From the Department of Molecular and Experimental
Medicine, The Scripps Research Institute, La Jolla, California 92037 and the ¶ Department of Microbiology and Immunology and Walther
Oncology Center, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Received for publication, October 5, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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The hematopoietic cell-specific ets
family transcription factor PU.1 regulates many lymphoid and myeloid
genes. We have determined that PU.1 is critical for lineage-specific
expression of the tyrosine phosphatase CD45. CD45 is expressed
exclusively in hematopoietic cells at all stages of development, except
for mature red cells and platelets. Although CD45 is normally expressed
in all leukocyte lineages, it is critically regulated by PU.1 only in
myeloid cells. Whereas myeloid cells from PU.1 null mice failed to
express CD45, lymphoid cells were CD45+ by flow cytometry.
Additionally, mRNA for CD45 was absent from PU.1-deficient myeloid
cells. To understand the molecular basis for these observations, we
characterized a transcriptional regulatory region of the murine CD45
gene containing exons 1a, 1b, and 2. Distinct transcriptional
initiation sites for CD45 were demonstrated in T and B cells
versus myeloid cells. A transcriptional initiation site in
exon 1b (P1b) was principally utilized by myeloid cells. A PU.1 binding
site was identified upstream of exon 1b by sequence analysis and DNA
binding assays. Using this region of the CD45 locus we demonstrated
that PU.1 directly transactivated reporter gene expression. Finally,
retrovirus-mediated restoration of PU.1 expression to PU.1-deficient
myeloid cells resulted in expression of cell surface CD45 and restored
phosphatase activity, confirming the role of PU.1 in the positive
regulation of this well known signaling molecule. We conclude that CD45
is regulated differentially in myeloid and lymphoid cells and that
sequences critical to direct myeloid expression include a PU.1 binding
site upstream of the P1b transcriptional initiation site.
The transmembrane tyrosine phosphatase CD45 is one of the most
abundant hematopoietic cell surface proteins (1, 2). CD45 is found
exclusively on hematopoietic cells, from primitive CD34+
cells through mature cells of all leukocyte lineages, except platelets
and mature erythrocytes. Expression of different CD45 isoforms is
generated by nontranscriptional means that include alternative usage of
a subset of the 32 coding exons and by post-translational modifications. The pattern of isoform expression is highly regulated and varies according to cell type during differentiation and activation (for a review see Ref. 1).
CD45 function has been studied almost exclusively in terms of its role
in signal transduction through the lymphoid antigen receptors.
Engagement of these receptors results in activation of members of the
Src family of tyrosine kinases
(SFKs)1 (3, 4). The activity
of these kinases has been shown to be dependent upon their
phosphorylation state, and certain family members are regulated by
CD45. Gene targeting of CD45 revealed its critical role in T cell
development. T cells of CD45 null mice were arrested at the
CD4+CD8+ stage without further maturation.
Although the myeloid compartment of the CD45 null mouse initially
appeared normal, subsequent studies of CD45 null macrophages have
revealed defects in integrin-mediated adhesion. The CD45-deficient
macrophages contained hyperphosphorylated SFKs Hck and Lyn, suggesting
that these were CD45 substrates in myeloid cells (5). Thus, the
regulation of integrin-mediated cell adhesion by CD45 via its
regulation of SFK activity appears to be its best documented function
in myeloid cells at present. Although it is not the only phosphatase
present, the abundance of CD45 in myeloid cells implies its probable
importance; in fact, CD45 accounts for 20% of total phosphatase
activity in resting neutrophils (6).
Developmental and cell type-specific expression of CD45 is well
established, but the transcriptional mechanisms conferring this
regulation remain unclear. Studies in T and B cell lines have
demonstrated that CD45 transcription can initiate at three mutually
exclusive positions in exon 1a (P1a), exon 1b (P1b), and downstream of
exon 1b (P2), but all use the same translational start site contained
in exon 2 (7-9). A single report has identified a pyrimidine-rich
nucleotide cluster just 5' of the P2 initiation site that possibly has
a role in both basal and activator functions in B and T cells (10).
