Sp1 and C/EBP are necessary to activate the lactoferrin gene promoter during myeloid differentiation.

In this study, we sought to identify factors responsible for the positive modulation of lactoferrin (LF), a neutrophil-specific, secondary-granule protein gene. Initial reporter gene transfection assays indicated that the first 89 base pairs of the LF promoter are capable of directing myeloid-specific LF gene expression. The presence of a C/EBP site flanked by 2 Sp1 sites within this segment of the LF promoter prompted us to investigate the possible role of these sites in LF expression. Cotransfection studies of LF-89luc plasmid with increasing concentrations of a C/EBPalpha expression vector in myeloid cells resulted in a linear transactivation of luciferase reporter activity. Electrophoretic mobility shift assays found that the C/EBP site is recognized by C/EBPalpha and that both LF Sp1 binding sites bind the Sp1 transcription factor specifically in myeloid cells. Mutation of either Sp1 site markedly reduced activity of the LF-89luc plasmid in myeloid cells, and neither Sp1 mutant plasmid was transactivated by a C/EBPalpha expression plasmid to the same extent as wild-type LF-89luc. We also transfected LF-89luc into Drosophila Schneider cells, which do not express endogenous Sp1, and demonstrated up-regulation of luciferase activity in response to a cotransfected Sp1 expression plasmid, as well as to a C/EBPalpha expression plasmid. Furthermore, cotransfection of LF-89luc plasmid simultaneously with C/EBPalpha and Sp1 expression plasmids resulted in an increase in luciferase activity greater than that induced by either factor alone. Taken together, these observations indicate a functional interaction between C/EBP and Sp1 in mediating LF expression. (Blood. 2000;95:3734-3741)

Characterization and localization to chromosome 7 of psihGABPalpha, a human processed pseudogene related to the ets transcription factor, hGABPalpha.

GABP is a heteromeric transcription factor complex which consists of the ets related protein, GABPalpha, and the Notch-related protein, GABPbeta. We isolated a human genomic DNA fragment which is highly homologous and colinear with human GABPalpha cDNA, but which lacks introns. This processed pseudogene, psihGABPalpha, is expressed as RNA in U937 human myeloid cells, but a mutation at the site that corresponds to the ATG start methionine codon prevents its translation into protein. The pseudogene was localized to chromosome 7 using a somatic cell hybrid mapping panel and it is not syntenic with authentic GABPalpha, which was localized to chromosome 21. We have identified psihGABPalpha, a novel, GABPalpha-related processed pseudogene which is expressed as a RNA transcript in human myeloid cells.

Activation of utrophin promoter by heregulin via the ets-related transcription factor complex GA-binding protein alpha/beta.

Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate that ets-related GA-binding protein alpha/beta transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein alpha/beta complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal-regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne's muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.

An enhancer located between the neutrophil elastase and proteinase 3 promoters is activated by Sp1 and an Ets factor.

The adjacent neutrophil elastase, proteinase 3, and azurocidin genes encode serine proteases expressed specifically in immature myeloid cells. Subclones of a 17-kilobase (kb) murine neutrophil elastase genomic clone were assessed for their ability to stimulate the neutrophil elastase promoter in 32D cl3 myeloid cells. Region -9.3 to -7.3 kb stimulated transcription 7-fold, whereas other genomic segments were inactive. This enhancer is located in the second intron of the proteinase-3 gene and so may regulate more than one gene in the myeloid protease cluster. Deletional analysis of the enhancer identified several segments which activated the neutrophil elastase and thymidine kinase promoters 3-6-fold. The most active segment was a 220-base pair region centered at -8.6 kb, which activated transcription 31-fold. This segment contains an Sp1 consensus site, which bound Sp1, flanked by two Ets family consensus sequences, which bound PU.1, GABP, and an Ets factor present in myeloid cell extracts. Mutation of the Sp1-binding site reduced enhancer activity 8-fold in 32D cl3 cells, and mutation of either or both Ets-binding sites reduced activity 3-4-fold. Sp1 activated the distal enhancer 5-fold, GABP 3-fold, and the combination 8-fold in Schneider cells.

Sp1 cooperates with the ets transcription factor, GABP, to activate the CD18 (beta2 leukocyte integrin) promoter.

CD18, the beta chain of the leukocyte integrins, plays a crucial role in immune and inflammatory responses. CD18 is expressed exclusively by leukocytes, and it is transcriptionally regulated during the differentiation of myeloid cells. The ets factors, PU.1 and GABP, bind to three ets sites in the CD18 promoter, which are essential for high level myeloid expression of CD18. We now identify two binding sites for the transcription factor, Sp1, that flank these ets sites. Sp1 is the only factor from myeloid cells that binds to these sites in a sequence-specific manner. Mutagenesis of these sites abrogates Sp1 binding and significantly reduces the activity of the transfected CD18 promoter in myeloid cells. Transfection of Sp1 into Drosophila Schneider cells, which otherwise lack Sp1, activates the CD18 promoter dramatically. GABP also activates the CD18 promoter in Schneider cells. Co-transfection of Sp1 and GABP activates CD18 more than the sum of their individual effects, indicating that these factors cooperate to transcriptionally activate myeloid expression of CD18. These studies support a model of high level, lineage-restricted gene expression mediated by cooperative interactions between widely expressed transcription factors.

