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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4138-4149
Selective Sp1 Binding Is Critical for Maximal Activity of the
Human c-kit Promoter
By
Gyeong H. Park,
Howard K. Plummer III, and
Geoffrey W. Krystal
From the Division of Hematology/Oncology, Departments of Medicine and
Microbiology/Immunology, Medical College of Virginia/Virginia
Commonwealth University and McGuire VA Medical Center, Richmond, VA.
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ABSTRACT |
The receptor tyrosine kinase c-kit is necessary
for normal hematopoiesis, the development of germ cells and
melanocytes, and the pathogenesis of certain hematologic and
nonhematologic malignancies. To better understand the regulation of the
c-kit gene, a detailed analysis of the core promoter was
performed. Rapid amplification of cDNA ends (RACE) and
RNase protection methods showed two major transcriptional initiation
sites. Luciferase reporter assays using 5 promoter
deletion-reporter constructs containing up to 3 kb of 5 sequence
were performed in hematopoietic and small-cell lung cancer cell lines
which either did or did not express the endogenous c-kit gene.
This analysis showed the region 83 to 124 bp upstream of the 5
transcription initiation site was crucial for maximal core promoter
activity. Sequence analysis showed several potential Sp1 binding sites
within this highly GC-rich region. Gel shift and DNase
footprinting showed that Sp1 selectively bound to a single site within
this region. Supershift studies using an anti-Sp1 antibody confirmed
specific Sp1 binding. Site-directed mutagenesis of the  93/84 Sp1
binding site reduced promoter-reporter activity to basal levels in
c-kit-expressing cells. Cotransfection into Drosophila
SL2 cells of a c-kit promoter-reporter construct with an Sp1
expression vector showed an Sp1 dose-dependent enhancement of
expression that was markedly attenuated by mutation of the 93/84
site. These results indicate that despite the fact that the human
c-kit promoter contains multiple potential Sp1 sites, Sp1
binding is a selective process that is essential for core promoter
activity.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
C-KIT IS THE HUMAN cellular
homologue of v-kit1 which was isolated from the
Hardy-Zuckerman 4 feline sarcoma virus.2 c-kit
encodes a receptor tyrosine kinase of the type III family which
includes platelet-derived growth factor (PDGF) and colony-stimulating factor 1 receptors and is characterized by an intracellular split kinase domain as well as a cysteine-rich extracellular region. c-kit maps to the murine White Spotting (W)
locus,3,4 and its ligand, known as mast cell growth factor,
Kit ligand, or stem cell factor5 (SCF) maps to the mouse
Steel (Sl) locus.6-8 Kit and SCF
interaction is involved in the development of melanocytes, germ cells,
and hematopoietic cells. Mutations of the mouse W locus and the
Sl locus lead to severe defects in hematopoiesis and
deficiencies in mast cells, as well as unpigmented coats and sterility.9 SCF in combination with other hematopoietic
growth factors supports the proliferation and differentiation of
multiple hematopoietic cell lineages from early
precursors.6,10,11 In addition to playing a preeminent role
in normal hematopoiesis, Kit may be important in regulating the growth
of some hematopoietic malignancies.12 Kit and SCF are
coexpressed in a variety of solid tumors including gynecologic
tumors,13 colon tumors,14 breast tumor
specimens and tumor cell lines,15
neuroblastomas,16 and over 70% of small-cell lung cancer
(SCLC) lines and tumors.17-20 Coexpression in SCLC results
in a functional autocrine growth loop.21
Sp1 is a ubiquitous transcription factor mostly associated with
TATA-less GC-rich promoters and is mainly thought to be
involved in basal promoter activity by interacting with transcription
activation factors, which may stabilize components of the
transcriptional machinery.22 Sp1 consists of three
contiguous Cys2His2 Zinc-finger domains that
bind to the decanucleotide consensus sequence 5 GGGGCGGGGC
323,24 and similar sequences that are referred to as
GC boxes. Family members Sp3 and Sp4 have similar structural features,
and the DNA binding domains of all three proteins are highly
conserved.25 Another family member, Sp2, seems to have somewhat different binding specificities.26 Functional
analyses have shown that Sp4, like Sp1, is a transcriptional activator, whereas Sp3 has been shown to both repress27 and activate
transcription.28,29 Studies have shown that Sp1-responsive
promoters usually contain multiple binding sites of differing DNA
sequence and Sp1 binding affinity,30-32 although Sp1
binding to a single site seems to be sufficient for promoter
activation.
Previous studies of the regulation of c-kit transcription have
characterized the mouse c-kit33 and human
c-kit34-36 5 flanking sequence and have
identified potential Sp1 binding sites in the proximal promoter region.
In studies of the mouse promoter, important cis-acting elements
required for cell type-specific expression were localized within 105 bp of the transcription initiation site (TIS), and no additional
elements in over 5 kb of 5 sequence significantly influenced
activity.33 The human c-kit promoter was found to
lack a typical TATA box and contained several potential Sp1 binding
sites, as well as putative binding sites for AP-2, Ets-domain proteins,
Myb, and GATA-1.35 This study also showed that sequences
mediating cell type-specific expression were contained within 120 bp
of the TIS, which was different from the TIS identified in the mouse
promoter, and that possible negative regulatory elements were located
in the region between 992 and 604.35 A more
recent study found that sequences up to 184 bp 5 of the
translational initiation codon were important for promoter activity,
but the 4100 to 5500 region contained cell type-specific
negative regulatory elements.36 With the possible exception
of the microphthalmia transcription factor identified by
genetic studies and specifically regulating c-kit expression in
mast cells,37 and Myb36,38 and
Ets38 proteins, which seem to have effects on expression only when they are overexpressed, no definitive binding of a
transcription factor to the c-kit promoter has been shown. With
this fact in mind along with the discrepancies over the sequences
necessary for appropriating c-kit expression, we sought to
further characterize the human c-kit promoter and specifically
determine the role of Sp1 in its regulation.
