(Received for publication, June 15, 1995; and in revised form, July 14, 1995)
From the
CD18 ( leukocyte integrin) is a
leukocyte-specific adhesion molecule that plays a crucial role in
immune and inflammatory responses. A 79-nucleotide fragment of the CD18
promoter is sufficient to direct myeloid transcription. The CD18
promoter is bound by the B lymphocyte- and myeloid-restricted ets factor, PU.1, and disruption of the PU.1-binding sites
significantly reduces promoter activity. However, PU.1 alone cannot
fully account for the leukocyte-specific and myeloid-inducible
transcription of CD18. We identified a ubiquitously expressed nuclear
protein complex of extremely low electrophoretic mobility that also
binds to this region of the CD18 promoter. This binding complex is a
heterotetramer composed of GABP
, an ets factor, and
GABP
, a subunit with homology to Drosophila Notch.
GABP
competes with the lineage restricted factor, PU.1, for the
same critical CD18 ets sites. The CD18 promoter is activated
in myeloid cells by transfection with both GABP
and GABP
together, but not by either factor alone. Transfection of
non-hematopoietic cells with the two GABP subunits together with PU.1
is sufficient to activate CD18 transcription in otherwise
non-permissive cells. Thus, GABP and PU.1 compete for the same binding
sites but cooperate to activate CD18 transcription.
Cellular differentiation is linked to tightly regulated gene expression, and most developmentally regulated genes are controlled at the transcriptional level(1) . In some tissues, e.g. erythroid cells(2) , cellular differentiation and gene expression are governed by lineage restricted transcription factors. However, many lineage-specific or developmentally controlled genes are regulated by transcription factors that are expressed in a broader range of cell types. In such cases, specific combinations of these more widely expressed factors, or their lineage restricted modification, may contribute to tissue-specific gene expression.
CD18 ( leukocyte integrin) is a cell surface molecule that plays a
crucial role in cell-cell and cell-matrix interactions. It is expressed
exclusively by lymphocytes and myeloid cells (monocytes and
granulocytes). During myeloid differentiation, CD18 expression
increases due to increased transcription(3, 4) . We
and others have cloned the gene encoding CD18 and isolated its
promoter(4, 5, 6, 7) . The CD18
promoter directs expression of a linked reporter gene when transfected
into myeloid cells(4, 6) . A 79-nucleotide (nt) (
)region of the CD18 promoter that is crucial for its
myeloid expression contains three potential binding sites for ets transcription factors. We have shown that this region of the
promoter is bound by PU.1, an ets-related transcription factor
that is expressed by B lymphocytes and myeloid cells. Mutagenesis of
these PU.1-binding sites in the CD18 promoter decreases promoter
activity and commensurately decreases PU.1 binding (8) .
CD18 and PU.1 are co-expressed in some cell types, e.g. myeloid cells and B lymphocytes. However, other transcription factors must contribute to the regulated transcription of CD18. T lymphocytes express CD18 in the absence of PU.1 expression(9, 10, 11) , and transfection of PU.1 is not sufficient to activate the CD18 promoter in non-hematopoietic cells. CD18 expression increases 4-fold during myeloid differentiation while PU.1 binding activity is not appreciably altered(8, 12) . Thus, PU.1 alone does not fully account for the leukocyte-specific and myeloid-inducible expression of CD18.
We have found, using the electrophoretic mobility shift assay
(EMSA), that multiple nuclear proteins bind to the crucial regulatory
region of the CD18 promoter. We characterized these factors in order to
better define the regulated transcription of CD18. The sequence GGAA,
which is the core binding site for several members of the ets transcription factor family, is present at three locations in the
CD18 -79-nt promoter. We identified a protein complex of
extremely low electrophoretic mobility that binds to these crucial
regulatory elements. This complex contains both GABP, an ets-related factor, and GABP
, a subunit with homology to Drosophila Notch(13, 14) . GABP
alone
does not bind to DNA, but together these proteins form a
heterotetrameric complex on the CD18 promoter. GABP
competes with
PU.1 for binding to regulatory elements in the CD18 promoter.