Whether multiple transcription initiation sites reflect the capacity
for lineage and specific cell type regulation and which if any
additional DNA elements control transcription of CD45 is not known. We
have now identified a required role for the Ets family transcription
factor PU.1 in the regulation of CD45 in myeloid cells. PU.1 is a
hematopoietic specific transcription factor found in primitive
progenitors and in myeloid and B cells (11-13). The ets
family of proteins share a common DNA-binding domain that recognizes
purine rich sequences, usually containing a 5'-GGAA-3' core (12, 14).
We and others have shown that PU.1 is implicated in the regulation of
multiple lymphoid- and myeloid-specific genes and the loss of PU.1 in
mice results in a number of myeloid and B cell lineage defects (Refs.
15-18; reviewed in Ref. 14).
In this report, we demonstrate that CD45 is transcriptionally regulated
by PU.1 in a cell lineage-restricted manner. CD45 expression was
detected in T but not myeloid cells obtained from PU.1 null mice.
Differential transcriptional start site usage in T and B cells
versus myeloid cells was found. Characterization of the
region of the CD45 locus containing exons 1a, 1b, and 2 identified exon
1b as the principal CD45 transcriptional start site in myeloid cells.
Moreover, our studies showed that PU.1 bound to a specific site
upstream of exon 1b and could transactivate reporter gene expression.
Finally, retrovirally mediated reintroduction of PU.1 in PU.1 null
myeloid cells restored both CD45 expression and activity, confirming
the essential role of PU.1 in the regulation of this important
signaling molecule.
Mice--
C57BL/6 × 129 PU.1 gene-disrupted mice were
produced as previously reported (17).
Cell Culture and Cell Line 503--
Hematopoietic cells from
neonate liver were isolated and cultured as described (15). Samples
labeled "normal" were either heterozygous or homozygous for the
normal PU.1 allele. The PU.1 null myeloid cell line 503 that was
originally isolated from neonatal PU.1 null liver has been described
(15, 16, 19). This cell line was utilized for previously described
retroviral transduction studies in which PU.1 was restored (19).
Antibodies and Flow Cytometry--
Cells were prepared and
stained for flow cytometric analysis as previously described (17). A
CD45 antibody recognizing all isoforms of the molecule was obtained
from Pharmingen (clone 30-F11, Pharmingen, San Diego, CA). CD3, CD4,
CD8, CD18, Gr-1, and irrelevant isotype control antibodies were
obtained from Pharmingen. All flow cytometric analysis was performed
using a FACScalibur (Becton-Dickinson, San Jose, CA) and CellQuest
software (Becton-Dickinson).
RNA Isolation and Reverse Transcriptase-PCR for CD45
Expression--
Total RNA was prepared, and 0.5 or 1.0 µg was
treated with DNase and subjected to reverse transcription and PCR as
previously described (15). Primers used to detect the common 3' end of CD45 have been previously reported (20). A control reaction for DNA
amplification in the absence of reverse transcription was included for
each sample and reaction.
Isolation of B and T Cells from Spleen and Thymus--
Spleen
and thymus were removed and processed from 6-week-old C57BL/6J mice as
previously described (15). To isolate T lymphocytes from the
splenocytes, the cells were stained with CD90 (Thy1.2) microbeads
(Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions, and the CD90-positive cells were isolated using a
magnetic column. The remaining CD90-negative cells were then stained
with CD19 microbeads (Miltenyi Biotec) in a similar fashion to isolate
a pure population of B lymphocytes. Cell purity was measured by flow
cytometry using B220, CD3, Thy1.2, CD8, CD4, and the appropriate
isotype control antibodies directly conjugated to
R-phycoerythrin or fluorescein isothiocyanate (Pharmingen, San
Diego, CA).