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MATERIALS AND METHODS |
Cell cultures.
Cell lines used were as follows: c-kit-expressing SCLC cell
lines H526, H510, and WB19 and the HEL
leukemia cell line33,35; and the nonexpressing SCLC cell
lines H146 and H8219 and the Jurkat T-cell
line.36 Cultures were grown in RPMI 1640 (Bio-Whitaker, Walkersville, MD) medium supplemented with 10% fetal calf serum (Life
Technologies, Bethesda, MD), 2 mmol/L L-glutamine, and 50 U/mL
penicillin-50 U/mL streptomycin. Drosophila SL2 cells were cultured at 26°C in Schneider's Drosophila medium (Life
Technologies) containing 10% fetal calf serum (FCS).
Cloning of the c-kit promoter region.
A 6-kb NotI fragment containing 5 c-kit promoter
sequence was isolated from a human lambda (FIXII) placental library
(Stratagene, La Jolla, CA) by screening with a radiolabelled 369-bp
SstI-HindIII fragment of a 1.2-kb 5 partial cDNA
clone (American Type Culture Collection clone #594931).
After primary screening, the positive plaques were purified through
quaternary platings using two different T4 polynucleotide kinase-labeled oligonucleotides (complementary to bases 41-61 and 62-81 in exon 11) as probes. The genomic NotI fragment
hybridizing to the exon 1 probes was excised and subcloned into the
pGEM5Zf(+) plasmid vector (Promega, Madison, WI).
Cloning and characterization of c-kit mRNA primer extension
products.
Cloning of c-kit mRNA primer extension products was
accomplished by using a modification of the rapid amplification of cDNA ends (RACE) protocol for polymerase chain reaction (PCR) amplification of the 5 ends of cDNAs.39 Two oligonucleotide
primers (0.6 µg) specific for portions of the 5 sequence of
c-kit (complementary to bases 62-81 and 358-376 1)
were annealed to 25 µg of WB total RNA by incubation for
16 hours at 55°C in 10 to 20 µL of 0.4 mol/L NaCl/8 mmol/L PIPES (piperazine-N-N-bis[2-ethansulfonic acid]) buffer (pH 6.7) in separate reactions. The primer extension was accomplished by diluting the annealing mixture to 100 to 200 µL with the addition of 1,000 U
of Moloney murine leukemia virus reverse transcriptase (MMLV-RT) or
MMLV-RT Superscript (Life Technologies) and 0.5 mmol/L deoxynucleoside triphosphate with the manufacturer-supplied buffer. The extension reaction was performed at 37°C for 40 minutes and terminated by heating to 90°C for 2 minutes. Ten micrograms of RNase A was added, and the incubation was continued for 15 minutes at 37°C. The
mixture was then made 0.5% sodium dodecyl sulfate, 30 µg of proteinase K was added, and the incubation was continued for 15 minutes at 42°C. The primer-extended species were then
phenol-chloroform extracted, ethanol precipitated, and purified by
binding to GlassMAX (Life Technologies). The cDNA was eluted from the
GlassMAX matrix in TE [10 mmol/L Tris(hydroxymethyl)
aminomethane-Cl, pH 7.4, 0.1 mmol/L Ethylenediamine Tetracetic acid]
buffer, tailed with dATP as described by Frohman et al,39
and repurified using GlassMAX.
One-half of the cDNA was diluted to 100 µL total volume and used in
the PCR reaction. The PCR reaction mixture contained a 0.6 to 0.8 mmol/L concentration of orientation-specific primer designed to
hybridize 3 of the primer used for cDNA synthesis (complementary
to bases 41-61 and bases 62-81, respectively1); a
0.9-mmol/L concentration of a (dT)17 primer-adapter
containing HindIII, SalI, and XhoI cloning
sites (5-GACTCGAGTCGACAAGCTTTTTTTTTTTTTTTTT-3); 50 mmol/L
KCl, 10 mmol/L Tris HCl (pH 8.8), 1.5 mmol/L MgCl2, 3 mmol/L dithiothreitol (DTT), 100 µg/mL bovine serum albumin; 0.2 mmol/L deoxynucleoside triphosphates; and 2.5 U of Taq
polymerase (AmpliTaq; Perkin Elmer, Norwalk, CT). Amplification was
performed in a programmable thermal reactor by first heating the
mixture to 95°C for 1 minute and then using a step program
(95°C, 1 minute; 50°C, 2 minutes; 72°C, 2 minutes) for 35 cycles, with a final extension for 15 minutes at 72°C. The
amplified cDNA products were then phenol-chloroform extracted, ethanol
precipitated, resuspended in TE buffer, and cut at the NarI
(within exon 1) and SalI (within primer-adapter) restriction
sites. These restriction-cut cDNA products were then ligated into
either pGEM7Zf() or pSP70 vectors (Promega) cut with
ClaI and XhoI restriction endonucleases. The cDNA
inserts were sequenced using the dideoxynucleotide chain termination
method as described in Davis et al.40
RNase protection.
A 3-kb BamHI-BamHI fragment containing the promoter
region was subcloned into a pGEM7Zf() vector and then cut at a
SalI restriction site 445 bases 5 of the BamHI
site within exon 1 to make a template for the generation of a
riboprobe. RNase protection assays were performed using 30 µg of
total RNA as previously described.41
Luciferase constructs.
