Regulation of the megakaryocytic glycoprotein IX promoter by the oncogenic Ets transcription factor Fli-1.

Glycoprotein (GP) IX is a subunit of the von Willebrand receptor, GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced promoter activity. However, the Ets protein(s) that regulated GPIX promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T kidney fibroblasts with a Fli-1 expression vector shows that the oncogenic protein Fli-1 can transactivate the GPIX promoter when an intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells. Comparative studies showed that Fli-1 was also able to transactivate the GPIbalpha and, to a lesser extent, the GPIIb promoter. Immunoblot analysis identified Fli-1 protein in lysates derived from platelets. In addition, expression of Fli-1 was identified immunohistochemically in megakaryocytes derived from CD34(+) cells treated with the megakaryocyte differentiation and proliferation factor, thrombopoietin. These results suggest that Fli-1 is likely to regulate lineage-specific genes during megakaryocytopoiesis.

The ets family member Tel binds to the Fli-1 oncoprotein and inhibits its transcriptional activity.

The tel gene, recently shown to be translocated in a spectrum of acute and chronic human leukemias, belongs to the ets family of sequence-specific transcription factors. To determine the role of Tel in normal hematopoietic development, we used the tel gene as the bait in the yeast two-hybrid system to screen a hematopoietic stem cell library. Two partners were identified: Tel binds to itself, and Tel binds to the ets family member Fli-1. In vitro and in vivo assays confirmed these interactions. In transient transfection assays, Fli-1 transactivates megakaryocytic specific promoters, and Tel inhibits this effect of Fli-1. Transactivation studies using deletion mutants of Tel, and the Tel-AML-1 fusion protein, indicate that the helix-loop-helix domain of Tel only partially inhibits transactivation and that complete inhibition requires the full-length Tel molecule, including the DNA binding domain. The Tel and Fli-1 proteins are expressed early in hematopoiesis, and the inability of Tel fusion proteins such as Tel-AML-1 to counteract Fli-1 mediated transactivation may contribute to the malignant phenotype in human leukemias where this fusion protein is present.

Analysis of the megakaryocyte glycoprotein IX promoter identifies positive and negative regulatory domains and functional GATA and Ets sites.

The glycoprotein (GP) Ib-V-IX multisubunit complex binds to von Willebrand factor and mediates the adhesion of platelets to the subendothelium of damaged blood vessels. Expression of the GPIX subunit is required for stability of the complex, and its absence in platelets is associated with the rare bleeding disorder Bernard-Soulier syndrome. Comparative analyses indicate that the four GPIb-V-IX subunits are members of the leucine-rich repeat family and suggest that GPIX resembles a possible primitive progenitor of this group. To characterize GPIX transcriptional regulation, a series of 5' deletion constructs was made linking the GPIX upstream flanking sequence to the luciferase marker gene, and promoter activity was measured in transiently transfected human erythroleukemia cells. This analysis identified two negative regulatory domains between -686 to -423 and -311 to -203 and two positive regulatory domains at -323 to -311 and -151 to -100 relative to the GPIX transcription start site. In addition, site-directed mutagenesis experiments and in vitro gel retardation assays identified Ets and GATA elements at -42 and -65, which positively regulate GPIX promoter activity and specifically bind nuclear factors derived from human erythroleukemia cells. DNase I protection experiments identified a protein-dependent "footprint" and hypersensitive site within the GPIX Ets sequence. These results provide a framework for comparison of the GPIX promoter with others of the GPIb-V-IX system, other megakaryocyte-specific genes, and other members of the leucine-rich repeat family.

The platelet glycoprotein Ib-V-IX system: regulation of gene expression.

Platelet glycoproteins (GPs) Ib-V-IX form the surface receptor for von Willebrand factor, and this receptor-ligand interaction mediates the shear-dependent adhesion of platelets to damaged arterial vessel walls. The receptor is a multicomponent structure consisting of four distinct polypeptides (heterodimeric GPIb: Ib alpha-Mr143k and Ib beta-Mr22k; GPV-Mr83k; GPIX-Mr20k), and each of the four cDNAs and genes has been cloned and characterized. The genes appear to have evolved from a common progenitor genomic sequence related to that encoding GPIX. They share simple structures with few introns and possess common consensus regulatory sequences (GATA, ets, Sp-1) in their 5' flanks. Both the GPIb alpha and the GPIX promoters have been analyzed by transfection of reporter constructs into hematopoietic and nonhematopoietic cells. The promoters function in a tissue-specific fashion, and gel shift and mutational analyses indicate that GATA and ets sequences regulate activity. In the case of the GPIX promoter, footprints confirm the role of the ets-related consensus region. Recent studies of GPIb beta transcriptional regulation suggest that an aberrant polyadenylation signal, located in the 3' end of the gene immediately upstream of the GPIb beta gene, allows in vitro expression of a rare extended fusion transcript encoding both the upstream protein and GPIb beta. Little detailed information is available in regard to expression of the GPV gene. In summary, the genes of the GPIb-V-IX system display features of other megakaryocyte/platelet genes, but the unique regulatory events that direct the selective expression of these genes in megakaryocytes remain to be defined.

Long-range restriction mapping of megabase-sized chromosomes that may be homologs in Trypanosoma brucei.

Trypanosoma brucei is a blood-borne pathogen that changes its variant surface glycoprotein coat, thus evading immune destruction. Restriction digestion, combined with probe hybridization studies, was used to construct long-range restriction maps of the 1.4 (M4) and 1.5 megabase (M3) chromosomes from the IsTaR1 serodeme of T. b. brucei. Comparison of the two chromosomes suggests that they are a homologous pair. Hybridization with a repetitive sequence probe also identifies several copies on the M4 chromosome and a relative paucity of cross-hybridizing repetitive sequence on the larger M3 chromosome.

Concerted stimulation of transcription by glucocorticoid receptors and basal transcription factors: limited transcriptional synergism suggests mediation by coactivators/adaptors.

Steroid receptors have been reported to stimulate transcription in a manner synergistic with other transcription factors. We have examined this synergism or functional cooperativity between glucocorticoid receptors and basal transcription factors in a variety of promoter and reporter gene contexts. A fragment containing a hormone response element from mouse mammary tumor virus was fused to well characterized promoters from the herpes virus thymidine kinase and mouse beta-globin genes and to related mutant promoters altered by inactivation of transcription factor-binding sites through point mutagenesis or deletion. These constructs were transfected into glucocorticoid-sensitive fibroblasts, and reporter gene activity was assessed with or without hormonal stimulation. In contrast to previous studies, we found little indication of synergistic interaction between elements mediating a hormone response and adjacent basal promoters. In fact, we observed that inactivating basal factor-binding sites, thereby decreasing promoter strength, actually increased hormone inducibility. We suggest that the inverse relationship between basal promoter strength and the induction ratio attained upon hormonal stimulation may be due to limitation of a common factor, an "adaptor" through which glucocorticoid receptor and basal transcription factors interact with the components of the RNA polymerase II complex to stimulate rates of transcription.