©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Repressor Activity of CCAAT Displacement Protein in HL-60 Myeloid Leukemia Cells (*)

Patricia M. J. Lievens (§) , Janae J. Donady , Cristina Tufarelli , Ellis J. Neufeld (¶)

From the (1) Division of Pediatric Hematology/Oncology, Children's Hospital, Dana Farber Cancer Institute, and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.


INTRODUCTION

All organisms require a means of preventing expression of genes for potentially costly or toxic functions where and when these genes are not needed. Terminal differentiation in many tissues is accompanied by acquisition of specialized, tissue-specific functions which are stringently repressed in immature cells or cells of other lineages. This is the case for the myeloid oxidase system, which is responsible for bacterial killing but also generates toxic oxygen metabolites. We have studied the mechanism of repression of the myeloid cytochrome heavy chain gene, gp91-phox, whose expression is limited to terminally differentiated phagocytic cells of the monocyte/macrophage or granulocyte lineages. Defects in the gp91-phox gene are responsible for the X-linked form of chronic granulomatous disease. Skalnik et al.(2) proposed that CCAAT displacement protein (CDP),() a DNA binding activity first described in sea urchin (1) , serves as a repressor of the promoter of the gp91-phox gene. To investigate this possibility, we previously purified CDP from HeLa cells and isolated human CDP cDNA (3) . The CDP gene encodes a ubiquitous homeodomain-containing protein of 180-190 kDa, closely homologous to the Drosophila protein Cut. We will refer to the human gene and its protein as CDP/cut. Investigators studying diverse developmental systems have suggested that CDP/cut serves as a repressor of developmentally regulated genes (1, 2, 4, 5, 6, 7, 8) . This hypothesis is in general accord with the proposal of Barberis et al.(1) that CDP binding to promoters of tissue-specific genes is limited to tissues or developmental stages in which the target genes are not expressed. With terminal differentiation, CDP/cut promoter binding is lost, and transcription of tissue-specific genes is activated. Genetic experiments in Drosophila show that Cut is involved in determination of cell fate in several tissue types (9-12), but direct transcriptional regulatory effects of Cut have not been reported.

CDP DNA binding to the gp91-phox promoter is observed in nuclear extracts of immature HL-60 promyelocytic or PLB-985 myelomonocytic leukemia cell lines in continuous culture. With terminal differentiation induced by treatment with retinoic acid/dimethyl formamide (RA/DMF) or phorbol 12-myristate 13-acetate (PMA), CDP DNA binding decreases, and transcription of the gp91-phox target gene ensues. The proximal promoter of the gp91-phox gene appears weak, but exhibits some myeloid specificity (13) . Deletion of the CDP binding site from this promoter increases transcription in transient assays using undifferentiated PLB-985 cells (2) .

We have taken two complementary approaches to examine the potential repressor role of CDP/cut. First, artificial promoters were prepared to study the ability of CDP/cut binding sites to serve as negative regulatory elements in reporters transiently introduced into fibroblasts. In this assay, we have tested CDP/cut binding sites from the human gp91-phox -globin promoters. Second, as a more appropriate in vivo assay, we have asked whether constitutive expression of transfected CDP/cut cDNA in cultured myeloid leukemia cells might inhibit expression of the putative target gene, gp91-phox. Results in both expression systems support the hypothesis of CDP/cut repressor function.


EXPERIMENTAL PROCEDURES

CDP Expression Constructs

For stable transformation experiments, CDP cDNA was cloned into the expression vector RC-CMV as follows: the cDNA was excised from pBluescript KS II with Asp-718 (KpnI site in polylinker 5` to cDNA) and Spe1 (position 5293 in the 3`-untranslated region of CDP cDNA; Ref 3). The overhanging ends were filled in with Klenow DNA polymerase (14) . EcoRI/NotI adapters (Pharmacia Biotech Inc.) were then added with T4 DNA ligase. This cDNA fragment was then digested with NotI and inserted into RC-CMV (Invitrogen) digested with NotI and treated with alkaline phosphatase (Promega Biotech). The CMV promoter was expected to direct synthesis of a 6.3-kb mRNA transcript from this construct. For transient co-transfection assays, full-length CDP or fragments thereof were cloned into the expression vector pMT2, driven by the adenovirus major late promoter (15). These effector constructs, shown in Fig. 1 , included full-length CDP in sense or antisense (``CDP anti'') orientation, the 5` 1.0-kb EcoRI fragment encoding 334 nucleotides at the N terminus (Nterm); a 2.9-kb 3` EcoRI fragment including cut repeats 2 and 3, and the homeodomain region (``CR2-Cterm'').