GABP cooperates with c-Myb and C/EBP to activate the neutrophil elastase promoter.

Neutrophil elastase (NE) is a serine protease that is transcriptionally regulated during early myeloid differentiation. The murine NE (mNE) promoter contains functionally important c-Myb, C/EBP, and ets binding sites. Deletion of the ets site reduced promoter activity by 90%. Although the ets transcription factor, PU.1, bound to this ets site, it only modestly activated the mNE promoter. Here, we show that a second transcription factor from myeloid cells-GABP-binds to the mNE ets site but strongly activates the mNE promoter. GABP is a heteromeric transcription factor complex that consists of GABP alpha, an ets factor, and GABP beta, a Notch-related protein. GABP alpha bound to the mNE ets site and, in turn, recruited GABP beta to form a transcriptionally active complex. GABP alpha and PU.1 competed with each other for binding to the mNE ets site. GABP increased the activity of the mNE promoter sevenfold in U937 myeloid cells. GABP cooperated with c-Myb and C/EBP alpha to activate the mNE promoter more than 85-fold in otherwise nonpermissive, nonhematopoietic NIH 3T3 cells. Thus, GABP binds to the crucial mNE promoter ets site and powerfully activates its expression alone and in cooperation with the transcription factors c-Myb and C/EBP.

GABP and PU.1 compete for binding, yet cooperate to increase CD18 (beta 2 leukocyte integrin) transcription.

CD18 (beta 2 leukocyte integrin) is a leukocyte-specific adhesion molecule that plays a crucial role in immune and inflammatory responses. A 79-nucleotide fragment of the CD18 promoter is sufficient to direct myeloid transcription. The CD18 promoter is bound by the B lymphocyte- and myeloid-restricted ets factor, PU.1, and disruption of the PU.1-binding sites significantly reduces promoter activity. However, PU.1 alone cannot fully account for the leukocyte-specific and myeloid-inducible transcription of CD18. We identified a ubiquitously expressed nuclear protein complex of extremely low electrophoretic mobility that also binds to this region of the CD18 promoter. This binding complex is a heterotetramer composed of GABP alpha, and ets factor, and GABP beta, a subunit with homology to Drosophila Notch. GABP alpha competes with the lineage restricted factor, PU.1, for the same critical CD18 ets sites. The CD18 promoter is activated in myeloid cells by transfection with both GABP alpha and GABP beta together, but not by either factor alone. Transfection of non-hematopoietic cells with the two GABP subunits together with PU.1 is sufficient to activate CD18 transcription in otherwise non-permissive cells. Thus, GABP and PU.1 compete for the same binding sites but cooperate to activate CD18 transcription.

Differential regulation of JunD by dihydroxycholecalciferol in human chronic myelogenous leukemia cells.

1,25-Dihydroxyvitamin D3 inhibits the proliferation of the chronic myelogenous leukemia cell line RWLeu-4 but not the resistant variant, JMRD3. Although these cells exhibit no detectable differences in the vitamin D receptor, alterations in the interaction of nuclear extracts with the osteocalcin-1,25-dihydroxyvitamin D3-response element are noted. It is shown herein that the 1,25-dihydroxyvitamin D3 receptor binds to the osteocalcin-1,25-dihydroxyvitamin D3-response element along with activator protein-1 (AP-1) complexes and that the DNA binding activities of members of the Jun and Fos proto-oncogene families, which make up the AP-1 transcription factor, are differentially regulated by 1,25-dihydroxyvitamin D3. It is shown that JunD DNA binding activity is enhanced by 1,25-dihydroxyvitamin D3 during cell cycle arrest in the sensitive cells but is decreased in the resistant cells. These results suggest that the level of JunD DNA binding activity may be a critical factor in the regulation of proliferation.

CD18 (beta 2 leukocyte integrin) promoter requires PU.1 transcription factor for myeloid activity.

Normal cellular differentiation is linked to tightly regulated gene transcription. However, the DNA elements and trans-acting factors that regulate transcription in myeloid cells are poorly defined. CD18, the beta chain of the leukocyte integrins, is transcriptionally regulated during myeloid differentiation. The CD18 promoter is active after transfection into myeloid cells. We demonstrate that a region of the CD18 promoter that contains two binding sites for the PU.1 transcription factor is required for activity in myeloid cells. These sites are bound by in vitro translated PU.1 and by PU.1 from myeloid nuclear extracts. Mutagenesis of these sites abrogates binding by PU.1 and substantially decreases promoter activity in myeloid cells. Thus, the leukocyte-specific transcription factor PU.1 is required for myeloid activity of CD18.

Cloning and analysis of the CD18 promoter.

