©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a gp91-phox Promoter Element That Is Required for Interferon -induced Transcription (*)

(Received for publication, October 6, 1994; and in revised form, January 19, 1995)

Elizabeth A. Eklund (§) David G. Skalnik (¶)

From the Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202-5225

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cytochrome b heavy chain (gp91-phox) is expressed nearly exclusively in terminally differentiating myelomonocytic cells, thereby providing a model to study the events of late myeloid differentiation. We describe a tissue culture assay for studying interferon induction of gp91-phox expression and a cis-element in the gp91-phox promoter that is necessary but not sufficient for this activity. In vitro assays reveal two DNA-binding proteins that interact with this cis-element. One factor is restricted to hematopoietic cells, is required for an interferon gamma response, and binds to an element similar to the Ets protein family consensus, although it does not correspond to known family members. The second factor is the ubiquitous CCAAT-binding protein CP1, which is dispensable for an interferon response. Single base pair mutations in the gp91-phox promoter that specifically abolish the binding of the hematopoietic-associated factor have previously been identified in chronic granulomatous disease patients (Newburger, P. E., Skalnik, D. G., Hopkins, P. J., Eklund, E. A., and Curnutte, J. T.(1994) J. Clin. Invest. 94, 1205-1211). The data reported here directly demonstrate the functional significance of the hematopoietic-associated factor for gp91-phox promoter activity and reveal the binding properties and tissue distribution of this novel DNA-binding protein.


INTRODUCTION

Pluripotent bone marrow stem cells can differentiate into members of various hematopoietic lineages. This process includes tissue-specific transcriptional regulation of a complement of genes that characterizes each lineage. Granulocytes and monocyte/macrophages comprise the category of mature, terminally differentiated phagocytic cells and are characterized by the ability to generate toxic free-radicals via the respiratory burst. Respiratory burst activity requires a group of signaling components and the catalytic unit, the NADPH-dependent oxidase(1, 2, 3) . The expression of some of the components of the respiratory burst apparatus is confined to terminally differentiated phagocytic cells. The phagocyte-specific cytosolic components p47-phox(4) , p67-phox(5) , and the heavy chain of the membrane bound cytochrome b (gp91-phox) (6) are transcriptionally inactive until myeloid maturation progresses beyond the promyelocyte stage and then are actively transcribed until cell death. In addition, it has been observed that gp91-phox expression increases in response to interferon (IFN-)(^1)(7) . Absence of these gene products leads to chronic granulomatous disease (CGD), a disorder of host defense(8) . The transcriptional regulation of these myeloid-specific genes provides a model for the events of terminal myeloid differentiation.

Recent work has provided insight into the tissue-specific expression of the gp91-phox gene(9, 10) . We have previously demonstrated that 450 base pairs (bp) of the 5`-flank of the human gp91-phox gene is adequate to direct expression of a reporter gene specifically in a subset of monocyte/macrophages in transgenic mice(10) . These data suggest that important regulatory elements that control lineage and differentiation-specific expression of gp91-phox are present within the proximal 450 bp of the gp91-phox promoter. Similar tissue distribution of transgene transcription is observed with constructs containing up to 2.6 kilobase pairs of the proximal gp91-phox promoter(10) . The absence of detectable reporter gene expression in the majority of myelomonocytic cells suggests that additional regulatory elements are required to direct appropriate transcription in the full complement of phagocytic cells.

The study of two kindreds with a novel form of X-linked CGD also suggests the existence of important cis-elements in the proximal promoter region of the gp91-phox gene. gp91-phox protein is absent in the majority of phagocytic cells, although a subset of phagocytes express gp91-phox normally in this variant form of the disease(11) . Both of these kindreds carry a point mutation in the proximal promoter region of the gp91-phox gene(12) . The point mutations in these kindreds are at -55 and -57 bp relative to the published transcription start site(13) , and each specifically disrupts an interaction between the gp91-phox promoter and a DNA-binding protein (12) .

In this study, we have examined in more detail the proximal gp91-phox promoter in an attempt to identify cis-elements and the DNA-binding proteins that interact with these elements, which mediate tissue-specific expression of gp91-phox in myelomonocytic cells. Previous studies relied on transgenic mice for functional analysis of gp91-phox promoter fragments. While this technique has the advantage of permitting promoter characterization in an authentic in vivo setting, the time and expense of this approach are disadvantages. We report here that IFN- induction of the gp91-phox gene can be studied using stable transfectants of the myeloid cell line PLB985(14) , thus providing a more rapid and convenient assay system with which to study this aspect of gp91-phox promoter function.

The studies reported here reveal a cis-element in the proximal gp91-phox promoter that is necessary but not sufficient for IFN- induction of transcription and that co-localizes with promoter mutations identified in CGD patients. The -50 to -57 region of the gp91-phox promoter (5`-GAGGAAAT-3`) contains on the lower strand a sequence similar to the consensus binding sequence for the Ets protein family. The core motif for binding of Ets family members is 5`-GGA(A/T)-3`, with binding specificity of the various family members conferred by additional flanking sequences(15) . Various Ets factors have recently been demonstrated to be essential for the transcription of myeloid and lymphoid-specific genes, including major histocompatibility class II I-Abeta(16) , CD18(17) , perforin(18) , macrophage scavenger receptor(19) , and the immunoglobulin heavy chain genes(20) . In vitro assays reveal that a DNA-binding protein required for normal gp91-phox promoter function is largely restricted to cells of hematopoietic origin, shares an overlapping binding site with a ubiquitous CCAAT-binding protein, and exhibits a binding specificity distinct from previously described members of the Ets protein family.