Transfection into myeloid cells of GABP
and GABP
together,
but of neither factor alone, activates the CD18 promoter. Transfection
of both GABP subunits along with PU.1 into non-hematopoietic cells is
sufficient to activate a CD18 reporter construct. Thus, two different ets-related transcription factors, the lineage restricted
factor PU.1 and the ubiquitously expressed factor GABP
, compete
for binding to crucial regulatory sites in the CD18 promoter, yet they
functionally cooperate to activate CD18 transcription.
Figure 2:
ets factors bind to CD18
promoter. A, the sequence of the CD18 promoter between
-89 and -30 is displayed. The three potential ets-binding sites and the orientation of the sequence GGAA are
indicated by arrows. Note that the GGAA sequence at
-72/-75 is encoded on the bottom strand. Double-stranded
probes used for EMSA are indicated by brackets. B, a
double-stranded oligonucleotide probe corresponding to
-85/-37 of the CD18 promoter was radiolabeled with
[P]ATP. EMSA was performed with 10,000
counts/min probe in 0.5
TBE. Proteins used in the binding assay
include unprogrammed reticulocyte lysate (RL), in vitro translated PU.1, Ets-1, Fli-1, Elf-1, purified, bacterially
expressed GABP
and GABP
, or nuclear extracts from U937
(myeloid), Raji (B lymphocyte), HeLa (cervical carcinoma), and Jurkat
(T lymphocyte) cell lines. Right arrow indicates the location
of binding by PU.1 (lanes 3, 9, and 10), and
the curved arrow indicates a binding species unique to Jurkat
cells.
Figure 1: CD18 promoter is active in leukocytes, but not in non-hematopoietic cells. CD18(-918/luc) or CD18(-79/luc) were co-transfected with CMV/hGH into U937 (myeloid), Jurkat (T lymphocyte), Raji (B lymphocyte), and HeLa (non-hematopoietic) cell lines. Luciferase activity is normalized to the CMV/hGH internal control and expressed as normalized relative light units (NRLU). Results represent the mean ± S.E. from three separate experiments.
The multimeric pattern of binding by GABP and GABP
to the
CD18 promoter resembles GABP binding to the HSV IE promoter and other
genes that contain multiple ets sites. Gel filtration,
sedimentation, and cross-linking studies were used to characterize the
nature of each of these binding species on the HSV IE
promoter(13) . We used UV cross-linking and mutagenesis of the
GABP subunits to confirm the multimeric nature of the binding complexes
on the adenovirus E4 promoter(17) . The two complexes formed on
the CD18 promoter by GABP
correspond to monomeric and homodimeric
forms of the protein (Fig. 3, lane 2, M and D). GABP
alone does not bind to CD18 (lane 3).
However, GABP
and GABP
together can generate three distinct
binding species. These correspond to the monomeric GABP
(lane
4, M), heterodimeric GABP
/GABP
(D, which co-migrates with homodimeric GABP
),
and heterotetrameric GABP
/GABP
(T) species demonstrated in previous
studies(13, 17) .
Figure 3:
GABP and GABP
are present in the
co-migrating nuclear binding species. EMSA was performed with
radiolabeled CD18 -85/-37 probe and GABP
alone (lane 2), GABP
alone (lane 3), GABP
and
GABP
together (lane 4), and U937 nuclear extract (lanes 5-8). Binding reactions were preincubated with
antibody against GABP
(lane 6), antibody against
GABP
(lane 7), or preimmune serum (lane 8). The
locations of monomeric, dimeric, and tetrameric binding species are
indicated by M, D, and T, respectively, and
the open arrow indicates the location of the supershifted
proteins.
Figure 4:
Localization of GABP-binding sites and
sensitivity of binding to mutations. The following EMSA studies were
performed in 0.25 TBE. A, EMSA was performed with
purified GABP
and GABP
and the following radiolabeled probes:
-85/-37 (lanes 1-3), -89/-58 (lanes 4-6), and -59/-31 (lanes
7-9). The locations of monomeric, dimeric, and tetrameric
binding species are indicated by M, D, and T, respectively. B, EMSA was performed with purified
GABP
and GABP
and the following radiolabeled probes: wild
type (wt) -89/-58 probe (lanes
1-3), -89/-58 probe containing GGAA
CCAA
mutation (mut) in the ets site (lanes
4-6), wild type -59/-31 probe (lanes
7-9), and -59/-31 probe with GGAA
CCAA
mutations in both of the ets sites (lanes
10-12).