CD18, the common beta chain of the leukocyte integrin adhesion proteins, is expressed exclusively by myeloid cells and lymphocytes. During myeloid differentiation, the increase in CD18 cell surface expression is paralleled by increased CD18 messenger RNA levels. Nuclear run-on studies show that CD18 expression is transcriptionally regulated during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 monocytic differentiation. The CD18 transcriptional start site was defined by primer extension and RNAse protection. The gene encoding CD18 was cloned and a fragment that overlaps the transcriptional start site was isolated. DNA sequence analysis of this promoter fragment identified potential AP-1 elements that may mediate the TPA transcriptional response. The CD18 promoter also contains a putative binding site for PU.1, a leukocyte-specific transcription factor. DNA elements resembling those found in other myeloid and integrin promoters were identified. The CD18 promoter fragment was linked to the luciferase reporter gene, electroporated into the U937 monocytic cell line, and its expression increased after exposure to TPA. Thus, CD18 may serve as a model for identifying the cis elements and trans-acting factors that regulate gene expression during myeloid differentiation.

Analysis of the mechanism of anagrelide-induced thrombocytopenia in humans.

Anagrelide is a new therapeutic compound recently demonstrated to have a rapid and selective thrombocytopenic effect in humans. The effects of anagrelide were evaluated in plasma clot and liquid suspension cultures of optimally stimulated normal human peripheral blood megakaryocyte progenitors in order to determine the mechanism of its thrombocytopenic activity. In plasma clot cultures, at clinically relevant, therapeutic concentrations (5 to 50 ng/mL), anagrelide exerted no significant inhibitory effect on megakaryocyte colony numbers or colony size. Only at anagrelide concentrations of 10 to 500 times therapeutic doses did anagrelide inhibit megakaryocyte colony development: an anagrelide concentration of 5 micrograms/mL reduced colony numbers by 57% and colony size by 31%. In contrast, lower, therapeutic anagrelide concentrations exerted profound effects in liquid culture on megakaryocyte cytoplasmic maturation, size, and DNA content. When present for the entire 12-day culture duration, anagrelide induced left-shifted megakaryocyte maturation and reduced both megakaryocyte ploidy and megakaryocyte diameter. Anagrelide, at concentrations of 5 to 50 ng/mL, shifted the modal cultured megakaryocyte morphologic stage from III to II, reduced the model ploidy value from 16N to 8N, and decreased the mean megakaryocyte diameter by up to 22%, from 27.6 microns to 21.6 microns. Megakaryocyte diameter was significantly reduced in most instances, even when analyzed as a function of morphologic stage. When anagrelide was added to the cultures after 6- and 9-day delays (during the final 6 and 3 days, respectively, of culture), similar inhibitory effects on megakaryocyte maturation stage and ploidy distribution were observed. However, the magnitude of the left-shift in ploidy appeared to be less as the duration of anagrelide exposure was reduced. Conversely, megakaryocyte diameter was not significantly affected by the shorter 3- and 6-day anagrelide exposures. These data indicate that therapeutic concentrations of anagrelide influence primarily the postmitotic phase of megakaryocyte development, decreasing platelet production by reducing megakaryocyte size and ploidy, as well as by disrupting full megakaryocyte maturation. Inhibition of megakaryocyte diameter appears to require more prolonged anagrelide exposure than inhibition of maturation stage and ploidy. The molecular mechanisms responsible for the inhibitory effects of anagrelide on megakaryocytopoiesis remain to be defined.

Characterization of the myeloid-specific CD11b promoter.

The CD11b/CD18 heterodimeric surface antigen is expressed exclusively on human monocytes, macrophages, granulocytes, and natural killer cells. During differentiation of myeloid cell lines, CD11b steady state messenger RNA levels increase significantly; we show here that CD11b transcription rates increase commensurately. A 1.7-kb fragment of CD11b 5' flanking sequence directs expression of a reporter gene specifically in myeloid cell lines. Deletion analysis localizes elements directing high levels of tissue-specific reporter gene expression to the 412 bp proximal to the transcriptional start site. This sequence contains two consensus binding sites for Sp1, a GATA motif, and a purine-rich sequence that presents potential binding sites for members of the ets family of genes. Analysis of this promoter should result in the isolation of myeloid-specific transcription factors and the development of methods to direct the myeloid-specific expression of heterologous genes.

Differential expression of CD11b/CD18 (Mo1) and myeloperoxidase genes during myeloid differentiation.

During the course of differentiation of early human myeloid cells toward monocytes and granulocytes, cell surface expression of the cell adhesion molecule, CD11b/CD18 (Mo1) increases dramatically and expression of myeloperoxidase (MPO), a bacteriocidal enzyme, decreases markedly. Using the inducible promyelocytic cell line HL-60 as a model, we studied the mRNA expression of these genes. Differentiation of these cells along both a monocytic and a granulocytic pathway demonstrated that the mRNA levels of the two subunits of CD11b/CD18 increased in a pattern temporally and quantitatively similar to the increase in cell surface expression of this heterodimer. In contrast, the expression of MPO mRNA decreased in a temporal and quantitative pattern similar to the known decrease in MPO protein during differentiation, suggesting that regulation of these myeloid-specific proteins may occur at the level of mRNA expression. These findings have important implications with regard to the nature of the block in differentiation in acute nonlymphocytic leukemia and the regulation of myeloid gene expression.