CD45 Transcript Initiation Analysis--
cDNAs were
synthesized using 5 µg of total RNA and a specific murine CD45 exon
2-3 junction primer (spanning a ~50-kilobase intron) as previously
described (10). 1-5 µl of a 20-µl reverse transcription reaction
was subjected to 25 cycles of PCR using the exon 2-3 primer and a 5'
primer hybridizing to sequences corresponding to previously described
potential CD45 transcription initiation sites P1a, P1b, and P2 (10).
Location and sequence of primers are shown in Fig. 4. One-fourth to
one-third of the PCR reaction was analyzed by agarose gel
electrophoresis with ethidium bromide. Controls for DNA contamination
and nonspecific amplification contained template without reverse
transcription and demonstrated no detectable bands.
CD45 5' Regulatory Region Cloning, Mutagenesis, and Transient
Transfection Analyses--
A portion of the murine CD45 locus
surrounding exons 1a, 1b, and 2 was amplified from T cell line EL-4
genomic DNA and cloned into the plasmid KS+ (Stratagene Inc., San
Diego, CA) for sequence analysis. For PCR amplification the following
primers were used: 5' end,
5'-GTCGACGAATTCACTGATGCACAGAGGAGAGTC-3', and 3' end,
5'-CTCGAGACCCATGGTCATATCTGGAGATCAGC-3'. The CD45 5' regulatory region
was next cloned into the luciferase reporter plasmid pXP2 (21) using
the SalI and XhoI sites. Mutation of the core GGA
residues in site 1 (shown in Fig. 4) was performed by overlap extension
PCR, as previously described (22). For transfections, HeLa cells were
maintained in Dulbecco's modified Eagle's medium with 5% Fetalclone
I (Hyclone Inc., Logan, UT). Cells were seeded into 6-well plates at
2 × 105 cells/well. After 48 h, cells were
transfected with Lipofectin (Life Technologies, Inc.). Briefly, 2 µg
of both the reporter plasmid and the PU.1 expression plasmid were mixed
with 6 µl of Lipofectin in a final volume of 100 µl of serum-free
Dulbecco's modified Eagle's medium. After 15 min at room temperature,
the cells were given 2 ml of fresh Dulbecco's modified Eagle's medium with 5% Fetalclone I and 100 µl of DNA-lipid solution. 48 h
later, the cells were harvested, and luciferase activity was measured using standard protocols and a Lumat LB 9501 luminometer.
Gel Electrophoresis DNA Binding Assay--
Competitive DNA
binding assay was performed as previously described (12).
Immunoprecipitation and Phosphatase Assay--
The method for
preparation of cell lysates for phosphatase assay was as described (23)
with addition of 1% Nonidet P-40 (Sigma) and 0.5% CHAPS (Bio-Rad) to
the lysis buffer. 1-2 × 106 cells were incubated at
4 °C in lysis buffer for 30 min. Extracts were precleared by
incubation with 20 µl of protein A/G PLUS-agarose conjugate (Santa
Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4 °C. After
pelleting agarose, supernatants were transferred to fresh tubes. 5 µg
of CD45 goat polyclonal antibody (Santa Cruz Biotechnology) was added,
and samples were rotated at 4 °C for 4 h. 20 µg of protein
A/G PLUS-agarose conjugate was subsequently added, and samples were
rotated overnight at 4 °C. Precipitates were collected by
centrifugation and washed four times with 10 mM Tris-HCl,
pH 7.5. Samples were diluted with 10 mM Tris-HCl, pH 7.5, to 2.5 × 104 cell equivalents/µl, based on initial
cell counts. Tyrosine phosphatase assay kit 2 was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY), and the assay of
phosphatase activity was performed as directed by the manufacturer
using 2.5 × 105 cell equivalents/test well. Samples
were run in triplicate, and the results were combined from three
separate experiments. Phosphatase activity of PU.1 null and
PU.1-restored cells was expressed as a percentage of the activity of an
equal number of neutrophils cultured from PU.1-normal neonatal mice.