MATERIALS AND METHODS

Plasmid Construction and Site-directed Mutagenesis

Cloning of the 450-bp proximal gp91-phox promoter fragment into a plasmid containing the genomic sequence for the human growth hormone (hGH) reporter gene has been previously described(9) . Site-directed mutagenesis was performed using a mutant oligonucleotide-directed polymerase chain reaction technique as described by Vallette(21) . Oligonucleotides were synthesized on an Applied Biosystems model 394 DNA synthesizer. The 450-bp fragment of the proximal gp91-phox promoter, cloned into pUC-19, was used as a template. Mutations were introduced by complementary synthetic oligonucleotide primers containing mutations of the promoter flanked by 20 nonmutated bases. The mutant oligonucleotide primers were used as the ``right'' and ``left'' inside primers for the first step reactions, the right and left outside primers were the universal sequencing primers for M13 DNA. Reaction products were isolated on native polyacrylamide gels in 90 mM Tris-HCl (pH 8.3), 90 mM borate, 2.5 mM EDTA (22) prior to the second-step polymerase chain reaction utilizing the two outside primers. Products of the second-step polymerase chain reaction were isolated on an agarose gel and subcloned into a plasmid vector. The nucleotide sequence of mutated 450-bp promoter fragments was determined on both strands using the Sequenase system (U. S. Biochemical Corp.).

Mutated gp91-phox promoter sequences were sub-cloned into the BamHI and XbaI sites of the hGH reporter gene plasmid vector (Nichols Institute, San Juan, Capistrano). A neomycin resistance gene driven by the thymidine kinase promoter was inserted into the SalI site (TKneo cassette generously provided by Stuart Orkin, Boston Children's Hospital) in both parallel and anti-parallel orientation relative to the reporter gene. The orientation of the selectable marker did not affect reporter gene activity (data not shown).

Plasmid for generating a riboprobe specific for the gp91-phox promoter-hGH reporter transgene mRNA was constructed by subcloning double-stranded synthetic oligonucleotides into the Bluescript KS (Clontech, Palo Alto, California) transcription vector. Complementary oligonucleotides were synthesized that contain the proximal 38 bases of the gp91-phox promoter and the first 33 bases of hGH sequence. The oligonucleotides were annealed and cloned into the HindIII and PstI sites of Bluescript KS. The chicken -actin probe was a generous gift of Celeste Simon (University of Chicago).

Oligonucleotides

Oligonucleotides used as double stranded competitors and probes are as follows: gp91-phox wild-type -30 to -68 bp (9) (5`-dctgctgttttcatttcctcattggaagaagaagcatagt-3`); gp91-phox CGD mutation (12) (5`-dctgctgttttccttttcctcattggaagaagaagcatatgt-3`); gp91-phox CCAAT-box mutation (5`-dctgctgttttcatttcctcaccggaagaagaagcatatgt-3`); C/EBP (23) (5`-daatgtcagttagggtgtggaaagtccccaggct-3`); Ets-1 (24) (5`-dttccagaggatgtggcttctgcgggagagctt-3`); PEA3 (25) (5`-dtcgagcaggaagttcga-3`); Elf-1 (IL-2) (26) (5`-dagaaaggaggaaaaactgtttcatacagaaggc-3`); Elf-1 (HIV-2) (27) (5`-dttaaagacaggaacagctat-3`); Elf-1 (CD4) (28) (5`-daaacaggaagtcctgccccc-3`); E74 (15) (5`-daataaccggaagtaactc-3`); GABP (29) (5`-dcggaacggaagcggaaacc-3`); PU.1 (16) (5`-dtgaaataacctctgaaagaggaacttggttaggta-3`); NF-1 (30) (5`-dtttggattgaagccaatatgataatgccctac-3`).

Cell Culture and Stable Transfections

All cell lines used in this study were of human origin. The promyelocytic cell line HL60 (31) , erythroleukemia cell line HEL(32) , chronic myelogenous leukemia cell line K562(33) , epithelial carcinoma cell line HeLa(34) , hepatoma cell line HepG2(35) , and T-cell leukemia line MOLT-4 (36) were all obtained from the ATCC (Rockville, MD). The myelomonoblastic cell line PLB985 (14) was generously provided by Thomas Rado (University of Alabama, Birmingham). The B-cell leukemia cell line CESS (37) was provided by Yu Chung Yang (Indiana University, Indianapolis). All cell lines were grown at 37 °C and 5% CO(2). With the exception of HepG2 and MOLT-4, all lines were carried in RPMI 1640 supplemented to 10% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 0.2 mM glutamine. HepG2 and MOLT-4 cell lines were grown in alpha-minimal Eagle's medium with supplements as above.

Myeloid cell lines were differentiated by treatment with 0.1 µM phorbol 12-myristate 13-acetate (PMA) (Sigma) for 48 h (macrophage differentiation), with 1 µM all-trans-retinoic acid (Sigma) for 24 h followed by 60 mM dimethylformamide for an additional 24 h (granulocyte differentiation), or treated with 500 units/ml human recombinant IFN- (Boehringer Mannheim) for 48 h. Stable transfectants carrying the neomycin resistance cassette were treated with 1000 units/ml human IFN- for 6 days with the cells kept continuously in log phase.

