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During Differentiation of the Monocytic Cell Line U937, Pur Mediates Induction of the CD11c ₂ Integrin Gene Promoter¹

Renal Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD11c is a member of the ₂ integrin family of adhesion molecules that, together with CD18, forms a heterodimeric receptor on the surface of myeloid, NK, dendritic, and certain leukemic, lymphoma, and activated lymphoid cells. Monocytic differentiation is associated with an induction of both CD11c and CD18 gene expression. The resulting CD11c/CD18 receptor mediates firm adhesion to the vascular endothelium, transendothelial migration, chemotaxis, and phagocytosis. Monocytic differentiation can be mimicked in vitro by treatment of the promonocytic cell line U937 with PMA. Recently, we reported that in U937 cells, expression of the CD11c gene is controlled by an unidentified transcription factor that binds ssDNA. This finding suggested that DNA secondary structure plays an important role in controlling the CD11c gene and prompted us to search for additional ssDNA-binding activities with which this gene interacts. In this study, we report that in U937 cells, expression of the CD11c gene is mediated by the ssDNA-binding protein Pur. During PMA-induced differentiation, the ability of Pur to activate the CD11c promoter in U937 cells increases, as does that of Sp1. Together, these increases in the functional activity of both Pur and Sp1 combine to induce CD11c expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The four heterodimers that comprise the ₂ integrin family of cell surface glycoproteins mediate a broad range of the adhesive functions of lymphoid and myeloid cells. They are composed of distinct subunits encoded by the CD11a, CD11b, CD11c, and CD11d genes noncovalently associated with a common subunit encoded by the CD18 gene (1, 2). The CD11c/CD18 heterodimer is expressed on NK, mature myeloid, and dendritic cells, as well as on some cytotoxic T cell clones and some activated B and T lymphocytes. In addition, CD11c/CD18 is expressed on the surface of some neoplastic T cells and certain chronic B lymphocytic leukemias and large cell lymphomas. In hairy cell leukemia, high level constitutive expression of the CD11c/CD18 heterodimer represents a diagnostic marker of the disease. Since the CD18 gene is active in all leukocytes, it is the more selective expression of the CD11c gene that dictates the specific cell types on which the CD11c/CD18 heterodimer is present.

Expression of the CD11c gene is regulated during cell activation and differentiation, being induced during B and T cell activation and the differentiation of progenitor myeloid cells into monocytes and granulocytes (3, 4). The CD11c/CD18 heterodimer that results on the surface of differentiating monocytic cells mediates their invasion of the vascular endothelium at sites of inflammation by interaction with CD54 (ICAM-1). Such differentiation can be mimicked in vitro by treatment of the promonocytic cell line U937 with phorbol esters. Using this system, we recently reported that expression of the CD11c gene is controlled by the transcription factor PyRo1 (5). While the identity of PyRo1 remains to be determined by molecular cloning, we analyzed its DNA-binding characteristics and found that it interacts preferentially with pyrimidine-rich DNA, which is single-stranded. This finding suggested that the secondary structure of the CD11c gene plays an important role in controlling its expression, and that it might interact with additional ssDNA-binding activities. In this study, we report the identity of one such activity as being Pur. Unlike PyRo1, which interacts with pyrimidine-rich ssDNA, Pur interacts with ssDNA, which is purine rich. However, Pur and PyRo1 are similar in that both are induced during U937 differentiation. We found that during this differentiation process, the ability of Pur to activate the CD11c promoter increases. Furthermore, we found that during U937 differentiation, the ability of Sp1 to activate the CD11c promoter also increases. Together, Pur and Sp1 combine to induce CD11c expression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of the 5' regulatory region of the CD11c gene

The oligonucleotide AA3 (5'-TGCCACTCTTCCTGCAACGGCCCAGGAGCTCAGAGCTCCACATCTG-3'), designed from the 5' end of a human CD11c cDNA clone (6), was used to screen a human genomic library (no. 57765; American Type Culture Collection, Manassas, VA). One positively hybridizing clone, CD11c, was isolated and a 16-kb NotI fragment ligated into the NotI site of pGEM11Zf⁺ (Promega, Madison, WI) to generate the subclone pZ11c-16. A 6.7-kb SacI fragment of CD11c was ligated into the SacI site of pGEM7Zf⁺ (Promega) to generate the subclone pZ11c-7.

