Functional Comparison of Egr3 Transcription Factor Isoforms: Identification of an Activation Domain in the N-Terminal Segment Absent from Egr3, a Major Isoform Expressed in Brain *Kevin J. O’Donovan, *Yechiel Levkovitz, *David Ahn, and *²Jay M. Baraban Departments of *Neuroscience and ² Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Abstract: Recent studies indicate that the Egr family of transcription regulatory factors plays a key role in ner- vous system development and plasticity. In prior studies, we demonstrated that multiple isoforms of the Egr3 tran- scription regulatory factor are expressed in brain and appear to be generated by use of alternative translation start sites. To compare the functional activity of these isoforms, we have examined their ability to stimulate transcription of a luciferase reporter construct driven by the Egr response element. Analysis of a series of N- terminal truncation constructs indicates that Egr3 con- tains two distinct activation domains: one located in the segment upstream of Met 106 , the start site of the major truncated isoform Egr3, and the other located C-termi- nal to all of the alternative translation start sites used to generate Egr3 isoforms detected in brain. We confirmed this inference by demonstrating that each of these seg- ments is able to drive transcription when fused to the GAL4 DNA binding domain. Taken together, these stud- ies indicate that the internal translation start sites present in Egr3 are used to generate Egr3 isoforms lacking the activation domain located N-terminal to Met 106 . Key Words: Egr family—Egr1—NAB1—Alternative translation start sites. J. Neurochem. 75, 1352–1357 (2000). Recent studies have emphasized the importance of the Egr family of transcription regulatory factors in nervous system development and plasticity (for review, see O’Donovan et al., 1999). Targeted deletion of the Egr2 gene causes severe disruption of hindbrain formation and peripheral nerve myelination (Wilkinson et al., 1989; Schneider-Manoury et al., 1993; Topilko et al., 1994), whereas the absence of Egr3 blocks formation of muscle spindles (Tourtellotte and Milbrandt, 1998). In addition to their critical role in early development, members of this transcription factor family are also robustly induced in adult brain neurons by synaptic or pharmacological stimulation (Moratalla et al., 1992; Bhat and Baraban, 1993). The demonstration that members of this family are induced in hippocampal neurons by long-term poten- tiation stimulation (Cole et al., 1989; Wisden et al., 1990; Worley et al., 1993; Yamagata et al., 1994; Williams et al., 1995) as well as in other synaptic or behavioral plasticity paradigms (Wallace et al., 1995; Okuno and Miyasita, 1996; Lin et al., 1997; Morris et al., 1998) has heightened interest in defining how these transcription factors regulate gene expression. The Egr family contains four members that share a highly conserved zinc finger DNA binding domain (DBD) located in the C-terminal portion of these proteins (Pavletich and Pabo, 1991; Crosby et al., 1992; Gashler and Sukhatme, 1995; Swirnoff and Milbrandt, 1995). In contrast, their N-terminal regions have diverged consid- erably. As part of an ongoing effort to understand the role of the Egr family in the brain, we have, in previous experiments, used gel-shift analysis to characterize the expression of Egr family members in hippocampus (O’Donovan et al., 1998). In those studies, we detected a previously unidentified Egr family gel-shift complex. Analysis of this complex revealed that it contains a truncated isoform of Egr3, Egr3, that is generated by use of an alternative translation start site located at Met 106 (O’Donovan and Baraban, 1999). Higher-resolu- tion analysis of the Egr3 gel-shift complexes revealed that in addition to the major band corresponding to Egr3, there were additional bands that correspond to distinct Egr3 isoforms generated by use of other internal start sites. Received April 11, 2000; revised manuscript received May 10, 2000; accepted May 11, 2000. Address correspondence and reprint requests to Dr. J. M. Baraban at Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, U.S.A. E-mail: jbaraban@jhmi.edu Abbreviations used: CMV, cytomegalovirus; DBD, DNA binding domain; ERE, Egr response element; -Gal, -galactosidase; HEK- 293, human embryonic kidney 293. 1352 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry