From the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, New York 10021
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ABSTRACT |
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The human HNP-defensin-1 gene encodes
a peptide antibiotic found exclusively in neutrophils and is key to
elimination of microbes. Expression is a marker for the granulocytic
lineage and for certain stages of differentiation and is not known to
be inducible in mature cells under physiological conditions. Low level
of transcription also occurs in HL-60 promyelocytic leukemia cells and
is greatly activated upon drug-induced granulocytic maturation and by
low doses of retinoic acid, in a strictly cell-specific manner (Herwig, S., Su, Q., Ma, Y., and Tempst, P. (1996) Blood 87, 350-364). We have analyzed a 10-kilobase pair region, upstream of the
defensin-1 cap site, for the presence of control elements,
and we describe a minimal promoter (position 83 to +82) required to
drive transcription in HL-60 cells in a quasi cell-specific manner. Our
data also suggest the presence of negative regulatory elements in the
416/
191 region that may further contribute to cell specificity in a
chromosomal context. The basal promoter contains two functionally
essential, ETS-like (GGAA core sequence) elements. The proximal site
(
22/
19) constitutively binds the PU.1 transcription factor in
vitro and could function, together perhaps with an adjacent
TA-rich sequence (
32/
25), in assembly of a myeloid-restricted,
basal transcription factor complex. The distal site (
62/
59)
interacts in vitro with an unidentified activity, distinct
from PU.1, ETS-1, PEA3, and ELK-1 (factors with definite binding site
similarities), and is greatly stimulated by phosphorylation during
granulocytic differentiation of HL-60 cells. Identification of this
protein will be important to resolve the molecular mechanisms
controlling temporal, granulocytic restricted gene expression.
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INTRODUCTION |
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Neutrophils are specialized scavenger blood cells, killing
microorganisms through a combination of reactive oxidants and
polypeptide antibiotics (1). Such peptides are stored in cytoplasmic
granules and released, whenever required, into the phagosomes that hold ingested microbes (2-4). Defensins (also termed "human neutrophil peptides," or HNP)1 are the
major components of this system and account for a large percentage of
total granular protein (4-6). Four different isoforms, HNP-1-4, have
been isolated (7-9), and analysis of cDNA clones has indicated
processing from larger precursor structures (10-13). The mature
peptides are 29-30 amino acids long and are defined by a conserved
cysteine backbone (4). "Defensin-like" peptides have also been
detected in epithelial linings of the tongue (14), respiratory tract
(15), and gut (16, 17). Expression of HNP-type defensins is believed to
be cell-specific, however, and the non-neutrophilic types are now
commonly known under names such as TAP, LAP, cryptdins, and
-defensins (18, 19).
Even though defensin peptides are abundantly present in differentiated neutrophils, transcripts have never been detected in peripheral blood but rather in unfractionated bone marrow (10, 11, 20, 21). More specifically, transcription seems restricted to a certain window in myeloid blood cell differentiation (11, 21). Consistent with these findings is the presence of defensin mRNA, albeit at trace levels, in the HL-60 human promyelocytic leukemia cell line (10, 21-25). HL-60 cells can be chemically induced to mature along various pathways, thus providing a model system for study of differentiation-specific gene regulation (26-28). For example, in the course of retinoic acid (RA) treatment, defensin transcription reaches peak levels during the resultant myelocyte and very early metamyelocyte stages of the granulocytic pathway, later followed by a complete down-regulation (25). By contrast, instant down-regulation to virtually undetectable levels was observed during phorbol ester-promoted differentiation toward macrophages (25). Similarly, defensin transcripts have never been found in either myeloblastic (KG-1), monoblastic (U-937), myeloblastic/erythroblastic (K-562), B-lymphoid or T-lymphoid cell lines, not even after extensive RA treatment (10, 25). Any studies aimed at understanding this unique granulocytic expression of defensin genes must converge, eventually, at the identification of genomic regulatory elements and their cognate transactivating factors.
Considerable efforts have been expended already at analyzing the
control regions of other myeloid-specific genes (29-31). Instead of
being strictly myeloid-specific, many of the transcription factors
involved are more commonly expressed, for instance Sp1 (32, 33), OCT-1
(33), PU.1 (31, 34), PEBP2/CBF (35), myb (36), C/EBP (37, 38), and HLH
factors (39). Not surprisingly then, lineage-specific gene activation
is controlled, in many cases, through unique combinations (30). For
example, PU.1 allows Sp1 to bind in a cell-specific fashion (31);
likewise, PU.1 together with one or more of C/EBP, AML-1, c-MYB, and
HLH factors function as combinatorial activators of myeloid genes
(39-43), as do c-MYB, together with C/EPB or with ETS-1 or -2 (44,
45). Furthermore, C/EBP and PU.1 are activated, or have their
transactivating potential enhanced, by phosphorylation, which may
impart an additional layer of cell specificity (46-48). Alternative
scenarios of myeloid-specific gene activation have ubiquitous factors
(e.g. CP1) drive transcription only when promoters are not
occupied by repressor proteins (e.g. CDP); here,
lineage/stage-specific derepression is the real switch to expression
(e.g. in case of gp91-phox) (49). In view of the published
data, it is quite possible that defensin transcription in
HL-60 cells is also controlled by one or more of the aforementioned transcription factors and repressors. However, inspection of the 1.2-kb
upstream sequence and of the first intron of the HNP
defensin-1 gene (taken from Ref. 50) did not reveal a presence of
the precise binding sites, as previously characterized for these
particular factors; neither could RA response elements (51) be
identified. Thus, there is no easy way to formulate a mechanistic model
for promyelocytic defensin expression at this time.