DNA sequencing

dsDNA was sequenced by the dideoxynucleotide chain termination method (7) using Sequenase (United States Biochemical, Cleveland, OH). A series of specific oligonucleotide primers based on the CD11c cDNA sequence (6) and the CD11c gene sequence, as it became known, was used to sequence the 5' end of the CD11c gene contained in pZ11c-16 and pZ11c-7. This nucleotide sequence was determined on both DNA strands and has been deposited in the GenBank database under accession no. L19440.

Cell culture

The cell line U937 was obtained from the American Type Culture Collection and grown according to their specifications. PMA was obtained from Sigma-Aldrich (St. Louis, MO) and used at a concentration of 100 ng/ml, where indicated. U937 cells were treated with PMA for 12–24 h. These lengths of treatment are sufficient to induce monocytic differentiation as assessed by the production of CD11b mRNA (data not shown).

Plasmid construction

The activity of the CD11c promoter was assessed using the expression vector pATLuc (8), which contains a promoterless firefly luciferase reporter gene. Initially, the PCR was used to generate one fragment of the CD11c gene representing nt -128 to +36 relative to the major 5' transcription initiation site (9). This fragment was then subcloned into the filled-in HindIII site of pATLuc to generate p11Wt. The construct p11Pur again represented nt -128 to +36 cloned into the filled-in HindIII site of pATLuc. However, this construct was produced by oligonucleotide-directed mutagenesis (10) and contained a substitution of the antisense sequence 5'-GGGGAAGGAAG-3' spanning nt -30 to -40, with the mutant sequence 5'-TTTTAATTAAT-3'. This substitution abolishes Pur binding (see Figs. 1 and 2). The correct orientation and nucleotide sequence of all constructs was verified by DNA sequencing. The Pur expression construct, pHAPur1, was kindly provided by E. Johnson (Mount Sinai School of Medicine, New York, NY) (11), and the empty vector equivalent, pHA, was produced by religation following liberation of the Pur sequence by RsrII and EcoRI digestion. The Sp1 expression construct, pCMV-Sp, was produced from the parent plasmid p588 (12). This parent plasmid contains the Sp1 coding region cloned downstream of the CMV promoter and upstream of a region encoding the nuclease domain of the restriction endonuclease FokI. To generate pCMV-Sp, the FokI coding region was removed from p588 by digesting with the flanking restriction endonucleases XhoI and HpaI, filling-in these sites, and then recircularization. The construct, pCMV, is identical to pCMV-Sp, except that it is empty of Sp1 coding sequences. This construct was produced by digesting p588 with NotI and HpaI, which flank the Sp1/FokI coding region, filling-in, and recircularization. The plasmid p588 was kindly provided by J. Chung (National Institutes of Health, Bethesda, MD).

View larger version (11K): [in this window] [in a new window]   FIGURE 1. The nucleotide sequence of the CD11c gene promoter. Arrowed are the major and minor sites of transcription initiation (9 ). The most 5' of the major sites of transcription initiation is marked above the sequence with an asterisk. Numbering is relative to this site. The four regions that exhibit the capacity to interact with the nuclear factor Pur are marked above the sequence with filled bars. Asterisks below the Pur binding region located between nt -41 and -29 indicate the G to T substitutions present in the antisense strand of the construct p11Pur. The three regions reported to interact with the transcription factor Sp1 are marked below the sequence with open bars (4 19 21 ). The Sp1 binding site present closest to the major 5' transcription initiation site has been reported to be located at nt -25 (4 ). In the absence of a definitive report of the stretch of nucleotides involved in binding Sp1 at this site, nucleotides that constitute a consensus Sp1 binding site are marked.

View larger version (79K): [in this window] [in a new window]   FIGURE 2. Electrophoretic mobility shift analysis of Pur binding to the CD11c promoter. A radiolabeled single-stranded oligonucleotide, CD11c PWt, representing the antisense strand of the -41/-29 region of the CD11c gene, was incubated with no nuclear extract (lane 1) or a nuclear extract prepared from U937 cells induced for 24 h with PMA. Binding reactions were performed with radiolabeled CD11c PWt in the absence (lane 2) or presence (lane 3) of a 200-fold molar excess of unlabeled CD11c PWt; in the presence of unlabeled CD11c PWt-R (lane 4), which represents the complementary sequence of CD11c PWt; in the presence of CD11c PMt (lane 5), representing a mutant version of CD11c PWt; or in the presence of the single-stranded oligonucleotide GG-Rep (lane 6), which contains repeats of the sequence GGN, in which N is anything but G. Binding reactions were also performed in the presence of the Ab 9C12 (lane 7), which specifically interacts with Pur (11 ), and the Ab Egr-1 (588) (lane 8), which specifically interacts with Egr-1 (Santa Cruz Biotechnology). The position of the complex containing DNA and Pur is marked with an arrow, as is the position of the protein-free DNA probe. The U937 nuclear extracts and Abs used in the depicted analysis were used previously to characterize the interaction of Pur with the CD43 gene promoter (38 ).

