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.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

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").

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

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.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Mapping of defensin-1 transcriptional start site. A, schematic map of the defensin-1 gene indicating genomic organization. Three exons are depicted as boxes, and the shaded areas represent the coding region, labeled ORF; two introns, with respective sizes (bp), are also shown. The arrow on the top of the first exon indicates the transcriptional start site; the primers used for the extension assays are indicated by half arrows under the corresponding positions of the exons. Note that the sizes are not drawn proportionally. B, primer extension analysis with two different, exon-specific primers (1 and 2), annealed to 14 µg of HL-60 cell total RNA or to yeast RNA (0). The size and the position of end-labeled markers is shown at the left; the arrows at the right indicate the position of the extended products (120 and 65 bp). For more details, see under "Experimental Procedures."

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Exon I and upstream sequences of the defensin-1 gene. The first exon and part of the second exon (gray shaded areas) are shown; the first intron is depicted by lowercase letters and a (not to scale) dotted line. The numbering is relative to the site of transcriptional initiation (+1). Restriction sites and exonuclease III truncation positions are indicated. The D box (see text) is boxed; positions of distal and proximal ETS-like sites are indicated by large black dots underneath, and the TA-rich sequence is underlined by a heavy black bar. The coding region and corresponding translation product in the three-letter code below it are shown as well. Locations of two antisense primers used for the primer extension assays are marked by arrows.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Cell-specific activity of the extended defensin-1 promoter in vivo. A, the 5'-flanking region (solid bar), first exon (stippled box) and 6 bp of the first intron (thin line) of defensin-1 have been inserted in front of the luciferase reporter gene (Luc). The transcriptional initiation site is indicated by an arrow. The ScaI restriction sites and the corresponding sizes are marked. B, the cell lines (HL-60 promyelocytic, KG-1 myeloblastic, and U-937 monoblastic) and the plasmid constructs, containing gradually 5'-truncated sequences (from 10 kb to 552; 3'-end fixed at +82), used in transient transfection assays are shown on the left; the results of the luciferase expression analyses are shown on the right. Promoter activity, measured as relative light units (RLU), is normalized for transfection efficiency by co-expressing human growth hormone (hGH), driven by a constant CMV promoter and measured by enzyme-linked immunosorbent assay. The results, the mean of at least three experiments, are expressed as RLU per ng hGH and are shown here as percent of luciferase expression driven by an SV40 early promoter (arbitrarily assigned 100% and also normalized by CMV-driven hGH co-expression) in the same cells. 0-Luc indicates promoterless luciferase gene. Further details can be found under "Experimental Procedures" and in the text.

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.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   A minimal defensin-1 promoter confers quasi-specific promyelocytic expression in vivo. A, transient transfection assays for defensin-1 promoter activity in HL-60 promyelocytic cells, and using gradually 5'-truncated sequences (from 552 to 30; 3'-end fixed at +82) inserted in front of the luciferase (Luc) gene. The results are the mean of at least three experiments, carried out as described in Fig. 3 and under "Experimental Procedures." RLU from SV40 promoter-driven expression was normalized per ng of secreted hGH, co-expressed under CMV promoter control, and arbitrarily assigned a value of 100%. 0-Luc indicates a promoterless luciferase gene. B, several of the above constructs were then also transiently transfected in U-937 monoblastic, KG-1 myeloblastic, K-562 erythroleukemic, Burkitt lymphoma (B-cell) and HeLa carcinoma (non-blood cell control) cells; and luciferase expression (per ng of hGH) was monitored as described in A.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Differentiation-stimulated, nuclear phosphoprotein from HL-60 cells binds D1 probe (83/54 of defensin-1 gene) in vitro. A, schematic representation of two overlapping, double-stranded oligonucleotide (D1 and D2) probes (solid bars) and their relative positions within the D box (83 to 35) of the defensin-1 promoter sequence. The sequences are listed under "Experimental Procedures." B, D1 and D2 were utilized as labeled probes in EMSA experiments, using nuclear extracts from HL-60 cells that had either not () been treated or treated (+) for 72 h with 1 µM retinoic acid (RA). Self-competition with a 200× excess of unlabeled oligonucleotide is indicated in the "competitor" row by +. Oligonucleotide probes containing AP1 and OCT-1 transcription factor binding sites were used as controls. Full experimental details are to be found under "Experimental Procedures." C, EMSA with the D1 probe as described above, using nuclear extracts from HL-60 cells treated for various periods (0-72 h) with 1 µM RA; or for 72 h with either HMBA or Me2SO. D, EMSA with the D1 probe, using nuclear extracts from 1 µM RA-treated (72 h) HL-60 cells. Extracts were either used directly or after further treatment with 0.08 units (U) phosphatase (PPtase; potato acid phosphatase type VII) in the presence of protease inhibitors and phosphotyrosine phosphatase inhibitor. Heat-denatured phosphatase (PPtase + heat) was used as a negative control for enzymatic activity.