The promoter deletion constructs were made by restriction endonuclease
digestion of the 6-kb NotI clone subcloned into the promoterless luciferase reporter plasmid pGL2:Basic (Promega). The
3 3 kb and 935 bp of the NotI fragment were ligated as
BamHI fragments into pGL2:Basic to make the 3-kb and the
935-bp constructs, respectively. A BglII-XhoI
fragment was ligated into pGL2:Basic to make the 409-bp
construct. A SmaI fragment was ligated into pGL2:Basic to make
the 124-bp construct. A BamHI-NaeI fragment was
ligated into pGL2:Basic to make the 83-bp construct. All plasmid
constructs were purified by double cesium chloride gradient centrifugation and verified by sequence analysis.
Mutagenesis of the Sp1 binding site.
The 2-bp mutation of 93/84 Sp1 site in the 124
construct (XN2mt) was performed using the QuikChange Mutagenesis
protocol (Stratagene). The oligonucleotides used for this site-directed mutagenesis were 5-GGGGAGGCGAGGAGGTTCGTGGCCGCGCG-3
and 5-CGCGCCGGCCACGAACCTCCTCGCCTCCCC-3. Reinsertion of the 93/84 Sp1 site upstream of the XN2mt
construct was performed by annealing two oligonucleotides, 5
CCGGGAGGGGCGTGGCCG 3 and 5 CCGGCGGCCACGCCCCTC 3,
which were then ligated into the 124/XN2mt construct cut with
XmaI.
Luciferase reporter assays.
Transfection was initiated by adding DNA to 8 × 106
cells suspended in 0.4 mL in 2-mm cuvettes that were incubated on ice
for 10 minutes. H526 cells were electroporated in phosphate-buffered saline (PBS) (pH 7.4) at 300 V and 500 µF, H146 cells in PBS at 300 V/1,000 µF, HEL cells in PBS at 200 V/1,500 µF, and Jurkat cells in
RPMI 1640 at 300 V/1,250 µF using a BTX Electro Cell Manipulator 600 (BTX Inc, San Diego, CA). The cells were transfected with 25 µg of
the pGL2:3-kb construct and equimolar amounts of the smaller
reporter plasmids. pSP70 plasmid was used as a filler plasmid to
maintain a final DNA concentration of 25 µg for each transfection.
Ten micrograms of pCMV  gal plasmid was cotransfected to
serve as a control for transfection efficiency. After electroporation, the cells were plated in 6 mL of RPMI 1640/10% FCS in 60-mm tissue culture dishes, cultured for 24 hours, and harvested according to the
Reporter Lysis protocol (Promega) using 400 µL of reporter lysis
buffer. Luciferase and -galactosidase activities were assayed according to the manufacturer's protocol (Promega). Luciferase activity was analyzed using 20 µL of cell extract mixed with 100 µL
of luciferase substrate (Promega), which was quantitated for 30 seconds
in a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad,
Germany). -galactosidase activity was used to
normalize for transfection efficiency. SL2 cells were transfected using the calcium phosphate coprecipitation technique, as described in Davis
et al.40 Luciferase and -galactosidase activity in the
SL2 cells were assayed as above.
Gel mobility shift analysis.
Whole cell extracts used in this assay were prepared in the following
manner. Approximately 5 × 107 cells were obtained,
washed with PBS (pH 7.4), and resuspended in 1 mL of extraction buffer
(20 mmol/L Hepes [pH 7.8], 450 mmol/L NaCl, 0.4 mmol/L EDTA, 0.5 mmol/L DTT, 25% glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride).
This suspension was frozen and thawed three times using a dry
ice/ethanol bath and a 37°C water bath. After a 10-minute
centrifugation at 13,000g at 4°C, the supernatant was
aliquotted and stored at 70°C until use. Protein concentration was determined by BCA assay (Pierce, Cleveland, OH).
Oligonucleotides used were as follows: Xma-Nae fragment
(124 to 83):
5-CCGGGCGGGCGCGAGGGAGGGGAGGCGAGGAGGGGCGTGGCC-3 and 5-GGCCACGCCCCTCCTCGCCTCCCCTCCCTCGCGC-3; XN
(125/97): 5-CCCGGGCGGGCGCGAGGGA-3 and
5-TCGCCTCCCCTCCCTCGCGCCCGC-3; XN2 (102/82):
5-GGGGAGGCGAGGAGGGGCGT-3 and
5-CGGCCACGCCCCTCCTCG-3; the XN2 oligonucleotide
with a 2-bp mutation in a putative Sp1 site, XN2mt:
5-GGGGAGGCGAGGAGGTTCGT-3 and
5-CGGCCACGAACCTCCTCG-3; and XN3
(96/82): 5-GGAGGGGCGTGGC-3 and
5-CGGCCACGCCCCT-3. Oligonucleotides were synthesized by the MCV-VCU Nucleic Acid Synthesis Core Facility (Richmond, VA), and
Integrated DNA Technologies (Coralville, IA). Consensus and mutant Sp1
oligonucleotides were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). The oligonucleotides were annealed; radiolabeled by fill-in
reaction using Klenow fragment, dATP, dGTP, dTTP, and a32P-dCTP (3,000 Ci/mmol; NEN, Boston, MA) or T4
polynucleotide kinase and  32P-ATP40; and
unincorporated label was removed using a Sephadex G-50 column. Ten
micrograms of H526 cell extract was incubated with 10,000 cpm of
labeled DNA in 20 µL of binding buffer (20 mmol/L HEPES [pH 7.9], 1 mmol/L DTT, 1 mmol/L EDTA, 10% glycerol, 1 µg poly dI-dC, and 50 mmol/L NaCl) for 20 minutes at room temperature. Assays with
recombinant human Sp1 (rhSp1; Promega) protein were performed in the
same manner using 1 footprinting unit (fpu) of rhSp1 added to 1 µg of
Drosophila nuclear extract (Promega). For competition assays,
unlabeled competitor oligonucleotides were added to the nuclear
extracts 20 minutes before addition of radiolabeled probe. Reactions
were electrophoresed at 100 V in a nondenaturing 4% polyacrylamide gel
in 0.5 × Tris-borate, EDTA (TBE) buffer40 at 4°C.
