with
restricted expression of high levels of IIb expression to developing
megakaryocytes (data not shown), and consistent with published
transgenic mice toxigene reporter studies.12
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| Figure 3.
Transgenic mice constructs studied.
The hIIb gene locus is shown at the top with the restriction sites
of interest and the HS sites shown in Figure 2 are indicated as
vertical arrows. Below that are the 3 constructs from which transgenic
lines were made. For each, 5 founder lines were established, and the
copy number of the transgene per haploid genome for each line is shown
to the right.
|
|
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| Figure 4.
RT-PCR analysis of platelet RNA for human and mouse
IIb.
(A) Agarose gel of size-fractionated nonquantitative RT-PCR
products for mIIb (top) or hIIb (bottom) from total platelet RNA
for the various transgenic lines. The mIIb samples were amplified
for 20 PCR cycles within the linear range of amplification for
comparison of relative cDNA levels. The hIIb samples were amplified
for 33 PCR cycles, beyond the linear range of amplification, to
facilitate detection of hIIb within all the diversely expressed
transgenic lines. Size markers (M) are indicated to the left and the
observed cDNA or genomic bands indicated at the right of each figure.
The observed genomic bands were variably present and only seen with the
2 lanes indicated where there appears to be little hIIb message, but
high transgene copy number, was present. Copy numbers are listed as
numbers above each lane. WT indicates wild-type mouse platelet RNA
control; W, water; mIIb, cDNA plasmid RT amplified; HP,
human platelet RNA control. (B) Analysis of semiquantitative
RT-PCR comparing relative expression of human to mouse IIb message
versus the copy number for a particular line. The open diamonds are the
2.5hIIb lines, the closed circles are the
7.1hIIb lines, and the closed squares are the
3'+hIIb lines. The experiments were done in triplicate
and repeated at least 3 times. A single SD is indicated for each
point.
|
|
To determine the relative level of platelet hIIb transgene
expression to the levels of endogenous mIIb, semiquantitative RT-PCR
was performed using platelet total RNA.18,20,22
Species-specific primer pairs were used to detect both species' IIb
transcripts. Samples from 6 consecutive PCR cycles taken during the
exponential range of amplification were obtained for both mouse and
human primer sets. In total, the levels of 180 RT-PCR amplification products were measured for each experiment (3 experiments
collectively) using fluorescent-modified antisense primers.
Additionally, values obtained were corrected for differences in primer
efficiency for the detection of their respective transcripts. The
summary of these studies are shown in Figure 4B, which indicates the
platelet hIIb mRNA relative expression versus native mIIb
gene expression, for each founder line plotted against its
corresponding copy number.
The range of expression for the 2.5 hIIb constructs was
from 7.5 × 103-fold to 1.5 × 102 less
than that of the endogenous mRNA level. These values were also not copy
number dependent. Indeed, the lowest copy line had the highest
expression. For the 7.1hIIb line, the range of expression was 9.6 × 103- to 6.5 × 102-fold less
than that of the native mIIb mRNA level. Unlike the 2.5hIIb construct, the 7.1 hIIb constructs
appears to show copy-number dependency, suggesting that the additional
5' sequence between 7.1 and 2.5 kb upstream of the hIIb gene
protects the transgenic gene locus from local chromosomal influences at
the site of insertion. Addition of more 3' sequence in the
3'+hIIb constructs led to a marked increase in absolute
expression levels, which ranged from 1.3-fold lower to 3.5-fold higher
than that of the endogenous message level. These expression levels were
linear with respect to change in copy numbers, again demonstrating
position-independent expression. For the 3+hIIb
constructs, each copy of the hIIb gene was expressed at
about 30% of the expression level of a single copy of the native
mIIb gene.