Figure 1: Effector constructs used for stable and transient transfections. Constructs were cloned into expression vectors RC-CMV (for stable transformants) and pMT2-ATG (for transient transfections) in the direction shown by the arrows, as described under ``Experimental Procedures.'' Domain positions (see Ref. 3) are shown as boxes. Nterm, 1.0-kb EcoRI fragment; CR2-Cterm, 3.0 kb to 3` end; CDP, 5.1 kb encompassing entire coding region.



Probes and Plasmids

-Actin cDNA (16) was a gift from Dr. M. C. Simon. p47 (17) and p67 cDNA (18) were kindly provided by Dr. S. Chanock.

Myeloid Differentiation

HL-60 cells were obtained from the American Type Culture Collection. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. To induce monocyte-macrophage differentiation, growing cells (5 10/ml) were suspended in 30 ml of medium containing 2 µM PMA (Sigma) in each of five P150 dishes. Cells adhered to the dishes within 24 h and were harvested by scraping into phosphate-buffered saline at 48 h of induction. For granulocytic differentiation, HL60 cells at a density of 5 10/ml were treated with 1.3% (v/v) dimethyl sulfoxide (MeSO) for 4 days or with 1 µM retinoic acid for 4 days plus 0.5% (v/v) dimethylformamide added to the medium 48 h before harvesting. Fresh medium containing inducing agent was added to the cells 48 h prior to harvesting as needed to keep the cell density 5 10/ml.

RNA Analysis

Total RNA from 0.5-1 10 induced or uninduced HL-60 cells was extracted with RNAzol (Tel-Test B; Friendswood Tx) as described (19) . RNA in 0.5% SDS was adjusted to 20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 1 mM EDTA, and subjected to two rounds of selection on columns containing 25 mg (0.2 ml bed volume) of oligo(dT)-cellulose (Pharmacia) as described (14) . RNA was quantified by UV absorbance, and equal amounts of each sample were loaded on 1% agarose formaldehyde gels. RNA was blotted onto Magnagraph nylon membranes as suggested by the manufacturer (Micron Technologies, Westborough, MA). Northern blots were performed in standard fashion (14). For sequential probing, each blot was stripped by washing two to three times in 0.1% SDS at 100 °C.

Quantitation

Northern blots were quantified by PhosphorImager analysis, using ImageQuant software (Molecular Dynamics). For each band analyzed, background PhosphorImager counts were subtracted from an adjacent area of the scan. -Actin signal was used to normalize for differences in gel loading.

Transient Transfection Reporter Constructs

Artificial promoter fragments were cloned into the human growth hormone reporter plasmid TATA-GH. This minimal promoter construct includes the rabbit -globin TATA box in -GH (20) . Complementary oligonucleotides for double-stranded fragments were synthesized on an ABI model 400 DNA synthesizer with overhanging ends to facilitate cloning. These sequences (upper strand shown for clarity) were as follows. ``'', human -globin promoter fragment: TGCCTTGACCAATAGCCTTGACAAGGCAAACTTGACCAATAGTCTTAGAGTATCCAGTG; 5` restriction site EcoRI, 3` site BamHI. ``gp91,'' gp91-phox promoter fragment: GCTTTTCAGTTGACCAATGATTATTAGCCAATTTCTGATAAAAGAAAAGGAAACCGATTGC; BamHI sites were at both ends. ``SP1,'' GAGGCGTAACATAGGGGCGTAGCATAGAGGCGGAA. 5` restriction site SalI, 3` site HindIII.

