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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-02-0568.
HEMATOPOIESIS
From the Clinical Research Institute of Montreal and
from the Departments of Pharmacology, Biochemistry, and Molecular
Biology, Université de Montréal, Quebec, Canada; the
Telethon Institute for Child Health Research, Center for Child Research
and Western Australian Institute for Medical Research, The
University of Western Australia, West Perth, Australia; the Molecular
Haematology Unit, Weatherall Institute of Molecular Medicine, John
Radcliffe Hospital, Headington, Oxford, United Kingdom; and the Howard
Hughes Medical Institute, The Children's Hospital, Harvard Medical
School, Boston, MA.
The combinatorial interaction among transcription factors
is believed to determine hematopoietic cell fate. Stem cell
leukemia (SCL, also known as TAL1 [T-cell acute
lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix
(bHLH) factor that plays a central function in
hematopoietic development; however, its target genes and molecular mode
of action remain to be elucidated. Here we show that SCL and the c-Kit
receptor are coexpressed in hematopoietic progenitors at the
single-cell level and that SCL induces c-kit in chromatin,
as ectopic SCL expression in transgenic mice sustains c-kit
transcription in developing B lymphocytes, in which both genes are
normally down-regulated. Through transient transfection assays and
coimmunoprecipitation of endogenous proteins, we define the role of SCL
as a nucleation factor for a multifactorial complex (SCL complex) that
specifically enhances c-kit promoter activity without
affecting the activity of myelomonocytic promoters. This complex,
containing hematopoietic-specific (SCL, Lim-only 2 (LMO2),
GATA-1/GATA-2) and ubiquitous (E2A,
LIM- domain binding protein 1 [Ldb-1]) factors, is
tethered to DNA via a specificity protein 1 (Sp1) motif,
through direct interactions between elements of the SCL complex and the
Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin
immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the
c-kit promoter in vivo. We therefore conclude that
c-kit is a direct target of the SCL complex. Proper
activation of the c-kit promoter depends on the
combinatorial interaction of all members of the complex. Since SCL is
down-regulated in maturing cells while its partners remain expressed,
our observations suggest that loss of SCL inactivates the SCL complex,
which may be an important event in the differentiation of pluripotent
hematopoietic cells.
(Blood. 2002;100:2430-2440) Members of the basic helix-loop-helix
(bHLH) family of transcription factors are crucial regulators
of diverse developmental processes such as hematopoiesis, neurogenesis,
and myogenesis.1 This family of proteins includes
ubiquitously expressed members (E2A, HeLa E-box binding
protein [HEB]); tissue-specific factors (stem cell
leukemia/TAL1 [SCL/TAL1]; myogenic determination
factor [MyoD]); and non-DNA binding proteins (idiotope 1-4 [Id1-4]). Tissue-restricted members can
regulate gene expression by binding to E-box DNA sequences
(CANNTG) following heterodimerization with ubiquitously expressed E2A
gene products (E12 and E47) or HEB. Different dimers exhibit
preferential binding to specific E-boxes, and this selectivity is
thought to be an important determinant in the spatio-temporal control
of gene expression. SCL is a prototypic tissue-specific bHLH factor
normally expressed in pluripotent hematopoietic precursors, vascular
endothelial cells, and the central nervous system (reviewed in Begley
and Green2) and acts as a master regulator of
hematopoietic development. Indeed, SCL As for many hematopoietic transcription regulators, the SCL gene was
originally identified by virtue of its involvement in a tumor-specific
translocation.2 In fact, chromosomal rearrangements causing aberrant activation of SCL are the most common molecular anomalies associated with childhood T-cell acute lymphoblastic leukemia
(T-ALL).2 Surprisingly, SCL rarely induces leukemia in
transgenic mice and requires collaboration with the LIM-only (LMO)
proteins LMO1 and LMO2, whose genes are also translocated in
human T-ALL, to induce aggressive T-cell tumors.8-11 This
interaction is also relevant to normal development as SCL and LMO2 are
coexpressed in normal hematopoietic cells, and the phenotype of
LMO2 We have previously shown that SCL levels determine c-kit
gene expression in the TF-1 hematopoietic cell line, suggesting that c-kit, which encodes a receptor tyrosine kinase essential
for normal hematopoietic development, is a potential downstream target of SCL.20 Here, we provide evidence that SCL and
c-kit are coexpressed in primary hematopoietic precursors
and that in vivo ectopic expression of SCL increases c-kit
expression in B-cell precursors. In addition, we define a
multifactorial complex formed on the c-kit promoter containing SCL, E2A, LMO2, Ldb-1, and GATA factors (SCL complex). A
novel partner, the specificity protein 1 (Sp1) zinc finger
protein, is also identified. Our data highlight how a key
tissue-specific transcription factor can serve to nucleate the assembly
of multifactorial complexes containing other tissue-specific or
ubiquitously expressed proteins.
