Net-targeted mutant mice develop a vascular phenotype and up-regulate egr-1.

The ternary complex factors (TCFs) Net, Elk-1 and Sap-1 regulate immediate early genes through serum response elements (SREs) in vitro, but, surprisingly, their in vivo roles are unknown. Net is a repressor that is expressed in sites of vasculogenesis during mouse development. We have made gene-targeted mice that express a hypomorphic mutant of Net, Net delta, which lacks the Ets DNA-binding domain. Strikingly, homozygous mutant mice develop a vascular defect and up-regulate an immediate early gene implicated in vascular disease, egr-1. They die after birth due to respiratory failure, resulting from the accumulation of chyle in the thoracic cage (chylothorax). The mice have dilated lymphatic vessels (lymphangiectasis) as early as E16.5. Interestingly, they express more egr-1 in heart and pulmonary arteries at E18.5. Net negatively regulates the egr-1 promoter and binds specifically to SRE-5. Egr-1 has been associated with pathologies involving vascular stenosis (e.g. atherosclerosis), and here egr-1 dysfunction could possibly be associated with obstructions that ultimately affect the lymphatics. These results show that Net is involved in vascular biology and egr-1 regulation in vivo.

Net-b, a Ras-insensitive factor that forms ternary complexes with serum response factor on the serum response element of the fos promoter.

The Ras signalling pathway targets transcription factors such as the ternary complex factors that are recruited by the serum response factor to form complexes on the serum response element (SRE) of the fos promoter. We have identified a new ternary complex factor, Net-b. We report the features of the net gene and show that it produces several splice variants, net-b and net-c. net-b RNA and protein are expressed in a variety of tissues and cell lines. net-c RNA is expressed at low levels, and the protein was not detected, raising the possibility that it is a cryptic splice variant. We have studied the composition of ternary complexes that form on the SRE of the fos promoter with extracts from fibroblasts (NIH 3T3) cultured under various conditions and pre-B cells (70Z/3) before and after differentiation with lipopolysaccharide (LPS). The fibroblast complexes contain mainly Net-b followed by Sap1 and Elk1. Net-b complexes, as well as Sap1 and Elk1, are induced by epidermal growth factor (EGF) stimulation of cells cultured in low serum. Pre-B-cell complexes contain mainly Sap1, with less of Net-b and little of Elk1. There is little change upon LPS-induced differentiation compared to the increase with EGF in fibroblasts. We have also found that Net-b is a nuclear protein that constitutively represses transcription. Net-b is not activated by Ras signalling, in contrast to Net, Sap1a, and Elk1. We have previously reported that down-regulation of Net proteins with antisense RNA increases SRE activity. The increase in SRE activity is observed at low serum levels and is even greater after serum stimulation, showing that the SRE is under negative regulation by Net proteins and the level of repression increases during induction. Net-b, the predominant factor in ternary complexes in fibroblasts, may both keep the activity of the SRE low in the absence of strong inducing conditions and rapidly shut the activity off after stimulation.

Locations of the ets subfamily members net, elk1, and sap1 (ELK3, ELK1, and ELK4) on three homologous regions of the mouse and human genomes.

Net, Elk1, and Sap1 are related members of the Ets oncoprotein family. We show by in situ hybridization on banded chromosomes with specific cDNA probes that their map positions on mouse and human chromosomes (respectively) are net, 10C-D1 and 12q22-q23 (now called ELK3), sap1, 1E3-G and 1q32 (ELK4), and elk1, XA1-A3 and Xp11.2-p11.1 (ELK1), as well as a second locus 14q32 (ELK2) unique to the human genome. The results for the mouse net, sap1, and elk1 and human ELK3 genes are new. The human elk1 mapping confirms a previous study. The human ELK4 localization agrees with data published during the preparation of the manuscript. Human ELK3 colocalizes with sap2, and we confirm that they are identical. These results firmly establish for the first time that Net, Elk1, and Sap1 are distinct gene products with different chromosomal localizations in both the mouse and the human genomes. Net, Elk1, and Sap1 are conserved and map to homologous regions of the mouse and human chromosomes.

