C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter

The murine neutrophil elastase (NE) gene is expressed specifically in immature myeloid cells. A 91-bp NE promoter region contains three cis elements which are conserved evolutionarily and are essential for activation of the promoter in differentiating 32D cl3 myeloid cells. These elements bound c-Myb (at -49), C/EBPalpha (at -57), and PU.1 (at -82). In NIH 3T3 cells, the NE promoter was activated by c-Myb, C/EBPalpha, and PU.1, via their respective binding sites. Cooperative activation was seen by any combination of c-Myb, C/EBPalpha, and PU.1, including all three together, again via their DNA-binding sites. In CV-1 cells, but not in NIH 3T3 cells, cooperation between Myb and C/EBPalpha depended on the integrity of the PU.1-binding site. In addition to C/EBPalpha, C/EBPdelta strongly activated the NE promoter, alone or with c-Myb, but C/EBPbeta was less active. Either of C/EBPalpha's two transactivation domains cooperatively activated the promoter with c-Myb, in both NIH 3T3 and 32D c13 cells. Synergistic binding to DNA in a gel shift assay between C/EBPalpha, c-Myb, and PU.1 could not be demonstrated. Also, separation of the C/EBP- and c-Myb-binding sites by 5 or 10 bp did not prevent cooperativity. These results suggest that a coactivator protein mediates cooperative activation of the NE promoter by a C/EBP and c-Myb. These factors, together with PU.1, direct restricted expression of the NE promoter to immature myeloid cells.     

Human proteinase-3 expression is regulated by PU.1 in conjunction with a cytidine-rich element

Human proteinase-3 is one of three serine proteinases present in the azurophil granules of polymorphonuclear leukocytes along with elastase and cathepsin G. Proteinase-3 gene expression is confined to the promyelocytic stage of polymorphonuclear leukocyte maturation. The present investigation identifies elements responsible for this highly controlled tissue- and developmental-specific expression of proteinase-3. Within the first 200 base pairs of the proteinase-3 promoter, two elements were identified as important for expression, these elements at -101 and -190 confer the majority of the activity. The element at -101 has a PU.1 consensus. It binds a myeloid nuclear protein of approximately 45 kDa that "supershifts" with PU.1 antibody and is competed by the CD11b PU.1 element. The element at -190 has a core sequence of CCCCGCCC (CG element). The cytidines but not the guanidine are essential for promoter activity. The CG element binds a second nuclear protein with a molecular mass of approximately 40 kDa that is found in cells of myeloid lineage as well as non-myeloid HeLa cells. However, the proteinase-3 promoter is not active in HeLa cells which suggests that the CG element alone is not sufficient for proteinase-3 gene expression. Maturation of promyelocytic cells results in an inhibition of proteinase-3 gene expression and a reduction in nuclear protein binding to the PU.1 and CG elements. Similar elements occur in the elastase and cathepsin G promoters. Using the elastase and cathepsin G PU.1 and CG-like elements as probes results in identical band-shift patterns to that obtained with proteinase-3 PU.1 and CG elements. These data suggest that there is cooperative interaction between a PU.1 and a CG element with a consensus of CCCCXCCC and that they are important control elements for tissue- and developmental-specific expression of azurophil serine proteinases of polymorphonuclear leukocytes.     

Functional organization of the cassava vein mosaic virus (CsVMV) promoter

Cassava vein mosaic virus (CsVMV) is a pararetrovirus that infects cassava plants in Brazil. A promoter fragment isolated from CsVMV, comprising nucleotides -443 to +72, was previously shown to direct strong constitutive gene expression in transgenic plants. Here we report the functional architecture of the CsVMV promoter fragment. A series of promoter deletion mutants were fused to the coding sequence of uidA reporter gene and the chimeric genes were introduced into transgenic tobacco plants. Promoter activity was monitored by histochemical and quantitative assays of beta-glucuronidase activity (GUS). We found that the promoter fragment is made up of different regions that confer distinct tissue-specific expression of the gene. The region encompassing nucleotides -222 to -173 contains cis elements that control promoter expression in green tissues and root tips. Our results indicate that a consensus as1 element and a GATA motif located within this region are essential for promoter expression in those tissues. Expression from the CsVMV promoter in vascular elements is directed by the region encompassing nucleotides -178 to -63. Elements located between nucleotides -149 and -63 are also required to activate promoter expression in green tissues suggesting a combinatorial mode of regulation. Within the latter region, a 43 bp fragment extending from nucleotide -141 to -99 was shown to interact with a protein factor extracted from nuclei of tobacco seedlings. This fragment showed no sequence homology with other pararetrovirus promoters and hence may contain CsVMV-specific regulatory cis elements.     

Estrogen receptor does not directly regulate the murine Muc-1 promoter

Muc-1 is a heavily O-glycosylated, type 1 membrane glycoprotein present on the surface of polarized secretory uterine epithelial cells. Previous studies have shown that treatment of ovariectomized mice with 17-beta-estradiol (E2) strongly induces Muc-1 mRNA expression in an estrogen receptor (ER)-mediated fashion in the uterus. In this study, the 5.4 kb Muc-1 gene promoter has been isolated from a mouse genomic library and the proximal 1.85 kb region has been sequenced. Sequence analysis revealed the presence of one potential full estrogen response element (ERE) (GCTCGCGGTGACC) located at -748 to -735 bp in the Muc-1 promoter and several potential ERE half sites. Electrophoretic mobility shift assays (EMSA) showed that neither ERalpha nor ERbeta bind efficiently to this sequence. Transient cotransfection assays using constructs containing various deletion mutations of the 5' Muc-1 flanking sequences showed that E2 had no direct stimulation on promoter-driven reporter in NMuMG cells or primary mouse uterine epithelial cells, but did stimulate a consensus ERE CAT-reporter gene activity. In addition, E2-treatment of Weg-ER cells, a mouse uterine epithelial cell line stably expressing human ERalpha, did not restore endogenous Muc-1 expression or activate Muc-1 promoter-driven CAT activity. These results indicate that regions of the Muc-1 gene promoter within -1838 to +43 bp do not respond to E2 and ER stimulation and that ER alone is not sufficient to restore Muc1 gene expression. Deletion analyses also revealed that the sequence between -73 and +43 bp of the Muc-1 promoter is the minimal promoter region required for maximal Muc-1 promoter activity. Collectively, these results demonstrate that ER does not directly regulate the 1.85 kb murine Muc-1 gene promoter. Therefore, E2 control of uterine Muc-1 gene expression is likely to be indirect, i.e. mediated by stromal cell-derived factors.     

Cloning and characterization of the human interleukin-3 (IL-3)/IL-5/ granulocyte-macrophage colony-stimulating factor receptor betac gene: regulation by Ets family members

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common beta chain (betac). Whereas the mouse has two homologous beta subunits (betac and betaIL-3), in humans, only a single beta chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor beta subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine betac/betaIL-3 genes. Surprisingly, we also found the remnants of a second betac chain gene directly downstream of betac. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from -103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5' element specifically binds PU.1, whereas the 3' element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances betac promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.