Myeloid cell lines were transfected by electroporation using a Bio-Rad gene pulser (Bio-Rad). PLB985 cells growing in log phase were resuspended at a density of 4 times 10^7 cells/ml in RPMI 1640 supplemented as above plus 20% fetal bovine serum. Fifty µg of circular plasmid DNA containing a gp91-phox promoter-hGH reporter gene construct and the TKneo cassette was added to 800 µl of cells in an electroporation cuvette and pulsed at 0.24 mV and 960 microfarads. The cells were incubated for 20 min on ice, expanded into 10 ml of RPMI 1640 supplemented as above plus 15% fetal bovine serum, and incubated overnight at 37 °C and 5% CO(2). The cells were selected for G418 resistance in the usual RPMI 1640 media supplemented with geneticin at an initial concentration of 0.5 mg of active drug/ml, increasing to 1.5 mg/ml over 2 weeks to select for high transgene copy number.

Stable transfection was confirmed by Southern blot analysis. Genomic DNA was isolated from 5-8 times 10^5 tissue culture cells using the Elu-Quick system (Schleicher and Scheull) and probed for both the TKneo cassette and the promoter-reporter gene construct using formamide hybridization and random primer labeled probes(22) . Probes were labeled with 50 µCi of [alpha-P]dCTP (10 mCi/ml, 3000 Ci/mmol, DuPont NEN) per 100 ng of DNA template.

Stably transfected cells treated with IFN- were compared with nontreated cells for expression of hGH. Stable pools were tested with IFN- stimulation over increasing time periods to determine an optimal time for stimulation of log phase cells. The IFN- response was maximal after 6 days of stimulation (data not shown). Titration of the dose of IFN- demonstrated a maximal effect (with a variety of lots) at 1000-1200 units/ml (data not shown). Multiple independent pools of cells were analyzed for each promoter construct.

Assays for hGH were performed using a radio-immunoassay system from Nichols Laboratories (San Juan Capestrano, CA). Results of hGH assays were normalized for the number of trypan blue-excluding cells/ml of culture.

In Vitro DNA-binding Protein Assays

Nuclear extracts were prepared from tissue culture cells growing in log phase by the method of Dignam (38) in the presence of 0.5 mM dithiothreitol, 0.5 mM phenylmethysulfonyl fluoride, 0.1 mM leupeptin, 5 µg/ml aprotinin, and 5 µg/ml pepstatin. Fli-1 protein was produced by in vitro transcription/translation using the Riboprobe Gemini System and the Rabbit Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's instructions. The plasmid for producing Fli-1 protein was kindly provided by Mike Klemsz (Indiana University, Indianapolis, IN). Protein assays were performed by the method of Lowry (39) using a Bio-Rad Protein Assay kit I (Bio-Rad, Hercules, CA). Complementary double-stranded oligonucleotides were routinely subcloned into plasmids. DNA probes were produced by end labeling restriction digest fragments with polynucleotide kinase (New England Biolabs Inc., Beverly, MA) and 50 µCi of [-P]ATP (3000 Ci/mmol, 10 mCi/ml, DuPont NEN) per µg of plasmid DNA (about 1 pmol). Probes were isolated on a native polyacrylamide gel and recovered by ethanol precipitation. For DNase I protection assays and methylation interference assays, individual probes were synthesized for each strand.

Electrophoretic mobility shift assays (40) (EMSA) were performed in a 20-µl total volume with 2-4 µg of nuclear extract protein or 0.5-1 µl of rabbit reticulocyte lysate in a solution containing 10 mM Tris-HCl (pH 7.5), 50 mM potassium glutamate, 5 mM magnesium chloride, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 50 µg/ml poly(dIbulletdC) (Sigma). Nuclear extracts were preincubated for 15 min on ice with approximately a 200-fold molar excess of unlabeled double-stranded oligonucleotide competitor (where indicated) followed by an additional 15 min on ice after the addition of 1-2 times 10^4 cpm of probe. Bound and free probe were separated by electrophoresis at 25 mA (constant current) on native 4% polyacrylamide gels (19:1) in 45 mM Tris-HCl (pH 8.3), 45 mM borate, 1.25 mM EDTA at 4 °C. Gels were dried onto Whatman No. 3MM paper (Whatman), and autoradiograms were exposed at -70 °C using intensifying screens.

For antibody binding assays, nuclear extracts or rabbit reticulocyte lysate were incubated on ice in a 20-µl binding assay with 1 µg of an anti-ETS domain antibody raised against peptide corresponding to residues 362-374 of ets-1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h either before or after the addition of oligonucleotide probe. Blocking assays were performed as above, but in the presence of 5-fold excess by weight of synthetic peptide, as per manufacturer's instructions. Antibodies directed against NF-Y (41) were preincubated with nuclear extract for 15 min prior to the addition of probe.