Transgenic mice
FDG staining, cell sorting, and RT-PCR analysis
Plasmid constructs All promoter segments were cloned into a modified pXPII reporter vector (called pXPIII), in which 2 E-boxes and 2 GATA motifs in the proximity of the multiple cloning site were destroyed. The human c-kit promoter (kit-1146)20 was subcloned into pXPIII with the use of HindIII and BstEII. Deletion mutants were generated as previously described.23 Point mutations in the Sp1 binding site replaced GGG GCG TGG with Gaa Gct TGt. The lower case signifies nucleotide mutations. All constructs were verified by sequencing. The pGEX-Sp1 and Gal4-Sp1 were gifts from Dr D. Kardassis,24 who had originally obtained them from Dr R. Tjian, University of California, Berkeley. Expression vectors for Ldb-1 and SCL mutants have been described previously.18,25 Expression vectors for E47/PAN1 and Gal4-E47, as well as pcDNA-LMO2, were generously provided by Drs J. Drouin (Institut de Recherches Cliniques de Montreal [IRCM], QC, Canada) and M. Minden (Ontario Cancer Institute, Toronto, ON, Canada), respectively. The c-fms and granulocyte-macrophage colony-stimulating factor receptor (G-CSFR) reporter constructs were kindly provided by Dr D. Tenen (Beth Israel Hospital, Boston, MA).Cell cultures and transfections NIH 3T3 and TF-1 cell culture conditions have been described previously.20 Calcium phosphate was used to transfect NIH 3T3 cells 24 hours after plating at 30 000 cells per milliliter. The amount of reporter was kept at 1.5 µg per well, and 100 ng cytomegalovirus- gal (CMV-gal) was added as an internal
control. Total DNA was kept constant at 4.5 µg per well with
pGem4. Unless specified otherwise, expression vector
doses were 150 ng for SCL, E2A, and GATA factors; and 750 ng for
LMO2 and Ldb-1. Luciferase and -gal activities were assayed after 36 hours. For all transfections, results are shown as the mean ± SD
of replicate determinations and are representative of 2 or
more independent experiments (depicted in figures).
Electrophoretic mobility shift assays TF-1 nuclear extracts were prepared as described previously.20 Binding reactions were allowed to proceed at room temperature for 15 minutes in 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.5), 50 mM KCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM dithiothreitol (DTT), 5% glycerol, 10 µg bovine serume albumin (BSA), 100 ng poly(dIdC), 50 000 cpm 32P-labeled probes, and 5 µg nuclear extract. For experiments using in vitro-synthesized proteins, 2.5 µL E47 and SCL mutants were kept at 37°C for 30 minutes before performing the binding reactions. Antibodies and competitor DNA were added to the reaction mixtures before the probes. Samples were resolved by 4% polyacrylamide gel electrophoresis (PAGE) at 17 mA in 0.5 × Tris (tris(hydroxymethyl)aminomethane)-borate-EDTA (TBE). The oligonucleotides used were T-cell acute lymphoblastic leukemia 1 (TAL1) consensus,26 kit-GC-box 5'-CGAGGAGGGGCGTGGCCGGCG-3' and reverse, kit-GC-box-mutant (mt) 5'-CGAGGAGaaGCtTGtCCGGCG-3'. The underlining indicates the position of the GC-box motif in the oligonucleotide probes, and the lower case signifies nucleotide mutations.Pull-down, immunoprecipitation, and chromatin immunoprecipitation assays Glutathione S-transferase (GST) and GST-Sp1 were purified from bacteria and coupled to glutathione Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ). SCL and SCL deletion mutants, as well as GATA-1, LMO2, Ldb-1, E47, and luciferase, were