Transient Transfection Assays

NIH 3T3 or HeLa cells (from ATCC) were plated in Costar six-well dishes at 5 10 cells/well. 24 h later, calcium phosphate-mediated transfection was performed as described (22) . In each case, 5 µg of reporter construct was added to each well. For effector plasmids, either 10 µg of pMT2 CDP or, as control, pMT2 alone or pBluescript II (Stratagene) were included to keep the total DNA amount to 15 µg. We used the plasmid pSV-gal, 2 µg/transfection, to control for transfection efficiency. 48 h after transfection, medium was collected, centrifuged briefly, and assayed for human growth hormone by radioimmunoassay (Nichols Institute, San Juan Capistrano, CA). Cells were collected by trypsinization, the trypsin was neutralized with serum, cells were lysed by freeze-thawing, and -galactosidase was assayed as described (14).

Selection of Stable Transformants

HL-60 cells were transfected with RC-CMV-CDP or with RC-CMV vector alone by electroporation at 960 µF in a Bio-Rad gene pulser as described (23) . After 24 h of recovery in 20% fetal calf serum, G418 (Life Technologies, Inc.), 1.8 µg/ml, was added to cultures for selection, and cells were plated at limiting dilution. Twenty clones from the CDP transformation were obtained. Of these, 10 had intact transgenes, and one, HL-P6, expressed the expected 6.3-kb transgene mRNA. This line, plus vector-only line HL-V4 were used for subsequent experiments.


RESULTS

CDP Binding Sites from gp91-phox and -Globin Promoters Are Negative Regulatory Domains When Assayed in Artificial Promoter Constructs in Fibroblasts

Skalnik et al.(2) demonstrated that deletion of the CDP binding site of the gp91-phox promoter led to increased transcriptional activity in transient assays in undifferentiated PLB-985 myeloid leukemia cells. To test the ability of CDP/cut binding sites to act as negative regulatory domains in non-myeloid cells, we created artificial ``active'' promoters into which we placed CDP/cut binding sites from the gp91-phox gene (2, 3) or from the human -globin gene (Ref. 6; see Fig. 2A). For a basal promoter, we chose the TATA-growth hormone plasmid described by Martin et al.(20) . For an activation domain, we used a triplet of consensus sites for the ubiquitous transcriptional activator SP1. Constructs were transfected into 3T3 murine fibroblasts, which contain abundant CDP/cut (not shown).


Figure 2: CDP binding site is a negative regulatory domain in artificial promoter constructs in non-myeloid cells. A, reporter constructs were prepared from double-stranded oligonucleotides in TATA-GH as described under ``Experimental Procedures.'' hGH, human growth hormone gene; TATA, TATA region from rabbit -globin gene; SP1, artificial triplet of consensus SP1 binding sites (21); gp91, CDP binding site from duplicated CCAAT box region of gp91-phox proximal promoter (``FP'' in Ref. 3); , CCAAT-box region of human -globin gene (Ref. 6 and ``Experimental Procedures''). B, results of transient transfections in 3T3 cells. Reporter plasmids as shown in A were transfected in combination with CDP in PMT-2 or control plasmid (pBluescript II), and human growth hormone assays were performed on duplicate or triplicate wells. To correct for transfection efficiency which varied among experiments, results are normalized to the SP1-TATA positive control (mean = 100%). Bars represent mean ± S.D. of three or more determinations. Note that constructs including the gp91 site are repressed with or without co-transfection of exogenous CDP. C, -globin CDP binding site depends on expression of exogenous CDP cDNA. The experiment was performed as in B. Effectors included CDP, antisense CDP cDNA (anti), a construct including the 3` half of the cDNA, encompassing CR2 through the homeodomain (CR2-Cterm) in sense and antisense orientation as shown in Fig. 1.



Full- or partial-length CDP/cut expression constructs used for co-transfection were prepared as described under ``Experimental Procedures,'' and are shown in Fig. 1 . Reporter constructs tested are shown in Fig. 2A. Growth hormone expression results of constructs containing the gp91-phox CDP site are shown in Fig. 2B. As expected, the TATA box alone (TATA) or gp91-TATA constructs resulted in minimal human growth hormone expression, and the positive control construct SP1-TATA exhibited abundant activity (set to 100%). Exogenous CDP effector plasmid had no effect on this reporter plasmid in the co-transfection assay. However, reporters containing the gp91-phox CDP binding site had substantially lower transcriptional activity, less than 25% of the relevant control without a CDP binding site. We infer that this decrease was due to binding of endogenous murine CDP/cut to the CDP binding site (Fig. 2B).

To demonstrate that this was the case, we repeated these transient transfection assays using the CDP site from the -globin promoter, which binds approximately 40-fold less tightly to CDP than does the gp91-phox site. In this context, repression of reporter constructs requires exogenous CDP expression. As shown in Fig. 2C, constructs SP1-TATA and SP1--TATA had similar expression in the absence of exogenous CDP/cut effectors. Co-transfection with CDP/cut cDNA or the truncated CR2-Cterm expression construct (Fig. 1) resulted in repression of transcription from SP1--TATA, while inactive antisense effectors (``CDP anti'' and ``CR2-Cterm anti'') had no effect on basal transcription. Similarly, the Nterm construct, which lacks DNA binding activity, had no effect on transcription from SP1--TATA.

gp91-phox mRNA Induction Is Reduced in an HL-60 Stable Transformant Constitutively Expressing CDP mRNA, While Induction of Other Terminally Differentiated Myeloid Genes Is Unaffected

Cell lines were selected as described under ``Experimental Procedures.'' RNA from a CDP-expressing stable transformant line of HL-60 cells, designated HL-P6, and a vector-only stably transformed control line (HL-V4) were subjected to Northern blot analysis to demonstrate expression of transgene CDP mRNA in the constitutively expressing line (Fig. 3A). The expected 6.3-kb transcript was found in HL-P6 (lanes 1-3, see arrow), but not in HL-V4 (lanes 4-6). We examined the induction of gp91-phox and two cytosolic components of the myeloid oxidase system, p47 and p67, in HL-P6 and the control line HL-V4. Three different inducing agents were tested as described under ``Experimental Procedures'': RA/DMF or MeSO (granulocyte differentiation) and PMA (monocyte/macrophage differentiation).


Figure 3: Constitutive CDP expression reduces gp91-phox mRNA induction in myeloid leukemia cells treated with phorbol ester or retinoic acid/dimethylformamide. HL-P6 stable transformants (lanes 1-3) or HL-V4 control (lanes 4-6) were grown in medium alone (lanes 1 and 4) or induced toward monocyte/macrophage differentiation with 2 µM PMA for 48 h (lanes 2 and 5) or with RA/DMF (1 µM retinoic acid for 4 days, plus 0.5% dimethylformamide for 48 h) (lanes 3 and 6). Oligo(dT)-selected mRNA, 2 µg/lane, was subjected to Northern blot analysis and probed sequentially with each of the following cDNA species. A, CDP 5` 1-kb EcoRI fragment. Endogenous CDP transcripts, constitutively expressed with induction, co-migrate with 28 S RNA and at 12 kb. The 3.4-kb band represents an alternatively spliced product of the CDP gene. The 6.3-kb transgene (arrow) is limited to HL-P6 stable transformants (lanes 1-3). B, gp91-phox mRNA. Arrow denotes the expected transcript. Induction is evident in HLV4 controls treated with PMA (lane 5) or RA/DMF (lane 6). Induction is substantially less in HL-P6 (lanes 2 and 3). C, p67 component of myeloid oxidase. Induction in HL-P6 and HL-V4 are similar. D, p47 component of myeloid oxidase. Arrow denotes the expected transcript. E, -actin, which serves as a control for RNA recovery and gel loading.