U937 cells were transfected by electroporation (13, 14, 15) in nonsupplemented medium at 280 V and 960 µF using a Gene Pulser system and 0.4 cm electrode gap Gene Pulser cuvettes (Bio-Rad, Hercules, CA). Cells in the early log phase of growth were harvested by centrifugation, washed once with medium, and resuspended at room temperature to a concentration of 4 x 10⁷ cells/0.4 ml. Aliquots of 0.4 ml cells were transfected with 23 µg luciferase test plasmid together with 2 µg of the plasmid pRSV- (Promega), which contains the lacZ gene. Each transfection of p11Wt and p11Pur was performed in parallel with a transfection of the promoterless luciferase plasmid pATLuc. In those experiments in which monocytic differentiation was induced, electroporated cells were treated with PMA at 100 ng/ml. Electroporated cells were incubated at 37°C for 16 h, pelleted, washed twice with PBS, and lysed in 200 µl reporter lysis buffer (Promega). One hundred microliters of this cell lysate were used in the assay of luciferase activity, and 50 µl were used in the assay of -galactosidase activity. Both luciferase and -galactosidase activities were assayed using reagents purchased from Promega. Luciferase activity, assessed as light output, was measured using a Moonlight 2010 luminometer, which integrated peak luminescence 10 s after injection of assay buffer. The levels of -galactosidase activity resulting from different transfections were taken as reflective of relative transfection efficiency and used to correct the measurements of luciferase activity. Trans activation by Pur was assessed by transfections in which 8 µg pATLuc, p11Wt, or p11Pur were mixed with 1 µg pRSV- and 16 µg of either pHAPur1 or the equivalent empty vector pHA (see Fig. 2). Trans activation conferred by Pur combined with Sp1 was assessed by transfections in which 8 µg p11Wt were mixed with 1 µg pRSV- and: 1) 8 µg both pHAPur1 and pCMV-Sp; 2) 8 µg both pHAPur1 and pCMV; 3) 8 µg both pHA and pCMV-Sp; or 4) 8 µg both pHA and pCMV (see Fig. 5B).

View larger version (32K): [in this window] [in a new window]   FIGURE 5. Functional expression of Pur increases during U937 differentiation. A, The radiolabeled single-stranded oligonucleotide, CD11c PWt, representing the antisense strand of the -41/-29 region of the CD11c gene, was incubated with no nuclear extract (lane 1) or nuclear extracts prepared from either untreated U937 cells (lanes 2 and 3) or U937 cells induced for 24 h with PMA (lanes 4 and 5). Binding reactions were performed in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of a 200-fold molar excess of unlabeled CD11c PWt. The position of the major protein-DNA complex, Pur, is marked with an arrow, as is the position of the protein-free DNA probe. B, The construct p11Wt was mixed with the -galactosidase expression construct pRSV- and one of four different combinations of plasmids: first, pHAPur1 and pCMV-Sp, which express Pur and Sp1, respectively; second, pHAPur1 and pCMV, which represents the empty vector equivalent of pCMV-Sp; third, pCMV-Sp and pHA, which represents the empty vector equivalent of pHAPur1; fourth, the empty vectors pHA and pCMV. These four DNA mixtures were then transfected into U937 cells that were either left untreated or treated overnight with PMA. Cells were harvested and lysed, and luciferase and -galactosidase assays performed. The levels of -galactosidase activity were taken as reflective of transfection efficiency and used to correct the luciferase assay results. Using these corrected values, the level of luciferase activity directed by p11Wt in the presence of the empty vectors pHA and pCMV was used to divide the levels of luciferase activity directed by p11Wt in the presence of constructs expressing Pur and/or Sp1. These calculations yielded the fold induction conferred on the CD11c promoter by Pur and Sp1 alone and in combination. The mean of these levels ± the SD resulting from three independent experiments are displayed as histograms.