labeled with 35S-methionine (Promega, Madison, WI). Labeled proteins (10-20 µL) were incubated with 2 µg immobilized GST fusion proteins in 400 µL binding buffer (50 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1% Nonidet P-40 [NP-40], 5 mM DTT, 10% glycerol) for 2 hours at 4°C with agitation and then centrifuged for 1 minute at 3000 rpm. Samples were washed 3 times with binding buffer, resolved by sodium dodecyl sulfate-PAGE (SDS-PAGE), and visualized with the use of phosphor storage plates.Coimmunoprecipitations were performed for 4 hours at 4°C with 1 mg TF-1 nuclear extract, 3 µg antibody, and 20 µL Protein G Plus agarose beads (Calbiochem, San Diego, CA) in 1 mL immunoprecipitation (IP) buffer (20 mM Tris-HCl [pH 8.0], 137 mM NaCl, 1% NP-40, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF]). Samples were washed 3 times with IP buffer and subjected to SDS-PAGE. Following transfer on poly(vinylidene difluoride) (PVDF) membranes, proteins were visualized by means of ECL Plus (Amersham Pharmacia Biotech). Chromatin immunoprecipitation (ChIP) assays were performed as described previously,27,28 with the use of 2 × 107 TF-1 cells per sample. Cells were fixed by adding formaldehyde (1% final) to the cultures for 10 minutes at room temperature. Formaldehyde was quenched with glycine at a final concentration of 0.125 M, and cells were washed for 15 minutes in Triton buffer (10 mM Tris-HCl [pH 8.0], 10 mM EDTA, 0.5 mM ethyleneglycoltetraacetic acid (EGTA), 0.25% Triton X-100) and 15 minutes in NaCl buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Cells were resuspended in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl [pH 8.0], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate) and sonicated (6 × 10-second bursts) to make soluble chromatin ranging in size from 500 to 1000 base pairs (bp). An aliquot of extract was kept for isolation of input DNA, while samples were precleared with Staph A cells (Calbiochem) for 30 minutes and then incubated overnight at 4°C with antibodies in RIPA buffer. DNA-protein complexes were collected with Staph A cells for 30 minutes at 4°C and sequentially washed twice with RIPA buffer, LiCl buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholate), and TE. Bound chromatin was eluted at 65°C for 15 minutes in 300 µL elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS). Samples were diluted by addition of 300 µL TE and heated overnight at 65°C to reverse cross-links. After RNA and protein digestion, DNA was phenol/chloroform extracted and precipitated with the use of 10 µg tRNA as carrier. PCR reactions were performed for 30 cycles (94°C, 1 minute; 62°C, 1 minute; 72°C, 20 seconds) in 50 µL PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl, 1.5 mM MgCl2, 5% dimethyl sulfoxide [DMSO], 0.2 mM deoxynucleoside triphosphate [dNTP], 1 µM each oligonucleotide, 1.25 U Taq DNA polymerase). PCR products were migrated on a 2% agarose gel, transferred on nylon membranes, and hybridized with internal probes. Oligonucleotide sequences are available upon request. Antibodies The mouse anti-E2A (YAE) and anti-GATA-2 (CG2-96), rat anti-GATA-1 (N6), rabbit anti-PU.1 (T-21), and goat anti-Sp1 (PEP2) were obtained from Santa Cruz Biotechnology (CA). Rabbit anti-LMO2 and anti-Ldb-1 antisera have been described previously.17 The mouse monoclonal antibodies against SCL, BTL73, and 2TL136 were generously provided by Dr D. Mathieu, Institut de Génétique Moléculaire, Montpellier, France.