Regulatory elements, and their binding factors, will have to be
uncovered and characterized without any preconception of identity.
Here, we describe a minimal defensin-1 promoter, required to drive transcription in HL-60 cells in a quasi cell-specific manner, that contains two essential, ETS-like elements, one binding the PU.1 transcription factor and the other binding a RA-stimulated activity in vitro.
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EXPERIMENTAL PROCEDURES |
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Materials-- All-trans-retinoic acid was purchased from Sigma (catalog number R2625); D-luciferin potassium salt was from Analytical Luminescence Laboratory (San Diego, CA), and poly(dI-dC)·poly(dI-dC) was from Amersham Pharmacia Biotech. Oligonucleotides were synthesized by the Sloan-Kettering Microchemistry Core facility. Purified TBP (TATA-binding protein) and rabbit polyclonal antibodies, specifically recognizing either PU.1 (sc-352X) or ELK-1 (sc-355X), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pCMV-hGH plasmids were a gift from Dr. Daniel Tenen (Harvard Medical School, Boston), and their use in transient transfection assays has been described (34). All other chemicals were from Sigma, unless otherwise indicated.
Cell Lines and Culture Conditions--
The human promyelocytic
leukemia cell line HL-60, the myeloblastic leukemia cell line KG-1, the
monocytic cell line U-937, and erythroleukemic line K-562 were obtained
from the American Type Culture Collection (ATCC, Rockville, MD);
Burkitt CA-46 lymphoma cells and S3 HeLa carcinoma cells were obtained,
respectively, from Drs. A. Zelenetz and J. Hurwitz (Sloan-Kettering,
New York); the retinoic acid-resistant cell line HL-60R was provided by
Dr. S. Collins (Fred Hutchinson Cancer Center, Seattle, WA). HL-60, HL-60R, U937, K-562, and Burkitt cells were grown in RPMI medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (HyClone
Laboratories Inc., Logan, UT), 5.0 units of penicillin, and 5 µg/ml
streptomycin (complete medium) and maintained at 37 °C in a
humidified atmosphere containing 5% CO2; HeLa S3 cells
were maintained in minimum Eagle's medium Joklin medium (Life
Technologies, Inc.) with 5% FCS supplement; KG-1 was cultured in
suspension in Iscove's modified Dulbecco's medium (Life Technologies,
Inc.) containing 10% FCS and 104 M
-thioglycerol. Cell cultures were always passaged twice a week to
maintain a cell density between 2 × 105 and 1 × 106 cells/ml. Cells were counted in a hemocytometer
chamber, and viability was assessed by exclusion of 0.1% trypan blue.
For induction experiments, cells were seeded at 2.5 × 105 cells/ml; inducers were added 24 h later and were
then left in the culture medium for 72 h, unless otherwise
indicated. The concentrations of the inducers were as follows: 1 µM all-trans-retinoic acid (RA); 160 mM dimethyl sulfoxide (Me2SO); 2 mM
hexamethylene-bisacetamide (HMBA).
Isolation and Characterization of Genomic Defensin
Clones--
To allow isolation of genomic clones containing large
portions (<10 kb) of the defensin-1 gene 5'-flanking
region, polymerase chain reaction (PCR) was first used to generate a
defensin-specific probe. Oligonucleotides DEF-38-S
(5'-AGATACAACCTGACCTGTGTC-3') and DEF-737-AS
(5'-TCCCGAGGACCTGGGGTCTAACCA-3') were designed based on the
published defensin genomic sequence (50) and used as primers. The PCR
reaction was done using a Gene Amp System 9600 (Perkin-Elmer),
Taq polymerase (Promega, Madison, WI), and 1 µg of HL-60
cell genomic DNA as template, 0.2 µM oligonucleotide primers, and the following cycling parameters: 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min for a total of 30 cycles. The resulting PCR product (722 bp) was then labeled using a random priming
labeling kit (Amersham Pharmacia Biotech). Briefly, 50 ng of PCR
products in 10 µl of nuclease-free water were boiled for 5 min to
denature the DNA, followed by submersion of the tube in ice water for 2 min and by adding 10 µl of labeling buffer (6 µg/m
hexadeoxyribonucleotides, 440 mM HEPES, pH 6.6, 110 mM Tris, pH 8.0, 11 mM MgCl2, 22 mM -mercaptoethanol, and 44 mM each of dATP,
dGTP, and dTTP), 5 µl of [
-32P]dCTP (NEN Life
Science Products), and 5 units of DNA polymerase I Klenow fragment.
After 1 h incubation at 37 °C, the probe was purified over a
Sephadex G-50 column.
Primer Extension Assay--
Primer extension assays were
performed using the appropriate reagent kit from Promega and following
the instructions provided by the manufacturer. Antisense primers,
DEF-1216-AS (5'-CTAGGCAGGGTGACCAGAGA-3') and DEF-2658-AS
(5'-AGAATGGCAGCAAGGATG-3') (positions indicated on Fig. 2), and X174
HinfI DNA marker (Promega) were separately kinase-labeled
with the [
-32P]ATP. Fourteen µg of total RNA from
HL-60 cells was annealed to 0.1 pmol of the labeled primer in the
buffer containing 50 mM Tris-HCl (pH 8.3 at 42 °C), 50 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM of each dNTP, and 0.5 mM spermidine. The components were gently mixed, heated to
58 °C for 20 min, and then cooled to room temperature for 10 min.