The probe corresponding to
-59/-31 of the CD18 promoter contains two potential ets sites (at -53/-50 and -47/-44, labeled
B and C in Fig. 2A). GABP alone
forms predominantly a monomeric species (lane 8, M),
with a small amount of homodimer (more apparent in Fig. 4B, lane 8). However, GABP
and
GABP
together generate all three species that form on the larger
-85/-37 probe (compare lane 9 to lane 3).
Thus, each of the three potential ets sites supports GABP
binding and can contribute to formation of tetrameric GABP binding on
the CD18 promoter.
Figure 5:
DNase I footprinting with GABP. A
probe corresponding to -148/+28 of the CD18 promoter was
prepared by polymerase chain reaction and radiolabeled on the
non-coding strand. DNase I footprinting was performed with 1 µl of
purified, bacterially expressed GABP
(labeled
), or
in the absence of added protein (N). Vertical bars indicate the location of the protected regions (labeled A-C which correspond to the ets sites in Fig. 2A). A prominent DNase I-hypersensitive site is
indicated by the asterisk. Numbers at left correspond
to promoter sequence relative to the dominant start site of
transcription.
Figure 6:
GABP and PU.1 directly compete for
binding to CD18 ets sites. A, EMSA was performed with
radiolabeled CD18 -85/-37 probe and purified bacterially
expressed GABP
(GST-E4TF1-60). GABP
binding (lane
2) is abrogated by 100-fold molar excess of unlabeled specific
competitor probe (lane 3), but not by nonspecific probe (lane 4). Addition of increasing amounts of GST-PU.1 (lanes 5-8) causes loss of GABP
binding, in
exchange for increased binding by PU.1. GABP
monomeric (M) and dimeric (D) binding species and PU.1 (PU.1) are indicated. A binding complex of high
electrophoretic mobility (indicated by the asterisk)
represents a proteolytic degradation product of GST-PU.1, as described
previously(8) . B, GST-PU.1 binding (lane 2)
is abrogated by a 100-fold excess of unlabeled specific competitor (lane 3), but not by nonspecific probe (lane 4).
Addition of increasing amounts of GABP
(GST-E4TF1-60)
recruits probe from PU.1 to GABP
complexes (lanes
5-9).
Similarly, EMSA with GST-PU.1 (Fig. 6B, lane 2, labeled PU.1) forms
a distinct binding species with the CD18 -85/-37 probe that
is abrogated by a 100-fold molar excess of homologous unlabeled probe
but not by nonspecific probe (lanes 3 and 4,
respectively). A species of high electrophoretic mobility (indicated by
the asterisks in Fig. 6, A and B) is
caused by binding to the probe by a proteolytic degradation product of
PU.1(8) . Addition of increasing amounts of GABP to a
fixed amount of PU.1 in the binding reaction recruits probe to the
GABP
complexes.
Figure 7:
GABP and GABP
together with
PU.1 activate CD18 transcription. A, U937 cells were
transfected with 20 µg of CD18(-918)/luc and no added
effector, 5 µg of GABP
(pCAGGS-E4TF1-60) alone, 5 µg
of GABP
(pCAGGS-E4TF1-53) alone, or 5 µg each of both
GABP
and GABP
. Each sample was transfected with the internal
control CMV/hGH and additional pGEM3zf
to bring the
amount of transfected DNA in each sample to 30 µg. Luciferase
activity was measured 14 h later and was normalized to growth hormone
expression. Data represent the mean ± S.E. from three separate
experiments. B, HeLa cells were transfected with 20 µg
CD18(-918)/luc and no added effector, 5 µg of GABP
(pCAGGS-E4TF1-60) alone, 5 µg of GABP
(pCAGGS-E4TF1-53) alone, 5 µg of PU.1 (in the mammalian
expression vector pECE) alone, or combinations of 5 µg each, as
indicated. Each sample was transfected with the internal control
CMV/hGH and additional pGEM3zf
to bring the amount
of transfected DNA in each sample to 40 µg. Luciferase activity was
measured 14 h later and was normalized to growth hormone expression.
Data represent the mean ± S.E. from three separate
experiments.