Colinearity of c-Kit receptor and SCL levels Both the SCL and the c-kit genes are essential for hematopoietic development, and our previous work demonstrated that SCL is required for c-kit expression and function in the hemopoietic cell line TF-1.20 To determine whether SCL and the c-Kit receptor are coexpressed at the single-cell level in primary hematopoietic cells, we monitored c-Kit surface expression through flow cytometry analysis of bone marrow cells taken from heterozygous SCLlacZ/w mice, in which the lacZ gene was knocked into the SCL locus.21 Cells expressing the lacZ gene were revealed through staining with the fluorogenic-gal substrate FDG. As illustrated in Figure 1A, the proportion of c-Kit+
cells in the LIN fraction (ie, negative for Mac-1, B220,
and TER119) was in the range of 15% to 30%. In
SCLlacZ/w mice, c-Kit-expressing cells were
mostly -gal+, while cells from WT littermates had only a
low background of -gal activity. The majority (95%) of
colony-forming cells were found in the
c-Kit+/-gal+ fraction (data not shown). This
population included multipotent (granulocyte, erythrocyte,
megakaryocyte, macrophage colony-forming unit [CFU-GEMM]),
erythroid (erythroid burst-forming unit [BFU-E]), and
granulocyte/macrophage (granulocyte/macrophage CFU [CFU-GM]) precursors (data not shown), shown previously to express
c-Kit29 and SCL.21,30 Within the
LIN fraction, c-Kit levels were found, strikingly, to be
proportional to the activity of the SCL locus (Figure 1B), as shown by
the mean fluorescence intensities (MFIs) of FDG staining for
c-Kithigh, c-Kitint, and c-Kitneg
populations, which were 9.5, 2.8, and 1.6, respectively. Within the
LIN+ fraction, 3% of the cells were -gal+
(Figure 1A), and the MFI of FDG staining was 1.5 (data not shown). This
staining was due mostly to the presence of erythroid cells (TER119+), and possibly a subset of B220+
cells, as pro-B cells are c-Kit+. Early B-cell precursors
expressing the B220 marker were therefore fractionated from WT mice
according to Hardy's protocol,31 and SCL and
c-kit mRNA levels were assessed by RT-PCR analysis. As shown
in Figure 1C, the 2 genes are coexpressed in pro-B cells. Interestingly, both SCL and c-kit mRNA levels were
down-regulated on B-cell maturation, while IL-7R was up-regulated.
Finally, SCL and c-kit were absent in mature B cells. In
summary, our data indicate that SCL and c-kit are
coexpressed within the hematopoietic precursor compartment, in
multipotent progenitors (CFU-GEMMs) and in more committed progenitors
(BFU-Es, CFU-GMs, and pro-B cells). Furthermore, high levels of SCL
locus activity correlate with high levels of c-kit
expression.
Since SCL and c-kit are down-regulated at the same stage
during B-cell development, we assessed whether ectopic SCL expression could induce inappropriate c-kit expression in bone
marrow-derived B cells from homozygous SIL-SCL transgenic mice
(SCLtg/tg), which express SCL ubiquitously.10
B220+ cells from WT and SCLtg/tg mice were
isolated by flow cytometry, and c-kit expression was assessed by semiquantitative RT-PCR. As shown in Figure
2A, enforced SCL expression induced a 3- to 4-fold increase in c-kit mRNA levels, whereas expression
of RAG-2 was not affected by the SIL-SCL transgene. To identify which
stage of B-cell differentiation c-kit was being induced, we
performed RT-PCR analysis on purified B-cell fractions, as described
above.31 As quantified in Figure 2B, enforced SCL expression induced a 2- to 3-fold increase of c-kit mRNA
within late pro-B cells (fractions B and C), in which c-kit
and SCL are normally down-regulated. Furthermore, c-kit
expression persisted in pre-B and mature B cells in
SCLtg/tg bone marrow but was turned off in WT mice. The
effect of the SIL-SCL transgene was specific to c-kit, since
the levels of IL-7 receptor
The c-kit promoter is activated through functional collaboration between SCL, E47, LMO2, Ldb-1, and GATA factors To directly address whether SCL and its known transcriptional partners regulate c-kit expression, we inserted 1146 bp of the c-kit proximal promoter upstream of the luciferase reporter gene (kit-1146) and optimized a transactivation assay in heterologous cells (NIH 3T3). Expression vectors for SCL and its partners were transfected separately or in combination at several doses ranging from 50 ng to 1500 ng in order to reveal any dose-dependent effects on c-kit promoter activity (data not shown). Kit-1146 activity was not enhanced upon cotransfection of E47 or GATA expression vectors (Figure 3A) even though E47 homodimers and GATA factors are strong transcriptional activators. Similarly, SCL/E47 did not affect kit-1146 basal activity (Figure 3A), a sharp contrast to TAL1 reporters, which are activated by E47 homodimers and, to a lesser extent, by SCL/E47 heterodimers.32 