In supershift assays, 2 µg of an antibody specific for Sp1, Sp2, Sp3,
Sp4 (Santa Cruz), or nonspecific rabbit IgG (Sigma Chemical Co, St
Louis, MO) was incubated with the gel-shift mixture for 20 minutes at room temperature after normal gel-shift incubation. The
4% nondenaturing gel was run at 325 V at 4°C in 0.5 × TBE.
DNA footprinting.
Footprinting assays were performed according to the Core Footprinting
system protocol (Promega). A fragment containing bases 124 to
+37 was cut out of the pGL2:409-bp construct using
HindIII and SmaI. It was subsequently subcloned into
the HindIII/SmaI sites of the pSP64 plasmid vector
(Promega). From this construct, a 172-bp fragment was cut out using
HindIII and EcoRI, kinased using
32P-ATP, restriction enzyme digested with SmaI,
leaving only the +37 end of the probe labeled. Cell extracts and
binding buffer were prepared as described above. The labeled probe was
incubated with 10 or 20 µg of H526 cell extract for 20 minutes at
room temperature in a total volume of 20 µL. This mixture was
digested with 1.2 U of DNase I for 1 or 2 minutes at room temperature
and then run on a 6% acrylamide, 8 mol/L urea sequencing gel at 1,500 V/60 W until the bromophenol blue dye-front reached the bottom of the gel. For the sequencing ladder, a 17-bp oligonucleotide,
5-AGCTTGGATCCGAGCTC-3, corresponding to the exact
5 end of the footprinting fragment was used as a primer for
sequencing of the pSP64 plasmid containing the
HindIII-SmaI insert.
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RESULTS |
Identification of the transcription initiation sites.
We initially used a modification of the RACE protocol to identify the
transcriptional initiation sites using RNA isolated from the
c-kit-expressing SCLC cell line WB. Oligonucleotide primers were annealed to WB total RNA, extended, tailed with dATP, amplified by
PCR, subcloned, and inserts were sequenced. The transcription start
site was identified as the base adjacent to the start of the terminal
transferase-generated deoxynucleotide tail. We sequenced 11 independent
clones generated from the RACE protocol. Seven of the 11 clones
identified the major initiation sites at the nucleotide G, 58 bases
upstream of the translational initiation codon, and the nucleotide A,
56 bases upstream (Fig 1). These TIS are
designated as +1 and +3 on the proximal c-kit promoter sequence
(Fig 2).
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| Fig 1.
RACE clones show two major transcriptional initiation
sites. The transcription initiation site was identified as the base
adjacent to the start of the poly T tail. (Left panel) Transcription
start site at nucleotide G, base +1 in Fig 2. (Right panel)
Transcription start site at nucleotide A, base +3 in Fig 2.
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| Fig 2.
Nucleotide sequence of the human c-kit promoter
from 124/+37. The sequence is numbered from the 5
transcription initiation site. The locations of the double-stranded
oligonucleotides (XmaI-NaeI, XN, XN2, XN3) used in gel
shift analysis (below) are indicated. The locations of potential Sp1
binding sites are indicated as lines above the sequence showing degree
of homology to the consensus decanucleotide. The TT in the XN2mt
oligonucleotide marks the location of an inserted 2-bp mutation
(below). The +37 nucleotide is the most 3 base of
c-kit promoter sequence before the pGL2 polylinker in the
reporter plasmids.
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To confirm the RACE study and determine the relative usage of these
transcriptional start sites, an RNase protection assay was performed. A
BamHI-SalI fragment extending approximately 400 bases
beyond the potential transcriptional start sites was used to transcribe
a riboprobe. This riboprobe was annealed to total RNA isolated from
cell lines that express c-kit mRNA (WB, H510, and HEL) as well
as a cell line that does not express c-kit mRNA (H82), and an
RNase protection assay was performed.
Both the WB and H510 SCLC cell lines, which express high levels of
c-kit mRNA, were found to contain protected bands corresponding to at least three different transcriptional start sites
(Fig 3). The two major bands correspond to
the bases +1 and +3 identified by the RACE protocol (Figs 1 and 2). The
third band corresponds to base +7, a base also identified by one of the
RACE clones. We found no protected bands when our riboprobe was
annealed to either RNA from the SCLC cell line H82, which does not
express c-kit, or yeast tRNA (Fig 3). HEL cells were found to
have the identical three bands, showing that these transcriptional
start sites are common to SCLC and hematopoietic cells (Fig 3).
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| Fig 3.
RNase protection analysis showed multiple transcriptional
initiation sites. Two major and one minor protected bands, indicating
the transcriptional start sites, were detected in the WB and H510 SCLC
cell lines as well as the HEL leukemia cell line. These respectively
correspond to base +1 (41 bp from 3 BamHI site), +3,
and +7 in Fig 2. The upper major band comigrated with a 41-base
RNA size marker made by transcribing BamHI-cut pGEM3 with
SP6 polymerase.