Reversion of Ras transformed cells by Ets transdominant mutants.

Considerable progress has been made in elucidating the components of the Ras signalling pathway, from both biochemical and genetic investigations. However little is known about the nuclear targets of the pathway, and in particular those that mediate the long-term changes in gene expression resulting from Ras transformation. Ets family members may be involved in these processes since Ras stimulates transcription through ets-DNA binding sites. We show that a mutated Ets protein, delta PU.1, inhibits Ras activation of transcription. Stable expression of delta PU.1 in Ras transformed NIH3T3 fibroblasts reverts the transformed phenotype by many characteristics, including morphology, anchorage independent growth, saturation density, growth in low serum, tumour formation in nude mice and to some extent sensitivity to apoptotic cell death. Similar trans-dominant mutants of c-Ets-1 and c-Ets-2, the most divergent members of the Ets-family to PU.1, also revert Ras transformed cells, as indicated by morphology, anchorage-independent growth, saturation density and doubling time in low serum. Reversion may result from a shared property of the mutants, such as binding to ets motifs in promoters. These results provide evidence for an important role for Ets proteins in Ras transformation.

Net, a new ets transcription factor that is activated by Ras.

Ras signaling appears to be mediated in part by transcription factors that belong to the ets gene family. To identify downstream targets for the Ras signal transduction pathway, we have used Ras-transformed mouse fibroblasts to isolate a new member of the ets gene family, net. Net has sequence similarity in three regions with the ets factors Elk1 and SAP1, which have been implicated in the serum response of the fos promoter. Net shares various properties with these proteins, including the ability to bind to ets DNA motifs through the Ets domain of the protein and form ternary complexes with the serum response factor SRF on the fos serum response element, SRE. However, Net differs from Elk1 and SAP1 in a number of ways. The pattern of net RNA expression in adult mouse tissues is different. Net has negative effects on transcription in a number of assays, unlike Elk1. Strikingly, Ras, Src, and Mos expression switch Net activity to positive. The study of Net should help in understanding the interplay between Net and other members of the Elk subfamily and their contribution to signal transduction through Ras to the nucleus.

Nucleotide sequence encoding a novel member of the hydratase/dehydrogenase family.

The nucleotide sequence encoding a novel member of the family of fatty acid oxidation enzymes has been determined. Clones were generated from porcine gastric corpus cDNA by application of the polymerase chain reaction (PCR) using oligodeoxyribonucleotides based on the amino acid sequence of a 78 kDa gastrin-binding protein isolated from porcine gastric mucosal membranes (Baldwin et al., J. Biol. Chem. 261 (1986) 12252-12257). Clones encoding the 3' and 5' ends of the cDNA were then isolated by conventional screening of a porcine liver cDNA library, and by application of anchored PCR, respectively. The composite cDNA of 2744 nucleotides encoded a protein of 763 amino acids, the sequence of which was related to a rat peroxisomal trifunctional enzyme, delta 3, delta 2-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and to other members of the same enzyme family. Northern blots indicated that the 3-kb mRNA encoding the novel protein was abundant in gastric corpus and liver, with lower amounts also present in gastric antrum and forestomach.

Structure and biological activity of the transcriptional initiation sequences of the murine c-myb oncogene.

To study the control mechanism(s) that govern the transcription of c-myb, genomic clones corresponding to the 5' region of the murine c-myb gene have been isolated and characterized structurally and functionally. Primer extension and nuclease protection analysis have revealed the presence of multiple transcriptional initiation sites, that are utilized in several hemopoietic cell lines (WEHI3B(D+). FDC-P1 and RB22.2). Some of the sites are used in all cell lines but others are unique; all are located in a region of the c-myb gene that is G-C rich, contains a number of potential Sp1 binding sites and lacks classical promoter consensus sequences. Experiments in which well characterized promoters controlling expression of a reporter gene have been replaced by fragments of c-myb DNA (including the observed cap sites) were performed in an attempt to demonstrate promoter activity in various cell types. It was shown that a region of the c-myb gene (approximately 1.0 kbp upstream from the splice donor site of the first exon) contains a weak promoter that has a low level of transcriptional activity in hemopoietic as well as in fibroblastic cells. These results support the suggestion that c-myb expression is not regulated primarily at the level of initiation of transcription.