DNase I protection assays were performed using a binding reaction that was identical to that used for EMSA with the following exceptions. The total volume was 50 µl, the amount of nuclear extract protein was 8-20 µg, and the amount of probe was 1-3 times 10^5 cpm/sample. After the binding reaction was complete, the sample was incubated at room temperature for 1 min and supplemented to 10 mM magnesium chloride and 5 mM calcium chloride. The reaction was incubated for 1 min at room temperature and supplemented to 0.2-0.8 µg/ml with DNase I (Sigma) and incubated for 1 min at room temperature. The DNase I digestion was stopped by supplementing to 10 mM EDTA and 5% glycerol. Free and bound probe were separated on a 5% native gel as above. After electrophoresis, a wet autoradiogram was performed for 4 h at room temperature. Free and shifted probe bands were eluted from the gel (22) and ethanol precipitated. The probes were resuspended in 80% formamide loading buffer and subjected to electrophoresis on a denaturing polyacrylamide sequencing gel with a Maxam and Gilbert G+A sequencing ladder(42) . The gel was fixed in 10% methanol, 10% acetic acid and dried, and autoradiography was performed at -70 °C with intensifying screens. Sequencing ladders for each strand were generated by piperidine (Fisher) cleavage of dimethyl sulfate (Aldrich)-methylated probe as described previously(43) .

Methylation interference assays were performed with partially methylated end-labeled probe(43) . Binding reactions were performed as for DNase I protection assays, and free and bound probe were separated on 3.5% native polyacrylamide gels, recovered, and precipitated as above. Piperidine cleavage at G and A residues was performed as described(42) , and the probes analyzed on denaturing polyacrylamide sequencing gels as described for DNase I protection assays.

RNase Protection Assays

Riboprobes were synthesized using the Riboprobe Gemini system (Promega, Madison, WI). Plasmid DNA was restriction digested, phenol/chloroform-extracted, and ethanol-precipitated prior to the in vitro transcription reaction. Probes were labeled with 50 µCi of [alpha-P]CTP (3000 Ci/mmol, 10 mCi/ml, DuPont NEN) per µg of initial plasmid DNA. After in vitro transcription, the probes were ethanol-precipitated and gel-isolated on a native polyacrylamide gel and recovered as described(44) . Probes were resuspended in hybridization buffer (40 mM PIPES (pH 6.5), 0.4 M NaCl, 1 mM EDTA, and 80% formamide).

Total RNA was extracted from tissue culture cells by the single-step extraction method as described previously (45) and resuspended in 100% formamide(46) . Annealing reactions were performed in hybridization buffer with 2-20 µg of total RNA (or yeast tRNA as a control) and 10^5 to 2 times 10^6 cpm of probe in a final volume of 30 µl and incubated for 16 h at 55 °C. Elevation above this annealing temperature (up to 75 °C) did not further reduce background. RNase digestion was performed at 37 °C for 15 min in a total volume of 400 µl containing 40 µg/ml of RNase A (Boehringer Mannheim) and 700 units/ml RNase T1 (Boehringer Mannheim) in 10 mM Tris-HCl (pH 7.5), 0.3 M NaCl, and 5 mM EDTA. RNase digestion was terminated, and the protected fragments were analyzed on a denaturing polyacrylamide sequencing gel as described(47) . The gel was fixed, and autoradiography performed as described above.


RESULTS

Functional Assay of the Proximal gp91-phox Promoter

Previous study of the gp91-phox gene demonstrated that 450 bp of proximal promoter is sufficient to direct transgene expression in a subset of monocyte/macrophages in transgenic mice(10) . To analyze the proximal gp91-phox promoter in more detail, stable transfectant pools of the human promyelocytic leukemia cell line PLB985 were generated. Pools of transfectants were analyzed to compensate for possible integration site effects on the transcriptional activity of transfected sequences. Multiple independent pools were analyzed for each construct. A background of reporter gene expression is seen in promoterless transfectants (data not shown). This background transcription has been previously reported for transient transfectants of PLB985 cells with an identical vector and is presumably due to cryptic transcription initiation sites within the vector(9) .

Reporter gene expression was analyzed following IFN- stimulation and compared with unstimulated transfectants. IFN- stimulation of myeloid cell lines has previously been demonstrated to increase transcription of the gp91-phox gene(48) . No induction of reporter gene expression occurs upon IFN- stimulation of transfectants that contain a promoterless reporter gene construct (0.4 ± 9.6%) (Fig. 1). However, stable transfectants carrying a 450-bp gp91-phox promoter construct demonstrate a statistically significant increase in hGH reporter gene expression upon stimulation with IFN- (117.1 ± 20.9%) (Fig. 1). Analysis of a construct containing 1.5 kilobase pairs of gp91-phox promoter gave similar results (data not shown). Importantly, no increase in promoter activity occurs following IFN- stimulation of HeLa cells carrying the 450-bp gp91-phox promoter-hGH construct (data not shown). Thus, the stimulation observed with the PLB985 transfectants reflects a myeloid-specific regulatory process and not a more general IFN response.


Figure 1: Effect of IFN- treatment on reporter gene expression in stably transfected PLB985 cells. Expression levels are presented as the percent change in reporter gene expression in response to IFN- stimulation. No promoter/hGH, human growth hormone vector alone; 450 bp promoter/hGH, proximal 450 bp of the gp91-phox promoter; 100 bp promoter/hGH, proximal 100 bp of gp91-phox promoter; 450 bp (-57 A to C) promoter/hGH, CGD mutation at -57 bp; 450 bp (CCGGT) promoter/hGH, mutation of the inverted CCAAT box region. NS, not statistically significant.