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Scanning of the sequence immediately upstream of the transcription
initiation sites (Fig 2) failed to reveal a TATA box. This region was
also scanned for an initiator (Inr) promoter element, which can also
mediate transcriptional initiation at unique sites.42 The
Inr overlaps transcriptional start sites, and has the core consensus
sequence Py Py A+1 N T/A Py Py.43 The
c-kit transcriptional start site at base +1 corresponds to this
sequence, except guanine is substituted for adenine at +1 (Fig 2). If
the c-kit transcriptional start site at base +3 is considered,
then the only differences are purines substituted for pyrimidines at
both ends of the consensus sequence.
Functional analysis of the human c-kit promoter.
To characterize functional regulatory elements within the c-kit
promoter, we used a series of 5-deletion fragments linked to the
luciferase gene and tested them in transient transfection assays. The
promoter fragments ranging from 83 bp up to 3 kb of 5 sequence
were cloned into the promoterless pGL2:Basic luciferase vector. The
deletion construct plasmids were electroporated into the H526 SCLC and
the HEL cell lines which express endogenous c-kit or the H146
SCLC and the Jurkat cell lines which do not express c-kit.
Reporter assays showed the same level of expression independent of
whether the cell lines expressed c-kit, indicating there are no
cell type-specific regulatory elements within sequences 3 kb upstream
of the TIS (Fig 4). The 83 construct
showed a basal level of activity. There was a fourfold to fivefold
increase in activity from the 83-bp construct to the
124-bp construct. Additional sequence beyond the 409
construct showed a decrease in reporter activity which may indicate
negative regulatory sequences. Based on this analysis, regulatory
elements responsible for maximal core promoter activity must lie within
the 83 to 124 region.
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| Fig 4.
Promoter-reporter activity of sequential 5
deletion constructs. Promoter activity of all promoter constructs
(diagrammed at left) was compared with that of pGL2:SV40 promoter with
transient transfection of the constructs into H526, H146, HEL, and
Jurkat cell lines. Cotransfection with pCMV gal plasmid was used to
control for transfection efficiency. After a 24-hour incubation, cell
extracts were assayed, normalized for transfection efficiency, and
relative reporter activity was determined. Error bars indicate standard
error (SE) calculated from at least six separate electroporations. All
activity values are relative to the pGL2:SV40 promoter, which was
assigned a value of 1.0. Relative reporter gene activity was calculated
as follows: luciferase activity divided by -galactosidase activity,
divided by the ratio obtained for the pGL2:SV40 promoter construct.
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Specific protein binding to the 93/84 site in the core
promoter.
A search for transcription factor binding sites within this 40 bp
yielded several potential consensus binding sites for Sp1 including a
9/10 consensus Sp1 site, GGGGCGTGGC (Fig 2). To characterize the
proteins that bind to this region of DNA, we performed gel mobility
shift analysis (Fig 5). When
oligonucleotides corresponding to the 5 and 3 halves of
the 40 bases were incubated with H526 cell extract, only the 3
half oligonucleotide (XN2) produced a strong shifted band. A further
gel shift using a smaller oligonucleotide corresponding to the 3
15 bp (XN3) showed that only a 15-bp fragment of DNA containing the
93/84 consensus Sp1 site is necessary for a gel shift.
This shifted band comigrates with the band obtained using a consensus
Sp1 oligonucleotide.
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| Fig 5.
Gel mobility shift analysis of protein binding to the
124 to 83 DNA fragment. Gel mobility shift analysis was performed
with the double-stranded DNA fragment containing the 124 to 83
region alone and with 10 µg of cellular protein from H526 cells. The
SC Sp1 oligonucleotide (Santa Cruz) contains a consensus Sp1 binding
site. The oligonucleotides corresponding to c-kit sequences
are: Xma-Nae (124/83), XN (125/97), XN2
(102/82), XN3 (96/82). The +/ indicates whether H526
extract was added to the gel shift mixture.
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To show specificity, a competition assay was performed. Incubating the
XN3 oligonucleotide with unlabeled competitor DNA containing a cognate
binding site before addition of labeled probe should compete for
protein binding and result in subsequent loss of a shifted band. Only
the oligonucleotides containing the 93/84 site and the
consensus Sp1 oligonucleotide were able to successfully compete for
binding (Fig 6). Neither the mutant
consensus Sp1 oligonucleotide
(5-ATTCGATCGGTTCGGGGCGAGC-3) nor the
oligonucleotide corresponding to the 5 half of the 40-bp (XN)
fragment was able to compete for binding, which shows specific binding
by the protein to the XN3 oligonucleotide.
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| Fig 6.
Competition assays using specific and nonspecific
competitor oligonucleotides. In the absence or presence of competing
oligonucleotides, 0.1 ng of 32P-labeled XN3 oligonucleotide
was incubated with H526 extract. Competing oligonucleotides were at
100-fold excess. The competing oligonucleotides corresponding to
c-kit sequences were: Xma-Nae (124/83),
XN (125/97), XN2 (102/82), XN3 (96/82). Labeled
DNA:protein complexes were only formed in the presence of competitors
that do not contain the 93/84 Sp1 site. Competition using the
consensus Sp1 oligonucleotide was used as a positive control, and the
oligonucleotide containing a mutant Sp1 site (SC mt Sp1) was used as a
negative control.
