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, angio genesis, and organogenesis during murine development. The ETS genes al so have a role in neoplasia, for example in Ewing's sarcomas and retro virally induced cancers. The ETS genes encode transcription factors th at bind to specific DNA sequences and activate transcription of variou s cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA d ifferential display technique. Previously, we have shown that the over expression of ETS1 and ETS2 genes effects transformation of NIH 3T3 ce lls 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 ce ll lines and converted it into cDNA. This cDNA was amplified by PCR an d displayed on sequencing gels. The differentially displayed bands wer e subcloned into plasmid vectors. By Northern blot analysis, several c lones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed b y 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 At-activating protein, and earl y growth response 1 (Egr1) genes. In the second approach, to isolate E TS target promoters directly, we performed ETS1 binding with MboI-clea ved genomic DNA in the presence of a specific mAb followed by whole ge nome PCR. The immune complex-bound ETS binding sites containing DNA fr agments 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 ide ntified. Three clones turned out to contain regulatory sequences deriv ed from human serglycin, preproapolipoprotein C II, and Egr1 genes. Th e ETS binding sites derived from these three regulatory sequences show ed specific binding with recombinant ETS proteins. Of interest, Egr1 w as identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.