No statistically significant change in reporter gene expression is observed, however, upon IFN- stimulation of transfectants that contain the proximal 100 bp of gp91-phox promoter (6.5 ± 10.8%). Although not sufficient for reporter gene expression, the proximal 100 bp of gp91-phox promoter contains a cis-element found to be mutated in kindreds with variant CGD(12) . A -57 bp A to C mutation that mimics one of the CGD lesions was introduced into the 450 bp gp91-phox promoter sequence, transfected into PLB985 cells, and assayed for reporter gene expression. No statistically significant increase in reporter gene expression is demonstrable with the mutant construct (3.1 ± 15.3%) (Fig. 1), suggesting that the CGD kindred mutations disrupt a cis-element that is necessary for IFN--induced expression of gp91-phox.

The proximal gp91-phox promoter also contains an inverted CCAAT-box at -45 to -49 bp. To investigate the functional contribution of this element, a mutation was introduced into the 450-bp gp91-phox promoter sequence that changes the element from CCAAT to CCGGT. This mutation has been previously reported to disrupt the binding of CCAAT box binding factors to other CCAAT box elements(9) . Assay of the mutant construct in PLB985 transfectants demonstrates an IFN--stimulated increase in reporter gene expression (127.4 ± 27.5%) similar to that observed with the wild-type 450-bp sequence (Fig. 1). This result indicates that the inverted CCAAT box element is not necessary for IFN--stimulated gp91-phox expression. As expected, a promoter that contains both the CGD and CCAAT box mutations fails to respond to IFN- (data not shown).

The transcription start site for the gp91-phox promoter-hGH transgene was determined by RNase protection assay (Fig. 2). Following IFN- stimulation, two protected bands are detected with RNA isolated from transfectants carrying the wild-type promoter sequence (Fig. 2, lane2) but are absent when RNA isolated from transfectants carrying the CGD-mutant promoter sequence is analyzed (Fig. 2, lane4). The position of the lower band corresponds to the published transcription initiation site of the gp91-phox gene(13) . Transfectants carrying the CCGGT-mutant promoter sequence yield an RNase protection pattern similar to that illustrated for the wild-type promoter (data not shown). These results are consistent with the observed IFN--induced increase in reporter gene product activity and indicate that increased reporter gene expression in response to IFN- is due to authentic gp91-phox promoter activity. RNase protection assays using a riboprobe for -actin were performed as an internal standard for the quantity of RNA in the hybridization reactions (Fig. 2, bottompanel).


Figure 2: RNase protection analysis of the transcription start site for the gp91-phox promoter/hGH-reporter gene in PLB985 transfectants. Lane1, 450-bp promoter/hGH; lane2, IFN--treated 450-bp promoter/hGH; lane3, -57 bp (A to C) CGD mutation 450- bp promoter/hGH; lane4, IFN--treated -57 bp (A to C) CGD mutation 450 bp promoter/hGH; lane5, yeast t-RNA. Toppanel, total RNA (20 µg) probed for the gp91-phox promoter/hGH transcript (see ``Materials and Methods''). Bottompanel, total RNA (1 µg) probed for the -actin transcript. Arrows indicate positions of protected bands.



DNA Binding Proteins Interacting with the Proximal gp91-phox Promoter

We have previously demonstrated the binding of two dominant protein complexes to the -30 to -68 bp region of the gp91-phox promoter(12) . Both the -57 and -55 single-bp mutations identified in two variant CGD kindreds abolish binding of the faster mobility complex(12) . A slower mobility complex is unaffected by these mutations. EMSA was performed to investigate the effect of myeloid differentiation on these two protein complexes (Fig. 3). No change in either complex is observed when nuclear extracts from PLB985 cells treated with retinoic acid and dimethylformamide to induce granulocyte differentiation, PMA to induce macrophage differentiation, or IFN- are used (Fig. 3, lanes1-4). Identical complexes are also generated with nuclear extracts from similarly treated promyelocytic HL60 cells (data not shown).


Figure 3: Tissue distribution of DNA-binding proteins that interact with the -30 to -68 bp region of the gp91-phox promoter. EMSA was performed as described under ``Materials and Methods'' using 2 µg of nuclear extract from a variety of cell lines. Lane1, undifferentiated PLB985 cells; lane2, PLB985 cells treated with retinoic acid and dimethylformamide; lane3, PLB985 cells treated with PMA; lane4, PLB985 cells treated with IFN-; lane5, CESS cells (B-cell); lane6, MOLT-4 cells (T-cell); lane7, K562 cells (chronic myelocytic leukemia in erythroleukemia blast crisis); lane8, HEL cells (acute myeloid leukemia, erythroleukemia type); lane9, HeLa cells (epithelial carcinoma); lane10, HepG2 cells (hepatocellular carcinoma). Arrows indicate positions of two specific complexes.



The tissue distribution of each binding activity was also examined. EMSA using nuclear extracts from the human hematopoietic cell lines CESS, MOLT-4, K562, and HEL demonstrate protein complexes of the same mobilities (Fig. 3, lanes5-8), indicating that both activities are present in a wide variety of hematopoietic cells. However, the faster mobility complex is much less abundant in nuclear extracts from the nonhematopoietic cell lines HeLa and HepG2 (Fig. 3, lanes9 and 10). We conclude that the slower mobility complex is ubiquitous, while the faster mobility complex is largely restricted to cells of hematopoietic origin, hereafter denoted hematopoietic associated factor 1 (HAF-1). Faint bands above the indicated doublet are not reproducible and may reflect multiple proteins bound to the probe.