Cotransfection of LMO2 and its cofactor Ldb-1 with SCL/E47 induced a modest dose-dependent increase in luciferase activity (Figure 3A). Similarly, the combined effects of GATA-2, E2A, LMO2, and Ldb-1 were modest. In contrast, a high level of synergistic transactivation of the c-kit promoter was achieved upon cotransfection of SCL with its partners, and this effect was dose dependent (Figure 3A). Therefore, c-kit promoter activation by SCL must rely on the formation of a multifactorial complex (SCL complex), within which SCL plays a crucial role. In this complex, GATA-1 could substitute for GATA-2, albeit with lower efficiency (Figure 3A). Western blotting indicated that all 5 factors were expressed in the pluripotent c-Kit+ hematopoietic cell line TF-1 but not in untransfected NIH 3T3 fibroblasts (Figure 3B, compare lanes 1 and 7). Transient transfection of expression vectors for SCL and its partners resulted in efficient expression in NIH 3T3 cells (lanes 2-6). Furthermore, the level of expression of each factor was similar whether the factor was transfected alone or in combination, indicating that the synergy was not due to cross-regulation of transgene expression. Finally, to assess whether transcription regulation by the SCL complex was specific to the c-kit promoter, we verified its activity on the promoters of the G-CSFR and c-fms cytokine receptor genes, chosen because of their specificity for myeloid cells that do not express SCL. As above, the SCL complex activated kit-1146, but did not activate the G-CSFR and c-fms promoters, nor did it affect transactivation of the G-CSFR promoter by C/EBP (Figure 3C). Furthermore, the empty pXPIII vector
included as a negative control was not affected by the SCL complex.
Finally, the SCL promoter (Figure 3C) was not activated by the SCL
complex. Together, our results demonstrate that SCL and its partners
functionally synergize to enhance the activity of the c-kit
promoter. The results also show that transactivation by the SCL complex
is highly specific for the c-kit promoter.
Importance of an Sp1 binding site for transcription activation by the SCL complex To identify the cis elements through which the SCL complex activates the c-kit promoter, we constructed a series of 5' deletion mutants. Activation of these reporter constructs by the SCL complex was then assessed in NIH 3T3 cells. As shown in Figure 4A, sequences upstream of position 122 could be deleted without affecting promoter activation by the SCL
complex. Further deletion up to position 83 abolished activation,
suggesting that a response element involved in recruitment of the SCL
complex was situated between positions 122 and 83 of the promoter.
This sequence lacks canonical E-boxes or GATA sites but contains a
consensus GC-box that is also conserved in the murine c-kit
promoter, which is a potential binding site for members of the Sp1
family of zinc finger proteins (Figure 4A). To determine which factor
associates with this sequence in hematopoietic cells, we performed gel
shift assays with TF-1 cell nuclear extracts. The major complex that
binds this GC-box was supershifted with an anti-Sp1 antibody but not
preimmune serum (Figure 4B, lanes 1-4), indicating that it contained
Sp1. Binding to the c-kit GC-box probe was efficiently displaced by an
unlabeled WT competitor oligonucleotide (lanes 5-6). In contrast, an
oligonucleotide mutated within the GC-box was unable to compete for Sp1
binding (lanes 7-8). These same point mutations were therefore
introduced into either the full-length promoter (kit-1146-Sp1mt) or
the most proximal promoter deletion mutant that still retained
responsiveness to the SCL complex (kit-122-Sp1mt). Mutating the Sp1
binding site completely abolished c-kit promoter activation
by the SCL complex (Figure 4A). In light of these results, we confirmed
by Western blotting that the Sp1 protein is expressed in NIH 3T3 cells
(Figure 3C) and that it was not affected by ectopic expression of SCL and its partners, indicating that transcriptional synergy was not due
to increased Sp1 expression. Finally, to determine whether transcription activation by all 5 partners was synergistic or additive,
in the context of both kit-1146 and the minimal kit-122, we
sequentially omitted each factor from our transfection mixtures. As
shown in Figure 4C, omission of either of the expression vectors abolished transactivation and reduced luciferase activity at or near
basal level. These results demonstrate that all 5 partners act
synergistically to activate both the full-length and the minimal c-kit promoter segments. Together, our observations indicate
that the Sp1 binding site is necessary for regulation of the
c-kit promoter by the SCL complex, which further suggests
that Sp1 can help to recruit these factors to their target
genes.