Approximately 250 million cells were collected by centrifugation, washed three times in ice-cold PBS, and resuspended in 4 ml ice-cold buffer 1 (10 mM NaCl, 0.4 M sucrose, 10 mM Tris-HCl (pH 7.8), 0.2 mM EDTA, 0.1 mM EGTA, 0.5% Nonidet P-40, 0.5 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 50 mg/ml antipain, and 1 mg/ml aprotinin). The resuspended cells were then incubated on ice for 30 min and layered over 4 ml ice-cold buffer 2 (buffer 1 containing 1.5 M sucrose and 0.5 mM DTT, but no Nonidet P-40). Nuclei were collected by centrifugation, washed with 4 ml ice-cold buffer 3 (buffer 1 containing 0.5 mM DTT, but no Nonidet P-40), and resuspended to a concentration of 1 x 10⁸ cell equivalents/300 µl in ice-cold buffer 4 (20 mM Tris-HCl (pH 7.8), 300 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 25% glycerol, and the protease inhibitor mixture listed above). Resuspended cells were then incubated at 4°C for 60 min and dialyzed overnight at 4°C against buffer 4. The dialyzed nuclear extract was clarified by centrifugation, frozen in liquid nitrogen, and stored at -80°C. The concentration of protein present in the nuclear extracts was determined using the Bio-Rad protein assay system (Bio-Rad).

EMSA

Oligonucleotides were radiolabeled at their 5' ends using T4 polynucleotide kinase and purified through G-25 Sephadex columns. To generate dsDNA, equimolar amounts of complementary oligonucleotides were combined. These oligonucleotides were then annealed by adding 5 M NaCl to a final concentration of 100 mM, heating to 90°C, and slowly cooling overnight to 4°C. DNA/protein-binding reactions were conducted in a 20 µl volume. First, nuclear extracts were incubated with or without a molar excess of unlabeled competitor probe at 4°C for 15 min in 70 mM KCl, 5 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 mM DTT, 10% glycerol, and 2.4 µg poly d(I:C)·poly d(I:C). Radiolabeled probe was then added, and the incubation continued for 30 min. The DNA/protein complexes were resolved by electrophoresis through 7% native polyacrylamide gels and visualized by autoradiography. Electrophoretic mobility supershift assay analyses were performed in the same way as the standard EMSA analyses, except that before the addition of DNA probes, nuclear extracts were preincubated for 15 min at 4°C with either 1 µl polyclonal Ab, which specifically interacts with Egr-1, or 1 µl mAb 9C12, which specifically interacts with Pur (11). The Egr-1 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the Pur Ab was kindly provided by E. Johnson (Mount Sinai School of Medicine). The oligonucleotides used in the analyses were: CD11c PWt, 5'-AGGGGAAGGAAGA-3'; CD11c PWt-R, 5'-TCTTCCTTCCCCT-3'; CD11c PMt, 5'-ATTTTAATTAATA-3'; CD11c P2, 5'-GTGTGGGAGGCCGAGC-3'; CD11c P3, 5'-GAGGGGGCGGGCAGAGT-3'; CD11c P4, 5'-AGAGAGGTGGCCAGGG-3'; GG-Rep, 5'-GGCTGGATGTGGTGGCTCAC-3'; and NS-1, 5'-TATTAATTAAAAT-3'.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The CD11c promoter interacts with two ssDNA-binding proteins

The human CD11c gene has been cloned and its proximal promoter region has been shown to direct a pattern of expression in vitro that mimics that of the endogenous gene in vivo (4). Recently, we determined that the activity of the CD11c promoter is controlled in part by a cis-acting element spanning nt -128 to -110, which interacts with the transcription factor PyRo1 (5). PyRo1 binds preferentially to pyrimidine-rich ssDNA, and its binding site within the CD11c promoter contains repeat sequences capable of forming structures with such conformations. In addition to the PyRo1 binding site, the region spanning nt -44 to -29 also contains repeat sequences that might form single-stranded structures (16). Since it was possible that the -44/-29 region contains such structures, we tested whether it could interact with ssDNA-binding proteins. Using single-stranded oligonucleotides in EMSA analysis, we demonstrated that this was indeed the case (Fig. 2). Specifically, this analysis demonstrated that the purine-rich antisense strand of the region spanning nt -41 to -29 interacts predominantly with a single nuclear factor. When the antisense strand is annealed with its complementary sequence to yield a double-stranded probe, no additional binding activities appear. However, upon the production of dsDNA, the intensity of the protein-DNA complex is significantly reduced. This suggests that the nuclear factor detected, while capable of binding dsDNA, preferentially binds DNA, which is single-stranded.