The sequence specificity of each DNA-binding activity was examined in more detail. Formation of the two dominant complexes is disrupted by an excess of homologous unlabeled double-stranded oligonucleotide competitor (Fig. 4A, lane2), while a competitor containing the -57 bp CGD mutation fails to disrupt the HAF-1 complex (Fig. 4A, lane3). Similar results were obtained using a competitor containing the -55-bp CGD promoter mutation (data not shown). Conversely, an oligonucleotide containing a mutation of the inverted CCAAT box fails to disrupt the slower mobility complex (Fig. 4A, lane4), suggesting that a CCAAT-binding factor is present in this complex. An isoform of C/EBP is highly expressed in myelomonocytic cells(49) . However, competition with C/EBP binding sites fails to disrupt the CCAAT factor complex (Fig. 4A, lane5, and data not shown). Studies with additional oligonucleotide competitors and antibodies for CCAAT-binding factors identify this activity as the ubiquitous factor CP1 (data not shown).


Figure 4: Characterization of HAF-1 DNA-binding properties. A, determination of HAF-1 binding site specificity. EMSA was performed as described under ``Materials and Methods'' using 2 µg of nuclear extract from undifferentiated PLB985 cells and a probe corresponding to the -30 to -68 bp region of the gp91-phox promoter. Oligonucleotide competitions were performed using a 200-fold molar excess of double-stranded unlabeled oligonucleotides. The arrows indicate the two specific complexes. Competitors are as follows: lane1, none; lane2, homologous; lane3, -57 A to C CGD mutation; lane4, CCAAT-box mutated to CCGGT; lane5, C/EBP binding site(23) ; lane6, Ets-1 binding site(24) ; lane7, PEA3 binding site (25) ; lane8, Elf binding site (IL2 gene)(26) ; lane9, E74 binding site(27) ; lane10, GABP binding site(28) ; lane11, PU.1 binding site(14) . Arrows indicate positions of two specific complexes. B, determination of whether Fli-1 binds to the -30 to -68 bp region of the gp91-phox promoter. Fli-1 protein was synthesized and EMSA was performed as described under ``Materials and Methods.'' Lanes1-5, Fli-1 binding site probe; lane1, control lysate; lane2, 2 µl of Fli-1 protein; lane3, 0.5 µl of Fli-1 protein; lane4, 0.5 µl of Fli-1 protein plus anti-ETS domain antibody; lane5, same as lane4 with addition of blocking ETS-domain peptide; lanes6 and 7, -30 to -68 bp gp91-phox promoter probe. Lane6, 2 µl of Fli-1 protein; lane7, control lysate. C, determination of whether HAF-1 contains an ETS-domain. Nuclear extract was isolated from PLB985 cells stimulated with IFN-, preincubated with the anti-ETS domain monoclonal antibody and then incubated with a -30 to -68 bp gp91-phox promoter probe. Lane1, PLB985 nuclear extract plus 10-fold excess blocking peptide; lane2, same as 1, but without blocking peptide. Arrow indicates position of HAF-1 complex.



Since a consensus binding site for the Ets protein family is found within the proximal gp91-phox promoter, and a subset of Ets proteins is expressed in hematopoietic cells, competition studies were performed with high affinity binding sites to known Ets proteins. No significant competition of the HAF-1 complex is detected using binding sites for the Ets family members Ets-1, Elf-1 (IL-2 gene), or PU.1 (Fig. 4A, lanes6, 8, and 11). In addition, no competition is observed with Elf-1 binding sites derived from the CD4 and HIV-2 promoters (data not shown). Only slight disruption of the HAF-1 complex is demonstrated with both a GABP binding site and a PEA3 element that is a binding site for many Ets proteins, including Ets-1 and Ets-2 (25) (Fig. 4A, lanes10 and 7). However, high affinity competition of the HAF-1 complex is observed with a binding site for Drosophila E74.

Ets proteins known to interact with the E74 site, such as Erg-1, Erg-2, Elk-1, Fli-1, Ets-1, Ets-2, SAP-1, Erp, PU.1, and Elf-1, are therefore candidates for HAF-1(15, 26, 29, 50, 51) . However, as described above, high affinity binding sites for PU.1, Ets-1, and Elf-1 fail to compete efficiently for HAF-1 binding. Elk-1 and SAP-1 are expressed poorly in spleen but to high levels in liver(52) , Erg-1 is expressed highly in some epithelial lines but at low levels in MOLT-4 cells(53) , Erg-2 is not present in HL60, HEL, and K562 cell lines(54) , and Erp is scarce in B-cell lines(29) . These distributions are inconsistent with that observed for HAF-1. Therefore, only Fli-1 was investigated in further detail. In vitro synthesized Fli-1 protein binds to an authentic Fli-1 binding site (15) (Fig. 4B, lanes2 and 3) but is unable to bind to the -30 to -68 bp gp91-phox promoter probe (Fig. 4B, lanes6 and 7). In summary, these data indicate that HAF-1 is not Fli-1.

