Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype.

The human leukemia cell line K562 can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) to differentiate along the megakaryocytic pathway, generating morphological changes and increased expression of lineage-specific surface markers. We report that TPA-treated K562 cells also express higher levels of FLI-1/ERGB, a member of the ETS family of transcription factors. Furthermore, introduction of a retroviral construct expressing human FLI-1/ERGB into K562 cells induces changes similar to those seen following TPA treatment, including increased adherence to the surface of the culture vessel and altered size and morphology. Infected cells exhibit higher levels of the megakaryocyte marker CD41a and, to a lesser extent, CD49b. These markers, as well as virally encoded FLI-1/ERGB-specific RNA and protein, are expressed at the highest levels in the attached cell population, while the growth rate of adherent cells is reduced, and the fraction of cells in G0-G1 is increased. FLI-1/ERGB virus-infected cells also exhibit increased expression of hemoglobin, a marker of erythroid differentiation. Our results suggest FLI-1/ERGB plays a role in controlling differentiation and gene expression along the megakaryocyte/platelet pathway, and further implicate ETS-related genes in the control of multiple developmentally regulated hematopoietic genes.

Signal requirements for interleukin 4 promoter activation in human T cells.

We have studied the signal requirements for human IL-4 promoter activation in Jurkat T cells by the use of DNA transfection assays with vectors carrying the IL-4 promoter linked to a reporter gene. Stimulation with calcium (Ca2+) ionophores (ionomycin), but not with phorbol esters (phorbol myristate acetate, PMA) or cyclic AMP elevating agents (prostaglandin E2, PGE2), induced the transcriptional activity of the IL-4 promoter by approximately 3-fold. Costimulation with ionomycin and PGE2 resulted in the same level of IL-4 promoter activity as the stimulation with ionomycin alone. In contrast, costimulation with ionomycin and PMA decreased the activity of the IL-4 promoter by approximately 40% compared to stimulation with ionomycin alone. Induction of Il-4 promoter by ionomycin was partially inhibited (approximately 50% inhibition) in the presence of as high as 2 microgram/ml cyclosporin A (CsA), an inhibitor of the Ca+/calmodulin-dependent phosphatase calcineurin. Under the same conditions, only 0.1 microgram/ml of CsA inhibited by >95% the transactivation of the IL-2 promoter in response to ionomycin and PMA. Transfection of a deletion mutant of the calcineurin catalytic subunit (delta CaM-AI) known to have Ca2+-independent, constitutive phosphatase activity increased IL-4 promoter activity by approximately 14-fold. Stimulation with ionomycin of cells transfected with low doses of delta CaM-AI, further induced IL-4 promoter activity by approximately 2-fold. These results identify the Ca2+-signaling system as a key component of the signal transduction pathway leading to IL-4 promoter activation in Jurkat T cells and suggest a major role of calcineurin in its regulation.

The Gag-Myb-Ets fusion oncogene alters the apoptotic response and growth factor dependence of interleukin-3 dependent murine cells.

Expression of the avian E26-derived Gag-Myb-Ets fusion oncogene in interleukin-3(IL3)-dependent murine hematopoietic cell lines results in a pattern of cell line dependent changes in growth factor-induced proliferation and apoptosis. A drug-selectable retrovirus expressing p135Gag-Myb-Ets induced an erythropoietin(Epo)-responsive phenotype in the cell lines FDC-P2, BaF3 and 32Dc123. Gag-Myb-Ets expression alone did not increase expression of GATA-1 or the Epo receptor(EpoR) in the presence of IL3, and infected cell lines express increased GATA-1 and EpoR only when IL3 was replaced by Epo in the culture media. Indicative of Epo-induced erythroid differentiation, these cells also began to express beta-globin after 3-5 days growth in Epo. Unlike control cells, infected FDC-P2 cells failed to undergo programmed cell death (apoptosis) when transferred from IL3- to Epo-containing media, although a fraction of the cells failed to proliferate following the media shift. Three other IL3-dependent cell lines showed no changes in growth behavior when induced to express the fusion oncogene. Our data shows that Gag-Myb-Ets can have different affects on growth factor pathways depending on the cell background, suggesting a model in which the p135gag-myb-ets fusion oncogene promotes these different responses through its affect on apoptosis.

ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation.