CD45 Is Not Expressed by PU.1-deficient Myeloid Cells--
We have
reported that freshly isolated liver or bone marrow from neonatal PU.1
null mice yields reduced numbers of hematopoietic progenitors and few
detectable mature myeloid cells (17). However, when cells isolated from
these sources are cultured in interleukin-3-containing medium for 5-14
days, chloroacetate esterase+, Gr-1+,
CD18+ polymorphonuclear cells (neutrophils) can be
identified. Myeloid cells with the same cell surface profile begin to
appear by days 3-5 in PU.1 null mice that are kept alive with
antibiotics (15-17). Hematopoietic cells freshly isolated from PU.1
null neonatal liver were examined for the expression of the
panleukocyte marker CD45 by flow cytometry using an antibody that
recognizes all forms of murine CD45 (see "Experimental
Procedures"). As might be expected based on the reported paucity of
leukocytes, CD45 was not detectable (data not shown). Surprisingly,
however, CD45 was not expressed by the myeloid cells cultured in
vitro from PU.1 null neonatal liver (Fig.
1A). In contrast, these cells
expressed the CD45 Transcription Is Differentially Regulated in Lymphoid and
Myeloid Cells in PU.1 Null Mice--
We previously demonstrated that
the CD45 isoform B220 is expressed on PU.1 null cells committed to B
cell development. B lymphopoiesis in these mice is highly aberrant,
does not progress beyond a very early stage, and generates few
detectable cells in the PU.1 null mouse (17). Detectable thymic
development in PU.1 null mice does not occur until 5-8 days after
birth, at which time TCR+, CD3+, CD4, and CD8
double- and single-positive cells can consistently be found in numbers
about 5-10-fold less than normal littermates (Ref. 17 and Fig.
2). T cells from 11- and 12-day-old PU.1
null mice were analyzed by flow cytometry. As shown, we found that essentially all thymocytes were also CD45+ as well as
CD3+ (Fig. 2). Thus, it appeared that CD45 transcription
might be differentially regulated in a lineage-specific fashion,
because this molecule was expressed by PU.1 null lymphoid cells but not myeloid cells. Intriguingly, although myeloid and B cells normally express PU.1, T cells have not been shown to express PU.1, suggesting that a distinct transcriptional mechanism for CD45 operates in this
lineage. Moreover, although PU.1 appeared to be critical for CD45
expression in myeloid cells, it did not appear to be required for B
cell CD45 expression.
Distinct CD45 Transcriptional Initiation Sites Are Utilized by
Lymphoid Cells and Myeloid Cells--
Three distinct CD45
transcriptional initiation sites have been identified in the
5'-untranslated region of this gene and are designated P1a, P1b, and
P2. P1a and P1b are located in exon 1a and 1b, respectively, and P2 is
found just 5' of exon 2 (Refs. 9 and 24 and shown in Fig.
3A). It has been shown by
primer extension analysis and S1 nuclease mapping that usage of exons 1a and 1b is mutually exclusive and is the result of alternative initiation of transcription. However, all CD45 transcripts utilize the
same ATG codon present in exon 2 to begin translation (9). Using a
PCR-based assay previously described by DiMartino et al. (10), we identified CD45 transcriptional initiation sites in primary B
cells, T cells, and myeloid cells. Thymic T cells derived from either
wild type or PU.1 null mice as well as wild type splenic Thy1.2+ T cells demonstrated initiation from all three
potential start sites (Fig. 3B). Similarly, wild type
CD19+ splenic B cells were able to initiate transcription
from P1a, P1b, and P2 (Fig. 3B). In contrast, wild type
neutrophils and macrophages did not utilize the P1a site but did use
the P1b and, inefficiently, the P2 site for initiation (Fig.
3B). As expected, PU.1 null myeloid cells demonstrated no
PCR products, consistent with their failure to express CD45 (Fig.
3B). Thus, in contrast to the three potential start sites
for transcriptional initiation that can be utilized by T and B cells,
myeloid cells have only two defined start sites.