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If the protein was bound to the 93/84 site, mutation of
this site should cause disruption of binding and loss of the shifted band. A 2-bp mutation (GGGGCGTGGC > GGTTCGTGGC)
was introduced into the 93/84 site in the XN2
oligonucleotide (Fig 2); gel shift analysis with this oligonucleotide
failed to produce a shifted band (Fig 7A).
This indicates that a protein from the H526 extract was specifically
recognizing the 2-bp mutated sequence as part of its binding site.
Furthermore, incubation of the XN2 oligonucleotide with H526 extract
yielded a shifted complex with the same mobility as that of the XN2
oligonucleotide incubated with recombinant human Sp1 protein (Fig 7B).
A Drosophila extract, which does not contain Sp1, failed to
produce a shifted band. These data further suggest that the binding
protein is Sp1.
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| Fig 7.
The protein in the 93/84 complex has the binding
specificity and mobility of Sp1. (A) A 2-bp mutation
(GGGGCGTGGC > GGTTCGTGGC) was introduced into the
93/84 Sp1 site of the XN2 oligonucleotide (XN2mt), and binding
ability was compared with the wild-type XN2 oligonucleotide. The
/+ indicates whether cellular extract was added to the gel shift
mixture. Ten thousand cpm of the XN2 and XN2mt oligonucleotides were
incubated with 10 µg of H526 extract. (B) Mobility shift of XN2
oligonucleotide with rhSp1. Mobility shift analysis using the XN2
oligonucleotide incubated with H526 extract (10 µg ) or with
recombinant human Sp1 (rhSp1, 1 fpu) and Drosophila nuclear
extract (1 µg). Incubation of the XN2 oligonucleotide with H526
extract (lane 2) yielded a band with a similar mobility pattern as that
obtained by incubating XN2 oligonucleotide with rhSp1 and
Drosophila extract (lane 3). Incubation of XN2 oligonucleotide
with Drosophila extract alone was used as a negative control
(lane 4).
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To confirm the location of protein binding and to screen for additional
binding sites, a DNase footprint analysis was performed. A DNA fragment
containing the 124/+37 region was 5 end-labeled, incubated with H526 cell extract, and then digested with 1.2 U of DNase
I. The digestion products were analyzed on a 6% sequencing gel. A
sequencing reaction of the identical region was run on the same gel to
identify the protected region. The region corresponding to the
93/84 consensus Sp1 was protected from DNase I digestion (Fig 8). This was the only
region shown to be protected under our assay conditions, but it is
possible that there are other protected sites at the 5 and
3 ends of the fragment which cannot be well visualized.
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| Fig 8.
DNase footprinting analysis of the 124 to
+37 region. The ability of proteins to bind and protect the core
promoter region of the c-kit promoter was analyzed. DNase I
digestion for 1 and 2 minutes of the 32P-labeled DNA
fragment was performed with no cell extract (lane 1) and in the
presence of 10 and 20 µg of cell extract (right panel). The digested
products were analyzed on a 6% sequencing gel. A sequencing reaction
of the DNA region was run in parallel (left panel). The sequence of the
protected region, corresponding to the 93/84 SP1 site, is
shown.
|
|
Sp1 specifically binds the 93/84 site.
The above data strongly suggest that an Sp family member binds to the
93/84 site based on the specificity of binding and mobility of gel-shifted species. To further determine if the protein binding to the 93/84 site was indeed Sp1, a supershift
assay was performed. If Sp1 was the binding protein, incubation of the protein:DNA complex with an anti-Sp1 antibody should result in a
complex of slower mobility than the original shifted band. Because the
other Sp family members also bind GC boxes, we also assayed the
abilities of antibodies to Sp2, Sp3, and Sp4 to bind to the protein:DNA
complex. Incubating 32P-labeled XN2 oligonucleotide with
H526 cell extract and Sp1-specific antibody resulted in a supershifted
band, whereas incubating with antibodies specific for Sp2, Sp3, Sp4, or
nonspecific rabbit IgG did not (Fig 9),
confirming that Sp1 specifically binds to this region of DNA.
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| Fig 9.
Supershift analysis of Sp1 family members. Interaction of
the XN2 fragment with H526 cell extracts in the absence and presence of
antibodies specific for Sp1, Sp2, Sp3, Sp4, and nonspecific rabbit IgG.
The DNA:protein complexes were analyzed on a 4% nondenaturing
polyacrylamide gel. The first lane contains no extract and no
antibody.
|
|
Mutational analysis of the 93/84 Sp1 site.
The reporter construct containing the 124/+37 region has maximal
activity, likely representing the core promoter. To correlate Sp1
binding with the potency of this region to promote transcription, we
introduced a 2-bp mutation into the 93/84 Sp1 site
(GGGGCGTGGC > GGTTCGTGGC) of the 124
construct (XN2mt). This mutation eliminated Sp1 binding by gel-shift
analysis (Fig 7). Reporter activity of the mutant construct transfected
into H526 cells was reduced to basal levels when compared with the
wild-type construct (Fig 10). To study
the effect of restoration of the Sp1 binding site, we inserted the
wild-type 93/84 consensus site directly upstream of the
XN2mt construct (XN2mt/Sp1), which restored much of the activity,
though it was less than wild-type. This lower activity may be related
to the altered position of the Sp1 binding site in this construct.
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| Fig 10.
Functional analysis of 93/84 binding site.