ERF (ETS2 Repressor Factor) is a novel member of the ets family of genes, which was isolated by virtue of its interaction with the ets binding site (EBS) within the ETS2 promoter. The 2.7 kb ubiquitously expressed ERF mRNA encodes a 548 amino acid phosphoprotein that exhibits strong transcriptional repressor activity on promoters that contain an EBS. The localization of the DNA-binding domain of the protein at the N-terminus and th repression domain at the C-terminus is reminiscent of the organization of ELK1-like members of the ets family; however, there is no significant homology between ERF and ELK1 or any other ets member outside the DNA-binding domain. The repressor activity of ERF can antagonize the activity of other ets genes that are known transcriptional activators. Furthermore, ERF can suppress the ets-dependent transforming activity of the gag-myb-ets fusion oncogene of ME26 virus. Although ERF protein levels remain constant throughout the cell cycle, the phosphorylation level of the protein is altered as a function of the cell cycle and after mitogenic stimulation. The ERF protein is also hyperphosphorylated in cells transformed by the activated Ha-ras and v-src genes and the transcription repressor activity of ERF is decreased after co-transfection with activated Ha-ras or the kinase domain of the c-Raf-1 gene, indicating that ERF activity is probably regulated by the ras/MAPK pathway. Consistent with the in vivo phosphorylation and inactivation by ras, ERF is efficiently phosphorylated in vitro by Erk2 and cdc2/cyclin B kinases, at sites similar to those detected in vivo. Furthermore, a single mutation at position 526 results in the loss of a specific phosphopeptide both in in vivo and in vitro (by Erk2) labeling. Substitution of Thr526 for glutamic acid also decreases the repression ability of ERF. Our data suggest a model in which modulation of ERF activity is involved in the transcriptional regulation of genes activated during entry into G1 phase. Obstruction of the ERF repressor function by the transactivating members of the ets family of genes (i.e.gag-myb-ets) may be essential for the control of genes involved in cell proliferation and may also underlie their tumorigenic effects.

Positive and negative factors regulate the transcription of the ETS2 gene via an oncogene-responsive-like unit within the ETS2 promoter region.

The DNA-protein interactions in the ETS2 promoter have been studied. Three distinct sequence motifs have been identified, each of which interacts with at least two distinctive protein complexes. The GC motif, possessing mirror symmetry, interacts with two ubiquitously identifiable complexes (S and S2); the PEA3 motif interacts with a ubiquitous (H1) and a tissue-specific (H3) complex; the H2 (an AP1-like) motif interacts also with a ubiquitous (H2a) and a tissue-specific (H2b) complex. Mutational analysis and correlation of the presence of defined complexes with the ETS2 mRNA levels indicate that the S, S2, H1, and H2b complexes have positive effects on ETS2 transcription, whereas the H3 and H2a have negative effects. The organization of the PEA3 with the AP1-like motif in the ETS2 promoter resembles the oncogene-responsive unit previously identified in the polyoma virus enhancer region. Our data suggest that cooperation between these two motifs is vital for ETS2 promoter function.

The human ETS1 gene: genomic structure, promoter characterization and alternative splicing.

Genomic clones encompassing the human ETS1 gene were isolated and utilized to define its molecular organization. This gene consists of eight exons spanning over 60 kb. The 5' end of the human ETS1 gene was subcloned and characterized. S1 nuclease, primer extension and RNAase protection analyses of human mRNAs showed multiple transcription initiation sites. DNA sequence analysis indicated a high G + C content in the promoter region and the absence of either a 'TATA' box or a 'CAAT' box. Six consensus recognition sequences for the transcription factor SP1, two AP1 consensus sequences and one consensus AP2 recognition sequence were identified, as well as two GC elements with dyad symmetry. A palindromic region similar to the serum response element of the c-fos gene and two octamer consensus recognition sequences were located upstream of the promoter region. A series of promoter deletion constructs positioned upstream from the bacterial chloramphenicol acetyl transferase gene were transfected into HeLa cells and their functional promoter activity assayed. The deletion constructs identified the 5' boundary for maximum promoter activity at 486 bp upstream of the first initiation site and suggest possible positive and negative regulatory regions. Polymerase chain reaction analysis of ETS1 cDNA identified several amplified products, indicating alternative splicing. In addition to the presence of mRNA products lacking exon VII, products lacking exon IV, as well as ones lacking both exons IV and VII, were found.

Molecular organization and differential polyadenylation sites of the human ETS2 gene.