PU.1 Binds Upstream of Exon 1b and Regulates Reporter Gene
Expression--
To determine whether PU.1 directly regulated the
murine CD45 gene, we cloned and analyzed the region of the CD45 genomic
locus including exons 1a, 1b, and 2. (9, 24). Within the first 140 base
pairs upstream of the P1b major murine transcriptional start site and
downstream of the P1a transcriptional start site, we identified by
sequence analysis two potential PU.1 binding sites, designated as sites
1 and 2 (Fig. 4A). Both sites
contained the critical core residues, 5'-GGAAGT-3' (12), and these
potential PU.1 binding sequences are conserved between the murine and
human CD45 genomic locus (24).
To determine whether PU.1 bound to either of these sites,
oligonucleotides for each site were generated, and band shift analyses were performed using in vitro translated PU.1 as well as
nuclear extracts from macrophages that express PU.1. These data showed that PU.1 had high binding affinity for site 1 (
Because we verified that PU.1 could bind to sites in the amplified CD45
5'-untranslated region, we next tested whether PU.1 could directly
regulate reporter gene activity through site 1. A wild type and site 1 mutated CD45 5' regulatory region were each cloned into the luciferase
reporter gene plasmid, pXP2. HeLa cells, which express neither CD45 nor
PU.1, were cotransfected with these amplified CD45 5' regulatory region
plasmids with and without the PU.1 expression plasmid PU.1PJ6 (12). As
shown, cotransfection of the wild type amplified CD45 5' regulatory
region with PU.1 resulted in an 8-9-fold increase in luciferase
activity (Fig. 4C). Cotransfection of the mutant CD45
reporter plasmid with PU.1 resulted in only a 2-fold increase over
background (Fig. 4C). Thus, these studies demonstrate that
PU.1 can bind to and transactivate through site 1 ( Restoration of PU.1 Is Sufficient to Restore Expression of CD45 in
PU.1-deficient Myeloid Cells--
To elucidate the role of PU.1 in
CD45 expression and myeloid development, we reintroduced PU.1 by
retroviral transduction into the PU.1 null myeloid cell line 503 (12,
15, 16). 503 is an interleukin-3-dependent PU.1 null
neonatal liver-derived myeloid cell line that expresses Gr-1
(40-50%), chloroacetate esterase (>90%), and CD18 (80%). 503 cells
have phenotypic and functional characteristics consistent with
aberrantly or incompletely matured neutrophils (16). Similar to the
myeloid cells that can be generated in short term interleukin-3
cultures of PU.1 null liver (Fig. 1), 503 cells do not express CD45
(Fig. 5A). However, in further
support for the role of PU.1 in the regulation of CD45 transcription in
myeloid cells, we observed both CD45 message (data not shown) and
surface protein expression following PU.1 restoration to 503 cells
(503-PU; Fig. 5A). Finally, phosphatase activity of
immunoprecipitated CD45 was restored in two PU.1-restored 503 clones at
94 and 80% of normal neutrophil activity, whereas PU.1-deficient 503 cells exhibited only 8% of normal activity (Fig. 5B). Our
combined results provide clear evidence that PU.1 is directly and
critically involved in the expression of CD45 specifically in myeloid
cells.
In this study we have investigated the role of PU.1 in regulating
transcription of the transmembrane tyrosine phosphatase CD45 in myeloid
and lymphoid lineages. Gene disruption of PU.1 in mice resulted in the
loss of CD45 expression on myeloid cells but not lymphoid cells. Of the
three transcriptional start sites initially described in T and B cells
(8, 9, 10), termed P1a, P1b, and P2, myeloid lineages primarily
utilized P1b, and to a lesser extent P2, for transcriptional
initiation. We have identified two potential PU.1 binding sites 5' of
the P1b and P2 CD45 transcriptional start sites, of which one appears
critical for directing CD45 gene expression in myeloid cells. Moreover, mutation of the strongest PU.1 binding site in the CD45 5' regulatory region abolished transactivation of a luciferase reporter in
transfection assays. These results indicate that the expression of
CD45, which is ubiquitously expressed on hematopoietic cells, is
differentially controlled in myeloid and lymphoid cells and demonstrate
that PU.1 is necessary for CD45 expression in myeloid cells.