In order to better define the molecular basis of myeloid
differentiation, we are studying the factors that regulate
transcription of the leukocyte integrin, CD18. A
79-nt fragment of the CD18 promoter is required for transcription in
myeloid cells(6, 8) , and we have previously shown
that the ets-related transcription factor PU.1 binds to this
region (8) . We now demonstrate that GABP
, a second ets factor, competes with PU.1 for binding to these sites in
the CD18 promoter. GABP
forms multimeric complexes with its
Notch-related partner GABP
, and together these factors activate
CD18 transcription in myeloid cells. Furthermore, we show that GABP and
PU.1 are sufficient to activate the CD18 promoter in non-hematopoietic
cells. Thus, although these ets factors compete for promoter
binding, they functionally cooperate to activate CD18 transcription.
The region of CD18 that is required for promoter activity contains
three potential ets sites. Because ets transcription
factors bind to similar core DNA sequences, we sought to determine if
other ets factors, besides PU.1, bind to CD18. We detected a
widely expressed nuclear factor of extremely low electrophoretic
mobility that binds to this region of the CD18 promoter.
Böttinger et al.(6) suggested
that GABP might bind to the CD18 promoter. We now demonstrate that
purified GABP binds to all three CD18 ets regulatory
elements. Purified GABP
alone forms monomers and homodimers on
these sites and its binding is sensitive to mutation (GGAA
CCAA)
of the consensus ets sequence. GABP
alone does not bind
to the CD18 promoter, but in the presence of GABP
it forms a
complex that co-migrates with the low mobility nuclear binding complex.
We used antibodies to confirm that this myeloid-binding complex
contains proteins that are immunologically related to both GABP
and GABP
.
GABP was originally identified as a factor from rat
nuclei that binds to critical regulatory elements in HSV IE
genes(13, 16) . GABP is composed of two distinct
polypeptides, which together are required for high affinity DNA binding
and transcriptional activation. The human equivalents of GABP and
GABP
were independently cloned as regulators of adenovirus E4 gene
transcription (E4TF1-60 and E4TF1-53/47, respectively) (14) and as nuclear respiratory factor 2 (NRF-2
and
NRF-2
/
, respectively) (18) GABP
is a DNA-binding
protein with homology to the ets family of transcription
factors. GABP
is unrelated to the ets family, but it has
homology to Drosophila Notch and Caenorhabditis elegans Glp-1 and Lin-1. Four ankyrin repeats in its amino terminus are
required for its interaction with GABP
, and a coiled-coil motif in
its carboxyl terminus permits GABP
homotypic
dimerization(13, 17) . Whereas DNA binding is mediated
by the GABP
subunit, transcriptional activation is controlled by
GABP
(17, 18, 19) .
GABP generates a
complex of extremely low electrophoretic mobility when it binds to CD18
promoter probes that contain at least two of its three ets-binding sites. Gel filtration, gradient sedimentation, and
cross-linking studies were used to conclusively demonstrate the
heterotetrameric nature of GABP complexes on a similar arrangement of
three ets sites on HSV IE genes(13) . We used UV
cross-linking and mutagenesis of the GABP subunits to confirm the
multimeric nature of the binding complexes to the adenovirus E4
transcriptional promoter(17) . In the present study, we used
mutagenesis of CD18 ets sites to demonstrate an identical
pattern of binding by GABP and GABP
. Other genes with
multiple ets-binding sites, including folate-binding
protein(20) , aldose reductase(21) , and cytochrome c oxidase subunits IV and 5b(22, 23) , are
bound by GABP in a similar manner. As with each of these genes, the low
mobility CD18 promoter binding complex represents
GABP
/GABP
heterotetramers.
The two ets factors that bind to the CD18 promoter, PU.1 and
GABP, are expressed in very different lineage-specific patterns.
PU.1 is expressed by B lymphocytes and myeloid
cells(9, 10, 11) ; in accordance with its
pattern of expression, we detect PU.1 binding activity only in these
cellular compartments. PU.1 appears to control the transcription of
several leukocyte-specific genes including CD11b (24) , M-CSF
receptor(25) , Fc
R1(26, 27) , and
macrophage scavenger receptor(28) , as well as
CD18(8) . PU.1 is required for myeloid colony
formation(29) , and its targeted disruption by homologous
recombination abrogates lymphopoiesis and myelopoiesis(30) .
