ETS target genes: identification of egr1 as a target by RNA differential display and whole genome PCR techniques.

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing's sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Identification of differentially expressed genes in breast cancer.

A number of genes differentially expressed in breast cancer were isolated using a subtractive cloning technique. DNA sequence analysis and GenBank searches of T4F10, T2H7, and T2E5 cDNA clones found them to be identical with E2A, MSS1, and SEC13R genes. Their expression in a variety of primary breast tumor and cancer cell lines was compared with c-ERB-B2 and pS2 by Northern blot analysis. In breast cancer cell lines, the genes that we isolated are more frequently expressed than the previously described c-ERB-B2 and pS2.

The EndoA enhancer contains multiple ETS binding site repeats and is regulated by ETS proteins.

EndoA is a type II keratin and with EndoB (type I keratin), constitutes intermediate filaments in various simple epithelial tissues. EndoA is developmentally regulated and has an enhancer that is located at the 3'- end of the gene. This enhancer contains two single and five dual Ets binding sites. Thus far, no other promoter or enhancer has been shown to contain as many potential clustered Ets binding sites. To study the transcriptional regulation of EndoA by the ETS family proteins, we amplified the EndoA enhancer fragment from mouse genomic DNA by PCR, and cloned it into the pBLCAT2 vector upstream from the CAT reporter gene. Several pBLCAT-ENDOA clones were sequenced to verify the presence of all the ETS binding sites. Clones that did not show any point mutations in the ETS binding sites were chosen to study the transcription regulation by ETS1, ETS2 and ERGB/FLI-1 gene products. EMSA results indicated that the ETS1, ETS2 and ERGB/FLI-1 proteins bind to the enhancer sequence, and DNase I protection data demonstrated that the ETS proteins protect all seven EBS core sequences. Cotransfection of the COS cells with the pBLCAT-ENDOA construct, along with increasing amounts of different ETS expression vectors, resulted in a significant induction of CAT reporter gene expression. Previously, we have shown that the overexpression of the ETS1 gene transforms NIH3T3, and these transformed cells (7AQS2.1) produce high levels of ETS1 protein (Seth & Papas, 1990). In this report, we show that the undifferentiated P19 EC cells do not express detectable levels of ETS1; however, an elevated level of ETS1 is expressed in differentiated derivatives of these cells. We therefore used these two cell lines to examine the activity of the EndoA enhancer with the ETS1 product. Transfection of the pBLCAT-ENDOA construct alone in undifferentiated P19 EC cells results in very low CAT gene expression; however, upon differentiation with retinoic acid the level of CAT gene activity increases dramatically. Similarly, an increase in CAT expression from the same construct (pBLCAT-ENDOA) was also observed in 7AQS2.1 cells. Our results therefore indicate that the EndoA enhancer is regulated by ETS proteins via interaction with multiple ETS-binding site sequences.

ETS family proteins activate transcription from HIV-1 long terminal repeat.

ets is a multigene family and its members share a common ETS DNA-binding domain. ETS proteins activate transcription via binding to a purine-rich GGAA core sequence located in promoters/enhancers of various genes, including several that are transcriptionally active in T cells. The ETS1, ETS2, and ERBG/Hu-FLI-1 gene expression pattern also suggests a role for these genes in cells of hematopoietic lineage. The HIV-1 LTR core enhancer contains two 10-base pair direct repeat sequences (left and right) that are required for regulation of HIV-1 mRNA expression by host transcription factors, including NF kappa B. Two ETS-binding sites are present in the core enhancer of all the HIV-1 isolates reported so far. In our studies, we utilized HIV-1 HXB2 and HIV-1 Z2Z6 core enhancers because the Z2Z6 strain has a single point mutation flanking the right ETS-binding site. We demonstrate that the ETS1, ETS2, and ERGB/Hu-FLI-1 proteins can trans-activate transcription from both the HXB2 and Z2Z6 core enhancer when linked to a reporter (cat) gene. In addition, we show that the DNA binding and trans-activation with the Z2Z6 core enhancer is at least 40-fold higher than that observed with the HXB2 core enhancer. Further, we provide evidence that the marked increase in binding and trans-activation with Z2Z6 core enhancer sequences is due to the substitution of a flanking T residue in HXB2 TGGAA) by a C residue in Z2Z6 (CGGAA) isolate, thus generating an optimal ETS-binding core (CGGAA) sequence.

Relative paucity of p53 gene-mutations in male breast carcinomas.

Mutations of the p53 suppressor -ene are the most common genetic lesion noted in human cancers and appear to be relatively common (30%) as somatic cell mutations in female breast cancer. p53 mutations have also been frequently reported in familial breast cancers as in Li-Fraumeni syndrome (LFS). Males with breast cancer are far rarer than females. We investigated the mutational spectra of the p53 gene in male breast cancers. Of 10 samples analyzed for p53 mutations in exons 5, 6. 7 and 8, only two showed point mutations corresponding to amino acid residues 248 and 290. One of the point mutations turned out to be a silent change, thus representing only DNA polymorphism. Although the number of male breast cancer samples thus far examined is small, the p53 mutations in male breast cancer (10%), unlike females (30%), does not appear to be as frequent.

Influence of nucleotides flanking the ggaa core sequence on ets1 and ets2 DNA-binding activity and the mechanism of ets1 autoregulation.

The Ets family of genes encode nuclear proteins that activate transcription by binding to a specific purine-rich (GGAA) ets binding sequence (EBS) present in promoters/enhancers of various genes. We have previously shown that over-expression of ets1 via transfection of ets1 expression vectors into NIH3T3 cells induced the expression of the endogenous Ets1 gene. Here we report that the autoregulation occurs as a result of the ets1 protein binding to the EBS-core located in its own promoter. In the present study, we have also identified Ets binding sites in the IL-4, G-CSF (granulocyte colony stimulating factor), and the 2'5' OAS (oligoadenylate synthetase) promoters by binding with Ets1 and Ets2 proteins using electrophoretic mobility shift assays. Interestingly, we have found that the EBS containing T nucleotides on either side of the GGAA core sequence, does not bind Ets1 or Ets2 proteins. Our findings demonstrate that the sequences surrounding the purine core - GGAA- have a profound influence on the binding of Ets proteins.