The extension reaction was carried out in the buffer described above in
the presence of 2.8 mM sodium pyrophosphate and 1 unit of
avian myeloblastosis virus reverse transcriptase in a total reaction
volume of 20 µl. The incubation was performed at 42 °C for 30 min.
An equal volume of a 2× loading buffer, containing 98% formamide, 10 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue,
was then added, the mixture heated to 90 °C for 10 min, cooled on
the ice, and a 10-µl aliquot loaded onto a 10% polyacrylamide gel
(19:1; 1× TBE, containing 8 M urea; 1.0-mm thick),
electrophoresed at 250 V constant voltage, at 15 °C, until the
bromphenol blue marker had reached the bottom of the gel.
Dephosphorylated
X174 HinfI DNA markers were
co-electrophoresed in adjacent lanes as size markers. The gel was then
transferred onto Whatman paper, vacuum-dried, and exposed to Hyperfilm
(Amersham Pharmacia Biotech) for the desired time at
80 °C with an
intensifier screen.
Northern Blot Analysis--
RNA extraction, agarose gel
electrophoresis, transfer of the RNA to Hybond-N+ membranes
(Amersham Pharmacia Biotech), and hybridization with a
defensin-specific RNA probe were all done exactly as described before
(25). Probe template was a 0.45-kb SphI-EcoRI
fragment derived from the HNP-1B cDNA clone (10), which was used to
generate a [-32P]UTP-labeled RNA, also as described
(25). Washed blots were exposed to Hyperfilm-MP (Amersham Pharmacia
Biotech), autoradiographs scanned, bands quantitated and normalized to
ribosomal RNA levels (determined from ethidium bromide-stained gels),
as described (25).
Plasmids for Transient Transfections--
A promoterless
luciferase reporter vector, "pGL3-Basic" (Promega), and an SV40
promoter-containing but otherwise similar luciferase plasmid,
"pGL3-Promoter," were used in the course of these studies. The
expression plasmid pCMV-hGH (human growth hormone gene under control of
a cytomegalovirus promoter) was also used throughout as an internal
control for transfection efficiency (34). Genomic clone "17" (see
above under "Isolation and Characterization of Genomic Defensin
Clones") was digested with NheI, and the resulting large
fragment was inserted into the pGL3-Basic vector. This plasmid was then
linearized with XhoI, partially digested with
ScaI, followed by "filling in" with Klenow enzyme and
self-ligation, to generate subclones pGL3basic-A, -B, -C, and -D with
approximate insert sizes of 10, 7, 5, and 2 kb, respectively. Clone
pGL3basic-A (10 kb) was subsequently taken through a second round of
partial digestion with ScaI, which resulted in subclones
pGL3basic-A1, -A2, and -A3, exhibiting approximate insert sizes of,
respectively, 5, 2, and 0.6 kb, and all having their insert 3'-ends
anchored at the "gtaagt" sequence immediately downstream of exon I
(arbitrarily numbered +82; see Fig. 2). Plasmid pGL3basic-A3
(552/+82) served to generate several smaller constructs, with all
inserts bracketed by the fixed ScaI site (+82) at their
3'-ends and by varying restriction sites at their respective 5'-ends as
follows: pGL3b-AvaI (
416), pGL3b-HinfI (
218),
pGL3b-Sau96I (
83), and pGL3b-Tru9I (
30). In addition, plasmids
pGL3b-Exo1 (
191/+82), pGL3b-Exo2 (
50/+82), pGL3b-Exo3 (
34/+82),
and pGL3b-Exo4 (+11/+82) were derived from exonuclease III digestion of
the linearized parental pGL3basic-A3, whereby the deletion was started
from the KpnI site in the vector sequence.
Site-directed Mutagenesis--
Mutagenesis of selected
nucleotides in the defensin regulatory sequences, contained within
plasmid pGL3b-Sau96I (83), was done as described by Zaret et
al. (52). For the first round of PCR, two pairs of oligonucleotide
primers were synthesized for each mutant to be constructed. First, the
vector sequence including the restriction site adjacent to the 5'-end
of the insert of interest was used as the sense primer; the antisense
primer was designed from the same region but carrying the nucleotide substitutions. For the second pair, the sense primer was again designed
from the same region and with the complementary nucleotide substitution, and the antisense primer was designed from the vector sequence adjacent to the 3'-end of the insert including the restriction site. The PCR reactions were performed separately, and their products then used in a second round of PCR by annealing the two overlapping PCR
products first, followed by the second reaction which used the sense
and antisense primers derived from the vector. The resulting PCR
product was then digested with the appropriate restriction enzyme and
ligated into the corresponding vector to generate the mutant
construct.