Lineage-specific mechanisms of transcriptional regulation of the same
gene are rarely reported but not unique. Very recently, characterization of the promoter of the RAG2 gene has shown that different transcriptional factors are responsible for B cell-specific versus T cell-specific expression of this lineage- and
developmental stage-specifically regulated gene. Whereas a similar
upstream region was required for promoter activity in both B and T
cells, mutations of this region affected transcriptional activity
differentially. Furthermore, B cell-restricted BSAP was critical for
RAG2 promoter activity in B lineage cells (25, 26). We found that
freshly isolated primary B and T cells initiate transcription from
three different sites in the CD45 genomic locus containing exons 1a, 1b, and 2, consistent with previous data from T and B cell tumor lines
(10). However, in primary neutrophils and macrophages, only the P1b
site and to a lesser extent the P2 site appear to be utilized for
transcriptional initiation. To date, other regulatory elements in the
CD45 genomic locus, such as TATA- and CCAAT-like sequences, have not
been identified. A single report suggests that a pyrimidine-rich
nucleotide cluster just 5' of the P2 initiation site might have a role
in both basal and activator functions in B and T cells (10). Whether
similar pyrimidine-rich sequences have a role for transcriptional
initiation of the more prominent start sites located at P1a and P1b is
unclear. Additionally, our results do not exclude the possibility that
more distal regulatory elements exist that have not yet been described.
Our previous studies of PU.1 null mice have demonstrated that CD45 was
expressed on cells committed to the B cell lineage (17). This
observation might reflect the preferential use of the P1a start site in
cells committed to the B cell lineage in the absence of PU.1.
Alternatively, given our demonstration of the use of the P1b and P2
start sites by B cells, it is tempting to speculate that other Ets
family members may be involved in CD45 expression in lymphoid cells.
Spi-B is most closely related to PU.1, sharing a 67% amino acid
homology in the DNA-binding domain. This protein is expressed in both T
and B cells (27, 28), unlike PU.1, which is not detectably expressed in
committed T cells (17). It has been documented that Spi-B null mice
express CD45 (B220) and do not have T cell abnormalities as do CD45
null mice (29, 30), suggesting that their expression of CD45 is normal
(28). This outcome might be expected if both Spi-B and PU.1 could be
used by B cells for CD45 expression. It is also possible that other
PU.1-related Ets proteins may be responsible in these lineages, such as
the recently described Spi-C, which is expressed in B cells (31, 32) or
other as yet undiscovered PU.1-like Ets proteins. Alternatively, it may
be that an entirely different class of transcription factors regulates
CD45 expression in B and T cells, similar to the example of RAG2 gene
regulation in B versus T cells cited above (25, 26). In this
regard, it will be important to determine whether the PU.1 site(s) are critical for CD45 expression from P1b in B and T cells.
The role of CD45 in hematopoietic cells is best understood in T and B
cell lineages. CD45 has been shown to regulate SFK-mediated antigen
receptor signaling in T cells and B cells (reviewed in Refs. 4 and 33),
and hyperphosphorylated SFKs Lck and Fyn were observed in T cells from
CD45 null mice (34). It was previously believed that dephosphorylation
of SFKs by CD45 was always a kinase-activating event that
counterbalanced the phosphorylation-induced inactivation by Csk;
however, this idea has recently been disputed (33). For example,
macrophages derived from CD45-deficient mice contained hyperphosphorylated and hyperactivated Hck and Lyn, indicating that
these SFKs were substrates that were normally negatively regulated by
CD45 in myeloid cells. Defects in integrin-mediated adhesion, which is
known to be critical for functions associated with normal innate
immunity, were demonstrated in CD45-deficient macrophages (5). We have
identified many functional deficits that exist in PU.1-deficient
myeloid cells, including abnormal adherence and chemotaxis,
phagocytosis, and killing of microbes (16). Exactly how the absence of
CD45 relates to these abnormalities is not yet clear.