Identification of Pur as the factor that binds the -41/-29 region of the CD11c gene

The -41/-29 CD11c gene sequence that interacts with the ssDNA-binding protein detected by EMSA contains two copies of the sequence GGN, in which N is not G. Repeats of this sequence represent the recognition element of the transcription factor Pur (17, 18). Pur, like the factor we have identified that interacts with the -41/-29 region, can bind dsDNA, but exhibits a distinct preference for DNA that is single-stranded. To determine whether the factor binding the CD11c gene was Pur, we first attempted to compete such binding with a single-stranded oligonucleotide containing GGN repeats. EMSA analysis demonstrated that this competition was entirely effective (Fig. 2, lane 6). Therefore, the factor binding the CD11c gene exhibits DNA-binding characteristics consistent with those of Pur. Next, we determined whether the factor interacts with the mAb 9C12 that specifically recognizes Pur (11). This analysis proved positive, demonstrating that the factor that binds the -41/-29 region of the CD11c gene is immunologically indistinguishable from Pur (Fig. 1, lane 7).

Interaction with Pur is critical to CD11c promoter activity

Repeats of the sequence GGN are found in all Pur binding sites. This is also the case for the Pur binding site within the CD11c gene, which contains the sequence GGGGAAGGAAG. When this sequence was mutated to TTTTAATTAAT, Pur-binding activity was lost (Fig. 2, lane 5). These same mutations were introduced into the -128/+36 promoter to assess the importance of Pur binding to its activity. Transfection of this mutant promoter into U937 cells subsequently treated with PMA demonstrated that it directs expression levels that are over 80% lower than those directed by the wild-type sequence (Fig. 3A). Therefore, Pur binding to the CD11c promoter appears to be of critical importance.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Contribution of the Pur site located within the -41/-29 region of the CD11c promoter to transcriptional activity in U937 cells. A, The wild-type CD11c promoter spanning nt -128 to +36 is present in the luciferase expression construct p11Wt, while p11Pur represents the specific replacement of the sequence GGGGAAGGAAG, extending from nt -30 to -40 within the antisense strand, with the sequence TTTTAATTAAT. This replacement abolishes Pur binding (Fig. 2). The constructs p11Wt and p11Pur were mixed with the -galactosidase expression construct pRSV- and transfected into U937 cells. Transfected cells were either left untreated or treated for 12 h with PMA then luciferase and -galactosidase assays were performed. The levels of -galactosidase activity were taken as reflective of transfection efficiency and used to correct the luciferase assay results. Corrected luciferase values were divided by the background activity conferred by the control plasmid pATLuc. Each histogram represents the mean ± the SD of three independent transfection experiments. B, The luciferase reporter constructs p11Wt and p11Pur were transfected into U937 cells mixed with either pHAPur1 or its empty equivalent pHA. The -galactosidase expression plasmid pRSV- was included in each transfection. Cells were then treated with PMA, and after 16 h luciferase and -galactosidase assays were performed. The levels of -galactosidase activity were taken as reflective of transfection efficiency and used to correct the luciferase assay results. Using these corrected values, the levels of luciferase activity directed by p11Wt and p11Pur in the presence of pHAPur1 were then divided by their expression levels in the presence of pHA. This calculation yielded the levels of induction conferred by Pur on the wild-type and the mutant CD11c promoters. The mean of these levels ± the SD resulting from three independent experiments are displayed as histograms.

Pur is capable of activating the CD11c promoter

Mutation analysis suggested that Pur binding is necessary for the activity of the CD11c gene promoter. However, it remained possible that the same mutations that disrupt Pur binding also disrupt promoter topology. In this situation, the effects of the mutations on promoter function might not truly reflect the importance of Pur. Therefore, next we sought to determine directly whether expression of Pur was sufficient to activate the CD11c promoter. This was achieved by cotransfecting the CD11c reporter construct p11Wt with the construct pHAPur1, which constitutively produces Pur driven by the CMV promoter (11). Under these conditions, in three independent experiments, p11Wt exhibits a mean expression level in PMA-treated U937 cells that is 3.5 times higher compared with when it is transfected with the CMV plasmid empty of Pur sequences (Fig. 3B). Therefore, Pur is capable of activating the CD11c promoter. Previous studies have demonstrated that in the myeloid cell line HL-60, the CD11c promoter can be activated by Sp1, Sp3, c-Jun, c-Fos, and C/enhancer-binding protein (19, 20, 21). With the exception of Sp3, the degree of activation conferred by these factors is lower than that conferred by Pur in U937 cells.

