In this study, we conducted experiments aimed at identifying a major ERE binding complex expressed in the brain, defining how it is generated, and understanding its role in regulating ERE-mediated transcription. In the first phase of the study, we confirmed our hypothesis that the unidentified ERE complex contained a truncated form of Egr3 missing the N terminus. This conclusion is based on its absence from extracts prepared from Egr3 knockout mice as well as studies with an antibody directed to an internal segment of Egr3. In the second phase of the study, we obtained evidence that the shorter isoform is generated by utilization of an internal translation start site located at M106 and that additional isoforms of Egr3 are generated by utilization of other Met residues as well. In the last phase of this investigation, we compared the functional activities of Egr3 isoforms that contain or lack the N-terminal segment and found that they display distinct transactivation properties.
Although we initially identified multiple Egr3 isoforms in hippocampal extracts, gel shift studies on extracts prepared from other brain regions indicate that they express a similar array of truncated isoforms. In addition, heterologous expression of Egr3 in hEK-293 cells generated a pattern of Egr3 binding complexes that was indistinguishable from the pattern displayed in the brain. In the periphery, Egr3 is induced by T-cell activation (23
). In preliminary studies, we have detected a similar pattern of Egr3 expression in a murine T-cell hybridoma, 2B4.11. Accordingly, coexpression of full-length and truncated Egr3 isoforms appears to be a general feature of this transcript.
Previous in situ hybridization studies have demonstrated induction of all four Egr family members in the hippocampus following electroconvulsant stimulation (2
). Thus, it is surprising that all of the major ERE binding complexes observed in gel shift studies contain only Egr1 and Egr3. The absence of prominent Egr2 and Egr4 binding complexes in these assays could in principle reflect low levels of protein expression or inhibitory factors that block binding activity. In any case, these findings suggest that Egr1 and Egr3 are the dominant family members mediating ERE-driven transcription in the hippocampus.
The demonstration that mutations targeting individual Met residues block expression of Egr3 isoforms provides compelling evidence that they are generated by utilization of internal translation start sites. Although viruses commonly utilize this mechanism to express variant proteins, it has been seldom implicated with regard to cellular proteins (32
). A priori initiation at internal start sites can be attributed either to internal entry of the ribosomal complex, instead of its attachment to the 5′ cap, or to leaky ribosomal scanning, i.e., ignoring potential start sites presumably because the flanking sequence deviates from the rules governing faithful initiation. As shown in Table , the leaky scanning mechanism may apply to Egr3. However, it is unclear if this fully accounts for the ability of the translation initiation complex to skip the first Met. Substituting nucleotides at key sites surrounding the initial start codon to comply with these rules enhanced usage of the Egr3 initial Met but did not prevent generation of truncated products.
TABLE 1 Nucleotide sequences surrounding Egr3 initiationsitesa
Although alternative splicing is widely recognized as a mechanism used to generate multiple protein isoforms from a single transcription factor gene, only a few instances in which alternative translation start sites are utilized have been reported. Interestingly, two members of the C/EBP family, C/EBPβ (9
) and C/EBPα (30
), employ this strategy to generate truncated versions with markedly different transcriptional properties. As the splicing mechanism is not available to these intronless genes, the alternative translation start site strategy appears to be used instead. This teleological rationale presumably applies to Egr3 as well, since it contains only two exons, as found for all Egr family genes (8
Our comparison of the functional activities of Egr3 isoforms that retain or lack the N-terminal segment indicates that deletion of this segment has little effect on Egr3’s ability to stimulate transcription of genes containing a single copy of the ERE in their promoter. In contrast, deletion of the N-terminal segment impairs its ability to stimulate transcription when two copies of the ERE are present. Accordingly, the N-terminal domain appears to play an important role in boosting expression of genes containing multiple EREs. Based on these findings, it is tempting to speculate that altering the ratio of Egr3α and Egr3β isoforms may provide a mechanism for modulating Egr target genes with multiple EREs such as platelet-derived growth factor A chain (15
), prohormone convertase 2 (13
), and synapsin I (36
) without affecting those containing a single ERE, e.g., platelet-derived growth factor B chain (14
), transforming growth factor (10
), luteinizing hormone beta chain (18
), and FasL (23
). In this scenario, shifts in the functional activities of full-length and truncated Egr3 isoforms could be achieved directly by changes in their protein levels. Indeed, preliminary findings (26a
) suggest that there is some developmental regulation of the pattern of Egr3α isoform expression. Egr3α1 is present at relatively high levels in embryonic day 17 rat cortex, and its levels decrease to near adult levels by postnatal day 8. In contrast, Egr3α2 DNA binding activity is low in embryonic day 17 cortex but its levels are significantly increased in postnatal day 8 cortex. Alternatively, as the N-terminal truncation does not impinge on the domain mediating suppression of Egr3 by NAB proteins (31
) located just N terminal to the DNA binding domain, it is conceivable that changes in the relative sensitivities of full-length and truncated Egr isoforms to NAB proteins could be used to alter their activities.
Although the Egr family DNA binding domain has been extensively characterized, little is known about how Egr family members act to stimulate the transcriptional apparatus. Our initial analysis of naturally occurring truncations of Egr3 has revealed interesting differences in the activation properties of these isoforms and underscores the need to conduct more extensive studies aimed at defining the activation domains contained within these proteins and how they interact with the transcriptional apparatus. Progress in this direction will be helpful in elucidating the functional significance of the Egr3 isoforms expressed in vivo as well as in understanding how the Egr family orchestrates changes in gene expression in response to cellular stimulation.