With this report, we have extended the list of PU.1-regulated genes
beyond myeloid growth- and function-specific genes, such as CD11b,
gp91phox, and myeloid CSF receptors, to include signal
transducing molecules such as the tyrosine phosphatase CD45. Tyrosine
kinases and phosphatases have been implicated in influencing cellular
events ranging from proliferation to motility to generation of
antimicrobial activity (reviewed in Refs. 35 and 36). We have
documented direct transcriptional regulation of CD45 by PU.1, and
significantly, this regulation occurs exclusively in myeloid cells.
Understanding the means by which this lineage-restricted gene
regulation is accomplished may provide critical insight into the
mechanisms that control the defining events of lineage development and
confer normal myeloid function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 integrin component CD18 (Fig.
1A), a well known marker of myeloid cells. To determine
whether the absence of cell surface CD45 could be explained by the loss
of gene transcription, PU.1-deficient myeloid cells were then analyzed
for the presence of CD45 mRNA by reverse transcriptase-PCR. We used
previously designed primers (20) that amplify a 3' portion of CD45
sequence known to be present in all forms of the molecule (2). As shown
in Fig. 1B, CD45 was undetectable in PU.1-deficient myeloid
cells at the mRNA level as well.
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Fig. 1.
CD45 is not expressed by PU.1-deficient
myeloid cells. A, CD18+ myeloid cells
cultured from the liver of neonatal PU.1 null mice fail to express CD45
as detected by a monoclonal antibody that recognizes all forms of the
molecule. Ig controls (IgG) consisted of irrelevant
isotype-matched antibodies. B, reverse transcriptase-PCR was
performed using primers designed to amplify the 3' portion of CD45 that
is common to all isoforms. CD45 mRNA was not detectable in PU.1
null myeloid cells (PU.1 Null Myeloid) but was present in
normal (homo- or heterozygous for normal PU.1 allele) myeloid cells
(Normal Myeloid) cultured in an identical manner and in the
T cell line BW5147. PCR amplification of the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase was included to verify
cDNA sample integrity and equivalence. Sizes of bands are given in
base pairs (bp). Controls for DNA amplification in the
absence of reverse transcription were negative (lanes labeled No
RT). The far right-hand lane (MW) contains a
100-base pair DNA ladder.
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Fig. 2.
Thymocytes from PU.1 null mice express
CD45. Thymocytes were isolated from 10-12-day-old PU.1 null mice
and their normal (homo- or heterozygous for normal PU.1 allele)
littermates and were analyzed as described under "Experimental
Procedures." CD4+ and CD8+ cells as well as
CD3+CD45+ cells were consistently present in
the thymus of PU.1 null mice over 8 days of age. Flow cytometry data
are presented from a representative PU.1 null and normal individual; at
least 12 PU.1 null mice have been examined to date. Ig controls
consisting of irrelevant isotype-matched antibodies were negative and
are not shown.
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Fig. 3.
Distinct CD45 transcriptional initiation
sites are utilized in lymphoid cells and myeloid cells.
A, a schematic drawing of the CD45 5' regulatory region
depicts the previously identified transcriptional initiation sites P1a,
P1b, and P2. All transcripts utilize a single ATG in exon 2 for
translational initiation. The intron between exons 2 and 3 is ~50
kilobases. B, cDNA was prepared using an exon 2-3
junction-spanning primer and subjected to PCR amplification using this
primer and a specific 5' primer designed to anneal to sequence in each
of the initiation regions P1a, P1b, and P2. Predicted PCR products of
247, 235, and 180 base pairs indicating initiation from all three sites
were seen in splenic CD19+ B cells and splenic
Thy1.2+ and thymic T cells. In contrast, myeloid cells
demonstrated initiation primarily from the P1b site with some P2
initiation as well. No PCR products were amplified from PU.1 null
myeloid cells that do not express CD45. Controls containing template
without reverse transcription contained no bands (data not
shown).
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Fig. 4.
PU.1 can bind and transactivate through a
site upstream of exon 1b in the CD45 genomic locus. A,
potential PU.1 binding sites were identified by sequence analysis at
positions 124 to
107 (site 1) and
68 to
51 (site 2) relative to
the major murine transcriptional start site and are indicated as
boxed regions with the core sequence lightly
underlined. Exons 1a, 1b, and 2 are indicated with
brackets, and the sequence of primers used to detect
transcriptional initiation from the three previously identified sites,
P1a, P1b, and P2 is shown (heavy underlining). The common
translational start site is indicated by capital letters.
B, competition analysis for DNA binding was performed using
labeled cloned oligonucleotide consisting of site 1 (wild type)
sequence. Cold competitor oligonucleotides included site 1 (45PU1),
mutated site 1 (m45PU1), or site 2 (45PU2) and were used at 50-, 100-, and 500-fold molar excess. The sample with labeled oligonucleotide but
no in vitro-translated PU.1 protein and no competitor is
indicated by a dash (
). The lane labeled PU.1 contains
labeled site 1 oligonucleotide and PU.1 protein with no competitor.
Note that an unlabeled site 1 oligonucleotide competed efficiently with
a labeled site 1 probe at 50-fold excess, whereas a mutant site 1 oligo
was unable to compete at any concentration tested. Site 2 oligonucleotides competed only at 500-fold molar excess. C,
the wild type (WT) and mutant CD45 5' regulatory regions
were cloned into plasmid pXP2 upstream of a luciferase reporter gene.
Non-CD45- or PU.1-expressing HeLa cells were transfected with and
without the PU.1 expression plasmid PU.1PJ6. Note the 8-9-fold
increase in luciferase activity only when the wild type CD45 5'
regulatory region and PU.1PJ6 plasmids were cotransfected.
124 to
107; Fig.
4A) and very weak affinity for site 2 (
68 to
51; Fig.
4A) (data not shown). To demonstrate specific binding to
site 1, competition analyses were performed using site 1, mutant site
1, and site 2 oligonucleotides as cold competitors (Fig.
4B). As shown, the site 1 oligonucleotide efficiently
competed with the labeled site 1 probe, but an oligonucleotide with a
mutation of the core GGA residues of site 1 was unable to compete. Site
2 oligonucleotides were able to compete efficiently only at 500-fold
molar excess, further confirming the lower affinity of PU.1 for this
sequence as compared with site 1. To further delineate the binding of
PU.1 to site 1, we performed methylation protection analysis and
observed that both core GG residues were protected from methylation
(data not shown), which is consistent with our previous studies on how PU.1 and other Ets domain proteins interact with DNA (12).
124 to
107; Fig.
4A), which is located upstream of the major point of
transcriptional initiation for the CD45 gene in myeloid cells.
View larger version (19K):
[in a new window]
Fig. 5.
Restoration of PU.1 expression in the
PU.1-deficient myeloid cell line 503 restores CD45 expression and
activity. PU.1 was reintroduced to PU.1-deficient 503 cells by
retroviral transduction as previously described (503-PU).
A, cell surface expression of CD45 protein was restored in
503-PU cells. The solid black area represents cells stained
with irrelevant isotype-matched antibody, whereas the heavy black
line represents staining with an CD45 monoclonal antibody.
B, CD45 was immunoprecipitated from cells and its
phosphatase activity was measured at 94 and 80% of wild type
neutrophil activity level in two different PU.1-restored 503 samples
(503-PU1 and 503-PU2). In contrast, CD45 activity
in PU.1-deficient 503 cells (503) was 8% of wild type
neutrophil activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Giano Panzarella and the animal care personnel at The Scripps Research Institute and Drs. William Raschke and Dong-Er Zhang for careful reading of the manuscript and helpful suggestions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK49886 (to B. E. T.) and CA71384 (to M. J. K.). This is Publication 13470-MEM from The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of National Institutes of Health Training Grant T328HLO7195-22.
To whom correspondence should be addressed: Depts. of
Molecular and Experimental Medicine, MEM55, Scripps Research Inst., 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.:
858-784-9123; Fax: 858-784-2121; E-mail: betorbet@scripps.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M009133200
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ABBREVIATIONS |
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The abbreviations used are: SFK, Src family kinase; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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