Transient Transfection Assays-- Transfection of tissue culture cells and luciferase assays were carried out as described by Pahl et al. (34). In brief, tissue culture cells were diluted into the corresponding growth media (see under "Cell Lines and Culture Conditions"), at densities of 4 × 105/ml, the day before transfection. After 18 h, cells (1 × 107 per transfection) were pelleted and washed with pre-warmed (37 °C) Iscove's modified Dulbecco's medium, centrifuged at 500 × g for 5 min at room temperature (RT), resuspended at a density of 1 × 107 cells in 0.4 ml of warm Iscove's modified Dulbecco's medium containing 2.5 µg of pCMV-hGH plasmids. This suspension was added into the electroporation cuvette already containing the luciferase expression DNA constructs (18 µg of pGL3-control plasmid in less than 20 µl volume; weight amounts of insert-containing plasmids were adjusted to be equimolar with the control). Cells and plasmids were then mixed with a pipette, incubated for 5 min at RT, followed by electroporation at 975 microfarads capacitance and 280 V using a Gene Pulser II (Bio-Rad), unless otherwise indicated. The cells were then transferred to 10 ml of warm Iscove's modified Dulbecco's medium with 10% FCS, the dishes swirled and incubated at 37 °C for 5 h, and the cells harvested in 15-ml tubes by centrifugation at 500 × g for 5 min at RT. One ml of supernatant from each experiment was stored in an Eppendorf tube for human growth hormone (hGH) assay (see below). Pellets were washed with 5 ml of phosphate-buffered saline at RT; 300 µl of lysis buffer (containing 1% Triton X-100, 25 mM Gly-Gly, pH 7.8, 15 mM MgSO4, 4 mM EGTA, pH 7.8, 1 mM DTT) were added, and pellets were then resuspended, transferred to Eppendorf tubes, vortexed for 5 s, and spun at full speed for 3 min at RT. Fifteen µl of the above lysate was then mixed with 300 µl of freshly made assay buffer, which contained 25 mM Gly-Gly, pH 7.8, 15 mM KPO4, pH 7.8, 15 mM MgSO4, 4 mM EGTA, pH 7.8, 2 mM ATP, pH 7.8, 1 mM DTT. Relative light units (RLU) were measured for 20 s in a model Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). The hGH was measured with the enzyme-linked immunosorbent assay kit from the Nichols Institute (San Juan Capistrano, CA) as per the manufacturer's instructions. Briefly, 100 µl of supernatant was mixed with an equal volume of antibody solution; the latter is a mixture of two monoclonal antibodies, each one specific for a different and distinct epitope on the hGH molecule, to form a soluble sandwich complex in the presence of hGH. One of the antibodies is 125I-labeled for detection, whereas the other antibody is coupled to biotin. The reaction was then mixed with an avidin-coated plastic bead and incubated for 90 min at RT while shaking (180 rpm). After two washes, the bead was counted in gamma counter (LKB 1272 Clinigamma; Wallac, Gaithersburg, MD) for 1 min.
Mini-preparation of Nuclear Extract--
Nuclear extract was
prepared as described (53), with modifications. Briefly,
108 cells were pelleted at 500 × g for 5 min at room temperature. The pellet was resuspended in 1.5 ml of
ice-cold phosphate-buffered saline and transferred to an Eppendorf tube
and spun for 10 s at full speed. The pellet was then resuspended
in 1× packed cell volume of cold buffer containing 10 mM
HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM
phenylmethylsulfonyl fluoride by flicking the tube and leaving it on
ice for 15 min. The reaction mixture was then passed five times through
a syringe with 23-gauge needle and spun for 20 s at full speed.
The supernatant was discarded and the pellet resuspended in two-thirds
packed cell volume of ice-cold buffer containing 20 mM
HEPES-KOH, pH 7.9, at 4 °C, 25% glycerol, 1.5 mM
MgCl2, 420 mM NaCl, 0.2 mM EDTA,
0.5 mM DTT, 0.2 mM phenylmethylsulfonyl
fluoride. After incubating the suspension on ice for 30 min while
stirring, it was centrifuged for 5 min at full speed at 4 °C. The
clear supernatant was then collected, quickly frozen in liquid
nitrogen, and stored at 80 °C until further use. Protein
concentrations were determined using the Bradford assay (Bio-Rad) and
bovine serum albumin standards (Sigma).
Wild Type and Mutant Oligonucleotides Used in EMSA-- The following double-stranded oligonucleotides were synthesized for use in the various EMSA experiments discussed in the text and figure legends. Underlined nucleotides have been changed from the wild type sequences. Only the sense sequences of each pair are listed here: D box, (5'-GACCCAACAGAAAGTAACCCCGGAAATTAGGACACCTCATCCCACAAGA-3'); D1 (5'-GACCCAACAGAAAGTAACCCCGGAAATTAG-3'); D1M1 (5'-GACCCAACAGAAACATTCCCCGGAAATTAG-3'); D1M2 (5'-GACCCAACAGAAAGTAACCCCAAGGATTAG-3'); D2 (5'-CCGGAAATTAGGACACCTCATCCCACAAGA-3'); D2M1 (5'-CCGGAAATTATTCAACCTCATCCCACAAGA-3'); D2M2 (5'-CCGGAAATTAGGACACCTCAGAGGACAAGA-3'); TA box (5'-CAAGACCTTTAAATAGGGGAAGTCCACTTG-3'); TAM1 (5'-CAAGACCTTTCTAGAGGGGAAGTCCACTTG-3'); TAM2 (5'-CAAGACCTTTAAATAGGGCCCGTCCACTTG-3'); PU.1 (SV40) (5'-TGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTA-3'); ELK-1 (probe L) (5'-TCCTGATCATCCACCGGAAGAGCTAATG-3'); ETS-1 (5'-GATCTCGAGCAGGAAGTTCGA-3'); TFIID (5'-GCAGAGCATATAAGGTGAGGTAGGA-3'); AP-1 (5'-TTCCGGCTGACTCATCAAGCG-3'); OCT-1 (5'-TGTCGAATGCAAATCACTAGAA-3'). Additional single base pair mutant derivatives of the double-stranded oligonucleotides "D1" and "TA box" have been constructed as discussed in the figure legends and in the text. All oligonucleotides were synthesized by the Sloan-Kettering Microchemistry Core Facility, except for ETS-1, TFIID, AP-1, and OCT-1, which were purchased from Santa Cruz Biotechnology.