Expression of hIIb protein by the transgenic lines
We examined the protein level of platelet hIIb in these
transgenic animals as a secondary measurement of expression. Immunoblot studies of low and high hIIb-expressing 7.1hIIb and
3'+hIIb lines were done with MAB1990, a murine monoclonal
antibody directed specifically against hIIb32 (Figure
5). Only in protein extracts from
platelets derived from the 3'+hIIb lines was hIIb
detectable. The relative level of protein expression in the 2 tested
3'+hIIb lines was comparable to their 4.5-fold difference
in hIIb mRNA, after accounting for lane loading differences (Figure
5, Coomassie panel). Not shown was that protein levels for all of the
2.5hIIb lines, as with the 7.1hIIb
lines, was undetectable even for the highest expressing
one copy founder line whose mRNA level was about 1% of native levels
(data not shown). Compared with the human platelet control in Figure 5,
it appears that the highest 3'+hIIb transgenic mice
platelets expressed only about 10% of the hIIb expressed by human
platelets on a per milligram basis, although it is unclear from these
studies how this level compares with the level of mIIb expressed.
Thus, it appears that although these platelets have about 3.5 times the
level of hIIb mRNA compared with mIIb mRNA, the protein level of
hIIb/m3 was fairly low as compared with human platelets.
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| Figure 5.
Immunoblot of platelet protein for hIIb.
Immunoblot of size-fractionated platelet proteins using the monoclonal
anti-hIIb antibody MAB1990 as the primary antibody (right panel).
Size markers are indicated on the sides of the blot as well as the
expected hIIb band and the mouse IgG band. C indicates human
platelet control. Bands below the expected band were due to partial
sample degradation. Equal loading into each lane was confirmed on a
parallel gel, where a comparable loading of each sample was done and
the gel stained using Coomassie blue (left panel).
|
|
We also measured platelet hIIb/m3 surface levels by flow
cytometry. Consistent with the immunoblot studies, hIIb/m3
complex was detected only on the surface of the highest expressing
3'+hIIb line platelets and not on the surface of
platelets obtained from the lower expressing 3'+hIIb line
or from the 7.1hIIb lines (Figure
6) or the 2.5hIIb lines
(data not shown). Also consistent with the immunoblots in Figure 5, the
level of hIIb/m3 surface expression on the 3'+hIIb
platelets was about 10% of that on the human platelet control.
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| Figure 6.
Flow cytometric analysis of hIIb/m3 on the surface of platelets.
The mixed species hIIb/m3 receptors were detected by flow
cytometry using MAB1990 as the primary antibody. Except for the 18 copy
3'+hIIb platelets, none of the other animals had
platelets showing demonstrable hIIb/m3 receptors on their
surface, and the intensity in this highest line was still only about
10% of the human platelet control.
|
|
| |
Discussion |
In vitro studies of the IIb gene, using transient
reporter systems, previously demonstrated that the proximal promoter
was sufficient to direct tissue-specific
expression.9,10,33 Studies in transgenic mice of the human
and mouse IIb proximal promoter driving the expression of a toxigene
have been consistent with these in vitro studies, but did not examine
relative IIb expression levels nor the effects of the site of
chromatin integration on transgene expression. Our studies had a
different focus. We were interested in understanding the molecular
basis of IIb gene expression, in vivo, without
concentrating on its immediate proximal promoter region, but rather
searching for additional regulatory regions. We were prompted to do
this because our previous structural studies indicated that the
platelet-specific IIb gene was surrounded by 2 ubiquitously
expressed genes, KIAA0553, positioned about 5.8 kb upstream
and the Granulin gene located about 18 kb
downstream.23 The proximity of these ubiquitous genes
suggested to us the need for intergenic regulatory constraints to act
as boundaries to prevent inappropriate cross-regulation between genes.
Our current observation, that the widely expressed hCP44813
gene is located about 8.5 kb downstream of the IIb gene and
before the Granulin gene (Figure 1), is still in agreement
with our original premise, but further narrows the downstream
intergenic region between the IIb gene and its nearest
downstream neighbor.
The studies presented here demonstrate that there are phylogenetically
conserved, noncoding, intergenic domains both upstream and downstream
of the IIb gene, that these regions overlap with DNase I HS
sites and that the inclusion of the upstream region in a transgenic
mice construct confers copy number-dependent expression to the
transgene, whereas inclusion of the downstream intergenic region
confers an approximate 103-fold increase in expression.