Members of the Ets family of transcription factors are characterized by homology in a highly conserved ETS-domain that is involved in binding site recognition(55) . Binding of Fli-1 is disrupted by the addition of an anti-ETS domain antibody (Fig. 4B, lane4), an effect that is blocked by the addition of an excess of ETS-domain peptide (Fig. 4B, lane5). However, no disruption of the HAF-1 complex occurs following preincubation of nuclear proteins with the ETS-domain antibody (Fig. 4C). Similar results were obtained using an antibody raised against Fli-1 (15) (data not shown). In additional control experiments, the anti-ETS domain antibody disrupted complexes formed following incubation of PLB985 nuclear extract with consensus binding sites for Ets-1 and PU.1 (data not shown). Hence, we are unable to demonstrate that HAF-1 contains an ETS-domain.

The binding characteristics of HAF-1 were further studied by DNase I footprinting and methylation interference assays. DNase I footprinting reveals that the HAF-1 complex protects a region between -45 and -62 bp of the gp91-phox promoter (Fig. 5A). Methylation interference analysis of the HAF-1 complex demonstrates contact bases consistent with the Ets family of DNA binding proteins (Fig. 5B)(54) . Both the DNase I footprinting and methylation interference data indicate that CP1 and HAF-1 exhibit overlapping but distinct binding sites in the proximal gp91-phox promoter (data not shown).


Figure 5: Characterization of the HAF-1 binding site. A, DNase I footprint analysis of HAF-1 interacting with the -30 to -68 bp region of the gp91-phox promoter. Nuclear extract was isolated from undifferentiated PLB985 cells as described under ``Materials and Methods.'' Lane1, G+A ladder of the coding strand; lane2, DNase I footprint of HAF-1 for the coding strand; lane3, DNase I digestion ladder of the coding strand; lane4, DNase I footprint of HAF-1 for the non-coding strand; lane5, DNase I digestion ladder of the non-coding strand; lane6, G+A ladder of the noncoding strand. B, methylation interference analysis of HAF-1 interacting with the -30 to -68 bp region of the gp91-phox promoter using nuclear extract from undifferentiated PLB985 cells. Lane1, coding strand probe shifted in the HAF-1 complex; lane2, unshifted coding strand probe; lane3, noncoding strand probe shifted in the HAF-1 complex; lane4, unshifted noncoding strand probe. Summary of HAF-1 binding characteristics is illustrated below. Bars indicate position of footprints, hatchmarks denote hypersensitive sites. Arrows indicate position of promoter mutations identified in CGD patients(12) . Weak contact bases are indicated with an opencircle, and strong contact bases are indicated with a filledcircle.




DISCUSSION

During terminal myeloid hematopoiesis, a number of phagocyte-specific genes, including the gene for gp91-phox, become transcriptionally active(6) . To understand the events involved in the tight lineage specificity of this process, we analyzed the proximal gp91-phox promoter for cis-elements important in IFN--induced transcription of this gene. Stable transfectants of PLB985 cells containing gp91-phox promoter-hGH constructs exhibit IFN--inducible transgene expression, thus permitting study of IFN- response elements in the gp91-phox promoter. Using this approach, we demonstrate that elements necessary for IFN--induced transcription reside between -100 and -450 bp of the gp91-phox promoter. We also demonstrate that, although the proximal 100 bp of promoter is not sufficient for the IFN- transcription response, there is a cis-element within the proximal 100 bp that is necessary for the IFN- response. RNase protection assays demonstrate a stimulation of transgene transcription from the authentic gp91-phox initiation site in response to IFN- treatment.

Study of phagocyte function has been facilitated by the existence of CGD kindreds with various respiratory burst defects(4, 5, 7) . We demonstrate that gp91-phox promoter mutations previously described in two kindreds with variant CGD (12) disrupt a DNA-protein interaction that is required, but is not sufficient, for IFN--induced gp91-phox transcription. This binding activity is largely restricted to cells of hematopoietic origin, and hence is denoted HAF-1.

The presence of HAF-1 in nuclear extracts in cell lines not transcribing gp91-phox further suggests that binding of HAF-1 is not sufficient to activate transcription of the gp91-phox gene in response to IFN-. However, the abundance of HAF-1 activity is not increased in nuclear extract isolated from IFN--stimulated myeloid cells and hence is not directly involved in the signal-transduction process. A distinct IFN- responsive element presumably resides elsewhere in the proximal gp91-phox promoter construct, consistent with the finding that elements between -100 bp and -450 bp are required for an IFN- response. Inspection of this DNA sequence, however, did not reveal consensus sequences for IFN- responsive elements(56, 57, 58) . Additional studies utilizing the functional assay system described in this report should permit an identification of the cis-elements that are necessary and sufficient for IFN--induced transcription of the gp91-phox gene.

Although not myelomonocytic-specific, HAF-1 binding may contribute to phagocyte-specific expression of gp91-phox through cooperation with other cis-elements and trans-factors. For example, we have previously demonstrated that binding of CCAAT-displacement protein (CDP) to an element at -120 bp of the gp91-phox promoter serves to repress promoter activity. We proposed that down-regulation of CCAAT-displacement protein DNA-binding activity during terminal differentiation serves to ``poise'' the gp91-phox promoter for activation upon binding of a number of ubiquitous or lineage-restricted transcriptional activating proteins(9) .