Thus, PU.1 is essential for normal hematopoiesis and may control
transcription of some myeloid genes.
In contrast to PU.1, GABP
and GABP
are widely expressed(16) . We detected binding to
CD18 by the heterotetrameric GABP species in all cell types examined.
Thus, GABP binding activity is present both in cells that express CD18
(myeloid cells and B and T lymphocytes) and in cells that do not
express CD18 (HeLa). Although most genes whose activity is controlled
by GABP are widely expressed, GABP also controls genes that are
expressed in a lineage-restricted manner. For example, the gene
encoding
4 integrin, which is expressed predominantly by
leukocytes(31) , and the F promoter of 6-phosphofructo-2 kinase (32) are transcriptionally regulated by GABP.
How can the
widely expressed GABP transcription complex control the
lineage-restricted expression of genes such as CD18 and 4
integrin? There are multiple isoforms of the
GABP
(14, 16, 22) , the subunit that
controls transcriptional activation by GABP(17, 18) .
These isoforms, which may be derived from alternative splicing of the
mRNA transcript differ primarily in the region of the protein that is
responsible for GABP
homodimerization. Alternate forms of
GABP
1 may affect formation of GABP complexes and their
interactions with other components of the transcriptional apparatus.
Recently, GABP
2-1, a distinct but related gene that is
encoded on a different chromosome, was described(33) . The two
distinct GABP
genes differ in the region of the protein that is
responsible for transcriptional activation. GABP
1 and GABP
2
can form heteromeric complexes with one another, thereby adding another
level of complexity to the regulation to GABP function(33) .
The cell type-specific and differentiation-associated expression of the
GABP
genes and their various isoforms have not been well defined.
Thus, subtle lineage-specific alterations in GABP
expression may
control GABP complex formation and transcriptional activation and
thereby contribute to the regulated expression of CD18. Cell
type-specific transcriptional activation by the ubiquitously expressed
GABP may also be influenced by lineage-restricted factors such as PU.1.
Other genes besides CD18 are also controlled by more than one ets factor. Immunoglobulin heavy chain enhancer function is
regulated by binding of different ets factors including PU.1,
Ets-1, and Erg-3(34, 35) . The 4 integrin
promoter may be bound by a second ets factor besides
GABP
(31) . Although we cannot exclude the possibility that
GABP and PU.1 physically interact on the CD18 promoter in
vivo, we identified no novel binding complexes that are formed by
these factors in the electrophoretic mobility shift assay. Rather,
titration of increasing amounts of PU.1 competes for GABP
binding
to the CD18 promoter and vice versa. Furthermore, antibodies
to GABP do not supershift the EMSA probes that are bound by PU.1 and
vice versa(8) . Thus, PU.1 and GABP
appear to
compete for the same CD18 promoter sites and their binding appears to
be mutually exclusive.
Jurkat (T lymphoid) cells express CD18, yet they lack PU.1 expression. These cells possess a unique nuclear binding species which does not co-migrate with other ets factors that are expressed by T lymphocytes (Fig. 2). Additional mutagenesis studies and supershift assays (not shown) suggest that this binding species likely represents an as yet unidentified ets factor. We propose that this T cell factor acts in lieu of PU.1 and contributes to T lymphoid expression of CD18. These findings suggest that at least one other ets factor may functionally cooperate with GABP to effect CD18 transcription.
Transfection of both
GABP and GABP
into myeloid cells activates CD18 reporter
activity more than 10-fold; we have found no such activation of CD18
myeloid transcription by PU.1. Furthermore, co-transfection of both
GABP and PU.1 activates CD18 transcription in non-hematopoietic cells.
This confirms our previous proposal that although PU.1 is necessary for
myeloid expression of CD18, it alone is not sufficient to control CD18
transcription. Although these two ets factors compete for the
same CD18 promoter-binding sites, they functionally cooperate to
activate its transcription. GABP and PU.1 may have different roles in
the process of transcriptional activation. For example, because PU.1
directly interacts with TATA-binding protein(36) , it may
recruit the basal transcriptional apparatus to the TATA-less CD18
promoter, while GABP enhances other aspects of transcriptional
activation. Further analysis of these factors should demonstrate the
means by which these two ets factors cooperate to control CD18
transcription.