These data extend what was previously known about the proximal IIb
promoter described above with its ability to drive tissue-specific
expression in vitro and in vivo. Our studies with the shortest
2.5 hIIb lines are in agreement with those studies and
demonstrate that a minimal 5' promoter can restrict IIb expression
to megakaryocytes and platelets. But, in addition, we now add to those
previous studies, by analyzing transgene expression relative to native
IIb mRNA levels. Our data reveal that 2.5 kb of 5'-flanking region,
though tissue specific, is insufficient to direct high levels of IIb
in vivo. We estimate that the transgenic mice in the earlier
toxigene reporter system studies using either 780 bp of the hIIb
promoter or about 2.7 kb of the 5' region of the mIIb promoter to
drive expression,12,39 most likely expressed their
transgenes at an approximate 0.1% to 1% of the native mIIb
gene expression. Perhaps that is why a toxigene reporter model was
successful for monitoring expression, as even low expression levels
could be phenotypically detected. We would also predict that in those
models expression levels would not have been copy number dependent (see
below). Whereas the 2.5 hIIb transgene lines did not show
any correlation between copy number and the relative hIIb to
mIIb message level, the 7.1hIIb transgene lines
demonstrated a clear correlation (Figure 4B). These data suggest
that there may be a regulatory element(s) between 2.5 and 7.1 kb
upstream, consistent with properties similar to an insulator element,
which by definition protects a gene from both positive and negative
influences of nearby chromatin, but may also impede enhancer action in
a directional fashion.40,41 It is possible that either the
tissue-specific HS I-II sites at 3.1 to 4.0 kb upstream of the
hIIb gene or the constitutive HS III and IV sites at 5.4
and 6.4 kb, respectively, upstream of the hIIb gene are
involved. Both are phylogenetically conserved and both are contained in
the 7.1hIIb transgenic construct. Further studies will
define which of these regions are needed to observe consistent
expression in transgenic mice studies.
The addition of 5.2 kb of downstream sequence in the
3'+hIIb construct led to an approximate
103-fold increase in hIIb expression above that from the
7.1hIIb construct. Within this 3' domain, we found
tissue-specific HS sites that overlap with a conserved domain and this
domain is a strong candidate for containing an enhancer, because other
enhancer elements for other genes have been shown to share these
structural features.42,43 The alignment of this conserved
domain for the human and murine loci is shown in Figure
7, and was analyzed for consensus-binding
sites of transcription factors known to be involved in
megakaryopoiesis. Unlike the -globin upstream HS sites, which contain a number of NF-E2-binding sites,44,45 there are
no NF-E2-binding sites in the analyzed IIb regions. Mice lacking NF-E2 have considerable megakaryocytic defects; however, these studies
did not examine IIb expression levels.45 The only
previously defined megakaryocyte-specific enhancer is upstream of the
PBP gene22 and it too has no NF-E2 sites. In
addition, neither region has conserved-binding sites for the AML-1
transcription factor, which is also involved in
megakaryopoiesis.46 However, both the IIb and PBP
enhancers have conserved GATA and Ets consensus-binding sites. Whether
these are of biologic importance and the mechanism(s) by which these
binding sites might lead to high level tissue-specific IIb
expression are unclear, but what is clear is that for the erythroid-specific -globin gene LCR, functionally
important GATA-binding sites are abundantly present in the upstream
DNase I-HS regions.47,48
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| Figure 7.
Conserved intergenic regions sequences.
Downstream intergenic, conserved sequences are compared with the human
sequence on top and the mouse below. A "|" refers
to an identical nucleotide. Conserved elements that may represent
important nuclear factor-binding sites are indicated in bold and the
nuclear factor and orientation of binding are shown.
|
|
One interesting observation from these studies was that the
3'+hIIb transgenic mice expressed only about 30% hIIb
message compared with native mIIb per genome copy. Although it is
possible that species-specific factors may limit the relative
expression of hIIb in mice models, an alternative explanation is
that there are additional upstream or downstream sequences that would
further increase the relative level of hIIb expression. Our studies
indicate that there are additional noncoding, phylogenetically
conserved sequences both upstream and downstream of the areas
investigated in this paper (Figure 1; also see supplemental Figure 1 provided online). Further, examples of genes in which regulatory
regions are found within and beyond neighboring genes have been
described.14,49,50 For instance, the flanking sequence of
the interleukin (IL) IL4, IL13, IL5 gene locus contains a
coordinate regulation element that exists between IL4
and IL13; however, the third gene, IL5, is
actually not continuous with these 2, having the RAD50 gene between itself and IL13. Larger transgenic constructs
containing more distal conserved flanking regions of the IIb locus
would have to be tested to see if expression level of the transgene would be further increased.