Electrophoretic Mobility Shift Assay (EMSA)--
EMSA was
performed as described by Skalnik et al. (49). In brief,
pre-binding of 10-18 µg of the nuclear extract to the poly(dI-dC)
was carried out at 30 °C for 10 min in buffer containing 4%
glycerol, 1 mM MgCl2, 0.5 mM EDTA,
0.5 mM DTT, 25 mM NaCl, 10 mM
Tris-HCl, pH 7.5, and 0.05 mg/ml poly(dI-dC)·poly(dI-dC). In case of
competition experiments, the competing oligonucleotides (5-200-fold
molar excess) were included in this preincubation mixture. Radiolabeled
oligonucleotide probe (3.5 fmol, ~2 × 104 cpm) was
then added to the reaction mixture and incubated at 30 °C for 20 min. One microliter of 10× gel loading buffer, containing 250 mM Tris-HCl, pH 7.5, 0.2% bromphenol blue, 0.2% xylene
cyanol, and 40% glycerol, was then added to the reaction for loading
onto a 4-6% native gel (which was pre-run for 90 min at 100 V in
0.5× nondenaturing TBE buffer) at 15 °C and 125-150 V for about
3 h. The gel was then transferred onto Whatman paper,
vacuum-dried, and exposed to Hyperfilm (Amersham Pharmacia Biotech) for
the desired time at 80 °C and with an intensifier screen. For
antibody "supershift" experiments, antibodies (0.1 µg in 1-µl
volume) were added to the reaction mixtures after the DNA probes had
been incubated with nuclear protein for 20 min at 30 °C, and the
DNA-protein complexes were resolved on 6% polyacrylamide gels, using
0.5× nondenaturing TBE buffer.
Phosphatase Treatment of Nuclear Extract-- Potato acid phosphatase (type VII, Sigma) was diluted in 10 mM sodium acetate, pH 5.2, to give concentrations ranging from 0.01 to 0.1 units/µl. Eighteen µg of nuclear extract (in 1.1-1.4 µl volume) was incubated with 1 µl of phosphatase for 20 min at 30 °C, in the presence of protease inhibitors (20 µg/ml each of aprotinin, leupeptin, pepstatin A, and Sigma trypsin inhibitor) and 10 mM Na3VO4. In negative control experiments, phosphatase was replaced by either phosphate-buffered saline alone or by heat-inactivated phosphatase (10 min at 100 °C). Phosphatase-treated nuclear extracts (3 µl) were added to 6 µl of pre-binding buffer and incubated for 10 min at 30 °C; the entire mixture was then mixed with 1 µl of labeled probe and incubated 20 min at 30 °C (as detailed above under "EMSA").
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RESULTS |
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Defensin-1 Transcriptional Start Site and Promoter Capacity in
HL-60 Cells--
Using the published sequence of human neutrophil
defensin-1 gene (50), two primers were designed for PCR
amplification of a 700-bp fragment, with its 3'-end located about 0.5 kb upstream of exon I. The PCR product was used to screen a human
fibroblast genomic library; positives were reprobed with a labeled
oligonucleotide corresponding to exon I. To determine which of the
clones contained authentic defensin-1 sequences, and not
those of the highly similar defensin-3 gene, we screened for
the presence of a unique MvaI site within the
EcoRI fragment (1170 to +425), a site which is missing
from the corresponding region in the defensin-3 gene. One of
the MvaI-positive clones (def-1, number 17) was selected for
further study as it contained the entire defensin-1 gene, plus about 10 kb of 5'-flanking sequence.
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The 83/
51 Region of the defensin-1 Promoter Contains a Positive
Regulatory Element and Binds an RA-stimulated, Nuclear Phosphoprotein
from HL-60 Cells--
The
552/+82 defensin-1 genomic DNA
fragment then served to generate a series of six 5'-truncation
products, utilizing either suitable restriction sites or exonuclease
III digestion (see "Experimental Procedures"; positions indicated
in Fig. 2), which were all inserted in front of the luciferase reporter
gene (in pGL3-basic vector) to again assess abilities for activating
transcription in vivo. Interestingly, as upstream sequences
were systematically deleted from
552 to
83, promoter activity in
HL-60 cells moderately (by 70%) increased to about 65-fold over the
promoterless control (Fig.