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."

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Distal GGAA (62/59) sequence in the defensin-1 promoter, essential for interaction with HL-60 nuclear factor in vitro and for transcriptional activation in vivo. A, position and sequences of the D box (83/35), D1 and D2 oligonucleotides, and of the various mutant analogs are listed. Nucleotide substitutions, introduced in oligonucleotide probes (B) and in the reporter constructs (C), are specifically indicated. B, D1, D2, and corresponding mutant oligonucleotide analogs were utilized as labeled probes in EMSA experiments with nuclear extract of 1 µM RA-treated (72 h) HL-60 promyelocytic cells. Details are under "Experimental Procedures." C, transient transfection assays for defensin-1 minimal (83/+82) promoter activity in promyelocytic HL-60 and myeloblastic KG-1 cells. The wild type (shown above the defensin-1 schematic drawing; top of panel) and variously mutated (shown underneath) sequences were inserted in front of the luciferase (Luc) gene. The results are the mean of at least three experiments, carried out as described in Fig. 3 and under "Experimental Procedures." RLU from SV40 promoter-driven expression was normalized per ng of secreted hGH, co-expressed under CMV-promoter control, and arbitrarily assigned a value of 100%. Luc. indicates a promoterless luciferase gene.

View larger version (54K): [in this window] [in a new window]   Fig. 7.   HL-60 nuclear factor binding to defensin-1 D1 probe (83/54) in vitro is distinct from the PU.1 and ETS-1/PEA3 transcription factors. A, EMSA experiments with a defensin-1 D1 oligonucleotide probe (D1, lanes 1-10) and a probe containing a characterized PU.1-binding site from the SV40 enhancer (PU1, lanes 11-20) (taken from Refs. 60 and 62), incubated with nuclear extract (NE) from 1 µM RA-treated (72 h) HL-60 promyelocytic cells. Probe sequences and all further details are under "Experimental Procedures." Binding of nuclear protein to labeled D1 and PU1 probes in the absence of competitors is shown in lanes 1 and 11, respectively. Self-competitions, by preincubation with increasing amounts (5, 50, 100, and 200 × molar excess) of the unlabeled oligonucleotides, are indicated by heavy black triangles above lanes 2-5 for D1 and lanes 12-15 for the PU1 probe. Cross-competitions with increasing amounts (5, 50, 10, and 200 × excess) of the reciprocal, unlabeled oligonucleotide are similarly indicated by the triangles, as shown above lanes 6-9 for the D1 probe (PU1 competition) and lanes 16-19 for the PU.1 probe (D1 competition). F (lanes 10 and 20) indicates free probe (no nuclear extract). B, EMSA supershift experiments, with (+) or without () specific anti-PU.1 antibody (PU1 Ab) present in the D1 and PU1 binding reactions to nuclear protein (NE) from RA-treated (72 h) HL-60 cells. The lower arrow indicates the position of the regular PU.1-specific complex (or lack thereof), and the upper arrow shows the supershifted band. C, EMSA competition experiments whereby 200-fold excess of unlabeled oligonucleotides D1 or ETS-1/PEA3 (oligonucleotide containing a characterized ETS-1/PEA3 binding site; taken from Ref. 63) were preincubated prior to the binding reactions of D1 probe with HL-60 nuclear protein. The minus () sign indicates the absence of competitor oligonucleotide. Comparative EMSAs using labeled D1 versus ETS-1 probes in binding reactions with HL-60 nuclear protein are shown in the panel on the right.

