Characterization of the Cooperative Function of Inhibitory Sequences in Ets-1

Abstract

DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.

Myb binding sites within the N-ras promoter repress transcription

“…This ®nding is of particular interest since it suggests that binding of the oncoprotein v-Myb can result in two di erent protein-DNA complex conformations, implying that v-Myb can function as a repressor or activator depending on the promoter to which it binds. Such a conformational change upon binding to DNA has been shown for other DNA binding proteins such as the yeast transcriptional activator Mcm1 (Tan and Richmond, 1990) and the c-Ets protein (Petersen et al, 1995;Jonsen et al, 1996). The idea that the c-Myb protein undergoes a conformational change upon binding to DNA has been proposed before (Dubendor et al, 1992) and has been supported by the recent ®nding that the negative regulatory domain at the c-Myb C-terminus might actually compete for binding of a cellular cofactor to the N-terminus (Dash et al, 1996).…”

“…Such a conformational change upon binding to DNA has been proposed for other DNA binding proteins. Examples are the yeast transcriptional activator Mcm1 (Tan and Richmond, 1990) and the oncoprotein Ets-1 (Jonsen et al, 1996). To test the conformational change hypothesis we used electrophoretic mobility shift assays in combination with di erent Myb-speci®c antisera which could result in a supershift or the disappearance of the slower migrating Myb-DNA complex (Figure 5a and b).…”

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor

“…The cooperative inhibition observed between these domains is highly reminiscent of the inhibitory mechanism used by Ets-1. In Ets-1, a-helices located N-and Cterminally to the ETS-domain are thought to interact with each other and form an inhibitory module (Jonsen et al, 1996;Skalicky et al, 1996). Further studies are necessary to ®nd out whether a similar mechanism is employed by PEA3 subfamily members.…”

“…Binding of a second protein and/ or phosphorylation is also required by several ETSdomain proteins to relieve intramolecular inhibitory mechanisms and thereby permit high a nity DNA binding. In the case of Ets-1, the inhibitory mechanism, involving an allosteric change mediated by cooperating C-and N-terminal modules from outside the ETS-domain is well characterized (Jonsen et al, 1996;Skalicky et al, 1996) although the regulatorỳ switch' is unknown. Further studies on the DNA binding properties of other family members are required to establish whether they possess distinct binding speci®cities and autoregulatory mechanisms.…”

A variant form of ETS1 induces apoptosis in human colon cancer cells

“…Both 42 kDa and 51 kDa ETS1 bind to DNA with similar a nity and dissociate at di erent rates (Fisher et al, 1994). P42-ETS1 lacks the inhibitory domain involved in the intramolecular folding (Wasylyk et al, 1992;Jonsen et al, 1996). Furthermore, phosphorylation of ETS1 has been shown to inhibit its DNA binding properties (Rabault and Ghysdael, 1995;Fisher et al, 1994).…”