Generation of altered transcripts by retroviral insertion within the c-myb gene in two murine monocytic leukemias.

Two murine monocytic leukemia cell lines, WEHI-265 and WEHI-274, were found to carry a rearranged c-myb gene. The rearrangements are due to insertion of a deleted Moloney murine leukemia virus (Mo-MLV) provirus in the 5' region of the c-myb gene and thus are similar to rearrangements in the ABPL tumors (G. L. C. Shen-Ong, M. Potter, J. F. Mushinski, S. Lavu, and E. P. Reddy, Science 226:1077-1080, 1984). In each cell line, the retroviral insertion has induced high levels of two aberrant RNA species, which, as in the ABPL tumors (G. L. C. Shen-Ong, H. C. Morse, M. Potter, and J. F. Mushinski, Mol. Cell. Biol. 6:380-392, 1986), contain both viral (Mo-MLV) and cellular (myb) sequences. Both species lack the sequences encoding the amino terminus of the c-myb protein and thus could encode a protein which, like the v-myb gene products (and the predicted ABPL myb proteins), is truncated at the amino terminus. We have found that the larger (5.3 kilobase [kb]) and more abundant of the tumor-specific myb RNAs was predominantly nuclear, while the smaller species (3.9 kb) was cytoplasmic. Furthermore, our data imply that the 3.9-kb RNA was derived from the 5.3-kb RNA by an additional splice which utilized a cryptic splice acceptor site within the viral gag sequences. On the basis of subcellular distribution and predicted translational potential, we conclude that the 3.9-kb RNA is probably the mRNA which encodes a truncated myb protein. We also show that, due to different insertion points in W265 and W274, the W274 myb RNAs contained sequences from a c-myb exon upstream of the exons represented in the W265 (and ABPL) RNAs. The significance of our findings with regard to transformation by myb in these tumors is discussed.

The structure and expression of the murine gene encoding granulocyte-macrophage colony stimulating factor: evidence for utilisation of alternative promoters.

Two overlapping genomic clones containing the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene have been isolated. On the basis of transfection experiments, we have established that a 9-kb BamHI fragment from one of these recombinants encodes biologically active GM-CSF. As deduced from nucleotide sequence analysis, the GM-CSF gene comprises four exons encompassing 2.5 kb of genomic DNA. Primer extension analysis of GM-CSF mRNA identifies a transcriptional initiation site 35 bp upstream of a single translational initiation codon in-frame with the GM-CSF coding sequences and 28 bp downstream of a TATA promoter consensus sequence. Pre-GM-CSF molecules encoded by mRNAs originating from this promoter would include a hydrophobic leader sequence typical for a secreted protein. Intriguingly, sequences present at the 5' end of a GM-CSF cDNA clone previously isolated in our laboratory are not contained within either of the genomic clones and must therefore be transcribed from a promoter located at least 10 kb 5' of the main body of the gene. mRNAs transcribed from this alternative upstream promoter possess an additional initiating codon and potentially encode a pre-GM-CSF polypeptide with an atypical NH2-terminal leader peptide. Comparison of the nucleotide sequence of the GM-CSF gene with that of other haemopoietic growth factor genes has revealed a common decanucleotide (5'-GPuGPuTTPyCAPy-3') within their respective 5'-flanking regions which may be involved in their co-ordinate regulation.

Hybridization of restriction fragments derived from calf satellite I DNA.

Two DNA fragments, the 643 base pairs (bp) and 621 bp long, obtained by endoR.Pst nuclease digestion of the 1350 bp basic repetitive unit of the calf satellite I DNA and cloning, do not hybridize with each other. Both of them, however, hybridize with the 970 and 1550 bp fragments, the sequence of which has been found to be homologous with that of the satellite I DNA.