The human ETS2 gene is a member of the ets gene family (ETS1, ETS2, ERG, ELK1 and ELK2) with amino acid similarity to the v-ets oncogene of the avian leukemia virus, E26. The ETS2 gene is composed of 10 exons, nine of which contribute to the open reading frame encoding 469 amino acids. The ETS2 gene directs the synthesis of three RNA transcripts, which differ from each other by the length of their 3' ends. This heterogeneity of 3' end is the major reason for the size differences between the transcripts, presumably reflecting utilization of three different polyadenylation signals/sites. The coding regions of all of the ETS2 RNA species are the same length and, thus, should contain the same open reading frame.

The human ETS-2 gene promoter: molecular dissection and nuclease hypersensitivity.

The human ETS-2 gene is a homolog of the v-ets oncogene of the E26 virus, coding for a 56 kilodalton nuclear protein. Herein we characterize the ETS-2 gene promoter, using a series of deletion constructs. Analysis of the nucleotide sequences from -3600 bp to +141 bp reveal that the region -159 bp to +141 bp is absolutely essential for maximum promoter activity. This region includes half of the lengthy polypyrimidine (CT) tract of the ETS-2 promoter, one Sp1 binding site and the GC element proximal to the initiation site. This CT tract is able to increase in an orientation independent manner, the transcription from alpha-globin promoter. Several S1 hypersensitive sites are found to be located in this promoter region, using chromatin and supercoiled DNA, in close proximity with cis regulatory elements. Our results indicate that an unusually long (approximately 250 bp) CT tract is necessary for ETS-2 transcription and this tract can also serve as a transcription activator using a heterologous promoter.

Molecular and functional characterization of the promoter of ETS2, the human c-ets-2 gene.

The 5' end of the human c-ets-2 gene, ETS2, was cloned and characterized. The major transcription initiation start sites were identified, and the pertinent sequences surrounding the ETS2 promoter were determined. The promoter region of ETS2 does not possess typical "TATA" and "CAAT" elements. However, this promoter contains several repeat regions, as well as two consensus AP2 binding sites and three putative Sp1 sites. There is also a palindromic region similar to the serum response element of the c-fos gene, located 1400 base pairs (bp) upstream from the first major transcription initiation site. A G + C-rich sequence (GC element) with dyad symmetry can be seen in the ETS2 promoter, immediately following an unusually long (approximately 250-bp) polypurine-polypyrimidine tract. A series of deletion fragments from the putative promoter region were ligated in front of the bacterial chloramphenicol acetyltransferase gene and tested for activity following transfection into HeLa cells. The 5' boundary of the region needed for maximum promoter activity was found to be 159 bp upstream of the major initiation site. This region of 159 bp contains putative binding sites for transcription factors Sp1 and AP2 (one for each), the GC element, one small forward repeat, one inverted repeat, and half of the polypurine-pyrimidine tract. The promoter of ETS2 (within the polypyrimidine tract) serves to illustrate an alternative structure that may be present in genes with "TATA-less" promoters.

Molecular analysis of the mouse class II gene, E q alpha.

The structure and transcription of the mouse E q alpha gene were investigated by structural analysis of genomic clones, transient and stable cell line transformation, and spleen cell nuclear transcription studies. The transcription rate of Eq alpha is comparable to that of the Ek alpha allele. The q allele is transcribed to a normal size mRNA, but the Eq alpha mRNA level in spleen cells is 100-fold less than that of Ek alpha and is increased after cycloheximide treatment. The promoter region of the q allele can stimulate transcription of the chloramphenicol acetyl transferase gene after treatment with gamma interferon in transient expression assays. The Eq alpha gene was cloned and sequenced from -1630 nucleotide to +5200 nucleotide, and it shows 98% sequence identity with Eq alpha. A frame shift in codon 89 causes premature termination of translation, which probably accounts for its low steady state level of mRNA.

Purification and properties of three cytosolic ribonucleases of mouse liver.

The ribonucleolytic activity of mouse liver cytosol is due to at least three different enzymes, whose purification is reported. Two of these enzymes, an alkaline and a neutral RNase, have specificities practically identical with that of pancreatic RNase. The third enzyme, an acid RNase, is highly specific for NpU bonds where N is A, G or C and also cleaves ApG bonds provided they are part of a GpApG sequence and preferentially a GpApGpA repeat.