The relative hIIb to mIIb message steady-state level in
circulating platelets would have suggested that in the
highest-expressing 3'+hIIb transgenic lines that platelet
surface level expression of hIIb/m3 receptors would have been
higher than that observed in Figures 5 and 6. If one assumes that the
density of the IIb/3 receptor is similar on mice and human
platelets, and that these mice have continued expression of mIIb at
normal levels, then the highest expressing hIIb line should have had
hIIb protein levels 3 times that of the endogenous protein. The
hIIb/m3 heterodimers should have accounted for about 75% of
total surface IIb/3. Instead, our flow cytometry studies showed
these platelets had only about 10% of hIIb/m3 on its surface
compared with human platelets. This would suggest that there was an
approximate 30-fold lower level of hIIb/m3 to mIIb/m3 from
expected. This deficit in hIIb/m3 receptor level may be due
to a decrease in translational efficiency of hIIb mRNA or
in posttranslational hIIb or hIIb/m3 protein stability
compared with its mouse counterparts. The IIb pairs with 3 as it
is trafficked through the Golgi. The misparing or nonpairing with its
heterodimer partner leads to retention and ubiquitin-mediated
destruction.51 One intriguing possibility is that human
and mouse IIb compete for a limited supply of m3 chains.52 The hIIb may be out-competed by mIIb for
m3, and the unpaired hIIb is removed. Such a model would fit with
the proposed limited supply of 3, which selectively binds IIb
over v.52 In thrombasthenic patients without IIb,
the level of v more than doubles. If the m3 is in limited supply,
then decreased levels of mIIb in the megakaryocytes would
theoretically increase detectable hIIb/3 expression, because
there would be less competing mIIb. Consistent with this model, we
have found in preliminary flow cytometry data, that the level of
hIIb/m3 increased 3- to 4-fold in transgenic animals that were
also heterozygotes for an mIIb gene targeted
disruption53 (data not shown).
The formation of IIb/3 heterodimer appears to involve the
N-terminal -propeller of IIb and the A domain of
3.54 These domains show about 80% amino acid
cross-species homology. Whether any of the remaining amino acid
differences account for the dearth of hIIb/m3 seen on the surface
of the transgenic mice platelets remains to be tested.
In summary, our studies have extended the analysis of the regulated
expression of the IIb gene to the intergenic flanking regions between the IIb gene and its nearest gene neighbors. These studies were consistent with others showing that the proximal IIb promoter is sufficient to drive megakaryocyte-specific
expression, but also defined 2 previously unrecognized distal
regulatory regions. The first is an upstream region that allows for
consistent position-independent IIb gene expression. And the
second is a downstream region that drives high-level IIb
gene expression in vivo. Inclusion of these regulatory regions appears
to account for at least 30% of total IIb expression levels. Further
studies of these regions and comparison with similar elements
regulating other megakaryocyte-specific genes may provide important
insights into the mechanism(s) by which these tissue-specific genes are
highly expressed during megakaryopoiesis.
| |
Acknowledgments |
We would like to thank Dr Edward Rubin and his colleague Jan F. Cheng, both at the Lawrence Berkeley National Laboratory (Berkeley, CA), who assisted us in setting up the VISTA analysis. The hIIb pWE15 cosmid clone was generously provided by Dr Susan L. Neuhausen at
the University of Utah. CHRF cells were provided by Dr M. Liebman at
the University of Cincinnati. P1 clones used to generate hIIb clones were provided by screening a P1 library made available through
Dr Nat Sternberg at Dupont (Glenolden, PA).
| |
Footnotes |
Submitted May 2, 2002; accepted June 26, 2002.
Prepublished online as
Blood First Edition Paper, July 25, 2002; DOI
10.1182/blood-2002-05-1307.
Supported in part by grant HL40387 (to M.P.), a grant from the
Schulman Foundation (to M.P.), and a gift from the Plummer Family (to
M.P.).
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.
Reprints: Mortimer Poncz, Children's Hospital of
Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA
19104; e-mail: poncz{at}emailchop.edu.
| |
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Abstract
Introduction