PMC:1084331 / 6487-7752 JSONTXT

We first defined an ETS transcription regulator that is able to replace the function of Er81 within proprioceptive afferents to direct projections into the ventral spinal cord. Er81, Pea3, and Erm constitute the Pea3 subfamily of ETS transcription factors, show a high degree of amino acid identity, and bind to very similar DNA target sequences [17,18,19]. Nevertheless, when introduced into the Er81 locus in analogy to a previously used targeting strategy (data not shown; [14]), neither Pea3 nor Erm could rescue Ia proprioceptive afferent projections to extensively invade the ventral horn of the spinal cord (data not shown). These findings prompted us to analyze mice in which we integrated EWS-Pea3, a break-point fusion product between the amino-terminal domain of the Ewing sarcoma (EWS) gene and the Pea3 DNA binding domain [20,21], into the Er81 locus (Figure 1). We found that in a luciferase-enzyme-based cell culture transfection assay, EWS-Pea3 showed stronger transactivation activity than Er81 or Pea3 (Figure 1J; data not shown), in agreement with previous studies [22,23,24]. Moreover, transactivation by EWS-Pea3 was abolished by mutation of ETS-binding sites in the reporter plasmid, demonstrating ETS-binding-site dependence (data not shown).

We first defined an ETS transcription regulator that is able to replace the function of Er81 within proprioceptive afferents to direct projections into the ventral spinal cord. Er81, Pea3, and Erm constitute the Pea3 subfamily of ETS transcription factors, show a high degree of amino acid identity, and bind to very similar DNA target sequences [17,18,19]. Nevertheless, when introduced into the Er81 locus in analogy to a previously used targeting strategy (data not shown; [14]), neither Pea3 nor Erm could rescue Ia proprioceptive afferent projections to extensively invade the ventral horn of the spinal cord (data not shown). These findings prompted us to analyze mice in which we integrated EWS-Pea3, a break-point fusion product between the amino-terminal domain of the Ewing sarcoma (EWS) gene and the Pea3 DNA binding domain [20,21], into the Er81 locus (Figure 1). We found that in a luciferase-enzyme-based cell culture transfection assay, EWS-Pea3 showed stronger transactivation activity than Er81 or Pea3 (Figure 1J; data not shown), in agreement with previous studies [22,23,24]. Moreover, transactivation by EWS-Pea3 was abolished by mutation of ETS-binding sites in the reporter plasmid, demonstrating ETS-binding-site dependence (data not shown).