Pur has the ability to interact with the CD11c promoter at multiple sites

The capability of Pur to trans-activate the CD11c promoter is significantly reduced by mutations that prevent Pur binding to the -41/-29 region. Consequently, Pur influences CD11c promoter activity through this region. However, mutations within the -41/-29 region fail to completely abolish Pur trans activation, suggesting Pur can interact with the CD11c promoter at additional sites. We analyzed the CD11c promoter for such additional sites and found that as well as the -41/-29 region, three other regions contain closely spaced repeats that conform to the consensus sequence recognized by Pur. These repeats are GGAGGC located between nt -109 and -104, GGCGGGC located between -66 and -72, and GGTGGC present between nt -22 and -27. The ability of these repeats to compete with the -41/-29 region for Pur binding was tested in EMSA analysis (Fig. 4). This analysis demonstrated that all three repeats effectively competed for Pur binding. Consequently, Pur has the capacity to interact with the CD11c promoter at a total of four sites, one located on the sense strand and three located on the antisense strand.

View larger version (56K): [in this window] [in a new window]   FIGURE 4. Pur has the capacity to interact with the CD11c promoter at a total of four sites. The radiolabeled single-stranded oligonucleotide CD11c PWt, representing the Pur binding site located on the antisense strand of the -41/-29 region of the CD11c gene, was incubated with no nuclear extract (lane 1) or a nuclear extract prepared from U937 cells induced for 24 h with PMA. Binding reactions were performed in the absence (lane 2) or presence (lane 3) of a 200-fold molar excess of unlabeled CD11c PWt, or in the presence of the unlabeled single-stranded oligonucleotides CD11c P2 (lane 4), CD11c P3 (lane 5), or CD11c P4 (lane 6). These represent the purine-rich strands of the regions of the CD11c promoter that span, respectively, nt -114 to -99, -77 to -61, and -32 to -17. A binding reaction was also performed in the presence of a 200-fold molar excess of the unlabeled single-stranded oligonucleotide NS-1 (lane 7), which does not contain a Pur consensus recognition site. The position of the major protein-DNA complex, Pur, is marked with an arrow, as is the position of the protein-free DNA probe.

The functional expression of Pur increases during U937 differentiation

Previous studies have shown Pur to be subject to developmental regulation in the mouse brain (22). In this study, induction of its expression mediates activation of the myelin basic protein gene promoter at the end of the first postnatal week. Our trans activation studies presented in Fig. 3B clearly demonstrate that, in addition to activating gene expression during brain development, Pur also has the capacity to activate expression during monocytic differentiation of U937 cells. Therefore, we were interested in determining whether, as in brain development, Pur is subject to regulation during U937 differentiation. First, we compared Pur-binding activity in nuclear extracts prepared from untreated U937 cells with that present in extracts prepared from U937 cells induced to differentiate along the monocytic pathway for 24 h with PMA. This comparison demonstrated that Pur binding to the oligonucleotide CD11c PWt is induced during U937 differentiation (Fig. 5A). Next, we sought to determine whether the capacity of Pur to effect trans activation of the CD11c promoter is also modulated during differentiation. In transfection studies thatemployed equivalent amounts of the Pur expression constructpHAPur1, the transcriptional activity of the CD11c promoter was induced to a significantly higher degree in PMA-treated U937 cells than in cells that were left untreated (Fig. 5B). Consequently, our data indicate that both the expression of Pur, as measured by its DNA-binding activity, and the ability of Pur to effect trans activation are induced during U937 monocytic differentiation.

During U937 differentiation, Pur combines with an increase in the trans activation capacity of Sp1 to effect induction of the CD11c promoter

The functional activity of Pur has been shown to be influenced either in a positive or negative manner by its interaction with a number of cellular proteins, including calmodulin, Sp1, YB-1, Pur, and the retinoblastoma protein Rb (11, 22, 23, 24, 25, 26). Of these proteins, only Sp1 has been reported to influence CD11c gene expression (4, 19, 20, 21, 27). Therefore, we sought to determine whether in the context of the CD11c promoter, Sp1 and Pur combine or compete to induce transcription. Trans activation experiments performed in U937 cells either untreated or treated with PMA demonstrated that recombinant Pur and Sp1 combine to induce CD11c promoter activity (Fig. 5B). In addition, we found that, as with Pur, expression of Sp1 was more effective in activating the CD11c promoter in PMA-treated U937 cells than in cells that were left untreated. These increases in trans activation capacity resulted in the combination of Sp1 and Pur being over 2.5-fold more effective in inducing the CD11c promoter in PMA-treated U937 cells than in untreated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, we determined that the transcriptional activity of the -128/+36 promoter region of the CD11c gene is regulated by its interaction with the ssDNA-binding factor PyRo1 (5). This finding indicated that DNA secondary structure most likely influences CD11c gene expression, and prompted us to examine the CD11c promoter for additional regions that might exist in a ssDNA conformation. Such examination revealed that between nt -44 and -29, there are repeats with the potential to form single-stranded loop structures. Based on this analysis, we thought it possible that the -44 to -29 region might interact with ssDNA-binding proteins. This possibility was tested by EMSA analysis, and indeed a nuclear factor was found to bind the purine-rich antisense strand spanning nt -41 to -29. Further analysis established that this factor is immunologically indistinguishable from Pur.

