Abstract
The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism. Many transcription factors are controlled by cis-acting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain. A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative manner. Moreover, the formation of ternary PEA3.USF-1.DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly.
Highlights
Eukaryotic transcription factors are grouped into families based on the type of DNA-binding domains that they possess
Mapping and Characterization of the Autoinhibitory Cisacting Modules in PEA3—DNA binding by members of the PEA3 subfamily of ETS-domain transcription factors has previously been shown to be regulated by autoinhibitory mechanisms (6 – 8)
Deletion to amino acid 432 did not lead to any further increases in DNA binding, simultaneous deletion of inhibitory domain II led to a further enhancement of DNA-binding activity (Fig. 1B, lanes 7 and 8)
Summary
Eukaryotic transcription factors are grouped into families based on the type of DNA-binding domains that they possess. Mechanisms must be in place to prevent promiscuous binding to inappropriate sites and provide specificity to ensure binding to the correct sites One such mechanism is autoinhibition, whereby proteins block their own DNA binding by employing cis-acting inhibitory modules (reviewed in Ref. 1). Sequences flanking this motif permit subtle variations in binding specificities for individual family members Due to this relatively low stringency of binding, many ETS-domain transcription factors possess autoinhibitory domains, including Elk-1 (4), PU. (5), and members of the PEA3 subfamily (6, 7, 8). The PEA3 group of ETS-domain transcription factors contains three different proteins, PEA3/ E1AF, ERM, and ER81 (reviewed in Ref. 21) Members of this group of proteins are conserved at both the sequence and functional levels in vertebrates as diverse as humans and zebrafish (6, 22). Our results point to an intriguing model in which the regions that autoregulate DNA binding by PEA3 participate in cooperative DNA binding with the partner protein USF-1, thereby coordinately regulating the loss of autoinhibition, with the stimulation of nucleoprotein complex formation
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