Since the transcriptional activity of the SCL complex requires recruitment of the LMO2 and Ldb-1 cofactors, as well as E2A and GATA transcription factors, we investigated the expression of SCL partners within purified B-cell precursors. In addition to E2A, known to be highly expressed in B cells, we observed that LMO2 and Ldb-1 mRNAs are present in all bone marrow B-cell fractions (Figure 4D). The GATA3 transcription factor, highly expressed in T cells, was found to be present at low levels in fractions A to C (data not shown) and could be due to the presence of cells that are not fully committed to the B lineage. Finally, Sp1 mRNA is expressed in the B lineage in which c-kit expression is induced by the SCL transgene (Figure 4D), suggesting that the induction of c-kit by SCL in vivo is associated with the presence of appropriate partners within target cells. Dispensability of SCL DNA binding Our observations indicate that activation of the c-kit minimal promoter by the SCL complex (1) occurred in the absence of canonical E-box binding sites and (2) required multiple partners. We therefore determined which domains of SCL were required to form a functional complex. SCL was previously shown to contain a proline-rich N-terminal transactivation domain lying between amino acids 117 and 175.33 In NIH 3T3 cells, this domain could be deleted without affecting SCL transcriptional activity (Figure 5A). In fact, SCL could be reduced to its bHLH domain and still remain functionally active. Also, SCL mutants that are unable to bind to DNA owing to a deletion ( bSCL) or
point mutations (SCL-RER) within the basic region (Figure 5B,
lane 3, and data not shown), were still functional (Figure 5A). These
results are consistent with the fact that c-kit promoter
activation by the SCL complex was independent of E-box binding sites
(Figure 4A). Finally, point mutations in the HLH domain
(SCL-FL) that disrupt heterodimerization with E47 (Figure 5B,
lane 4) completely abrogate c-kit promoter activation by the
SCL complex (Figure 5A). Therefore, our observations reveal that the
HLH protein interaction motif of SCL is critical for the function of
this complex, while the DNA binding and putative transactivation
domains are dispensable. These data support the notion that SCL can be
recruited to regulatory regions via protein interactions with partners
such as Sp1. This mechanism helps to explain why transactivation by the
SCL complex occurs in the absence of E-boxes and why DNA binding by SCL
is not strictly required for its function in
vivo.18,19
Sp1 physically interacts with multiple elements of the SCL complex Since transcriptional synergy may result from direct interactions between transcription factors, we sought to determine, via several approaches, whether Sp1 physically associates with members of the SCL complex. First, we performed in vitro pull-down assays with immobilized GST-Sp1 and in vitro-translated, 35S-labeled SCL, GATA-1, LMO2, Ldb-1, and E47. GST columns and in vitro-translated luciferase were used as negative controls for the binding assays. Interestingly, GST-Sp1 columns specifically retained SCL, GATA-1, LMO2, and Ldb-1, but not E47 or luciferase (Figure 6A). These interactions were not affected by the presence of ethidium bromide (200 µg/mL) in the binding reactions, demonstrating that they were direct and not due to bridging by contaminant DNA molecules (data not shown). Quantification of in vitro pull-down results indicates that the associations between Sp1 and partners of the SCL complex were in the range of 1% to 5% of input proteins. These in vitro pull-down results indicate that Sp1 entertains direct physical interactions with 4 elements of the SCL complex.
