Fig. 7. Evidence that MNR2 can function as a transcriptional repressor. (A) Deletion isoforms of MNR2 tested for Lim3-inducing activity in vivo. Approximate activity of these proteins, assessed by induction of Lim3⁺ cells, is indicated on the right, by comparison with wild-type MNR2. Electroporation of each of these constructs was performed at stage 10-12, and stage 25 embryos were analyzed for expression of Lim3. +++, more than 20 Lim3⁺ cells per 12 µM section; ++, 10-12 Lim3⁺ cells per section; +, 10 cells per section; –, no ectopic Lim3⁺ cells per section. Six to ten embryos were analyzed for the activity of each construct. (B) Deletion of the C-terminal domain of MNR2 does not abolish Lim3 inductive activity. Expression of the MNR2 HD alone has no inductive activity. (C) The N-terminal domain of MNR2 functions as a potent transcriptional repressor when fused to a Gal4 DNA-binding domain in vitro. The repressive activity of the MNR2 domain is similar to that of a Gal4-Engrailed repressor domain fusion. A MyoD-Gal4 DNA-binding domain fusion acts as a potent transcriptional activator. COS-1 cells were co-transfected with Gal4-fusion constructs and a Gal4-E1b-luciferase reporter plasmid. In controls, a Gal4-MyoD construct activated E1b-luciferase activity 19.2±3.6-fold (s.e.m.) overexpression of Gal4 alone. A Gal4-EnR construct repressed E1b-luciferase activity 7.7±4.5-fold (s.e.m.). A fusion of Gal4 to protein sequence N-terminal to the MNR2 HD repressed E1b-luciferase activity 8.3±1.3-fold (s.e.m.). (D) Detection of an eh1 domain in all Mnx class HD proteins. MNR2 protein sequence is represented as a black bar. The HD is depicted as a white box and an N-terminal eh1 motif is depicted with a gray box. The sequence containing the eh1 motif in MNR2 has been aligned with other Mnx class HD proteins to illustrate the high degree of conservation of this motif. An alignment with the eh1 motif in Engrailed reveals several identical residues, highlighted by black circles. Eh1 motifs characterized in Nkx proteins are aligned below Engrailed. Black circles below the Nkx6.1 sequence highlight residues in either Nkx2.2 or Nkx6.1 that are identical to Engrailed. Black diamonds above MNR2 and below Nkx6.1 highlight acidic residues conserved between Mnx class and Nkx HD proteins. cMNR2, chick MNR2; hHB9, human HB9; AmphiHB9, Amphioxus homolog of HB9; dHB9, Drosophila homolog of HB9; dEn, Drosophila Engrailed; mNkx2.2, mouse Nkx2.2; rNkx6.1, rat Nkx6.1. (E) Expression of an MNR2 isoform lacking the 14 N-terminal residues retains wild-type Lim3 inducing activity. (F) Negligible Lim3 inductive activity of an MNR2 HD-Engrailed repressor domain fusion protein. Images are representative of over 10 electroporated embryos.