4A). Further truncation of the
5'-end by another 33 bp (to
50/+82) resulted in a more than 10-fold
reproducible reduction of measured luciferase activity. Clearly, an
important positive regulatory element, or elements, must be contained
within the
83/
51 region of the defensin-1 promoter
acting in HL-60 cells. To examine cell specificity of this element, the
same constructs were also transiently transfected into different
myeloid, myeloblastic/erythroblastic (K-562), and lymphoid (Burkitt
B-cell lymphoma) leukemia cell lines and into HeLa carcinoma cells. The
results shown in Fig. 4B indicate that, although the
capacity of the
83/+82 defensin-1 promoter to drive
transcription in vivo is not entirely exclusive to
promyelocytic cells, it certainly is more efficient in HL-60 than in
the other cell lines. Whereas its activity exceeds that of an SV40
promoter (arbitrary 100% activity) in HL-60 cells (250%), it is
consistently less in U-937/KG-1 (both ~80%), K-562 (35%), Burkitt
(30%), and HeLa (25%) cells. It appears therefore that
83/+82
defensin-1 promoter activity is directly correlated to the
extent that cell lineage and differentiation stage resemble the
promyelocytic phenotype; about 3-fold lower activity was measured in
myeloid cells of a lesser (KG-1) or more advanced (U-937) maturation stage, 8-fold lower in B-cells, and about 10-fold lower in non-blood (HeLa) cells. Moreover, defensin transcription is strongly activated during granulocytic differentiation of HL-60 cells but virtually uninducible in any other cell line tested so far (25). Promoter (
83/+82) leakiness, in terms of cell specificity, may have to do with
deletion of upstream negative regulatory elements, as extending the
5'-end of the promoter region construct from position
83 to
552
resulted in a reduction of in vivo activity by more than
5-fold in KG-1 and Burkitt cells but only by half in HL-60 cells.
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A Distal GGAA (62/
59) Sequence in the defensin-1 Promoter
Essential for in Vivo Transcription and Interaction with a Putative ETS
Family Nuclear Phosphoprotein in HL-60 Cells--
To narrow down the
sequences in region
83/
51 that are (i) essential for
transcriptional activation in vivo and (ii) involved in
interaction with nuclear proteins in vitro, the effects of selected mutagenesis were analyzed. We also did not know whether the
two elements were fully separated, overlapping, or identical. A
computer-aided search of the D box sequence for the presence of
transcription factor binding sites, using the MatInspector algorithm
(57), indicated a core consensus sequence (GGA(A/T)) found within the
binding sites of several members of the ETS family of transcription
factors, such as ETS-1, ELK-1, and PU.1 (58-60). Thus, we synthesized
mutant D1 oligonucleotide probes, having 5'-GGAA-3' replaced by AAGG
(labeled D1M2 in Fig. 6A), and
we constructed luciferase reporter plasmids containing a similarly modified
83/+82 defensin-1 promoter region (Fig.
6C). Likewise, two additional mutant oligonucleotide probes,
and associated mutant reporter constructs, were generated. GGAA
(
62/
59) replacement in the D1 probe completely abolished formation
of the upper complex in gel shifts using nuclear extracts of RA-treated
HL-60 cells (Fig. 6B). The exact same mutation resulted in a
12-fold reduction of in vivo transcriptional activity after
transient transfection in untreated cells, the equivalent effect of
deleting the entire D box (
83/
35) region (Fig. 6C).
Replacement of a slightly more 5'-located tetranucleotide (
70/
67),
on the other hand, resulted in the loss of the lower band from the EMSA
pattern, and in a less than 3-fold reduction of luciferase activity
after transfection (Fig. 6, B and C). D box
mutations just downstream from the GGAA sequence in the D2 probe and in
reporter constructs, namely of tetranucleotides (
54/
51), did not
result in attenuation of nuclear factor binding nor of in
vivo transcriptional capacity. In fact, the single, lower band in
EMSA was more intense, and luciferase activity was also slightly
increased as compared with the D2 probe and wild type promoter sequence
controls, respectively (Fig. 6, B and C).
Transient transfection of all the above mentioned constructs in
untreated KG-1 cells resulted in similar trends of decreased, and
increased, normalized luciferase activity. As can also be seen in Fig.
6C, constructs (
34/+82) missing the entire D box, or
containing a promoterless luciferase gene, yielded reproducibly higher
luciferase activities when transfected in KG-1 than in HL-60 cells, for
reasons unknown to us at this time. In sum then, the GGAA (
62/
59)
tetranucleotide sequence in the defensin-1 promoter is
essential for in vivo transcription and in vitro
binding of RA-inducible nuclear factor(s) in HL-60 cells. Because
another GGAA sequence is located between the D box and the first exon of the defensin-1 gene, at position
22/
19, we will refer
to the 5'-most located one as the "distal GGAA."
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Proximal GGAA (22/
19) and TA-rich (
32/
25) Sequences in the
defensin-1 Promoter Implicated in Transcriptional Activity in HL-60
Cells and in Vitro Binding of PU.1--
Even though the ability of the
defensin-1 promoter (
83/+82) to activate transcription in
transiently transfected HL-60 cells was severely impaired when D box
sequences (
83/
35) were deleted, we still measured luciferase
activities on the order of 25% of SV40 promoter-driven transcription
(Fig. 6C). However, the remaining activity was almost
entirely lost upon further deletion of promoter 5'-sequences to
position +11 (Fig. 9B).
Inspection of the
35/+82 region indicated a TA-rich sequence TTTAAATA
(
32/
25), already postulated as a candidate TATA box, and of a
second GGAA sequence (
22/
19), from here on referred to as the
"proximal GGAA." To demonstrate possible functional importance of
the TA box and the proximal GGAA, two separate trinucleotide mutations
were introduced in the
83/+82 promoter, and the changes of in
vivo transcriptional activity was assessed. The combination of
three point mutations (A
29
C, A
28
T, and T
26
G) caused a 5-fold reduction in promoter
activity, and changing of GAA (
21/
19) to CCC resulted in a 2.5-fold
decrease. Significantly, when assessed in combination with a mutated
distal "GGAA" site (see Figs. 6C and 9B), the
effect of the proximal AAAT
CTAG modification
was a total loss (>50-fold reduction; equal to negative control) of
in vivo promoter function in HL-60 cells (Fig.