We then sought to identify the domain of SCL that interacts with Sp1 by performing pull-down assays with in vitro-translated SCL mutants. Figure 6B displays the binding efficiencies of these SCL mutants for GST-Sp1. Deletion of either N-terminal or C-terminal domains of SCL did not affect its association with Sp1, whereas point mutations in the HLH domain (SCL-FL) abrogated this interaction. Furthermore, the bHLH domain of SCL was sufficient for physical interaction with Sp1. These findings are in complete agreement with our transactivation results in which the bHLH domain of SCL was sufficient to form a functional complex that drove c-kit promoter activation (Figure 5A). To assess whether SCL and Sp1 interact within transfected cells, we optimized a mammalian 2-hybrid assay in which Sp1 or E47 was fused to the Gal4 DNA-binding domain (Gal4 DBD-Sp1 or Gal4 DBD-E47), while SCL was grafted to the Gal4 activation domain (Gal4 SCL-AD). As shown in Figure 6C, Gal4 DBD-Sp1 and Gal4 SCL-AD interacted in vivo to produce a 5-fold activation of a reporter construct driven by multimerized upstream activator sequences (5XUAS-tk109-luc). This interaction was of similar magnitude to that observed with Gal4 DBD-E47 and Gal4 SCL-AD. By contrast, neither of these fusion proteins activated the reporter construct when expressed alone. This result indicates that a physical association between Sp1 and SCL takes place within transfected cells. Finally, to ascertain whether these interactions occur in vivo with
endogenous proteins present in c-kit-expressing
hematopoietic cells, we performed coimmunoprecipitations using TF-1
cell nuclear extracts. Antibodies against GATA-1, SCL, Sp1, and E2A
efficiently precipitated their respective proteins (Figure
7, lanes 4, 6, 10, and 17). Under these
conditions, SCL most effectively coprecipitated with
anti-LMO2, anti-Ldb-1, and anti-E2A (Figure 7, lanes 8-9, and
data not shown), demonstrating a strong association between SCL and
these factors. In addition, the anti-SCL antibody efficiently brought
down E2A (lane 16) and, to a lesser extent, GATA-1 (lane 3). These data
demonstrate that previously identified partners of the SCL complex are
indeed associated in pluripotent hematopoietic cells. In addition,
antibodies directed against Sp1 consistently coprecipitated GATA-1 and
SCL (lanes 2 and 13). Although weak, SCL/Sp1 coimmunoprecipitation was
consistently observed, whereas SCL coprecipitation did not occur when
either anti-GFP (lane 11) or anti-PU.1 (lane 14) was used as
control species-matched antisera. Therefore, our data reveal that Sp1
is indeed associated with partners of the SCL complex in vivo, and
suggest that Sp1 is a novel component of this complex. The
coimmunoprecipitation efficiencies were highest for SCL, LMO2, Ldb-1,
and E2A, suggesting that these factors may form a core complex with
which other partners such as GATA-1 and Sp1 can associate. Combined,
our results demonstrate that Sp1 directly interacts with partners of
the SCL complex both in vitro and in vivo, and explain the functional
collaboration observed between Sp1 and the SCL complex.
Sp1, SCL, and E2A occupy the c-kit promoter in vivo While our results demonstrate that Sp1 and the SCL complex regulate c-kit promoter activity in vitro, true proof of principle would have to come from a demonstration that these factors occupy the c-kit promoter in vivo in hematopoietic cells. To this end, we performed chromatin immunoprecipitation assays using formaldehyde cross-linked TF-1 cells. Chromatin extracts were subjected to immunoprecipitation with anti-SCL, anti-E2A, anti-Sp1, and species-matched control antibodies (anti-HA and anti-PU.1). Cross-linking was reversed, and DNA fragments that were specifically retained were purified. These samples were then subjected to PCR amplification with the use of specific primers that target different regions in the c-kit locus (Figure 8). We used serial dilutions of template DNA to ensure that the different samples could be compared within their linear range of amplification. In addition, PCR products were hybridized with 32P-labeled internal oligonucleotide probes to confirm the specificity of the amplified fragments. As shown in Figure 8, antibodies to SCL, E2A, and Sp1 efficiently recovered a 150-bp fragment of the c-kit proximal promoter, encompassing the Sp1 site. In contrast, species-matched control antibodies (anti-PU.1 and anti-HA) were unable to bring down this sequence. Furthermore, all antibodies precipitated a negligible amount of DNA located either 2 kb upstream or 13 kb downstream of the transcription initiation site. These results demonstrate that Sp1, SCL, and E2A directly and selectively associate with the c-kit proximal promoter region containing the functional Sp1 binding site in hematopoietic cells.
