To examine the possibility that ETS family transcription factors, PU.1, SPI-B, ELF-1, ERG-3, ETS-1 and TEL, and homeodomain proteins, HOXA10, HOXC13, MEIS1 and PBX1B, function cooperatively, we investigated their interactions. In luciferase assays, HOXA10 and HOXC13 augmented the activity of PU.1 and SPI-B while diminishing that of ELF-1 and ERG-3. MEIS1 diminished the activity of ETS-1. No clear effects were observed for other combinations. Immunoprecipitation assays showed protein-protein interactions among the combinations exhibiting functional interactions. A mutation of HOXC13, which abolished binding to ELF-1, also abolished the diminishing effect on ELF-1. The results suggest functional interaction through physical interactions.

The two-hybrid system was used to isolate cDNA clones encoding polypeptides that interact with the N-terminal region (activation domains A, B and C) of the Sp1 transcription factor. Among the 65 collected clones, 43 contained cDNA fragments with open reading frames. They corresponded to 13 genes encoding proteins of known function and to 15 genes, the proteins of which have no known function. Six overlapping cDNA clones corresponded to the Hsc70 protein. Host cell factor (HCF-1) and the KIAA0461 gene (encoding a putative Zn-finger protein of unknown function) were both identified through the isolation of three overlapping cDNA clones. Two cDNA fragments encoding the same region of the SREBP-2 transcription factor were independently selected and two overlapping cDNA clones corresponded to the splicing factor SF3A120. Two different cDNA clones encoded the N- and C-terminal region of the Oct-1 transcription factor. Transcription factors Elf-1 and TIEG, as well as HSph2, the putative human homologue of a murine polyhomeotic gene, were each represented by a single clone. Noticeably, for the four identified transcription factors, the DNA-binding domain was excluded from the selected polypeptides. In vitro binding of the selected polypeptides to the Sp1 protein was demonstrated for the four transcription factors and for the SF3A120, Hsc70, HCF-1, HSph2 and pKIAA0461(245) proteins. Four other cDNA clones encoding polypeptides of unknown function were tested in the in vitro binding assay. All four polypeptides were found to interact with Sp1 in this assay.

The AML1 and ETS families of transcription factors play critical roles in hematopoiesis; AML1, and its non-DNA-binding heterodimer partner CBFbeta, are essential for the development of definitive hematopoiesis in mice, whereas the absence of certain ETS proteins creates specific defects in lymphopoiesis or myelopoiesis. The promoter activities of numerous genes expressed in hematopoietic cells are regulated by AML1 proteins or ETS proteins. MEF (for myeloid ELF-1-like factor) is a recently cloned ETS family member that, like AML1B, can strongly transactivate several of these promoters, which led us to examine whether MEF functionally or physically interacts with AML1 proteins. In this study, we demonstrate direct interactions between MEF and AML1 proteins, including the AML1/ETO fusion protein, in t(8;21)-positive acute myeloid leukemia (AML) cells. Using mutational analysis, we identified a novel ETS-interacting subdomain (EID) in the C-terminal portion of the Runt homology domain (RHD) in AML1 proteins and determined that the N-terminal region of MEF was responsible for its interaction with AML1. MEF and AML1B synergistically transactivated an interleukin 3 promoter reporter gene construct, yet the activating activity of MEF was abolished when MEF was coexpressed with AML1/ETO. The repression by AML1/ETO was independent of DNA binding but depended on its ability to interact with MEF, suggesting that AML1/ETO can repress genes not normally regulated by AML1 via protein-protein interactions. Interference with MEF function by AML1/ETO may lead to dysregulation of genes important for myeloid differentiation, thereby contributing to the pathogenesis of t(8;21) AML.

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

We previously isolated different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specific blk promoter. We now report that NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 bound to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 led to cooperative transactivation of the blk promoter, whereas the interaction between AML1 and NERF-1a led to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, we determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia.

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

We previously isolated different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specific blk promoter. We now report that NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 bound to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 led to cooperative transactivation of the blk promoter, whereas the interaction between AML1 and NERF-1a led to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, we determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia.

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are structurally related retroviruses which both cause AIDS in humans. Although both viruses establish latency in quiescent human-peripheral-blood T cells, the asymptomatic phase of HIV-2 infection may be more prolonged than that of HIV-1. The latent phases of both HIV-1 and HIV-2 infection have been shown to be disrupted by T-cell activation, a process that requires host cell transcription factors. In the case of HIV-1, the transcription factor NF-kappa B is sufficient for inducible transcriptional activation. In contrast, factors in addition to NF-kappa B are required to activate HIV-2 transcription in infected T cells. In this report, we demonstrate that a novel Ets-related transcription factor, Elf-1, binds specifically to two purine-rich motifs in the HIV-2 enhancer. Mutagenesis experiments demonstrated that these Elf-1 binding sites are required for induction of HIV-2 transcription following T-cell-receptor-mediated T-cell activation. Moreover, Elf-1 is the only factor present in activated T-cell nuclear extracts that binds to these sites in electrophoretic mobility shift assays. Thus, Elf-1 is a novel transcription factor that appears to be required for the T-cell-receptor-mediated trans activation of HIV-2 gene expression. These results may explain differences in the clinical spectra of diseases caused by HIV-1 and HIV-2 and may also have implications for the design of therapeutic approaches to HIV-2 infection.