Reporter assays were performed as in Fig 4. A construct containing a
2-bp mutation of the 93/84 Sp1 (GGGGCGTGGC > GGTTCGTGGC) site, 124 XN2mt, displayed basal reporter
activity. The 124 XN2mt/9/10 construct, which has the 93/84
Sp1 site inserted 40 bp upstream in the 124 XN2mt construct,
restores reporter activity to near wild-type levels.
|
|
The above data correlates the ability of Sp1 to uniquely bind to the
93/84 site in vitro with the ability of this site to activate transcription in vivo. To correlate in vivo Sp1 binding with
the ability of this site to activate transcription we turned to the
Drosophila SL2 expression system.44 Courey and
Tjian44 devised this system to analyze the functional
domains of Sp1 because it is extremely difficult to observe the effects
of exogenously expressed Sp1 on the background of the high constitutive
levels of Sp1 in mammalian cells. Drosophila cells lack
endogenous Sp1, yet transfected reporter plasmids containing Sp1 sites
show a dose response to exogenously expressed Sp1, indicating that the conserved transcriptional machinery is capable of interacting with the
transcriptional activation domain of Sp1.44 To correlate in
vivo Sp1 binding with activation of c-kit transcription, we cotransfected into SL2 cells either the wild-type 124 reporter construct or the construct containing the mutant 93/84
site with increasing amounts of a plasmid encoding Sp1 under the
control of the Drosophila actin promoter (pPacSp1;
kindly donated by R. Tjian).44
Figure 11 illustrates that
transcriptional activation of the wild-type 124 construct showed
a dramatic dose response to Sp1 expression. Transfection of the empty
Drosophila expression vector, pPacU, had no effect
on expression. The mutant construct also showed an Sp1 dose-dependent
increase in transcription, but the levels of reporter expression
approached only 10% to 20% of those of the wild-type construct. The
Sp1-dependent transcription of the mutant construct suggests that
low-level transcriptional activation may be obtained through Sp1
binding to alternative sites. However, it is clear from all the
reporter assays that selective Sp1 binding to the 93/84
site and not to the other potential binding sites is required for
maximal promoter activity.
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| Fig 11.
In vivo transcriptional activation mediated through the
93/84 binding site correlates with Sp1 expression. Five
micrograms of either the 124 promoter-reporter construct (WT) or the
124 XN2mt construct (mut) were cotransfected into SL2 cells in
triplicate along with the indicated amounts of either the
pPacSp1 expression plasmid or the empty pPacU
expression vector. The pCMV gal plasmid was also cotransfected to
normalize for transfection efficiency. Relative reporter activity was
calculated as in Fig 4, with the activity of the pGL2:SV40 promoter
plasmid assigned a value of 1; error bars indicate ± SE.
|
|
| |
DISCUSSION |
In this study, two major transcriptional initiation sites for the human
c-kit gene were identified using the RACE protocol and RNase
protection assays, and these sites were shown to be common to both
hematopoietic and nonhematopoietic cell lines. The human c-kit
transcriptional initiation site identified in this study as base +1
(Fig 2) is homologous to the mouse c-kit transcriptional start
site33 and with one of the major start sites previously
identified in the human promoter.35 In addition, Yamamoto
et al35 identified a transcriptional initiation site 4 bases upstream of this site. We also identified multiple
transcriptional initiation sites, but in addition to the site at +1, we
localized the others to +3 as well as a minor site at +7 (Fig 3).
Yamamoto et al35 used an M13mp18 sequencing ladder to
determine the size of the primer-extended and S1 nuclease-protected
bands. We believe the RACE method used in this study, which allows for
direct sequencing of the primer-extended species, gives a more accurate
localization of the transcription initiation sites. This accurate
localization is a necessary prerequisite for the identification of
elements that regulate transcriptional initiation.
Initiator elements are known to localize transcription initiation sites
and mediate the action of some upstream activators in TATA-less
promoters.43 The human c-kit promoter has sequences centered around bases +1 and +3 that are very similar to the initiator consensus sequence (Py Py A+1 N T/A Py Py).43
Only one difference from the Inr consensus sequence is observed for
transcription starting at base +1, substituting the guanine for adenine
at the initiation site. For transcription starting at base +3, the
pyrimidines at both ends of the consensus Inr sequence are replaced by
purines. Although substituting a guanine for adenine at base +1
substantially weakens the Inr element,43 having two
overlapping Inr sequences may compensate and allow transcriptional
initiation at either the +1 or +3 position.
As mentioned earlier, there have been several studies performed on both
the mouse and human c-kit promoter. The initial study of the
mouse promoter found that as little as 105 bp upstream of the TIS was
enough to show strong promoter activity in cell lines that express
c-kit but not in nonexpressing cell lines.33 In a
similar study of the human c-kit promoter, sequences 120 bp
upstream of the TIS were shown to activate the c-kit promoter in a cell type-specific manner.35 In the study of the
mouse promoter,33 the HL-60 cell line was used as the
nonexpressing cell line. We also initially used HL-60 cells but found
transfection efficiencies to be very low, making expression studies
from any reporter plasmid difficult to interpret. Therefore, we
switched to the H146 and Jurkat cell lines, which, in our hands, had
much higher transfection efficiencies, and we found no cell
type-specific elements in the proximal promoter. We suggest that some
of the differences between our data and the previous studies may be
caused by the use of c-kit nonexpressing cells with low
transfection efficiencies.