In the present study, we show that SCL and c-kit are coexpressed in primary hematopoietic precursors and that SCL up-regulates c-kit expression in vivo in developing B-cells. Our study reveals Sp1 as a novel member of the SCL complex that tethers SCL and its partners to the c-kit promoter. Reinforcing this hypothesis is the finding that SCL DNA-binding defective mutants are still functional within the larger protein complex. Finally, we demonstrate that SCL and E2A, essential partners within the SCL complex, co-occupy the c-kit promoter with Sp1 in vivo in hematopoietic cells. Colinearity of SCL and c-kit expression and function During embryonic development, hematopoiesis occurs in 2 waves: a first wave of primitive hematopoiesis takes place in the yolk sac, and a second wave of definitive hematopoiesis initiates in the fetal liver and later moves to the bone marrow. In the embryo proper, hemopoietic cells are also found in the aorta, gonad, and mesonephros (AGM) region. Within all of these hemogenic sites, the expression of c-kit and SCL closely overlaps. From embryonic day 7.5 (E7.5) to E9.5, c-kit and SCL are both expressed in yolk sac blood islands; from E9.5 to E11.534,35 in the AGM region; and, from E10.5 to E11.534-37 in the fetal liver.34,35,37,38 We now show that in adult bone marrow cells, the SCL locus is transcriptionally active in cells that express the c-Kit receptor, a population that includes almost all hematopoietic progenitor colony-forming cells. Our results demonstrate that c-kit and SCL remain expressed in committed progenitors, BFU-Es, CFU-GMs, and pro-B cells, which is consistent with published studies.22,30,39 Our previous work also indicates that SCL and c-kit are coexpressed in early pro-T cells.11 Finally, both genes are down-regulated with terminal differentiation in the granulocyte-macrophage, B-, and T-lymphoid lineages.2 Thus, the mapping of c-kit and SCL expression by a variety of techniques indicates a close temporal association within developing hematopoietic cells.In mice, severe mutations in the c-kit gene (W
mutants) cause prenatal lethality at 13 to 15 days of gestation owing
to the interruption of fetal liver hematopoiesis,40
demonstrating the essential role of the c-Kit tyrosine kinase in
definitive hematopoiesis. Similarly, SCL is critical not only for
primitive but also for definitive hematopoiesis, as revealed by the
study of SCL c-kit as a target gene of the SCL complex The survival and differentiation of hematopoietic cells is critically dependent on the action of hematopoietic growth factors and their receptors. Therefore, genes encoding cytokine receptors represent critical targets for hematopoietic transcription factors. For instance, it has been shown that the major erythroid transcription factor GATA-1 activates the erythropoietin receptor (EpoR) promoter,43 whereas the myeloid regulator PU.1 regulates promoters of the c-fms and GM-CSFR-chain genes.44-46
Despite the critical importance of c-kit for the survival of
multipotent progenitor cells, the mechanisms controlling
c-kit gene transcription in hematopoietic cells has not been
studied as extensively as those of other cytokine receptor genes.
Furthermore, potential binding sites for the SCL complex were
previously identified in other hematopoietic genes, but the functional
importance of these sites and their in vivo association with SCL and
its partners has not yet been demonstrated. In this study, we combine
in vivo approaches with in vitro protein interaction and
transactivation assays, and provide direct evidence that the SCL
complex indeed activates the c-kit promoter. This activation
is selective for c-kit promoter sequences, as the SCL complex has no effect on 2 myeloid promoters, c-fms and G-CSFR. Finally, we show via chromatin immunoprecipitation that SCL, E2A, and
Sp1 occupy the c-kit promoter in vivo in TF-1 cells. We
conclude therefore that c-kit is a direct target of
transcription regulation by SCL and its partners. By activating target
genes such as c-kit, the SCL complex may ensure the survival
of undifferentiated pluripotent hematopoietic cells, a role that has
been formerly attributed to the c-Kit receptor.47
Sp1, a novel member of the SCL complex Analysis of the c-kit promoter has allowed us to demonstrate novel functional and physical interactions between Sp1 and the SCL pentameric complex. Sp1 is the founding member of a family of zinc finger-containing proteins (Sp/XKLF) that bind GC- or GT-boxes.48 Sp1 is ubiquitously expressed and is involved in chromatin remodeling and maintenance of methylation-free islands. Functionally important Sp1 binding sites have been identified in the regulato | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
A- -A, point
mutations within helix 1 of the HLH domain that disrupt dimerization
with E2A; RER
