ETS-1 Target Genes: Whole Genomic PCR methodology for the isolation of ETS-1 target genes and analysis of the Egr-1 gene promoter.

Abstract

The ETS gene products represent a family of transcriptional regulatory proteins containing a highly-conserved DNA-binding domain, termed the ETS domain. Several ETS family proteins bind to DNA as monomers; however, it has been shown that the DNA-binding activity of some (e.g., ETS-1) is enhanced in the presence of other factors. Whole genome PCR was utilized to isolate novel ETS target genes, by amplifying MboI cleaved genomic DNA bound to the ETS1 protein in the presence of a specific monoclonal antibody. The immune complex bound DNA fragments were amplified, subcloned into the pBS SK- plasmid, and subjected to DNA sequence and computer analysis. Sequence analysis of the forty-two clones revealed thirty-nine unique sequences, while three clones showed close homology with the regulatory regions of the human serglycin, apolipoprotein and Egr-1 genes. The regulatory regions of human serglycin and Egr-1 showed multiple ETS binding sites. In the Egr-1, the ETS-binding site (EBS) was found to be part of the serum response element (SRE). The EBS derived from these three regulatory sequences showed specific binding with recombinant ETS1 and FLI-1 proteins. Electrophoretic mobility shift assays (EMSA) show that the FLI-1 and EWS-FLI-1 proteins form ternary complexes with SRF on this Egr-1SREI. The data also suggest a difference in the binding affinity between the FLI-1 and EWS-FLI-1 proteins.