Pur binds to repeats of the sequence GGN. Therefore, these were mutated within the -41/-29 region to determine the functional significance of Pur interaction with the CD11c promoter. This analysis demonstrated that when mutations are introduced into the -41/-29 region, promoter activity in PMA-treated U937 cells is reduced by over 80%. Consequently, the binding of Pur appears critical to CD11c promoter activity. The binding site for Pur overlaps sequences that in HL-60 and HeLa cells have been shown to interact with the Ets family of transcription factors (28). This family recognizes DNA in its double-stranded conformation. However, when the single-stranded Pur site is made double-stranded, Pur binding is inhibited, but no additional complexes are observed in PMA-treated U937 cells. Therefore, in contrast to HL-60 and HeLa cells, in PMA-treated U937 cells, Ets factors appear not to bind the -41/-29 region. As a result, the mutations within the -41/-29 region that we have tested in PMA-treated U937 cells appear to reflect only the contribution of Pur to CD11c promoter activity.

Mutation of the -41/-29 region indicates that Pur binding is necessary for the functional activity of the CD11c promoter. Next, we sought to determine whether Pur binding is sufficient to induce such activity. This proved to be the case with Pur expression, causing a 3.5-fold induction of promoter activity in PMA-treated U937 cells. This degree of trans activation compares favorably with that conferred by the four other proteins that individually have been shown to be able to trans-activate the CD11c promoter in myeloid cells. Within the myelomonocytic cell line HL-60 Sp3 causes a 4.3-fold induction, while Sp1 induces 3.4-fold, c-Fos 2.5-fold, and c-Jun 1.8-fold (19, 21). In addition, in HeLa epithelial cells, C/enhancer-binding protein can induce the CD11c promoter by a factor of two (20). Whether Pur contributes to the expression of the CD11c gene in cell types such as HeLa that are not monocytic in origin remains to be determined. The ability of Pur to activate the CD11c promoter in U937 cells was significantly reduced by point mutations that abolish its binding to the -41/-29 region. Therefore, it appears that this region participates in the induction of CD11c expression by Pur. However, since disruption of the -41/-29 region failed to completely abolish Pur trans activation, other regions most likely also mediate its function. Indeed, in support of this hypothesis, we have identified three other regions within the CD11c promoter with the capacity to bind Pur. While it remains to be determined whether these sites function independently or in concert, it is of interest that Pur has the capacity to form homodimers. Consequently, it seems plausible that a Pur protein bound at one site within the CD11c promoter may interact with another bound at a different site. Such interaction could impart a particular architecture to the CD11c promoter facilitating its activation.