9B).
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DISCUSSION |
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Cell differentiation involves the orderly expression of numerous genes. Among the transcription factors involved are those governing differentiation itself and others regulating promoters of genes specific for the functions of mature cells. Defensin antibiotics are such key components in neutrophils. In fact, human HNP-type defensins are found exclusively in these cells. Contrary to the insect immune system (65), HNP defensin synthesis is not noticeably induced by microbial challenge but, instead, occurs in maturing bone marrow cells. Its expression is therefore uniquely restricted; it is a genuine marker for neutrophilic lineage and for differentiation stage. As acute myelogenous leukemia cells derive from a block in differentiation, they provide a freeze-frame to analyze molecular events at a given stage. Low level defensin transcription occurs in HL-60 promyelocytic cells but is greatly activated upon (i) drug-induced granulocytic differentiation and (ii) treatment with retinoic acid at doses too low to bring about morphological changes (25). The underlying molecular mechanisms of the basal expression and of the activation processes are unknown. Thus, we sought to analyze the molecular control of defensin transcription and how it is affected by chemotherapeutic drugs. This information may also provide novel insights into the pathogenesis of promyelocytic leukemia and may lead to a better mechanistic understanding of chemotherapeutic interventions as well.
Here, we show the capacity of extended defensin-1 promoter
regions, located at 552/+82, to drive gene expression in
vivo in a quasi cell-specific manner; promoter activity in
promyelocytic (HL-60) cells is 5-30-fold higher than in related
monoblastic (U-937) and, respectively, myeloblastic (KG-1) cell lines.
Truncation from position
552 onward to a minimal promoter, located at
83/+82, results in enhanced transcriptional activity in HL-60 cells
and even more so in other cells, suggesting deletion of negative
regulatory elements that may contribute to cell-specific expression in
a regular chromosomal context. The existence of possible repressors binding to those elements was not studied further here. Instead, we
provide data that demonstrate the presence (and near location) of two
positive regulatory elements within the 83-base pair minimal region
required for defensin-1 expression in HL-60 cells.
Functional significance is implicit from the fact that disruption of
the proximal or distal sites results in, respectively, a 5- or 10-fold loss of basal promoter activity; more than that, the double knock-out entirely abolishes all function.
The proximal control element contains a TA-rich sequence TTTAAATA
(32/
25) that, by established criteria of weighted consensus sequence and location (54), fulfills all requirements of a vertebrate "TATA box." A trinucleotide mutation
(TTTCTAGA), created within this limited sequence,
had a profound negative effect on minimal (
83/+82) promoter activity
in promyelocytic cells, consistent with the predicted functional role.
However, an oligonucleotide containing a bona fide TATA box sequence
failed to compete for binding of HL-60 nuclear proteins to an EMSA
probe comprising this TA-rich regulatory sequence ("TA probe";
39/
10), raising doubts about the precise role in
defensin-1 expression. Two sets of complexes formed with
proteins from HL-60 and U-937 cells. The faster migrating "ladder"
complex almost certainly contains transcription factor PU.1, binding to
the GGAA core sequence (
22/
19) just downstream from the TA site;
its banding pattern in mature myeloid cells is the result of
differential phosphorylation or represents degradation products (69).
Since PU.1 expression is limited to myeloid and B-cells (31, 33, 61,
66, 67), it explains the absence of this complex from HeLa cells; early myeloid KG-1 cells could be either too immature to express PU.1 or may
have other disregulated expression or activity, perhaps a reason for
failure to differentiate (68). The PU.1-binding site is functionally
active as well, since its disruption resulted in reduced promoter
efficacy in vivo. It appears therefore that defensin-1 is yet another myeloid gene target regulated, at
least in part, by PU.1 (31). This brings into perspective the
possibility that defensin-1 could indeed have a TATA-less
myeloid promoter, relying instead on the proven capacity of nearby
bound PU.1, and possible associated proteins, to tether TBP and TFIID
and assemble a basal transcription factor complex within reasonable
distance from the cap site (70, 71). If indeed the case, it still
cannot alone account for granulocytic specificity of
defensin-1 expression.
Additional control is most likely exerted by a more distal element,
also containing the ETS family GGAA signature binding site (62/
59;
see Fig. 2). A mere switch of the sequence within this tetranucleotide
(to AAGG) caused a substantial loss of minimal (
83/+82) promoter
activity in vivo, the same reduction, in fact, that resulted
from truncating the 5'-end by 53 bp (to
30/+82) and arguing for major
functional significance. We have termed this distal positive regulatory
site REDE (regulatory element of
defensin expression). A minor EMSA complex
formed with an REDE-containing oligonucleotide (D1 or REDE probe) but
markedly gained in intensity after HL-60 cells had been treated with RA
or HMBA for at least 2 days, indicating differentiation stimulated
binding activity, largely the result of phosphorylation. The GG
AA
nucleotide exchange, when introduced into this probe, abrogated all
binding of HL-60 nuclear protein in vitro. A second, faster
migrating band was sometimes observed and may represent the
unphosphorylated (or underphosphorylated) form of the major REDE
binding activity. The phosphorylation-dependent interaction
with specific nucleic acid sequences has broader implications for
possible factor identity since several ETS family members require
RAS-dependent phosphorylation to reach optimal
transactivation potential (72, 73), whereas others are negatively
regulated in this manner (74, 75). Our data convincingly rule out that
either PU.1 or ETS-1 interact with the wild type REDE probe, and we
speculate, on theoretical grounds, that ELK-1 is absent from the
complex as well. Indeed, no serum response element can be located
within reasonable distance from REDE, suggesting that if ELK-1 binds to
REDE it must do so autonomously and not in its typical capacity of
ternary complex factor (59, 64, 76).