In HL-P6 cells induced toward monocyte/macrophage differentiation with PMA (Fig. 3B, lane 2) or toward granulocyte differentiation with RA/DMF (Fig. 3B, lane 3), gp91-phox induction was reduced compared to the HL-V4 control (Fig. 3B, lane 5, PMA and lane 6, RA/DMF). However, induction of mRNA for the cytosolic oxidase components p47 and p67, which are not thought to be controlled by CDP, was equivalent in the two cell lines (Fig. 3, C and D). The differences in gp91-phox mRNA levels were not due to differences in RNA loading on the Northern blot, as shown by actin recovery (Fig. 3E). Results from the Northern blot in Fig. 3 and from duplicate or triplicate experiments were quantified by PhosphorImager analysis as described under ``Experimental Procedures.'' For RA/DMF, gp91-phox induction in HL-P6 was 44% ± 11% of that observed in HL-V4 cells (mean ± S.D. of three experiments). Compared to the results with RA/DMF, PMA induction of gp91-phox was more curtailed in HL-P6 (16 ± 3% of HL-V4 levels; mean of duplicate experiments). The difference between PMA and RA/DMF induction in HL-P6 cells was significant by two-tailed t test (p < 0.05). p47 and p67 induction as quantified by PhosphorImager revealed slightly greater induction in both cytosolic oxidase components' mRNA in HL-P6 compared to control HL-V4 (p67: 108% of control to PMA (n = 1), 148% to RA/DMF (n = 3); p47: 122% of control to PMA (n = 2), 137% of control induction to RA/DMF (n = 2). These differences in p47 and p67 induction between HL-V4 and HL-P6 were not statistically significant.

Since the difference in gp91-phox induction between RA/DMF and PMA induction might have been due to the differences in granulocyte versus monocyte differentiation, we also measured gp91-phox and p67 induction in response to dimethyl sulfoxide. MeSO promotes granulocytic induction, as does RA/DMF. However, the degree of inhibition of gp91-phox induction with MeSO was equivalent to that seen with PMA and more pronounced than that seen with RA/DMF (gp91-phox induction 14% ± 2% of HL-V4 level, mean of duplicate experiments; autoradiograph not shown).

Morphologic Differentiation Is Normal in HL-P6 Stable Transformants

We used standard morphologic criteria to assess differentiation of HL-P6 and HL-V4 cells in response to inducing agents. For PMA induction toward monocyte-macrophage lineage, differentiation refers to adhesion to the culture dish and extension of processes. For RA/DMF induction, differentiation was assessed as progression from promyelocytic phenotype to myelocytes and metamyelocytes on Wright-stained Cytospin preparations from the induced cultures. The degree of differentiation was similar for HL-P6 and HL-V4. For RA/DMF treatment, approximately 70% of cells in both cell lines had progressed along granulocytic development from promyelocytic appearance to the myelocyte or metamyelocyte stages. For PMA induction along the monocyte/macrophage pathway, floating cells were removed prior to analysis, so that essentially all of the cells analyzed had begun to differentiate. We conclude that constitutive expression of CDP/cut in the HL-P6 cell line had no observable effect on morphologic differentiation but had a specific negative regulatory effect on the expression of its target gene, gp91-phox.


DISCUSSION

We have demonstrated that constitutive expression of CDP cDNA in a myeloid leukemia cell line inhibits expression of the target gene, gp91-phox, upon terminal myeloid differentiation. Furthermore, the CDP/cut binding site from the gp91-phox promoter acted as a negative regulatory domain in artificial promoter constructs transfected into non-myeloid cells. Constitutive CDP/cut expression did not prevent normal morphologic differentiation, nor did it limit induction of other myeloid oxidase components which are not thought to be regulated by CDP/cut. We infer, therefore, that CDP/cut expression is likely to act directly on the gp91-phox promoter, and that lack of induction of gp-91phox in the stably transformed cells was not due to a general failure of terminal differentiation. These data provide direct evidence of CDP/cut repressor function and demonstrate that decreased CDP/cut DNA binding is not a ``master switch'' for terminal myeloid differentiation.