In addition to homotypic interaction, Pur has been shown to interact with a range of different cellular proteins (11, 22, 23, 24, 25, 26). Such interactions may account for the complex nature of the supershift obtained upon analysis of the Pur complex that interacts with the -41/-29 region (Fig. 2, lane 7). Heterotypic interactions can either promote or inhibit Pur functional activity. Of the proteins known to interact with Pur, the only one reported to control CD11c expression is Sp1. This factor binds the CD11c promoter at three sites (4, 19, 20, 21, 27). Interestingly, each of these three Sp1 binding sites overlaps or is adjacent to one of the four Pur binding sites that we have identified (Fig. 1). Such close proximity of all the identified Pur and Sp1 binding sites suggested to us that these factors could either combine or compete to activate the CD11c promoter. Our trans activation experiments demonstrated that Sp1 and Pur in fact act together to induce CD11c expression. However, this finding raised a potential paradox since Sp1 binds dsDNA, while Pur exhibits a marked preference for binding ssDNA. Consequently, Pur and Sp1 binding to the same DNA sequence would appear to be mutually exclusive events that compete, not combine, to induce the CD11c promoter. Two answers to this paradox appear possible. The first is that at any given time, Pur and Sp1 bind the CD11c promoter at different sites. The second answer stems from the observation that in the mouse brain, the interaction of Pur and Sp1 has only been observed in the absence of DNA. Such interaction was not seen on a DNA target containing overlapping Sp1 and Pur binding sites (22). Rather, under these circumstances, the interaction of Sp1 with Pur enhanced the ability of Pur to interact with this target. If this situation occurs in the context of U937 cells, then it suggests a mechanism by which Sp1 and Pur cooperate in driving CD11c expression. In this mechanism, Sp1 and Pur would interact away from their overlapping target sequences, causing an increase in the ability of Pur to interact with single-stranded structures within the CD11c promoter. If such a mechanism exists, it must be subject to induction during monocytic differentiation. This must be the case, since we have found that, in combination, Sp1 and Pur are much more effective in activating the CD11c promoter in differentiating U937 cells than in nondifferentiating cells. Two observations indicate how such regulation during U937 differentiation might occur. First, we have demonstrated that the expression of Pur, as assessed by its ability to bind DNA, increases during U937 monocytic differentiation. Second, it has been reported that Pur binds a phosphorylated form of Sp1, not Sp1 that is unphosphorylated. Expression of this phosphorylated form is induced upon both megakaryocytic differentiation of K562 cells and monocytic differentiation of HL-60 cells (29, 30). Consequently, a mechanism can be envisaged during U937 differentiation in which Sp1 becomes phosphorylated, and this phosphorylated form of Sp1 then interacts with induced levels of Pur, causing an increase in the ability of this factor to bind and activate the CD11c promoter.

The expression of a number of mammalian genes is known to be controlled by Pur. This list is composed of the genes encoding myelin basic protein, TGF-1, smooth muscle actin, c-Myc, neuropeptide Y, the ₄ subunit of the nicotinic Ach receptor, the neuron-specific protein FE65, and CD43 (18, 25, 31, 32, 33, 34, 35, 36, 37, 38). The CD11c and CD43 genes are the first described to be controlled by Pur which are expressed specifically in leukocytes and which produce molecules directly responsible for leukocyte adhesion during inflammation. CD43 is a large, highly charged mucin-like transmembrane molecule that maintains resting leukocytes in the circulation by preventing intercellular interaction (39, 40, 41, 42, 43, 44, 45). During inflammation, there is a dramatic down-regulation of CD43 expression mediated by proteolytic cleavage events at the cell surface and repression of transcription directed by the CD43 gene promoter (38, 46, 47, 48, 49, 50). This down-regulation of the antiadhesion molecule CD43 coupled to up-regulation of proadhesive molecules such as CD11c is critical to the inflammatory process, since these changes help leukocytes acquire the adhesive phenotype capable of extravasation. Recently, we have shown that Pur represses expression of the CD43 promoter during U937 differentiation (38). Consequently, Pur is capable of both inducing expression of the proadhesion molecule CD11c and repressing the expression of the antiadhesion molecule CD43. Therefore, it appears that Purrepresents a means by which leukocytes coordinately regulate the expression of proadhesive and antiadhesive forces. A role for Pur in inflammation has previously been implied by its control of TGF-1 expression and its increase in infiltrating eosinophils and activated endothelium during allergic reactions in the rat lung (51). Calcium mobilization is characteristic of the cellular activation that occurs during inflammation. In this regard, it is worth noting that the DNA-binding activity of Pur is increased by its interaction with the calcium-binding protein calmodulin (25).

In conclusion, we have demonstrated that during monocytic differentiation of U937 cells induction of the CD11c promoter is mediated by the combined action of Pur and Sp1. Since Pur binds preferentially to ssDNA, our data suggest that the secondary structure of the CD11c promoter plays a critical role in its regulation.

	Acknowledgments

 
We thank Alexander Georgakis and Fotini Vavva for expert technical assistance. In addition, we thank Edward Johnson and Sharon Barr for helpful discussions during the preparation of this manuscript and for providing the mAb 9C12 and the expression vector pHAPur1. Finally, we thank Jay Chung for providing the Sp1 expression construct p588.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI28465, DK43351, and DK50779, and a research grant from Ortho Biotech (Raritan, NJ). Additional support was provided by Grant DAMD17-00-1-0255. In regard to this grant, the U.S. Army Medical Research Acquisition Activity (Fort Detrick, MD) is the awarding and administering acquisition office.

² Address correspondence and reprint requests to Dr. C. Simon Shelley, Renal Unit, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail address: shelley{at}receptor.mgh.harvard.edu

³ Current address: Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02115.

⁴ Current address: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.

Received for publication October 30, 2001. Accepted for publication February 6, 2002.

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