In agreement with the studies mentioned above, our analysis of the
human c-kit promoter has shown that a construct containing 124 bp of promoter sequence was able to maximally activate the c-kit promoter, albeit not in a tissue-specific manner. This
finding agrees with Vandenbark et al,36 who concluded that
the DNA region to 183 (from the translational initiation codon;
equivalent to 125 in our numbering scheme) was important for
c-kit promoter activation, but did not confer cell
type-specific transcription. Their data suggested that a distal
negative regulatory DNA segment between 4100 to 5500 was
the main, though not the only, cis-acting DNA region controlling cell
type-specific transcription. They also showed that Myb binding to a
consensus site at approximately 1300 had a negative regulatory
effect. This is in contrast to a recent study by Ratajczak et
al,38 who showed that Myb and Ets-2 binding to consensus
sites located between 179 and 471 cooperatively enhanced
transcription. However, in the latter study overexpression of Myb and
Ets-2 was required to observe these effects; deletion of all Myb and
Ets consensus sequences had no significant effect on expression in the
presence of endogenous levels of these two transcription factors. Thus,
as has been previously shown,36 the tissue-specific
regulation of c-kit is likely to be complex. We believe that
the difficulty in reconciling all the published data indicates that
regions of DNA containing major tissue-specific regulators of
transcription have yet to be identified. Recently, two studies of the
Wsh mutation found an inversion disrupting
tissue-specific positive regulatory elements controlling c-kit
expression,45,46 suggesting that potential c-kit
upstream regulatory elements may lie near the breakpoint, located
between the PDGFR and c-kit loci, within 100 to 200 kb
5 of c-kit. Our study did not show any cell
type-specific elements within the proximal 3 kb of promoter sequence.
All proximal promoter-reporter studies performed thus far agree that
deletion of the region between 124 and 83 from the TIS
results in a drop in promoter function to basal levels in c-kit-expressing cells.33,35,36,38 This crucial
region contains the 93/84 Sp1 binding site we have
characterized, which is completely conserved in the mouse
promoter.33,37 Transfection studies of promoter-reporter
plasmids containing a mutation which eliminated in vitro Sp1 binding
resulted in a drop in transcriptional activity to basal levels, showing
that the 93/84 site is largely responsible for the
activity of the whole 124/83 fragment. However,
overexpression studies in SL2 cells did demonstrate that Sp1 was still
able to weakly transactivate in spite of mutation of the
93/84 site, indicating that one of the upstream consensus
sites may have some functional activity. In fact, gel-shift studies
(Fig 5) have suggested weak in vitro binding of Sp1 to the XN
oligonucleotide, which contains a perfect 6/6 Sp1 core consensus
sequence (GGGCGG; 122/117) that other investigators have
identified as the likely site of Sp1 binding within the
124/83 fragment based on computer homology algorithms.33,35 Mutation of this site in promoter-reporter constructs resulted in only a 20% to 25% decrease in transcriptional activity (not shown), consistent with our conclusion that the vast
majority of Sp1-mediated transcriptional activation occurs as a result
of binding to the 93/84 site.
There are many examples where Sp1 plays an essential role in core
promoter activity.47,48 In addition, in the human adenosine deaminase promoter, Sp1 not only controls basal level activation but is
also involved in enhancer-mediated activation.49
Furthermore, in some promoters Sp1 can discriminate between different
consensus Sp1 binding sites,50 indicating that certain Sp1
binding sites contribute more to the overall activity than other sites.
An example of a promoter that relies predominantly on only one of
multiple potential Sp1 sites, in addition to the c-kit
promoter, is the promoter of the transforming growth factor type I
receptor51 (TGF-RI). Deletion and site-specific mutation
analyses explored the importance of multiple Sp1 sites throughout the
TGF-RI promoter and established that one downstream site at position
63 to 54 contributes heavily to basal promoter activity.
In the nicotinamide adenine dincucleotide phosphate cytochrome P-450
oxidoreductase gene, promoter deletion studies indicated that loss of
the seven distal GC boxes had minimal effect on transcriptional
activity, but deletion of the two proximal Sp1 sites resulted in 90%
loss of promoter activity.48 In addition, some cell
type-specific promoters require cooperative activity of Sp1 with cell
type-specific transcription factors, including Egr-1 for
thrombospondin,52 AP2 for the acetylcholine receptor
3 gene,53 and GATA 1 for the -globin
gene.54 Although a potential AP2 site exists on the
124 to 83 fragment, no evidence for binding of factors
other than Sp1 was found by either gel-shift or DNase footprint
analysis.
In conclusion, we have localized the major transcriptional initiation
sites of the human c-kit gene to two bases 58 and 56 bases
upstream of the translational initiation codon. Two overlapping weak
Inr consensus sites may mediate transcriptional initiation at these
specific sites. All the c-kit promoter studies to
date33,35,36,38 agree that maximal promoter activity is
contained within approximately 125 bp of the transcriptional initiation
sites, and that deletion of the region between bases 124 and
83 results in a marked loss in activity. This region contains
several possible binding sites for the transcription factor Sp1, but
Sp1 seems to bind only to the 93/84 site with high
affinity, and mutation of this site results in a drop in promoter
activity to basal levels. Taken together, the data presented here and
previously published suggest that selective Sp1 binding to the
93/84 site must cooperate with presumed upstream factors
to control c-kit promoter activation in a cell type-specific
manner. Characterization of the binding sites and identification
of these upstream factors should be a subject for future
investigation.
| |
ACKNOWLEDGMENT |
We thank Barbara Armstrong and Julie Litz for excellent technical
assistance during the course of this study, Dr Robert Tjian for
donating the pPacU and pPacSp1 plasmids, and Dr
Richard Moran for donating the SL2 cells.
| |
FOOTNOTES |
Submitted February 19, 1998;
accepted July 31, 1998.
Supported by a Merit Review Award from the Department of Veterans
Affairs.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Geoffrey W. Krystal, MD, PhD, Room 5A-128,
McGuire VA Medical Center, 1201 Broad Rock Blvd, Richmond, VA 23249.
| |
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Abstract
Introduction