Homologs of Drosophila cut have now been described in several eukaryotes. The nomenclature in these reports and those concerning CDP/cut is not uniform, and the basis of cloning in each case has been different. summarizes and compares the nomenclature, experimental systems, and postulated function of CDP/cut in published reports from several laboratories. In some, but not all of these cases, CDP/cut has been shown or postulated to act as a repressor protein. It remains to be determined how CDP might act to increase or decrease transcriptional activity. The CDP/cut protein contains four DNA binding sites with overlapping but distinct sequence specificities (25, 26, 27) . It is likely that CDP/cut recognizes a wide variety of target genes, and the protein might repress or activate transcription based on any of several factors: (i) which of its DNA binding sites are in contact with promoter or enhancer elements; (ii) which other proteins contact CDP/cut or adjacent elements of target DNA; (iii) regulatory modifications, which might vary from cell type to cell type. At least two types of potential regulatory modifications have been reported. CDP/cut is a phosphoprotein (Ref. 24)() and changes in phosphorylation may alter DNA binding or function. The mechanism by which CDP DNA binding decreases with myeloid differentiation is unknown. Endogenous CDP mRNA levels do not change substantially with differentiation (Fig. 3A), so regulation of DNA binding is not at the level of CDP transcription or message stability. CDP mRNA also exhibits complex alternative splicing, with at least three major species, and perhaps several minor splice variants, whose expression with differentiation has not been studied. Variations among the cut homology family members in rodents (4, 24) and dog (8) may represent splice variants as well.

The degree of inhibition of gp91-phox expression in HL-P6 cells compared to control was greater for PMA and MeSO induction (gp91-phox induction approximately 15% of control) than for RA/DMF induction (about 40% of control). Although the mechanism for this difference is unknown, it suggests that different inducing agents may utilize different cellular signaling pathways to accomplish gp91-phox induction.

In numerous attempts with several different vectors, we isolated only a single cell line with stable, constitutive expression of CDP/cut: HL-P6. The difficulty in selecting additional lines is apparently unique to myeloid cells. Using several different expression vectors and two selectable markers, we found that with the exception of HL-P6, screening for stable lines by drug resistance yielded either cells with rearranged or deleted CDP/cut transgenes or cells which failed to express transgene CDP from an apparently intact construct. This occurred not only in HL-60 cells, but in the human myelomonocytic leukemia cell line PLB-985. On the other hand, stable transformants of human erythroid-like K562 cells expressing high levels of CDP mRNA and protein were readily selected (data not shown). We postulate that high level expression of CDP/cut is either toxic to myeloid cells or leads to slow growth. The results with line HL-P6 are unlikely to be due to a nonspecific toxic effect of the transformation or stable selection process, because myeloid differentiation is otherwise normal in this line.

The present results illustrate one way in which a ubiquitous, general repressor can participate in tissue-specific gene expression, acting on only a subset of myeloid-specific genes, not on the overall differentiation program. We propose that in myeloid and non-myeloid cells, de-repression of a subset of genes controlled by CDP could serve as a final step toward fully differentiated function in cells already committed to a particular lineage.

One may also speculate on the relationship between control of cell growth and CDP/cut function. In the systems for which CDP de-repression has been implicated (), terminal differentiation is accompanied by the arrest of cell growth. Cessation of cell growth alone, either by serum starvation or pharmacologic cell cycle arrest, is not sufficient for CDP de-repression of gp91-phox in myeloid leukemia cells in culture. However, in normal differentiation, growth control and CDP/cut de-repression may be controlled by similar signals. Understanding what these signals may be has particular relevance to the study of myeloid leukemias, in which terminal differentiation fails and unregulated growth results.

  
Table: Reported Studies of CDP/cut: numerous designations and postulated transcriptional regulatory roles



FOOTNOTES

*
This research was supported by National Institutes of Health Grants DK01977 and HL49196. 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.

§
Present address: Dept. of Neuropathology, Instituto Carlo Besta, Milan, Italy.

To whom correspondence and reprint requests should be addressed: Division of Hematology, Enders 720, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-8183; Fax: 617-355-7262.

The abbreviations used are: CDP, CCAAT displacement protein; PMA, phorbol 12-myristate 13-acetate; RA/DMF retinoic acid/dimethylformamide; MeSO, dimethyl sulfoxide; CMV, cytomegalovirus; kb, kilobase(s).

P. M. J. Lievens and E. J. Neufeld, unpublished results.


ACKNOWLEDGEMENTS

We thank Barbara Aufiero for performing the binding assay comparison of -globin and gp91-phox CDP binding site affinities and for helpful discussions. We gratefully acknowledge the support and advice of Stuart Orkin throughout the course of these studies.


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