View larger version (45K): [in this window] [in a new window]   Fig. 8.   Nuclear factor(s) from HL-60 and other myeloid blood cells binding to defensin-1 D1 probe (83/54) in vitro. A, EMSA with the D1 probe as described under Fig. 5, using nuclear extracts from KG-1 (KG), K-562 (K5), U-937 (U9), Burkitt lymphoma (Bu), or HeLa (He) cells, either treated or not treated with 1 µM RA (+/), and in the presence or absence of a 200-fold excess, self-competing D1 oligonucleotide (+/). B, EMSA with the D1 probe using nuclear extracts (NEs) from untreated (no RA) KG-1, K-562, U-937, Burkitt lymphoma, or HeLa cells; nuclear extracts were either treated or not treated with potato acid phosphatase (PPtase) as described under Fig. 5 and under "Experimental Procedures." C, EMSA with a defensin-1 D1 oligonucleotide probe, and single point mutation (at positions 65 to 56) derivatives thereof (total of 10 mutant probes), incubated with nuclear extract from 1 µM RA-treated (72 h) HL-60 promyelocytic cells or untreated KG-1 myeloblastic cells. The specific nucleotide exchanges, each one characteristic for a particular mutant probe, are indicated (wild type  changed to).

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 (A²⁹  C, A²⁸  T, and T²⁶  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).

View larger version (9K): [in this window] [in a new window]   Fig. 9.   Proximal TA-rich (32/25) and GGAA (22/19) sequences in the defensin-1 promoter are functionally important for promyelocytic expression in vivo. Transient transfection assays for defensin-1 minimal (83/+82) promoter activity in HL-60 promyelocytic cells. The wild type and variously mutated (singly or doubly; shown below the wild type sequence in A) promoter sequences were inserted in front of the luciferase (Luc) gene. The transcriptional start site is indicated by an arrow, and the box represents the first exon, as labeled (A). The left side portion of B represents the plasmid constructs used in the transfection assays; the solid bar represents the wild type sequence, and the empty areas represent the triple or quadruple base substitutions. The boxes indicate the first exon of defensin-1, and those open on the right represent the reporter gene, labeled Luc. The constructs labeled 34 and +11 contain defensin-1 promoter sequences located at positions 34/+82 and +11/+82 (missing 10 bp of exon 1), respectively. The results are the mean of at least three experiments, carried out as described in Fig. 3 and under "Experimental Procedures." RLU from SV40 promoter-driven expression was normalized per ng of secreted hGH, co-expressed under CMV promoter control, and arbitrarily assigned a value of 100%.

View larger version (62K): [in this window] [in a new window]   Fig. 10.   Proximal TA-rich/GGAA sequences (at position 39 to 10) of the defensin-1 promoter interact with HL-60 nuclear PU.1 and other factors in vitro. A, position and sequences of the TA probe (39/10) and of two mutant analogs. Nucleotide substitutions, introduced in oligonucleotide probes TAM1 (=M1) and TAM2 (=M2), are specifically indicated. B, EMSA experiments using a defensin-1 TA oligonucleotide probe (TA), and probes containing three point mutations each, incubated with nuclear extract (NE) from untreated HL-60 promyelocytic cells. Details are under "Experimental Procedures." Competition experiments, by preincubation of proteins with a 200-fold molar excess of various unlabeled oligonucleotides, are also shown; as well as an EMSA supershift experiment, whereby specific anti-PU.1 antibody (PU.1 Ab) was present (+) in the binding reaction of nuclear protein to the TA probe. Oligonucleotides termed D1, PU1, ELK, and ETS have been described in Figs. 5, 7, and 8 and in the text; the sequence of oligonucleotide TFIID, containing a bona fide TATA box, is given under "Experimental Procedures." C, EMSA with labeled TA (see A) or PU.1 (see Fig. 7) probes, using nuclear extracts from untreated (no RA) KG-1, K-562, U-937, and Burkitt lymphoma cells. D, EMSA with a defensin-1 TA oligonucleotide probe and single point mutation (at positions 32 to 18) derivatives thereof (total of 15 mutant probes), incubated with nuclear extract from untreated HL-60 promyelocytic or KG-1 myeloblastic cells. The specific nucleotide exchanges, each one characteristic for a particular mutant probe, are indicated (wild type  changed to).