Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

Auto-inhibition modulates a variety of transcription factor activities (21). In this regulatory mechanism, inhibitory sequences act in cis to repress such functions as DNA binding, transcriptional activation, nuclear localization, and ligand interaction. The picture emerging from the study of several transcription factors suggests that protein partnerships can counteract auto-inhibition. For example, serum response factor counteracts the auto-inhibition of Elk-1 DNA binding (36). The DNA binding of Pip, which contains an auto-inhibitory domain, requires interactions with PU.1 (9), and Pbx activates the DNA binding of its partner protein Hoxb (10, 11). Similarly, DNA binding by the yeast repressor a1 requires formation of a ternary complex with α2 (63). Examples within basal transcriptional machinery include the auto-inhibition of the ς subunit of Escherichia coli RNA polymerase. Interaction with core RNA polymerase counters this inhibitory effect (13). Also, the inhibitory function of the amino terminus of TATA-binding protein is abrogated by interactions with SNAPc (40). In each of these DNA binding scenarios, auto-inhibition generates a tighter control switch from the off to on state. To decipher the molecular basis of the interplay between auto-inhibition and protein partnerships, we focus on Ets-1, for which auto-inhibition is well characterized at the mechanistic and structural levels (21). Auto-inhibition, together with protein partnerships, can provide specificity within a family of highly related transcription factors (22). Ets-1 belongs to the ets gene family, which includes over 50 genes throughout the metazoa. The ets proteins display common DNA binding properties due to conservation in the ETS domain (22, 56). Nevertheless, each ets protein appears to have a specific function, presumably by regulating unique target genes. For example, Ets-1 is in the mesodermal compartment of several tissues in the mouse embryo (33) and in T and B lymphocytes, natural killer cells, endothelial cells, and the brain of the adult mouse (4, 7, 17, 18, 73). Targeted disruption of ets-1 in the mouse results in abnormal T- and B-cell function, as well as defective natural killer cell development (4, 8, 42). However, there are other ETS domain proteins found in these tissues. For example, T lymphocytes contain the ets proteins TEL, PU.1, Elf-1, Fli-1, Erg, Ets-2, and GABP, in addition to Ets-1 (1). Thus, some level of control beyond DNA binding must determine specificity. Auto-inhibition of DNA binding provides a regulatory framework with which to generate this specificity. We have developed a structural and mechanistic model of Ets-1 auto-inhibition based on biophysical and biochemical studies (21) (Fig. ​(Fig.1).1). Ets-1 binds DNA through the ETS domain, which consists of three α helices and four β strands folded into a winged helix-turn-helix motif (5, 15, 32, 41, 72). Two regions that flank the ETS domain work together to repress the DNA binding activity of Ets-1. Deletion of either region leads to derepression, resulting in DNA binding affinity that is 10- to 20-fold higher than that measured for full-length Ets-1 (25, 27, 35, 51, 71). Nuclear magnetic resonance analyses indicate that the flanking inhibitory regions consist of three α helices, HI-1 and HI-2 amino terminal and H4 carboxyl terminal to the ETS domain (15, 59). These data support a model in which the three inhibitory helices and H1 of the ETS domain pack together in a four-helix bundle to form an inhibitory module. Upon DNA binding, helix HI-1 unfolds, causing a disruption of the inhibitory module (27, 51). We have proposed that this conformational change is responsible for the relatively low DNA binding affinity of the native Ets-1. In this study, we tested whether a partnership with a second DNA binding protein counteracts the auto-inhibition of Ets-1. FIG. 1 Ets-1 domains and conformational change. (A) Schematic of the structural and functional domains of full-length Ets-1, Ets-1ΔN280, and Ets-1ΔN331. The asterisk indicates the mutation in helix H4 of Ets-1ΔN280;L429A. (B) Schematic ... We have chosen the Ets-1–core-binding factor (CBF) partnership as a model system. CBF represents a small family of heterodimeric proteins (62). Three genes in vertebrate genomes (in humans, CBFA1, CBFA2 [AML1], and CBFA3) encode α subunits that contain a conserved DNA binding domain, termed the Runt domain (28). A single vertebrate gene, CBFB, encodes the non-DNA-binding subunit, CBFβ (2, 3, 28, 34, 48, 49, 70). CBFα2 is found in most hematopoietic lineages and is required for hematopoiesis (45, 46, 50, 53, 54, 68, 69). Thus, both Ets-1 and CBF are important proteins in mammalian immune system development and function. Experimental approaches demonstrate the functionality of the Ets-1 and CBF partnership. Several cellular and viral enhancers contain adjacent Ets-1 and CBF binding sites, including the T-cell receptor (TCR) α and β enhancers, immunoglobulin μ heavy-chain enhancer, osteopontin promoter, and enhancers of the polyomavirus and Moloney murine leukemia virus (Mo-MLV) (16, 24, 29, 39, 55, 74). Mutation of the Ets-1 and CBF binding sites in the Mo-MLV enhancer reduces viral enhancer activity and alters viral disease specificity (60, 61). In vivo footprinting assays show occupancy of the Mo-MLV Ets-1 and CBF binding sites in T cells, thus confirming the in vivo function of these adjacent sites (20). Ets-1 and members of the CBF family synergistically activate the TCRα and TCRβ enhancers, the immunoglobulin μ heavy-chain enhancer, the osteopontin promoter, as well as the Mo-MLV enhancer in vivo (16, 19, 31, 39, 55, 65, 74). Cooperative DNA binding between Ets-1 and CBFα2 is detected in in vitro studies of the TCRα and TCRβ enhancers (19, 31, 39, 74). Furthermore, the accompanying report quantifies the level of cooperativity and demonstrates that enhancement of Ets-1 and CBFα2 DNA binding is reciprocal (23). These findings suggest that DNA binding cooperativity is a mechanism for transcriptional synergy. This study links the auto-inhibition phenomenon of Ets-1 to the DNA binding cooperativity of the Ets-1 and CBFα2 partnership. The regions of Ets-1 necessary for cooperativity and auto-inhibition overlap. In addition, disruption of the inhibitory module abrogates the effect of CBFα2 on Ets-1 DNA binding. Finally, the binding sites can be topologically uncoupled and widely spaced, suggesting that through-DNA effects do not mediate cooperativity. These findings support a mechanistic model of the Ets-1 and CBF protein partnership in which protein interactions counteract the auto-inhibition mechanism.