Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif.

Abstract

The proto-oncogene ets-1 is the founding member of a new family of eukaryotic transcriptional regulators. Using deletion mutants of murine ets-1 cDNA expressed in Escherichia coli, we show that the DNA-binding domain corresponds closely to the ETS domain, an 85-amino-acid region that is conserved among ets family members. To investigate the specificity of DNA binding of the ETS domain, we mapped the DNA contacts of a monomeric Ets-1 fragment by chemical protection and interference assays. DNA backbone interactions span a 20-nucleotide region and are localized on one face of the helix. Close phosphate and base contacts are restricted to 10 central nucleotides. Contacts map to the major groove in the center of the site. Flanking minor groove interactions also are predicted. To determine the sequence preference in the close contact zone, we selected a pool of high-affinity binding sites using a purified Ets-1 carboxy-terminal fragment. Our Ets-l-selected consensus, 5'-A/GCCGGAA/TGT/C-3', differs from the binding consensus for the Drosophila ETS domain protein E74A, suggesting that specificity of action of ets family members is mediated by the ETS domain. Compared to other well-characterized classes of DNA-binding proteins, Et8-1 produces a unique pattern of DNA contacts. These studies demonstrate that the ETS domain proteins bind DNA in a novel manner.

ETS1 lowers capillary endothelial cell density at confluence and induces the expression of VE-cadherin

“…rEts1 bound to EBS2 and EBS4 but failed to bind to EBS1, EBS3 or EBS5, although they had the correct GGAA/T core sequence. These results support the previous observations that an A in position 73 relative to the ®rst G of the core sequence and a G in position +5 favor binding of Ets1 to DNA (Fisher et al, 1991;Nye et al, 1992;Woods et al, 1992).…”

A novel transcription factor, ELF5, belongs to the ELF subfamily of ETS genes and maps to human chromosome 11p13–15, a region subject to LOH and rearrangement in human carcinoma cell lines

“…Elf5 bound the E74 oligonucleotide (containing a GGAA-core) (lane 1), but not to the E74m1 oligonucleotide (which had been mutated to an AGAA-core) (lane 2). The ®rst G-residue of the core has been demonstrated to be a physical point of DNA contact for ETS1, and consequently essential for DNA binding (Fisher et al, 1991;Nye et al, 1992). Thus, Elf5 displays sequence speci®c binding to a consensus ETS binding site, binding that is disrupted by a mutation known to similarly a ect other ETS family members.…”

Transforming activity of EWS/FLI is not strictly dependent upon DNA-binding activity

“…Hybridizations were carried out as previously described (Kinzler et al, 1988) as monomers (Wasylyk et al, 1993). While the ETS domain has been extensively characterized as a modular domain that mediates binding to the core DNA consensus motif GGAA/T (Nye et al, 1992;Seth et al, 1992), a number of recent studies with ETS family proteins have shown that this domain may also mediate distinct functions such as interactions with heterologous proteins (Treisman, 1994), associations with other ETS proteins (Carrere et al, 1998), and even RNA binding activity (Hallier et al, 1996). Thus, it is possible that the ETS domain may mediate critical protein-protein interactions, independently of DNA binding activity, that are required for transformation by EWS/FLI.…”