The DNase I footprint of HAF-1 covers a region of gp91-phox promoter that contains the core binding site for the Ets family of transcription factors(15) . The minimal six-bp recognition sequence for the Ets family members Ets-1, Ets-2, and PU.1/Spi-1 is 5`-(A/C)(1)G(2)G(3)A(4)(A/T)(5)(C/G)(6)-3` (15) . Binding of the Ets protein Fli-1 has additional requirements outside of the Ets core motif (5`-C(1)(C/A)(2)G(3)G(4)A(5)A(6)G(7)T(8)-3`), binding poorly in the absence of either C(1) or T(8) and not at all in the absence of both(15) . Although the gp91-phox promoter sequence is not identical to any of the defined Ets family member high affinity binding sites, there are striking similarities. The gp91-phox promoter sequence (antisense strand) from -50 to -57 is 5`-G(1)A(2)G(3)G(4)A(5)A(6)A(7)T(8)-3` (bases mutated in the two CGD kindreds are underlined(12) ). Methylation interference analysis of the HAF-1 complex demonstrates that the G(3)G(4) bases are important for HAF-1 binding, consistent with binding requirements demonstrated for several Ets family members.

Several lines of evidence indicate that HAF-1 does not correspond to previously described Ets factors. Abundant HAF-1 binding activity is found in all of the hematopoietic cell nuclear extracts tested (K562, HEL, CESS, MOLT-4, PLB985, and HL60), but not in nonhematopoietic lines (HeLa and HepG2), and is not altered by treatment of PLB985 or HL60 cells with PMA, retinoic acid, or IFN-. This tissue distribution narrows the potential Ets proteins to consider. Of the Ets proteins known to be expressed predominately in hematopoietic cells, all but Fli-1 can be ruled out based on binding site specificity. However, Fli-1 has a somewhat more limited tissue distribution than that observed for HAF-1. Although mRNA for Fli-1 has been detected in many immature and mature T- and B-cell lines and in the erythroleukemia cell line HEL, mRNA levels are undetectable in K562 cells(59) . Also, although mouse bone marrow macrophages have been demonstrated to express Fli-1 message, detectable Fli-1 mRNA is abolished by stimulation of macrophages with PMA or IFN-(59) . In addition, in vitro synthesized Fli-1 is unable to bind to the -30 to -68 bp gp91-phox promoter element. These results indicate that HAF-1 is not Fli-1, and we conclude that the tissue distribution and binding site specificity of HAF-1 do not conform to known Ets proteins.

In addition, the HAF-1 complex is not disrupted by an antibody directed against the conserved ETS-domain. We cannot exclude the possibility that HAF-1 contains an ETS domain unavailable for interaction with the monoclonal antibody due to protein-protein interactions or stearic hindrance, or that HAF-1 contains a divergent ETS-domain that is not recognized by the antibody. Alternatively, HAF-1 may represent a novel transcription factor with a binding specificity similar to an Ets protein. Search of a transcription factor binding site data base (60) failed to identify additional consensus binding sites within this promoter element.

Binding studies indicate that the ubiquitous CCAAT-binding factor CP1 interacts with a binding site that overlaps the HAF-1 site within the proximal gp91-phox promoter. Functional analysis indicates that CP1 binding at this site is not required for IFN--induced gp91-phox transcription. It remains possible, however, that this element is functionally relevant to gp91-phox promoter activity in vivo, perhaps in cooperation with other cis-elements and trans-factors in the full complement of myelomonocytic cells.

The data presented in this study indicate that binding of HAF-1 to the proximal gp91-phox promoter is necessary but not sufficient for IFN- induction of gp91-phox expression. The finding that loss of HAF-1 binding is associated with a variant form of CGD is suggestive of an important role for HAF-1 in the regulation of gp91-phox expression. The characterization of HAF-1 binding properties reported here should aid in efforts to obtain molecular clones for this factor. Identification of the HAF-1 protein(s) will allow detailed study of the function and regulation of this factor, including the relationship of HAF-1 to the Ets family, and may shed light on the molecular mechanisms involved in terminal myeloid differentiation.


FOOTNOTES

*
This work was supported in part by the Riley Memorial Association and by the following grants (to D. G. S.): a National Institutes of Health FIRST award (CA58947), an American Cancer Society Junior Faculty Award (421), a National Leukemia Association grant, and a grant by the Project Development Program, Research and Sponsored Programs, Indiana University at Indianapolis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Walther Oncology Center, a National Institutes of Health Clinical Investigator Development Award (K08 HL03139), and an American Medical Association Florence Carter fellowship. Present address: Wallace Tumor Inst., 1824 South 6th St., Birmingham, AL 35233.

To whom correspondence and reprint requests should be addressed: Wells Center for Pediatric Research, Rm. 2600, Riley Hospital for Children, 702 Barnhill Dr., Indianapolis, IN 46202-5225. Tel.: 317-274-8977; Fax: 317-274-8679.

(^1)
The abbreviations used are: IFN-, interferon ; bp, base pair(s); HAF-1, hematopoietic-associated factor 1; CGD, chronic granulomatous disease; EMSA, electrophoretic mobility shift assay; PMA, phorbol 12-myristate 13-acetate; hGH, human growth hormone; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Darlene Barnard and Beverly Pero for outstanding technical assistance, Riley Cancer Research for Children for supporting the oligonucleotide facility, Alan Bernstein for providing a Fli-1 antibody, Diane Mathis for providing NF-Y antibodies, and Mike Klemsz for providing the Fli-1 expression construct and for helpful discussions.


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