This leaves two key questions: (i) what is the precise identity of this activity, and (ii) how does it get stimulated during granulocytic differentiation? Although the first question can be approached experimentally by analysis of all known ETS family members for REDE binding and for abilities to drive transcription of reporter constructs in non-myeloid cells (after co-ectopic expression), a more direct and unbiased way is by purification and structural chemical analysis. Until then, we will refer to this activity as IRD (increased REDE binding during differentiation). We know for sure that IRD is different from the protein incorporated in the low mobility EMSA complex that forms with the proximal TA probe, for that band is obtained with extracts from untreated HL-60 cells and cannot be competed with an excess of cold REDE probe. Nuclear proteins from one lymphoid and a few myeloid cell lines formed identical complexes with the same REDE probe in the absence of drug treatment; exposure to RA did not significantly alter those patterns. For lack of IRD-specific reagents, we cannot yet determine whether the same activity is constitutively present in all those cells. However, several lines of evidence indicate that this may actually not be the case. Contrary to the heretofore implicated HL-60 nuclear phosphoproteins, binding activities from the other cells could not be readily eliminated by phosphatase treatments. Furthermore, various band shift patterns resulting from point mutational analyses of the REDE probe indicated subtle differences in preferred nucleic acid-binding sites between proteins from myelocytic and other cells. Assuming that IRD has transactivating ability in HL-60 cells, it could be that those REDE-binding proteins from other cell types are inactive forms of IRD, for reasons of differential modifications and/or conformational changes (not affecting DNA binding). Alternatively, repressors recognizing the REDE sequence may bind to the probe in vitro and perhaps antagonize putative activity of IRD in vivo by occupying the recognition site in the defensin promoter. In previous reports, ETS repressor factors have already been shown to specifically suppress ETS transcription factor associated activities (77). Yet another possibility is the in vivo incorporation of a suppressor into a bigger complex, also containing bona fide IRD, that might prove unstable during gel shift analysis. And finally, a functional IRD-REDE combination may still fail to drive transcription without a requisite co-regulator, most likely PU.1, bound to the proximal regulatory site, an argument applicable to KG-1 cells for instance.
Regarding defensin and IRD induction by RA or HMBA in
promyelocytes, we established a direct correlation over a 3-day period between (i) transcriptional rates and steady state mRNA levels in vivo (25) and (ii) density of IRD binding to the REDE
probe in vitro (this study), suggesting a scenario in which
the appearance of nuclear IRD might be the final stage of drug-induced
signaling toward defensin-1 transcriptional activation, most
likely involving activation of a protein kinase. This hypothesis is
consistent with an earlier view renouncing granulocytic expression of
this gene as the simple consequence of a direct, ligand-mediated RA receptor (RAR) binding to specific elements in its control region but
instead predicting a role for RAR more upstream in the pathway (25).
Retinoids must also partake to some extent in basal transcription, for
the minimal promoter functions 5-fold less effectively2 in
an HL-60 cell subclone, HL-60R, that harbors a dominant-negative RAR
mutation and is completely resistant to RA- and
HMBA-dependent induction of both differentiation and
defensin transcription (25, 78). As expected, basal IRD activity was
not RA-inducible in these mutant cells either (data not shown).
In summary, defensin-1 expression in promyelocytic leukemia cells is very likely controlled by a combination of at least two nuclear proteins, acting through two functional, ETS-like elements in the minimal promoter. The proximal element binds PU.1 in vitro, and the distal one contains a binding site for a differentiation-stimulated, possibly novel phosphoprotein IRD. Identification and molecular characterization of IRD will be crucial to resolve the molecular mechanisms of defensin-1 activation during drug-induced differentiation and perhaps also during normal granulopoiesis. Protein structural chemistry and mass spectrometric analysis will likely feature prominent roles in achieving the first of these goals.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Daniel Tenen and members of his laboratory (Harvard Medical School, Boston) for expert advice on transient transfection of myeloid cells and for the gift of CMV-hGH expression plasmids; to Dr. Susanne Herwig for sharing unpublished data at the onset of these studies; and to John Philip for help with the figures.
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FOOTNOTES |
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* This work was supported in part by National Cancer Institute Research Fellowship F32 CA71163 (to Y. M.), and the Hirschl Trust (to P. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Memorial
Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Fax: 212-717-3604; E-mail: p-tempst{at}mskcc.org.
1 The abbreviations used are: HNP, human neutrophil peptide; bp, base pair(s); CMV, cytomegalovirus; Me2SO, dimethyl sulfoxide; EMSA, electrophoretic mobility shift assay; hGH, human growth hormone; HMBA, hexamethylene bisacetamide; IRD, increased REDE-binding during differentiation; kb, kilobase(s); RA, retinoic acid; RAR, retinoic acid receptor; REDE, regulatory element of defensin expression; RLU, relative light unit(s); PCR, polymerase chain reaction; SV, simian virus; TBP, TATA-binding protein; DTT, dithiothreitol; RT, room temperature; FCS, fetal calf serum.
2 Y. Ma and P. Tempst, unpublished observations.
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REFERENCES |
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